2 material und methoden - opus 4 · at least three glnr binding sites were identified in the...

The GlnR-dependent

nitrogen regulatory network

of

Mycobacterium smegmatis

Der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität Erlangen-Nürnberg

zur

Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von

Nadja Jeßberger

aus Werneck

Das GlnR-abhängige

Netzwerk zur Stickstoffregulation

in

Mycobacterium smegmatis

Der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität Erlangen-Nürnberg

zur

Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von

Nadja Jeßberger

aus Werneck

Als Dissertation genehmigt

von der Naturwissenschaftlichen Fakultät

der Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 28.07.2011 Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink Erstberichterstatter: Prof. Dr. Andreas Burkovski Zweitberichterstatter: Prof. Dr. Yves Muller

Danksagung

Die vorliegende Arbeit wurde am Lehrstuhl für Mikrobiologie der Friedrich-Alexander

Universität Erlangen-Nürnberg durchgeführt.

Mein besonderer Dank gilt Herrn Professor Andreas Burkovski für die Bereitstellung des

interessanten Arbeitsthemas und die kompetente Betreuung. Weiterhin bedanke ich mich bei

der Universität Bayern e.V. für die Finanzierung der Arbeit.

Herrn Professor Yves Muller möchte ich für die freundliche Übernahme des Zweitgutachtens

danken.

Ein ganz herzlicher Dank gilt Frau Doktor Sophia Sonnewald und vor allem Stephen Reid

vom Lehrstuhl für Biochemie für die Durchführung der microarray Experimente. Ich möchte

mich auch besonders bei Amon für die vielen Ratschläge und Diskussionen bedanken.

Ein riesengroßens Dankeschön geht natürlich an die Mädels aus dem Labor: Kristin, die wir

alle sehr vermissen, Lisl, die kleine Eva und Naddel4-∞ für eine unglaublich tolle

Arbeitsatmosphäre und das gemeinsame Erledigen von jeder Menge „Unikram“. Eva, ein

ganz besonderer Dank für deine Tapferkeit und dein Durchhalten beim Thema

Sensorhistidinkinasen.

Meinen Eltern danke ich für die große Unterstützung während des Biologiestudiums und der

gesamten Doktorandenzeit.

Mein größter Dank gilt Mikko, der vor allem in der letzten Zeit sämtliche Anfälle von mir

ertragen musste und sich dann auch noch durch die gesamte Arbeit gequält hat. Paljon

kiitoksia!!!

Content

Content

1 Zusammenfassung/Summary ............................................................................... 1

2 Introduction ............................................................................................................ 3

2.1 The genus Mycobacterium ........................................................................................... 3

2.1.1 Taxonomy and characteristics .............................................................................. 3

2.1.2 Mycobacterium smegmatis ................................................................................... 6

2.2 Nitrogen metabolism and regulation ............................................................................ 8

2.2.1 Uptake and assimilation of nitrogen sources ......................................................... 8

2.2.2 Nitrogen control in Escherichia coli ....................................................................... 9

2.2.3 Nitrogen control in actinomycetes ........................................................................11

2.3 Aims of this study .......................................................................................................16

3 Materials and methods ........................................................................................ 17

3.1 Bacterial strains and plasmids ....................................................................................17

3.2 Cultivation of bacteria .................................................................................................21

3.2.1 Culture media for E. coli, C. glutamicum and M. smegmatis ................................21

3.2.2 Antibiotics ............................................................................................................22

3.2.3 Growth conditions ................................................................................................23

3.3 Procedures in molecular biology .................................................................................24

3.3.1 Procedures to work with DNA ..............................................................................24

3.3.1.1 Isolation of plasmid DNA from E. coli ............................................................24

3.3.1.2 Isolation of plasmid DNA from M. smegmatis ................................................24

3.3.1.3 Gel electrophoresis and extraction of DNA from agarose gels ......................24

3.3.1.4 Preparation of chromosomal DNA from M. smegmatis ..................................25

3.3.1.5 Polymerase chain reaction (PCR) .................................................................26

3.3.1.6 Two-step PCR ...............................................................................................27

3.3.1.7 Purification and enrichment of DNA ..............................................................27

3.3.1.8 Restriction of DNA.........................................................................................27

3.3.1.9 Ligation of DNA fragments ............................................................................28

3.3.1.10 Sequencing of DNA .....................................................................................28

3.3.1.11 Gel retardation and competition assays ......................................................29

3.3.1.12 Electrophoretic mobility shift assay (EMSA) ................................................31

3.3.1.13 Southern blot analysis .................................................................................32

3.3.2 Procedures to work with RNA ..............................................................................34

3.3.2.1 Isolation of total RNA from M. smegmatis .....................................................34

3.3.2.2 RNA gel electrophoresis ...............................................................................35

3.3.2.3 Synthesis of digoxigenin-labeled RNA probes ...............................................35

3.3.2.4 RNA hybridization analysis (Dot blot) ............................................................36

3.3.2.5 Quantitative real time RT PCR ......................................................................37

3.3.2.6 cDNA microarrays .........................................................................................39

Content

3.3.3 Procedures to work with proteins .........................................................................39

3.3.3.1 Preparation of total cell extract ......................................................................39

3.3.3.2 Protein purification via affinity chromatography .............................................40

3.3.3.3 Quantification and enrichment of proteins .....................................................41

3.3.3.4 SDS polyacrylamide gel electrophoresis .......................................................42

3.3.3.5 Native polyacrylamide gel electrophoresis ....................................................43

3.3.3.6 Staining with Coomassie Brilliant Blue ..........................................................44

3.3.3.7 Western blot analysis ....................................................................................44

3.3.3.8 Pull down assays ..........................................................................................45

3.3.3.9 Radioactive phosphorylation of proteins ........................................................46

3.4 Genetic manipulation of bacteria.................................................................................47

3.4.1 Preparation of competent E. coli cells ..................................................................47

3.4.2 Transformation of competent E. coli cells .............................................................47

3.4.3 Preparation of competent E. coli Rosetta2 cells and TSS transformation .............48

3.4.4 Preparation of electrocompetent M. smegmatis cells ...........................................48

3.4.5 Transformation of electrocompetent M. smegmatis cells ......................................49

3.4.6 Generation of genomic insertion and deletion mutants of M. smegmatis ..............49

3.4.7 Fluorescence measurements ...............................................................................50

3.4.8 Fluorescence microscopy ....................................................................................50

3.4.9 β-galactosidase assays ........................................................................................51

3.4.10 Determination of urease activity .........................................................................52

4 Results .................................................................................................................. 53

4.1 General analyses of nitrogen metabolism and control in M. smegmatis ......................53

4.1.1 Utilization of different nitrogen sources ................................................................53

4.1.2 Relation between growth behavior and initiation of nitrogen response .................61

4.1.3 Uptake and assimilation of urea ...........................................................................64

4.1.4 Generation of an in vivo reporter system for nitrogen response ...........................65

4.1.5 Purification of GlnR ..............................................................................................68

4.1.6 Generation of a GlnR-specific antibody ................................................................71

4.1.7 DNA binding ability of purified GlnR .....................................................................72

4.2 Characterization of the GlnR regulon ..........................................................................73

4.2.1 Global approach using DNA microarray analyses ................................................73

4.2.2 Verification of the microarray data ........................................................................83

4.2.3 Binding of GlnR to promoter sequences of its target genes ..................................89

4.2.4 Determination of binding properties of GlnR ........................................................91

4.2.5 Autoregulation of GlnR .........................................................................................96

4.3 Activation of GlnR .......................................................................................................97

4.3.1 Search for GlnR interaction partner ......................................................................98

4.3.1.1 Global pull down analysis ..............................................................................98

4.3.1.2 Analyses of deletion and insertion mutants ...................................................99

Content

4.3.1.3 Correlation between phosphate and nitrogen metabolism ........................... 104

4.3.1.4 Analyses of protein-protein interactions and phosphorylation of GlnR ......... 105

4.3.2 Investigation of phosphorylation residues .......................................................... 112

4.4 Nitrate and nitrite metabolism in M. smegmatis ......................................................... 118

4.5 Investigation of a conserved mode of action of GlnR in mycobacteria ...................... 124

5 Discussion ......................................................................................................... 128

5.1 GlnR as the global regulator of nitrogen metabolism in M. smegmatis ...................... 128

5.1.1 GlnR-dependent utilization of different nitrogen sources .................................... 128

5.1.2 Characterization of the GlnR regulon ................................................................. 131

5.2 GlnR as OmpR-type transcriptional regulator............................................................ 138

5.3 Activation of nitrogen response ................................................................................. 142

5.4 Ammonium assimilation in M. smegmatis ................................................................. 146

5.5 Nitrate and nitrite metabolism in M. smegmatis ......................................................... 149

5.6 Role of AmtR in M. smegmatis.................................................................................. 152

5.7 Nitrogen response in M. smegmatis as a possible model for M. tuberculosis ............ 154

6 References ......................................................................................................... 157

7 Appendix ............................................................................................................ 173

7.1 Plasmid constructions ............................................................................................... 173

7.2 Complete data obtained from DNA microarray analyses ......................................... 1800

7.3 Putative sensor histidine kinases activating GlnR ..................................................... 193

7.4 Abbreviations and units ............................................................................................ 195

7.5 Publications .............................................................................................................. 199

Zusammenfassung 1

1 Zusammenfassung

Mycobacterium smegmatis ist ein saprophytisch lebender und schnell wachsender Vertreter

der Gattung Mycobacterium und wird oft als Modellorganismus für verwandte pathogene

Spezies wie Mycobacterium tuberculosis verwendet. Zentrales Thema dieser Arbeit war der

Stickstoffmetabolismus in M. smegmatis, insbesondere die Antwort auf Stickstoffmangel auf

transkriptioneller Ebene, wobei der Regulator GlnR im Mittelpunkt der Betrachtungen stand.

Es wurde gezeigt, dass das Bakterium verschiedenste Substrate (Aminosäuren, Basen und

anorganische Stickstoffverbindungen) als Stickstoffquellen nutzen kann. Analysen eines

glnR Deletionsstammes verdeutlichten, dass GlnR maßgeblich an der Verwertung einiger

dieser Substrate beteiligt ist. Darüber hinaus wurden durch Transkriptomanalysen 125 neue

putative GlnR-Zielgene identifiziert. In darauffolgenden Experimenten wurde für 32 dieser

Gene eine GlnR-abhängige Steigerung der Transkriptmenge bestätigt, darunter Gene, die an

der Aufnahme sowie Assimilation von Aminosäuren, Peptiden und anorganischen

Stickstoffverbindungen beteiligt sind. Dies verdeutlicht die Rolle von GlnR als globalen

Stickstoffregulator in M. smegmatis. Weiterhin standen auch Gene, die nicht dem

Stickstoffmetabolismus angehören, unter GlnR-Kontrolle, was auf eine umfassendere

Funktion des Regulators hindeutet. Außerdem wurde eine untergeordnete Rolle von AmtR in

der Regulation einiger stickstoffrelevanter Gene beobachtet.

Zusätzlich wurden verschiedene Methoden zur Reinigung von GlnR etabliert und in vitro

Untersuchungen zum DNA-Bindeverhalten des gereinigten Proteins durchgeführt, wobei eine

direkte Bindung an Promotorfragmente der Hälfte aller identifizierten Zielgene beobachtet

wurde. Dies führte zu dem Schluss, dass auch indirekte GlnR-Kontrolle über weitere

Transkriptionsregulatoren oder bisher unbekannte Mechanismen eine Rolle für die

Expression einiger Gene spielt. Außerdem wurden in der Promotorregion von amtB

wenigstens drei GlnR-Bindestellen ausgemacht, was das Modell der „galoppierenden“ DNA-

Bindung von OmpR aus E. coli unterstützt. Als ein Vertreter der OmpR-Familie von

Transkriptionsregulatoren soll GlnR durch eine zugehörige Sensorhistidinkinase

phosphoryliert werden. Obwohl diese im Rahmen dieser Arbeit nicht ermittelt werden konnte,

wurden hochkonservierte Aspartatreste als Phosphorylierungsstellen von GlnR identifiziert.

Erste Experimente mit M. smegmatis und M. tuberculosis GlnR deuteten trotz der

identischen C-terminalen DNA-Bindedomänen und N-terminalen Phosphorylierungsdomänen

der beiden Proteine auf klare Unterschiede in der DNA-Bindung und der Aktivierung der

Stickstoffantwort hin. Dennoch kann das in dieser Arbeit entwickelte Modell der

Stickstoffkontrolle in M. smegmatis durchaus als ein erstes Schema für M. tuberculosis

verwendet werden. .

Summary 2

1 Summary

Mycobacterium smegmatis is a saprophytic and fast-growing member of the genus

Mycobacterium and is often used as a model organism for related pathogenic species such

as Mycobacterium tuberculosis. Focus of interest in this study was nitrogen metabolism and

especially transcriptional response to nitrogen limitation in M. smegmatis, while the

regulatory protein GlnR was the focus of attention.

It was shown that the bacterium is able to use different substrates (amino acids, bases and

inorganic nitrogen compounds) as nitrogen sources. Analyses of a glnR deletion strain

revealed that GlnR is substantially involved in the utilization of several of these substrates.

Moreover, 125 new putative GlnR target genes were identified in transcriptome analyses. In

subsequent experiments, a GlnR-dependent increase of transcripts of 32 of these genes was

verified, including genes involved in uptake and assimilation of amino acids, peptides and

inorganic nitrogen sources. This confirms the role of GlnR as global nitrogen regulator in

M. smegmatis. Moreover, also genes not involved in nitrogen metabolism were identified as

GlnR targets, suggesting a more global regulatory function of GlnR. A GlnR subordinate role

in regulation of some genes involved in nitrogen metabolism was also monitored for AmtR.

In addition to that, different methods for purification of GlnR were established and in vitro

analyses of DNA-binding behavior of the purified protein were carried out. Direct binding to

promoter regions of half of the identified target genes was monitored, leading to the

conclusion that indirect GlnR-control via further transcriptional regulators or so far unknown

mechanisms is also involved in gene expression. At least three GlnR binding sites were

identified in the promoter region of amtB, supporting the idea of a “galloping” DNA binding

model which is described for E. coli regulator OmpR. As a member of the OmpR-family of

transcriptional regulators, GlnR is supposed to be phosphorylated by a corresponding sensor

histidine kinase. Although this kinase could not be identified within this study, highly

conserved aspartate residues were distinguished as phosphorylation sites of GlnR.

First experiments with M. smegmatis and M. tuberculosis GlnR pointed to distinct differences

in DNA binding and activation of nitrogen response, although the two GlnR proteins share

identical C-terminal DNA binding and N-terminal phosphorylation domains. Nevertheless, the

model of nitrogen control in M. smegmatis developed in this study can indeed be used as a

first pattern for similar processes in M. tuberculosis.

Introduction 3

2 Introduction

2.1 The genus Mycobacterium

2.1.1 Taxonomy and characteristics

Within the order Actinomycetales five different phylogenetic families exist. Actinomycetes are

generally characterized by their filamentous, thready and branched growth, which can be

compared to a fungal mycelium. Their name is derived from Greek aktis (ray) and mykes

(fungus). The five families are indicated as Actinomycetaceae, Corynebacteriaceae,

Dermatophilaceae, Nocardiaceae and Mycobacteriaceae. The only genus in the family

Mycobacteriaceae is Mycobacterium, which contains more than 110 different species

(Hartmans et al., 2006). The first of these species to be identified was Mycobacterium

tuberculosis, which was discovered by the German physician Robert Koch in 1882 and

reported as “tubercle bacilli” (Philip, 1932). The name Mycobacterium was first used by

Lehmann and Neumann (1896). The securest method to quickly identify mycobacteria is the

acid-fast Ziehl-Neelsen stain. Here, cells are stained red by slow heating using the dye

fuchsin. After washing in H2O, cells are stained with methylene blue. Acid-fast organisms

such as mycobacteria occur red, non-acid-fast ones blue (Barksdale and Kim, 1977).

Apart from this, mycobacteria are generally characterized by their rod shape, the high GC

content of their DNA (60-70 %) and their rough, “mycelic” colony morphology (Ojha et al.,

2000). Furthermore, mycobacteria are considered Gram-positive, although they can hardly

be stained according to Gram. They are facultative aerobic, non-sporulating and non-motile

bacteria with a size of 0.2-0.6 x 1-10 µm (Traag et al., 2010). Moreover, they are resistant

against many antibiotics.

A closer look to the structure of the mycobacterial cell wall gives an explanation why these

organisms are hardly stained by Gram, why they are acid-fast and why they possess a high

resistance to antibiotics. The so called CMN group (corynebacteria, mycobacteria and

nocardia) are the only organisms known to be able to synthesize mycolic acids (Embley and

Stackebrandt, 1994; Rastogi et al., 2001) and developed a unique and very complex cell

envelope which is unusual for Gram-positive bacteria. The Gram-positive cell wall usually

consists of a thick peptidoglycan layer with associated teichoic acids lacking the outer

membrane of Gram-negative bacteria. In the mycobacterial cell wall mycolic acids, which are

groups of hydroxy lipids, are covalently bound to arabinogalactan (a polysaccharide

consisting of arabinose and galactose) complexes at the peptidoglycan layer. Mycolic acids

are large branched chains of 20-90 carbon atoms, which give the cell wall its typical thick,

Introduction 4

A

C

B

waxy and hydrophobic consistency (Barry et al., 1998). Although mycobacteria are classified

as Gram-positive, recent studies revealed the existence of a second, outer bilayer (figure 1).

This mycobacterial outer membrane (MOM) is about 8 nm thick, morphologically symmetrical

and organized in a native three-dimensional structure (Niederweis et al., 2010).

Fig. 1: The mycobacterial cell wall. A. Cryosectioning of the cell wall of Mycobacterium bovis. MOM: mycobacterial outer membrane. CM: cytoplasmic membrane. B. Cryo-electron tomography. Blue: arabinogalactan-peptidoglycan polymer between CM and MOM. C. Model of the mycobacterial cell wall. L1: unknown periplasmic layer. L2: part of the peptidoglycan-arabinogalactan polymer, partly unknown. Yellow: hydrophobic compounds. Blue: porin. Adapted from Niederweis et al. (2010).

A primary classification of mycobacteria was introduced by Ernest Runyon (1959), based on

growth rate, morphology and pigmentation of the bacteria. He separated fast growing

mycobacteria (group IV) with a generation time of less than five hours and formation of

visible colonies after less than seven days from slow growing ones. The slow growers were

described by having a generation time of up to 20 hours and by forming visible colonies only

after more than seven days. Furthermore, Runyon separated them again by their

pigmentation into photochromogens (pigmentation when exposed to light, group I),

scotochromogens (pigmentation in light or dark, group II) and nonphotochromogens (not

pigmented, group III). This separation into fast and slow growing organisms was proven by

sequence comparison of macromolecules. One of the most commonly used macromolecules

for phylogenetic analyses is the 16S ribosomal RNA because of its universal existence

(Rogall et al., 1990; Stahl et al., 1990). The affiliation of mycobacteria to the Gram-positive

branch of eubacteria was also supported with this method (Pitulle et al., 1992).

Introduction 5

Generally, fast growing mycobacteria are considered non- or facultative pathogenic. Most of

them are harmless inhabitants of soil or water living saprophytic from decomposed organic

material (Kamala et al., 1994). They are also known as the MOTT group of mycobacteria,

which stands for “mycobacteria other than tuberculosis”. Nevertheless, some species can

cause diseases to animals or humans with immune deficiencies. Some considered non-

pathogenic fast growers are Mycobacterium agri (Tsukamura et al., 1981), Mycobacterium

confluentis (Kirschner et al., 1992), Mycobacterium phlei (Lehmann and Neumann, 1899),

Mycobacterium smegmatis (Lehmann and Neumann, 1899) and Mycobacterium

thermoresistibile (Tsukamura, 1966). Pathogenic examples of this group are Mycobacterium

chelonae (Bergey et al., 1923), Mycobacterium fortuitum (Da Costa Cruz, 1938) and

Mycobacterium mucogenicum (Springer et al., 1995).

On the contrary, the group of slow growing mycobacteria is very often associated with or

even the cause of serious animal or human diseases. They are often obligatory pathogenic.

Most popular examples are Mycobacterium africanum (Castets et al., 1969), Mycobacterium

bovis (Karlsen and Lessel, 1970), Mycobacterium microti (Reed et al., 1957) and

M. tuberculosis (Lehmann and Neumann, 1896), which form the “Mycobacterium

tuberculosis complex”, as well as Mycobacterium leprae (Lehmann and Neumann, 1896).

There are also some members of the MOTT group among the slow growing mycobacteria,

which can cause serious diseases. These are e.g. Mycobacterium kansasii (Hauduroy, 1955)

and Mycobacterium marinum (Aronson, 1926). A few untypically non-pathogenic organisms

also exist in this group. These are e.g. Mycobacterium gastri and Mycobacterium terrae

(Wayne, 1966).

Of all mycobacteria, the biggest attention in research is paid to M. tuberculosis (figure 2A and

B). This bacterium with a generation time of 15-20 hours is extremely slow growing and

consequently difficult to investigate in vitro. M. tuberculosis is an intracellular parasite that is

easily transferred by respiration. After inhalation of M. tuberculosis, a primary infection starts

by settling and growth of the bacteria in the lungs. A subsequent immune response results in

accumulation of activated macrophages. M. tuberculosis is able to survive and grow within

modified phagosomes of macrophages (van Crevel et al., 2002). Because of the high lipid

content of its cell wall, it is very resistant to chemical agents and antibiotics. It blocks immune

response of macrophages to infection by preventing the formation of phagolysosomes

(Flannagan et al., 2009). In humans with weak immune resistance, the infection becomes

acute leading to an enormous destruction of lung tissue (figure 2C) followed by spread of the

bacteria over the whole body and finally death. In humans with good immune resistance, the

infection remains local and finally ends, but the patient keeps hypersensitivity against the

bacteria. Another exposure to M. tuberculosis can lead to chronic infection and destruction of

lung tissue (van Crevel et al., 2002). Over one third of the world’s population is infected with

Introduction 6

B C

tuberculosis, while 1.6 million humans die every year (Dye et al., 1999). The first treatment of

tuberculosis was achieved using the antibiotic streptomycin. Another very effective drug is

isoniazid, which blocks the synthesis of mycolic acids leading to cell wall instabilities and

finally to death of the bacteria (Engbaek et al., 1973). But with using these drugs, more and

more resistant M. tuberculosis strains appeared (Ozturk et al., 2005; Chaoui et al., 2009). For

that reason, research on M. tuberculosis is still of major priority.

Fig. 2: M. tuberculosis. A. Scanning electron microscopy (de.academic.ru/dic.nsf/dewiki/986655). B. Ziehl-Neelsen staining (de.academic.ru/dic.nsf/dewiki/986644). C. X-ray photograph of infected lungs (http://valuestockphoto.com/downloads/38482-2/tuberculosis-screening-chest-xrayDSC_7427. jpg).

2.1.2 Mycobacterium smegmatis

As described in section 2.1.1, research on pathogenic mycobacteria plays a very important

role. Due to the fact that many of these bacteria are complicated to investigate in vitro (high

pathogenicity and slow growth; M. leprae e.g. shows no growth in vitro at all), certain model

organism are used. One very commonly known model organism for mycobacteria is

M. smegmatis (figure 3). This organism is characterized by its fast growth (generation time of

approximately three to four hours) and by its general non-pathogenicity. M. smegmatis is a

saprophytic living soil bacterium (Kamala et al., 1994). Nevertheless, it was first discovered

in 1884 by Lustgarten in syphilitic chancres and shortly after that it was also found in genital

secretions (see Wallace et al., 1988). So, after M. tuberculosis (Koch, 1882, see section

2.1.1), M. smegmatis was the second mycobacterium identified. It plays an important role in

research on anti-tuberculosis drugs (Wang and Marcotte, 2007) and also in identifying

virulence factors of M. tuberculosis. The gene Rv3810 (erp) e.g. was determined to be

specifically involved in virulence of M. tuberculosis, but after the genome sequence of

M. smegmatis with an Rv3810 homolog was identified, this gene turned out to be a common

mycobacterial housekeeping gene, which is still of great importance for mycobacterial

multiplication (Reyrat and Kahn, 2001).

http://de.academic.ru/dic.nsf/dewiki/986655





Introduction 7

B C

In some rare cases, M. smegmatis was reported to be pathogenic itself. Richardson (1971)

showed clinical mastitis in sheep after infusion and Grooters et al. (1995) investigated

systemic granulomatous lesions in an immunocompromised dog. Talaat et al. (1999)

revealed the pathogenicity of M. smegmatis to goldfish. Human diseases caused by

M. smegmatis are barely reported so far, except one nontuberculous mycobacteriosis, a

chronic carnificating pneumonia caused by infiltration of the organism into bronchial tubes of

a man (Schreiber et al., 2001).

In this study, a modified M. smegmatis strain, namely SMR5, was used. SMR5 is a derivative

of M. smegmatis mc2 155, carrying a mutation in the rpsL gene, which makes it resistant to

streptomycin. The strain mc2 155, which has a very high transformation efficiency, was

generated as a cloning host for genes from virulent mycobacteria (Snapper et al., 1990).

Recapitulating this information, it can be determined that M. smegmatis is an organism of

increasing importance for medical and biological research, on the one hand because of its

own increasing pathogenic features, on the other hand because of its close relationship to

slow growing pathogenic mycobacteria. M. smegmatis is also used to investigate general

mycobacterial metabolic pathways, such as the assembling of the typical cell wall or carbon

and nitrogen metabolism (Ortalo-Magne et al., 1996; Faller et al., 2004; Pimentel-Schmitt et

al., 2007; Amon et al., 2008; Niederweis et al., 2010).

Fig. 3: M. smegmatis. A. Electron microscopy (University of Pittsburgh, Pennsylvania, USA). B. Typical mycelic colony morphology (ASM MicrobeLibrary.org, © MacDonald, Adams and Smith). C. Microscopy of GFP-expressing M. smegmatis cells (this study).

Introduction 8

2.2 Nitrogen metabolism and regulation

2.2.1 Uptake and assimilation of nitrogen sources

The focus of this study was set on nitrogen metabolism. Following carbon, nitrogen is a very

recurrent element in cells. Particularly microorganisms struggle in their natural habitat with

often varying environmental conditions. For that reason, they are able to utilize a multitude of

different nitrogen sources. The Gram-negative model organism Escherichia coli uses

different amino acids such as aspartate, glycine, glutamate or glutamine (Goux et al., 1995;

Newman et al., 2003a) and a variety of inorganic nitrogen sources. This is also well

investigated for Corynebacterium glutamicum, an actinomycete which is often used as a

model organism (Burkovski, 2005), whereas in M. smegmatis little is known about these

processes.

These three organisms as well as the majority of bacteria prefer ammonium as nitrogen

source (Leigh and Dodsworth, 2007). In solution, the protonated ammonium ion (NH4+) is in

equilibrium with uncharged ammonia (NH3). Ammonia is able to pass bacterial cell

membranes by diffusion, which happens under high ammonia/ammonium concentrations

outside the cell. When this concentration decreases, passive diffusion is no longer sufficient

to guarantee cell growth. Subsequently, bacterial ammonium transport systems are

synthesized. These transporters work with high affinity to ammonium and depend on

membrane potential (Walter, 2007; Walter et al., 2008). Inside the cell, ammonium is

assimilated under high concentrations by glutamate dehydrogenase (GDH), a low affinity

enzyme that catalyzes the reductive amination of 2-oxoglutarate to L-glutamate in an

NADPH-dependent reaction. At low ammonium concentrations, glutamine synthetase and

glutamate synthase (GS and GOGAT) are used. In a first reaction, ammonium is bound to

glutamate by GS, resulting in glutamine. This enzyme has a much higher substrate affinity

than GDH and dissipates one molecule ATP per reaction. Four different types of bacterial

glutamine synthetases are known so far: the common form GSI encoded by glnA, the second

form GSII encoded by glnII, which is found in Agrobacterium, Rhizobium or Streptomyces

strains, the third form GSIII encoded by glnN and found in Bacteriodes or Synechocystis

strains and a fourth form identified in Rhizobium strains and named GlnT (Merrick and

Edwards, 1995). GSI activity is additionally regulated by adenylylation (inactive form) or

deadenylylation (active form) by adenylyltransferase GlnE (van Heeswijk et al., 1993; Nolden

et al., 2001a). Subsequently, glutamine, together with 2-oxoglutarate, is converted in a

second, NADPH-dependent reaction to two molecules glutamate by GOGAT (for an

overview, see figure 4 and Merrick and Edwards, 1995).

Introduction 9

B. Nitrogen limitation

A. Nitrogen surplus

2-oxoglutarate + NH4+ + NADPH+H+ L-glutamate + NADP+GDH

L-glutamate + NH4+ + ATP L-glutamine + ADPGS

GOGATL-glutamine + 2-oxoglutarate + NADPH+H+ 2 L-glutamate + NADP+

Fig. 4: Assimilation of ammonium in bacteria. A. Assimilation by glutamate dehydrogenase (GDH) under nitrogen surplus. B. Assimilation by glutamine synthetase (GS) and glutamate synthase (GOGAT) preferentially under nitrogen limitation.

Although it is very energy consumptive, the GS/GOGAT-system is the preferred way of

ammonium assimilation under nitrogen limitation. While some organisms such as Bacillus

subtilis use only the GS/GOGAT-system for ammonium fixation (Belitsky and Sonenshein,

1998), GDH-mediated ammonium assimilation is sufficient for most bacteria under nitrogen

surplus. Under these conditions, some GS activity can also be detected. As described

before, synthesis and activity of GS and GOGAT demand a high amount of energy.

Consequently, these processes are strictly regulated on transcriptional, posttranscriptional

and even posttranslational level. These mechanisms depend on nitrogen availability and are

summarized as “nitrogen control”.

2.2.2 Nitrogen control in Escherichia coli

Mechanisms of nitrogen control are best investigated in Gram-negative enteric bacteria, such

as E. coli or Salmonella enterica serovar Typhimurium (for an overview see Merrick and

Edwards, 1995; Leigh and Dodsworth, 2007). The four most important components of the

global nitrogen regulatory system of E. coli are uridylyltransferase GlnD, PII-type signal

transduction protein GlnB, histidine kinase NtrB and its corresponding response regulator

NtrC. Indicator of nitrogen availability is the intracellular concentration of glutamine and

2-oxoglutarate. Under nitrogen surplus, the glutamine level is high compared to

2-oxoglutarate, whereas this ratio changes under nitrogen limitation. This ratio determines,

whether the PII protein is present in the cell in uridylylated or deuridylylated form (Engleman

and Francis, 1978; Jiang et al., 1998). Responsible for (de)uridylylation of PII is

Introduction 10

uridylyltransferase (UTase) GlnD. Under nitrogen surplus, UTase binds glutamine, which is

accumulated in the cell. This leads to a conformational change of the protein allowing

deuridylylation of PII. The PII protein itself is able to bind ATP and 2-oxoglutarate. Under low

nitrogen concentrations, binding of 2-oxoglutarate induces also a conformational change

resulting in uridylylation of PII (Kamberov et al., 1995; Javelle et al., 2004). UMP is attached

by UTase to the tyrosine 51 residue of each of the three subunits of the PII protein (Son and

Rhee, 1987; Ninfa and Atkinson, 2000). Consequently, the PII protein responds directly to

the intracellular concentration of 2-oxoglutarate and indirectly to the intracellular

concentration of glutamine. PII modulates activity of the cytoplasmic sensor histidine kinase

NtrB. This kinase is part of the two-component system NtrB/NtrC controlling nitrogen-

dependent transcriptional response (Merrick and Edwards, 1995). Under nitrogen limitation,

PII-UMP is not able to interact with NtrB, which leads to autophosphorylation of the kinase at

histidine 139 residue. In a subsequent reaction, NtrB phosphorylates its corresponding

response regulator NtrC at a conserved aspartate 45 residue (Keener and Kustu, 1988). In

its unmodified form, the PII protein binds to NtrB leading to dephosphorylation and

inactivation of NtrC under nitrogen surplus. NtrC contains a typical helix-turn-helix motif at its

C-terminal end and is able to bind DNA even without activation. Nevertheless, it is able to

activate transcription of its target genes exclusively in its phosphorylated form. It is supposed

that only phosphorylated NtrC proteins are capable of forming oligomers, and only NtrC

complexes of at least two dimers can activate transcription by interacting with RNA

polymerase (Merrick and Edwards, 1995).

The genes encoding NtrB and NtrC are located in an operon named glnAntrBC, in which

glnA encodes GSI. Transcription of these genes is strictly regulated by the usage of different

promoters. There are two promoters upstream of glnA and under nitrogen surplus,

transcription starts from glnAp1 leading to a termination structure of the RNA and to the

expression of ntrBC from its own promoter ntrBCp. Under low nitrogen concentrations, the

transcriptional regulator NtrC is activated as described above and inactivates transcription

from glnAp1 by binding. This leads to the expression of glnA and simultaneously ntrBC from

glnAp2. This mechanism guarantees the cell a low level of GSI, NtrB and NtrC under

nitrogen surplus, as well as a higher amount of these proteins under nitrogen limitation

(Merrick and Edwards, 1995). Besides others, genes regulated by NtrC are glnAntrBC,

genes encoding transport systems for arginine, glutamine and histidine and genes involved

in nitrate and nitrite uptake and assimilation (Merrick and Edwards, 1995).

Introduction 11

Furthermore, PII has great influence on an adenylyltransferase (ATase, encoded by glnE;

Merrick and Edwards, 1995), which regulates GSI activity by (de)adenylylation at tyrosine

394 residue. Under nitrogen surplus, PII activates adenylylation of GSI, which is as a

consequence inhibited in its ammonium assimilating function. Nitrogen limitation leads to

activation of the deadenylylating function of ATase by PII-UMP and thus, GSI is active in its

unmodified state (Ninfa and Jiang, 2005).

Additionally, a second PII-type signal transduction protein, namely GlnK, exists in E. coli. The

corresponding gene glnK is located in an operon with amtB, coding for an ammonium

transporter. This system is important for regulation of nitrogen response at low ammonium

concentrations. As described above, under these conditions the response regulator NtrC

activates transcription of its target genes, which include amtB and glnK. The GlnK protein is

directly uridylylated and consequently not able to bind AmtB. At ammonium concentrations

higher than 50 µM, GlnK is deuridylylated and blocks AmtB activity by binding to it (Javelle et

al., 2004; Forchhammer, 2008).

2.2.3 Nitrogen control in actinomycetes

For many years, the well investigated system of nitrogen control in E. coli and related enteric

bacteria (section 2.2.2) was considered as the paradigm of nitrogen regulation in bacteria.

More recent studies of nitrogen control in other, especially Gram-positive bacteria revealed a

great variety of regulation processes, while the relevant enzymes (GDH, GS, GOGAT,

ATase, UTase and PII) are highly conserved. Concerning the order actinomycetes,

regulation of nitrogen metabolism is best understood in C. glutamicum (Burkovski, 2007;

Hänßler and Burkovski, 2008) and Streptomyces coelicolor (Reuther and Wohlleben, 2007),

while little is known about these mechanisms in mycobacteria (Amon et al., 2010).

C. glutamicum is a non-sporulating, aerobic and immobile member of the order

Actinomycetales. This organism, named according to its characteristic club shape (Greek:

koryne), has become extremely relevant for industry and research. This is due to the fact that

modified strains are used for extensive biotechnological production of amino acids such as

L-glutamate, L-lysine, L-serine and L-threonine (Leuchtenberger et al., 2005). Furthermore,

C. glutamicum undergoes extensive research as a model organism for related pathogenic

species such as Corynebacterium diphtheriae or Corynebacterium jeikeium (Coyle and

Lipsky, 1990; Hadfield et al., 2000). Compared to E. coli, nitrogen control in C. glutamicum

includes adenylyltransferase GlnD, the only PII-type signal transduction protein GlnK (Nolden

et al., 2001b) and the transcriptional regulator AmtR (Jakoby et al., 2000). Whereas in E. coli

glutamine is described as the main nitrogen indicator and GlnD as the sensor (Merrick and

Introduction 12

Edwards, 1995), this could not be revealed for C. glutamicum so far (Rehm et al., 2010).

Nevertheless, nitrogen-dependent transcriptional response is very well characterized. Under

nitrogen surplus, the bifunctional ATase GlnD is responsible for deadenylylation of GlnK; in

contrast, under nitrogen limitation it adenylylates the trimeric PII protein by attaching AMP to

the tyrosine 51 residue of each of the three subunits (Strösser et al., 2004). Only modified

GlnK is able to interact with AmtR (Beckers et al., 2005). Consequently, the transcriptional

regulator is not affected under nitrogen surplus and represses transcription of its target

genes. Furthermore, unmodified GlnK is recruited to the membrane where it interacts with

the ammonium transporter AmtB and is eventually degraded (Strösser et al., 2004). Under

nitrogen limitation, GlnK-AMP binds AmtR leading to a conformational change and finally to

desertion of the repressor from its target DNA. This results in derepression of at least 40

nitrogen-dependent genes, which encode transport systems for ammonium (amtA, Jakoby et

al., 2000; amtB, Meier-Wagner et al., 2001), glutamate (gluABCD, Kronemeyer et al., 1995),

urea (urtABCDE, Beckers et al., 2004), creatinine (crnT, Bendt et al., 2004) and others, as

well as ammonium assimilation systems (gdh for GDH, Beckers et al., 2005; glnA for GSI,

Nolden et al., 2001a; gltBD for GOGAT, Beckers et al., 2001; ureABCEFGD for urease,

Beckers et al., 2004; codA for creatinine deaminase, Bendt et al., 2004; and others). Even

the transcription of glnK and glnD is regulated by AmtR, which is surprising, as they encode

the two signal transduction proteins involved in AmtR activation or deactivation. These two

genes are located in an operon with amtB. Additionally, some genes of unknown function

(Beckers et al., 2005) and the vanABK operon, which is regulated indirectly (Merkens et al.,

2005), belong to the AmtR regulon.

Like in E. coli, GSI activity is additionally regulated by post-translational modification, i.e. by

(de)adenylylation via GlnE. This enzyme seems to work independently from the regulatory

system described above to guarantee fast adaption to changing glutamine concentrations

(Burkovski, 2003). At high nitrogen concentrations, ATase GlnE adenylylates GSI at tyrosine

405 residue leading to inactivation of the enzyme. In case of nitrogen limitation, GlnE again

deadenylylates GSI, which results in new glutamine synthetase activity (Nolden et al.,

2001a). The signal activating or deactivating GlnE could not be detected so far.

C. glutamicum owns a second GS encoded by glnA2, which is not regulated by

(de)adenylylation and which is of so far unknown function (Nolden et al., 2001a).

Although related to C. glutamicum, a very different type of nitrogen regulation is reported in

S. coelicolor. This non-motile soil bacterium is of great relevance for research, particularly

because of its morphological differentiation. S. coelicolor grows vegetatively forming a

filamentous mycelium. Under nutrient limitation, aerial branches are generated, followed by

hyphae differentiation and formation of spores (Chater, 2001). During these steps, the

organism switches from primary to secondary metabolism (Reuther and Wohlleben, 2007),

Introduction 13

resulting in the production of various secondary metabolites such as antibiotics (Newman et

al., 2003b; Bérdy, 2005). The mechanism of nitrogen response in this organism is not as well

understood as in E. coli or C. glutamicum. Like in C. glutamicum, a single PII-type protein,

encoded by glnK, was identified, which is deadenylylated under nitrogen surplus and

adenylylated at tyrosine 51 residue under nitrogen limitation by ATase GlnD (Reuther and

Wohlleben, 2007). If the modified GlnK protein is involved in further signal transduction has

not been proven yet. There is also evidence for adenylylation of GSI at tyrosine 397 residue

leading to inactivation under nitrogen surplus and deadenylylation leading to activation of this

ammonium assembling enzyme under nitrogen limitation by ATase GlnE, but the GlnK/GlnD

signal transduction system seems not to be involved in this mechanism either (Fink et al.,

1999). The component signaling GlnE to (de)adenylylate GSI, as well as the signal inducing

expression of nitrogen-related target genes has not been identified yet. Nevertheless, GlnR

is known as the global regulator of nitrogen metabolism in S. coelicolor. This protein was first

described by Wray and Fisher (1993) as the regulator of glnA gene expression. Furthermore,

GlnR directly regulates the expression of the amtB-glnK-glnD operon, glnII encoding GSII,

gdhA encoding GDH, nirBD encoding nitrite reductase, ureA encoding a urea assimilation

system and others (Fink et al., 2002; Tiffert et al., 2008). Unlike C. glutamicum AmtR (see

above) but as E. coli NtrC (see section 2.2.2), GlnR works in a bifunctional manner, which

means that it activates transcription of e.g. glnA and nirB, whereas it represses transcription

of e.g. gdhA or ureA under nitrogen limitation (Tiffert et al., 2008). Tiffert et al. (2011) suggest

that the role of GlnR might not be restricted to nitrogen metabolism; putative target genes

belonging to carbon metabolism, biosynthesis of antibiotics and amino acids or stress

response were also identified. No influence of GlnR to its encoding gene, glnR, was seen,

although it is repressed in the presence of ammonium, whereas the expression level rises

under nitrogen limitation (Fink et al., 2002).

In S. coelicolor, five genes encoding GS or GS-like proteins exist, namely glnA, glnII, glnA2,

glnA3 and glnA4 (Reuther and Wohlleben, 2007). glnA encodes GSI, the housekeeping

glutamine synthetase in this organism, which is constantly expressed in all growth phases,

whereas glnII encodes GSII, a primary typical eukaryotic enzyme, whose transcription is

increased during mycelia differentiation. No further, posttranslational regulation of GSII

occurs. The remaining three glnA genes seem not to be involved in nitrogen assimilation and

glutamine synthesis (Rexer et al., 2006). GlnR binds to upstream promoter regions of glnA

and glnII (Tiffert et al., 2008). Furthermore, glnRII, encoding a second regulatory protein, was

identified located three ORFs downstream of glnII. The resulting GlnRII protein shows 31 %

sequence homology to GlnR and binds equally to upstream sequences of glnA, glnII and

amtB-glnK-glnD (Fink et al., 2002). However, it is not a functional homolog of GlnR and its

particular role still needs to be investigated (Reuther and Wohlleben, 2007). Homologs of

Introduction 14

glnRII exist in various Streptomyces strains, but not in corynebacteria or mycobacteria. On

the contrary, homologs of glnR are widely spread among the actinomycetes (see figure 5).

They are highly conserved and show 60-80 % sequence identity. The conclusion can be

drawn that most actinomycetes (with the exception of corynebacteria) have evolved a

specific regulatory network depending on GlnR to respond to changing nitrogen conditions,

more or less unattached to their different lifestyles.

Compared to other members of the actinomycetes, nitrogen metabolism in mycobacteria is

barely investigated. For a long time, focus of research was glutamine synthetase I encoded

by glnA1, which is essential for growth and pathogenicity of M. tuberculosis (Tullius et al.,

2003; Harth et al., 2005) and M. bovis (Chandra et al., 2010). It is suspected that GSI is

involved in the formation of the typical poly-L-glutamate-glutamine cell wall structure and thus

a very interesting drug target (Harth et al., 2000). Furthermore, the essential role of glnE was

extensively studied in M. tuberculosis. As in other bacteria, GlnE is responsible for

posttranslational modification and activation of GSI by (de)adenylylation (Parish and Stoker,

2000; Carroll et al., 2008).

At the beginning of this study, little was known about nitrogen metabolism in M. smegmatis.

A genome-wide analysis of M. smegmatis and related species revealed the existence of

various popular components of nitrogen metabolism such as amtB-glnK-glnD, glnE and

gltBD (Amon et al., 2009). Some of them, including M. smegmatis, showed genes encoding

nitrate, nitrite and urea transporters and assimilation systems. Moreover, only in the genome

of M. smegmatis additional ammonium transporters, several copies of glutamate synthase-

encoding gens and several genes encoding different types of GDH and GS were identified.

Recent studies revealed the changing activities of glutamine synthetase, aminating and

deaminating GDH in response to changing ammonium concentrations in the cells (Harper et

al., 2010). Obviously, M. smegmatis possesses a variety of different genes involved in

nitrogen metabolism rather typical for other bacteria such as Agrobacterium, Burkholderia or

Pseudomonas species (Amon et al., 2009). Most interestingly, genes encoding two different

major regulatory proteins of nitrogen metabolism in actinomycetes were identified in

M. smegmatis (for an overview see figure 5). A protein showing 42 % sequence homology to

C. glutamicum AmtR (see above) was recognized, as well as a homolog of S. coelicolor

GlnR (see above) with 60 % identity (Amon et al., 2009). The study also exhibited that

M. smegmatis GlnR is closer related to homologs in Nocardia farcinia or Rhodococcus sp.

than to homologs in other mycobacteria. The assumption that GlnR is the major regulator of

nitrogen metabolism and an activator of transcription of its target genes in M. smegmatis was

verified in first experiments with a glnR deletion strain. This strain was no longer able to grow

under addition of MSX, a glutamate analog that blocks GS activity (Berlicki, 2008). Moreover,

the glnR deletion strain did not show enhanced transcripts of glnA, amtB and amt1 under

Introduction 15

S. coelicolor

Bifidobacterium sp.

T. fusca

P. acnes

A. naeslundii

C. glutamicum

C. efficiens

C. diphtheriae

M. smegmatis

M. tuberculosis

M. bovisM. avium

N. farcinica

Rhodococcus sp.

A. mediterranei

S. avermitilis

C. pseudogenitalium

C. accolens

S. griseus

S. roseum

Frankia sp.

C. michiganensis

M. abscessus

S. arenicolaR. salmoninarum

…

…

…

GlnRAmtR

nitrogen limitation (Amon et al., 2008). Additionally, a conserved sequence upstream of these

genes was identified, suggesting an influence of GlnR on the transcription of its target genes

by binding and thus activating RNA polymerase (Amon et al., 2009). Nothing is known about

signal transduction and activation of the regulator protein so far, even though GlnR was

associated by sequence analyses to the OmpR-family of transcriptional regulators (Amon et

al., 2008). The OmpR/EnvZ two-component system is very well investigated in E. coli,

regulating osmolarity response. Depending on changing osmolarity, membrane-bound

sensor histidine kinase EnvZ is autophosphorylated and thus able to (de)phosphorylate

OmpR at a specific aspartate residue (Mattison et al., 2002). OmpR is responsible for

osmolarity-dependent gene expression. This happens by binding of the regulator to specific

sequences in the promoter regions of its target genes. Phosphorylated OmpR-P binds to its

target DNA in a “discontinuous, galloping manner” (Yoshida et al., 2006). OmpR-like

structures such as an N-terminal phosphorylation domain with highly conserved aspartate

residues and a C-terminal DNA binding domain were identified for GlnR in M. smegmatis

(Amon et al., 2008). A corresponding sensor histidine kinase could not be detected so far, as

its gene is not located close to glnR, which is actually typical for members of bacterial two-

component systems.

Fig. 5: Distribution of GlnR and AmtR as putative transcriptional regulators for nitrogen metabolism in actinomycetes. Figure according to Amon (2010). Experimentally verified species are highlighted bold. References for these are: Jakoby et al. (2000); Fink et al. (2002); Nolden et al. (2002); Yu et al. (2006); Amon et al. (2008). Full species names are: Actinomyces naeslundii, Amycolatopsis mediterranei, Bifidobacterium longum, Clavibacter michiganensis ssp. michiganensis, Corynebacterium accolens, Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium glutamicum, Corynebacterium pseudogenitalium, Frankia alni, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium smegmatis, Mycobacterium tuberculosis, Nocardia farcinica, Propionibacterium acnes, Renibacterium salmoninarum, Rhodococcus erythropolis, Rhodococcus jostii, Rhodococcus opacus, Salinispora arenicola, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, Streptosporangium roseum and Thermobifida fusca.

Introduction 16

2.3 Aims of this study

Nitrogen metabolism in mycobacteria is not well investigated, although it is a fundamental

system of these often pathogenic organisms. This study obtains a detailed characterization

of nitrogen regulation in the facultative non-pathogenic species M. smegmatis, while the

regulatory protein GlnR is the focus of attention.

Intensive studies of the genome of M. smegmatis revealed uptake and assimilation systems

for various nitrogen sources (Amon et al., 2009). According to that, it is a first objective of this

study to identify different substrates that can be used as single nitrogen source and to

determine the involvement of GlnR in the corresponding processes. As not many procedures

are established so far for investigating nitrogen metabolism and control in mycobacteria, the

generation of a functional in vivo reporter system to measure nitrogen- or GlnR-dependent

promoter activities of different target genes is another important part of this study.

As the global regulator GlnR is the center of attention, this work concentrates on the one

hand on the protein itself, including protein purification, the generation of a GlnR-specific

antibody and DNA binding studies. Binding of purified GlnR protein to promoter sequences

upstream of its target genes needs to be investigated in detail, so that eventually accurate

binding sequences can be determined. With these sequences, the “galloping” DNA binding

theory (Yoshida et al., 2006) can either be proven or disproved for GlnR. Moreover, an exact

binding sequence allows the search for further GlnR target genes in the genome of

M. smegmatis. On the other hand, global analysis methods are used to enlarge the GnR

regulon.

According to sequence homologies, GlnR is associated to the OmpR-family of transcriptional

regulators, which feature conserved aspartate residues as phosphorylation sites and a

corresponding sensor histidine kinase (Amon et al., 2008). Thus, another aim of this study is

to identify the kinase and to verify phosphorylation of GlnR. While a bioinformatic approach

searching for putative kinases was already performed (Amon, 2010), no data are available in

respect of the phosphorylation residues.

As the main objective of this study is a detailed characterization of GlnR, the global

regulatory protein of nitrogen metabolism in M. smegmatis, this work might also contribute to

a better understanding of nitrogen metabolism in mycobacteria in general, as the system

investigated for M. smegmatis might be transferred to the related pathogen M. tuberculosis.

To investigate this, first tests are carried out in this study using M. tuberculosis GlnR.

Materials and methods 17

3 Materials and methods

3.1 Bacterial strains and plasmids

E. coli, C. glutamicum and M. smegmatis strains used in this study are listed in table 1. Table

2 shows all plasmids.

Tab. 1: Bacterial strains with characteristics and references. StrR: resistance to streptomycin.

CamR: resistance to chloramphenicol.

Strain Genotype, phenotype Reference

E. coli

BL21 F- ompT gal [dcm] [Ion] hsdSB (rB

- mB

-)

Studier et al., 1990

DH5mcr endA1 supE44 thi-1 - recA1 gyrA96 relA1 deoR

Δ(lacZYA-argF) U169 80ΔlacZ ΔM15mcrA Δ(mmr

hsdRMS mcrBC)

Grant et al., 1990

Rosetta2 (DE3) BL21 with plasmid pRARE; lacYZ-deletion, no Lon and

OmpT proteases, CamR

Novagen, Darmstadt

C. glutamicum

ATCC 13032 wild type

Abe et al., 1967

M. smegmatis mc2

155

SMR5 rpsL-mutant of M. smegmatis mc2 155, high

transformation efficiency, StrR

Sambrook et al., 1989

Sander et al., 1995

MH1 SMR5 with deletion of glnR

M. Höller,

personal communication

Δmsmeg_1918 SMR5 with deletion of msmeg_1918

this study

Δmsmeg_1918 kin SMR5 with deletion of msmeg_1918 kinase domain

this study

Δmsmeg_5241 SMR5 with deletion of msmeg_5241

this study

Δmsmeg_1918

Δmsmeg_5241

SMR5 with deletion of msmeg_1918 and msmeg_5241 this study

Imsmeg_0426 SMR5 with insertion of pML814 into msmeg_0426

this study


this study

InarB SMR5 with insertion of pML814 into msmeg_2837

(narB)

this study


this study


InarG SMR5 with insertion of pML814 into msmeg_5140

(narG)

this study


this study

ML371

M. smegmatis mc2 155 with plasmid pSJ27 (bxb1 site

at the 3’-end of groEL1)

Kim et al., 2003

DSM 43756 wild type DSMZ; Lehmann and

Neumann, 1899

Tab. 2: Plasmids with characteristics and references. AmpR: resistance to ampicillin.

Plasmid Characteristics Reference

pASK-IBA5plus F1-IG, AmpR, tet repressor, ori, tet

P/O,

Strep-tag® II, polylinker, tIpp

IBA BioTAGnology,

Göttingen

pASK-IBA5+1918 pASK-IBA5plus with msmeg_1918 for

overexpression of Msmeg_1918 with

N-terminal Strep-tag II

Hiery, 2009

pASK-IBA5+5241 pASK-IBA5plus with msmeg_5241 for

overexpression of Msmeg_5241 with

N-terminal Strep-tag II

Hiery, 2009

pASK-IBA5+glnR pASK-IBA5plus with glnR for overexpression

of GlnR with N-terminal Strep-tag II

Hiery, 2009

pASK-IBA5+glnRAsp43Ala pASK-IBA5+glnR with point mutation to

Asp43Ala

this study

pASK-IBA5+glnRAsp43Asn pASK-IBA5+glnR with point mutation to

Asp43Asn

this study

pASK-IBA5+glnRAsp43Glu pASK-IBA5+glnR with point mutation to

Asp43Glu

this study


Asp48Ala

this study


Asp48Asn

this study


Asp48Glu

this study

pASK-IBA5+glnRAla45Ser pASK-IBA5+glnR with point mutation to

Ala45Ser

this study


Asp52Ala

this study


Asp52Asn

this study


Asp52Glu

this study

pASK-IBA5+glnRAsp43/48/52Ala pASK-IBA5+glnR with three point mutations

to Asp43/48/52Ala

this study


pASK-IBA5+senX3* pASK-IBA5plus with truncated senX3 for

overexpression of SenX3* without N-terminal

transmembran domain

this study

pMal-c2 ptac, AmpR, pBR322 ori, M13 ori, malE, lacI

q,

lacZ, E. coli vector for protein purification

Guan et al., 1987

pMal-c2-1918 pMal-c2 with msmeg_1918 for purification of

an MBP-Msmeg_1918 fusion protein

this study

pMal-c2-5241 pMal-c2 with msmeg_5241 for purification of

an MBP-Msmeg_5241 fusion protein

this study

pMal-c2-glnR pMal-c2 with glnR for purification of an MBP-

GlnR fusion protein

this study

pMal-c2glnR M.tub. pMal-c2 with Rv0818 for purification of an

MBP-GlnR (M. tuberculosis) fusion protein

this study

pML814 COLE1 ori, FRT-hph-FRT, rpsL, AmpR M. Niederweis,


pML814-D1918 pML814 with 1000 bp fragments upstream

and downstream of msmeg_1918, for

deletion mutagenesis

Bräu, 2008

pML814-D1918kin pML814 with 1000 bp fragments upstream

and downstream of msmeg_1918 kinase

domain, for deletion mutagenesis

this study

pML814-D5241 pML814 with 1000 bp fragments upstream

and downstream of msmeg_5241, for

deletion mutagenesis

this study

pML814-I0426 pML814 with 500 bp fragment of

msmeg_0426, for insertion mutagenesis

this study



this study

pML814-I2837 (narB) pML814 with 500 bp fragment of


this study



this study

pML814-I5140 (narG) pML814 with 500 bp fragment of


this study



this study

pMN234 aph, rpsL, pimyc-flpe, pBR322 ori, PAL5000

ori

M. Niederweis,


pMN016 COLE1 ori, ori myc, hph, psmyc-mspA,

pAL5000

M. Niederweis,


pMN016control pMN016, mspA gene removed

Bräu, 2008

pMN016glnR pMN016control with psmyc-glnR

(M. smegmatis)

Bräu, 2008


pMN016glnR M. tub. pMN016control with psmyc-glnR

(M. tuberculosis)

Bräu, 2008

pMN016glnRAsp43Ala pMN016glnR with point mutation to

Asp43Ala

this study

pMN016glnRAsp43Asn pMN016glnR with point mutation to

Asp43Asn

this study

pMN016glnRAsp43Glu pMN016glnR with point mutation to

Asp43Glu

this study


Asp48Ala

this study


Asp48Asn

this study


Asp48Glu

this study

pMN016glnRAla45Ser pMN016glnR with point mutation to

Ala45Ser

this study


Asp52Ala

this study


Asp52Asn

this study


Asp52Glu

this study

pMN016glnRAsp43/48/52Ala pMN016glnR with three point mutations to

Asp43/48/52Ala

this study

pMN016glnR-his pMN016glnR with C-terminal his-tag (6 x His)

this study

pMN016his-glnR pMN016glnR with N-terminal his-tag (6 x His)

this study

pMN-gfpuv pMN016control with gfpuv

Y. Lu,


pMN016-gfpuv pMN016, mspA removed, with psmyc-gfpuv

Y. Lu,


pMN-amt1p-gfpuv pMN-gfpuv with 500 bp fragment upstream of

amt1

this study

pMN-amtAp-gfpuv pMN-gfpuv with 500 bp fragment upstream of

amtA

this study

pMN-amtBp-gfpuv pMN-gfpuv with 500 bp fragment upstream of

amtB

this study

pMN-gdhp-gfpuv pMN-gfpuv with 500 bp fragment upstream of

gdh

this study

pMN-glnAp-gfpuv pMN-gfpuv with 500 bp fragment upstream of

glnA

this study

pMN-glnRp-gfpuv pMN-gfpuv with 500 bp fragment upstream of

glnR

this study


pMN-lacZ pMN016control with lacZ

this study

pMN016-lacZ pMN-lacZ with psmyc-lacZ

this study

pMN-amtAp-lacZ pMN-lacZ with 500 bp fragment upstream of

amtA

this study

pMN-amtBp-lacZ pMN-lacZ with 500 bp fragment upstream of

amtB

this study

pMN-amtRp-lacZ pMN-lacZ with 500 bp fragment upstream of

amtR

this study

pMN-gdhp-lacZ pMN-lacZ with 500 bp fragment upstream of

gdh

this study

pMN-glnAp-lacZ pMN-lacZ with 500 bp fragment upstream of

glnA

this study

pMN-glnRp-lacZ pMN-lacZ with 500 bp fragment upstream of

glnR

this study

pQE70 ptac, AmpR, ori COLE1, 6 x His, E. coli

vector for protein purification

Qiagen, Hilden

pQE70-glnR pQE70 with glnR, for expression of a GlnR-

6 x His fusion protein

this study

pUC19-his-glnR

pUC19 (AmpR, lacZα) with his-tag and glnR,

for expression of a 6 x His-GlnR fusion

protein

M. Höller,


pWH948 COLE1, AmpR, lacZ, galK

J. Bürger,


3.2 Cultivation of bacteria

3.2.1 Culture media for E. coli, C. glutamicum and M. smegmatis

Generally, LB (Luria-Bertani) medium (table 4) was used for growth of E. coli. For production

of agar plates, 15 g/l Bacto-Agar (Oxoid, Heidelberg) were added. C. glutamicum was

cultivated in BHI and CgC medium (Keilhauer et al., 1993).

Middlebrook 7H9 Broth (Becton/Dickinson, USA) was used as defined minimal medium for

M. smegmatis. For production of 7H10 agar plates, 15 g/l Bacto-Agar (Oxoid, Heidelberg)

were added. Cells were incubated in 7H9-N medium to cause nitrogen starvation. SOC

medium was used to cultivate cells after electroporation. To induce phosphate starvation,

cells were transferred from ST to ST-P medium (according to Gebhard et al., 2006). All

media used in this study are listed in table 3.


Tab. 3: Media used for cultivation of E. coli, C. glutamicum and M. smegmatis strains.

Medium

Ingredients (per l)

LB (Luria-Bertani)

10 g tryptone, 5 g yeast extract, 10 g sodium chloride. Sterilization by autoclavation at 121°C for 20 minutes.

BHI

37 g Brain Heart Infusion (Difco, Detroit, USA and Oxoid, Heidelberg). Sterilization by autoclavation at 121°C for 20 minutes.

CgC

42 g MOPS, 20 g (NH4)2SO4, 5 g urea, 0.5 g KH2PO4, 0.5 g K2HPO4, pH (NaOH) 7.0. After autoclavation addition of: 10 ml 100 mM CaCl2, 10 ml 1 M MgSO4, 200 µg biotin, 1 ml trace element solution, 80 ml 50 % (w/v) glucose.

7H9

0.5 g ammonium sulfate, 0.5 g L-glutamic acid, 0.1 g sodium citrate, 1 mg pyridoxine, 0.5 mg biotin, 2.5 g disodium phosphate, 1 g monopotassium phosphate, 0.04 g ferric ammonium citrate, 0.05 g magnesium sulfate, 0.5 mg calcium chloride, 1 mg zinc sulfate, 1 mg copper sulfate, 4 ml 50 % (v/v) glycerin, pH 6.6. After autoclavation at 121°C for 20 minutes 2.5 ml 20 % (v/v) Tween80 were added.

7H9-N

Composition as 7H9 medium, absence of nitrogen sources ammonium sulfate, L-glutamic acid and ferric ammonium citrate.

SOC

20 g tryptone, 5 g yeast extract, 0.5 g sodium chloride, 2.5 ml 1 M potassium chloride, 10 ml 1 M magnesium chloride, 10 ml 1 M magnesium sulfate, 1.8 ml 20 % (w/v) glucose. Sterilization by filtration.

ST

0.5 g magnesium sulfate, 2 g citric acid, 1 g L-asparagine, 0.3 g potassium chloride, 2 ml 50 % (v/v) glycerin, 320 µl 0.5 M ferric chloride, 1 ml 100 mM ammonium chloride, 9.13 ml 3 M dipotassium phosphate. After sterilization by autoclavation at 121°C for 20 minutes 2.5 ml 20 % (v/v) Tween80 were added.

ST-P

Composition as ST medium, absence of phosphate source dipotassium phosphate.

Trace element solution

28.5 g FeSO4 x 7H2O, 16.5 g MnSO4 x H2O, 6.4 g ZnSO4 x 7H2O, 764 mg CuSO4 x 5H2O, 128 mg CoCl2 x 6H2O, 44 mg NiCl2 x 6H2O, 64 mg Na2MoO4 x 2H2O, 48 mg H3BO3, 50 mg SrCl2, 50 mg BaCl2 x 2H2O, 28 mg KAl(SO4)2 x 12H2O, pH (H2SO4) 1.

3.2.2 Antibiotics

For selection of resistant bacteria, antibiotics were added to the media as shown in table 4.

Tab. 4: Antibiotics used to select resistant bacteria.

Antibiotic Final concentration E. coli Final concentration M. smegmatis

Ampicillin 100 µg/ml -

Chloramphenicol 25 µg/ml -

Hygromycin B 200 µg/ml 50 µg/ml

Kanamycin 60 µg/ml 25 µg/ml

Streptomycin - 400 µg/ml


3.2.3 Growth conditions

All E. coli and M. smegmatis strains were cultivated at 37°C and 125 rpm in a rotary shaker

(SM30, Edmund Bühler GmbH, Tübingen), while baffled flasks were used to provide

abundant agitation. C. glutamicum was treated equally at 30°C. Growth was observed by

measuring the optical density at a wavelength of 600 nm (oD600) in plastic cuvettes (bio one,

Greiner, Essen). For this purpose, the photometer Ultraspec 2100 pro (Amersham

Biosciences, USA) was used.

For isolation of plasmid DNA (section 3.3.1.1), E. coli cells were cultivated overnight in 4 ml

LB medium with an adequate antibiotic. E. coli used for preparation of chemically competent

cells (section 3.4.1) were treated equally. For protein purification from E. coli, 800 ml LB

medium with an adequate antibiotic were inoculated to an oD600 of 0.1. For growing cells with

an MBP-tagged protein, 2 % glucose was added to the medium. At an oD600 of 0.4-0.6,

overexpression of the protein of interest was induced by adding either 1 mM IPTG or 1 µg/ml

doxycycline. Afterwards cells were further incubated for four hours at 37°C (with doxycycline

in the dark) and treated as described in section 3.3.3.2.

For cultivation of M. smegmatis, the media were inoculated following a standard scheme to

assure growth of the clotting cells. First, 1 ml 7H9 medium was inoculated in a reaction tube

and vortexed intensely. After that, the cells were incubated for five minutes at 37°C under

strong agitation and then used to inoculate 20 ml 7H9 medium as overnight culture. After

growth of at least 12 hours, fresh 7H9 medium was inoculated to an oD600 of 0.3. The oD600

was measured every two hours. In order to cause nitrogen starvation, cultures that reached

exponential growth phase (approximately oD600 of 0.8) were centrifuged for 10 minutes at

4,000 x g and 4°C, resuspended and incubated in 7H9-N medium for 30 minutes. For

preparation of total RNA or enzyme activity, fluorescence and β-galactosidase

measurements, cells were also harvested by centrifugation (10 min, 4,000 x g, 4°C) and

further treated as described in sections 3.3.2.1 and 3.4.7-10. To generate growth curves, the

oD600 was measured every two, later every four hours. For preparation of chromosomal DNA,

cells were grown in 100 ml 7H9 medium overnight. For preparation of total cell extract, 500

ml 7H9 medium were inoculated to an oD600 of 0.3 and cells were grown until exponential

growth phase. All strains used in this study were stored as permanent cultures in Roti®-Store

tubes (Roth, Karlsruhe) at -80°C.


3.3 Procedures in molecular biology

3.3.1 Procedures to work with DNA

3.3.1.1 Isolation of plasmid DNA from E. coli

E. coli cells carrying the plasmid of interest were grown overnight in 4 ml LB medium

containing the corresponding antibiotic. Cells were harvested by centrifugation (1 min,

11,000 x g) and further treated using the NucleoSpin® Plasmid kit (Macherey-Nagel, Düren)

as recommended by the supplier.

For preparation of higher amounts of plasmid DNA, E. coli cells were grown overnight in 100

ml LB medium and then harvested by centrifugation (10 min, 6,000 x g). Plasmid preparation

was carried out using the QIAprep®Midipräp kit (Qiagen, Hilden) following the instructions of

the supplier. Subsequently, plasmids were analyzed by agarose gel electrophoresis (section

3.3.1.3).

3.3.1.2 Isolation of plasmid DNA from M. smegmatis

Plasmid preparation from M. smegmatis was carried out using the NucleoSpin® Plasmid kit

(Macherey-Nagel, Düren) as recommended by the supplier. Because of the resistance of the

mycobacterial cell wall, 2 µg/µl lysozyme (Merck, Darmstadt) were added to buffer A1. After

incubation for three hours at 37°C the samples were treated as described in section 3.3.1.1.

3.3.1.3 Gel electrophoresis and extraction of DNA from agarose gels

Plasmid DNA or DNA fragments were analyzed by gel electrophoresis. Depending on the

size of DNA, 0.8-4 % agarose gels were used in 1 x TAE buffer (Sambrook et al., 1989). 6 x

loading dye was added to the samples which were dispersed by electrophoresis at 10 V/cm

of gel length. Additionally, DNA marker (peqGOLD DNA ladder, peqlab Biotechnologie

GmbH, Erlangen) was applied to the gel to determine the size of DNA fragments. After that,

ethidium bromide solution was used to stain the DNA which was subsequently detected

using the Doc Print 1000 Gel documentation (peqlab Biotechnologie GmbH, Erlangen). If

necessary, DNA was excised and isolated from the agarose gel using the NucleoSpin®

Extract kit (Macherey-Nagel, Düren) and following the instructions of the supplier. DNA


concentrations were determined at a wavelength of 260 nm (ε = 5 cm2/mg) in the Nanodrop®

spectrometer ND-100 (peqlab Biotechnologie GmbH, Erlangen). Purity was analyzed by

determination of the quotient of A260 / A280, which is 1.8-2.0 for pure DNA.

1 x TAE buffer:

40 mM Tris, 0.5 mM EDTA, pH (acetic acid) 7.5

6 x loading dye:

0.25 % (w/v) bromphenol blue, 0.25 % (w/v) xylene cyanol FF, 40 % (w/v) sucrose

3.3.1.4 Preparation of chromosomal DNA from M. smegmatis

For preparation of chromosomal DNA phenol chloroform extraction was used. For this

purpose, M. smegmatis strains were cultivated in 20 ml 7H9 medium overnight. On the next

day, 100 ml 7H9 medium were inoculated, which were again incubated overnight. On the

following day, cells were harvested by centrifugation (15 min, 3,000 x g, 4°C) and the cell

pellet was resuspended in 3 ml TE buffer. 300 µl lysozyme solution (10 µg/µl) and 30 µl

RNAseA solution (10 µg/µl) were added, followed by 90 minutes of incubation at 37°C and

low agitation (400 rpm, “HeizThermoMixer” MHR20/23, HLC BioTech, Bovenden). After that,

210 µl 20 % (w/v) SDS and 60 µl proteinase K solution (10 µg/µl) were added. The samples

were inverted and incubated for two hours at 65°C. 600 µl 5 M NaCl and 480 µl 10 % (w/v)

CTAB solution were added, followed by further 30 minutes of incubation at 65°C. 4.5 ml of a

phenol-chlorophorm-isoamyl alcohol mixture (25:24:1) were added. After centrifugation at

8,900 x g and 4°C for 15 minutes, the upper phase was transferred into a fresh reaction tube

containing 4.5 ml of a chlorophorm-isoamyl alcohol mixture (24:1) and again centrifuged at

8,900 x g and 4°C for 15 minutes. Again, the upper phase was transferred into a fresh

reaction tube containing 3 ml isopropanol. After inversion of the tubes, the visible DNA was

transferred into a new reaction tube. The DNA was washed twice in 800 µl 70 % (v/v) ethanol

by centrifugation at 6,500 x g and 4°C for 10 minutes and subsequently dried for 10 minutes

in the vacuum centrifuge Speed Vac® (Savant, USA). To dissolve the DNA pellet, 500 µl TE

buffer were added and the sample was incubated at room temperature overnight.

TE buffer:

100 mM Tris, 0.1 mM EDTA, pH (HCl) 8

http://dict.leo.org/ende?lp=ende&p=Ci4HO3kMAA&search=subsequently&trestr=0x8004


3.3.1.5 Polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) is the favored method for amplification of specific DNA

fragments (Mullis et al., 1986). In this study, either genomic or plasmid DNA containing the

fragment of interest was used as template. Primers were designed by MWG (Ebersberg) and

diluted in H2O to a final concentration of 100 pmol/µl. The melting temperature of each primer

was calculated including 2°C for breaking of hydrogen bonds between adenine and thymine

and 4°C for guanine and cytosine. Thus, the specific annealing temperature for each PCR

reaction was set to melting temperature minus 4°C. The reactions were carried out in a

Mastercycler® gradient (Eppendorff, Hamburg) or Primus 96 (peqlab Biotechnologie GmbH,

Erlangen) instrument. For amplification of small DNA fragments, PuRe Taq Ready-To-Go

PCR beads (GE Healthcare, Munich) were used as recommended by the supplier. For

amplification of genes or DNA fragments bigger that 1000 bp, Phusion DNA polymerase

(NEB, Schwalbach), which has an additional proofreading activity, was used.

Composition of a 25 µl PCR sample with Taq beads:

1 µl chromosomal or plasmid DNA

1 µl primer forward

1 µl primer reverse

22 µl H2O

Composition of a 20 µl PCR sample with Phusion DNA polymerase:

4 µl 5 x Phusion DNA polymerase buffer (NEB, Schwalbach)

1 µl chromosomal or plasmid DNA

1 µl primer forward

1 µl primer reverse

1 µl 10 mM dNTP mixture (peqlab Biotechnologie GmbH, Erlangen)

1 µl Phusion DNA polymerase (NEB, Schwalbach)

11 µl H2O

In an initial step, template DNA was denatured for five minutes at 95°C (Taq) or 98°C

(Phusion). Subsequently, 30 repeats of denaturation, primer annealing and DNA synthesis

were performed. Therefore, 15-30 seconds at 95°C or 98°C were followed by 15-30 seconds

at 55°C to 68°C depending on the primer sequence, and one minute per kb (Taq) or 30

seconds per kb (Phusion) at 72°C. Synthesis of DNA fragments was finished in a final

incubation step at 72°C for five minutes. PCR products were analyzed by agarose gel

electrophoresis (see section 3.3.1.3) and, if necessary, purified with the NucleoSpin® Extract

kit (Macherey-Nagel, Düren) as recommended by the supplier.


3.3.1.6 Two-step PCR

Two-step PCR was carried out to induce specific point mutations into the glnR gene.

Therefore, a primer carrying a specific base exchange was used in a first PCR reaction,

which was performed as described in section 3.3.1.5. Then, 10 µl of the first PCR product

carrying the desired exchange were added to a second PCR replacing the forward primer in

a 25 µl reaction. Subsequently, the eventual PCR product carrying the desired base

exchange was purified as described in section 3.3.1.3.

3.3.1.7 Purification and enrichment of DNA

Ethanol precipitation was performed to purify DNA and to increase the concentration. For this

purpose, 1/10 volume 3 M sodium acetate pH 4.8 was given to the DNA sample. After

vortexing, 2.5-fold volume 100 % ethanol was added. After inversion, the precipitated nucleic

acids were centrifuged (15 min, 13,000 x g). The DNA pellet was washed with 2.5-fold

volume 70 % (v/v) ethanol (5 min, 13,000 x g) and dried for 10 minutes in the vacuum

centrifuge Speed Vac® (Savant, USA). Finally, the pellet was suspended in H2O or TE buffer.

TE buffer:

100 mM Tris, 0.1 mM EDTA, pH (HCl) 8

3.3.1.8 Restriction of DNA

Assumption for cloning of different DNA fragments into vectors is the existence of compatible

ligation sites. Therefore, vectors and DNA fragments were treated with one or two restriction

enzymes as recommended by the supplier (NEB, Schwalbach). The samples were incubated

for one to three hours at 37°C or room temperature depending on the restriction enzyme. If

appropriate, 1 µl CIP (calf intestine phosphatase, Roche, Penzberg), was added to

dephosphorylate 5’-ends of vector DNA. A 20 µl restriction sample was composed as follows:

x µl DNA (200-2000 ng)

2 µl NEB buffer

2 µl 10 x BSA (NEB, Schwalbach, optional)

0.5 µl restriction enzyme I

0.5 µl restriction enzyme II (optional)

x µl H2O


After treatment with restriction and dephosphorylating enzymes, DNA was purified by gel

electrophoresis and using the NucleoSpin® Extract kit (Macherey-Nagel, Düren) as described

in section 3.3.1.3.

3.3.1.9 Ligation of DNA fragments

For ligation of restricted DNA fragments and vectors, T4 DNA ligase (NEB, Schwalbach) or

Quick DNA ligase (NEB, Schwalbach) was used.

Composition of a 10 µl ligation sample with T4 ligase:

x µl vector (100 ng)

x µl DNA fragment (5-10 x overage)

1 µl 10 x T4 ligase buffer (NEB, Schwalbach)

0.5 µl T4 ligase

x µl H2O

Composition of a 20 µl ligation sample with Quick ligase:

x µl vector (100 ng)

x µl DNA fragment (5-10 x overage)

10 µl 2 x Quick ligase buffer (NEB, Schwalbach)

1 µl Quick ligase

x µl H2O

Ligation was carried out for two hours at room temperature or at 15°C overnight. The

complete sample was used for transformation of 100 µl competent E. coli cells as described

in section 3.4.2.

3.3.1.10 Sequencing of DNA

According to the chain termination method described by Sanger et al. (1977), DNA fragments

were sequenced. A sequencing sample was composed of 400-700 ng DNA, 10 pmol of an

oligonucleotide primer and 3 µl Terminator Ready Reaction Mix (Applied Biosystems, USA).

This mix contained didesoxynucleotides labeled with fluorescent dye as chain terminators.

Each sample was filled with H2O to a final volume of 10 µl. Sequencing reactions were

performed using the following PCR program:


The resulting DNA fragments were precipitated as described in section 3.3.1.7, solved in 12

µl formamide and investigated with the ABI PRISM Genetic Analyser 310 (Applied

Biosystems, Freiburg). Data were further analyzed with the Clone Manager software (Sci-Ed,

North Carolina, USA).

For further sequence analyses, samples containing 30 µl of plasmid DNA (30-100 ng/µl) and

10 µl of an appropriate sequencing primer (10 pmol/µl) were sent to GATC Biotech

(Konstanz). Data were again analyzed using the Clone Manager software (Sci-Ed, North

Carolina, USA).

3.3.1.11 Gel retardation and competition assays

To detect binding of proteins like GlnR to their target DNA, gel retardation experiments were

carried out. For this, a digoxigenin-labeled DNA fragment containing a putative GlnR binding

site was incubated with purified GlnR protein. Subsequently, the mixture was loaded to a

native gel. Binding of the protein should slow down passing of the DNA through the gel.

The 200-300 bp target DNA for the gel shift assays was synthesized by PCR (refer to section

3.3.1.5) and purified by gel electrophoresis (section 3.3.1.3). For labeling of DNA and setup

of the reaction mixture for gel shift assays, the DIG Gel Shift kit, 2nd Generation (Roche

Diagnostics, Mannheim) was used. First, the purified DNA fragments were diluted 1:1 in TEN

buffer, incubated for 10 minutes at 95°C and shortly centrifuged after cooling down to

15-25°C. The following composition was mixed on ice:

10 µl diluted DNA fragment

4 µl DIG labeling buffer

4 µl cobalt dichloride

1 µl DIG ddUTP solution

1 µl terminal transferase

Incubating the samples for 15 minutes at 37°C ensured that the enzyme attached the

digoxigenin-labeled ddUTPs to the 3’-ends of the DNA. To stop the reaction, 2 µl 0.2 M

EDTA (pH 8) and 3 µl H2O were added. To test the functionality of the generated DNA

probes, these were applied to a positively charged nylon membrane (Roche Diagnostics,

96°C 10 s

25 x 50-65°C 5 s

60°C 4 min


Mannheim) in various dilutions. Labeling efficiency was detected by comparing the signal

strengths to a control DNA. For gel shift assays, DNA dilutions of 1:100-1:200 were used. As

protein sample, total cell extract of M. smegmatis or purified proteins were used (see

sections 3.3.3.1 and 3.3.3.2). A 10 µl reaction was composed as follows:

2 µl 5 x binding buffer

1 µl dilution of the labeled DNA fragment

0.5 µl poly lysine (if required)

1-3 µl competitor DNA polyd[I-C] (if required)

x µl protein or cell extract

x µl H2O

Samples were incubated for 15 minutes at room temperature, when protein-DNA complexes

were formed. After that, 2.5 µl of DNA loading dye (Roche Diagnostics, Mannheim) were

added to the samples. Separation by gel electrophoresis was performed in native 6 %

polyacrylamide gels (Anamed Electrophorese GmbH, Darmstadt) for 1.5 hours at 80 V. As

running buffer 0.5 x TBE buffer (Sambrook et al., 1989) was used. Subsequently, the labeled

DNA was blotted onto a positively charged nylon membrane (Roche Diagnostics, Mannheim)

by electroblotting for one hour at 300 mA and 30 V. The DNA was fixed to the membrane by

UV radiation (2 x Autocrosslink, UV-Stratalinker® 1800, Stratagene, Heidelberg). Blocking of

the membrane was conducted by incubation in 1 x blocking solution (Roche Diagnostics,

Mannheim) for 30 minutes. Then, alkaline phosphatase conjugated anti-digoxigenin Fab

fragments (Roche Diagnostics, Mannheim) were added in a 1:10.000 dilution. After 30

minutes of incubation, the membrane was washed for 2 x 15 minutes in washing buffer.

Accordingly, the membrane was equilibrated in detection buffer for three minutes. The

membrane was subsequently covered and incubated with 10 µl CSPD reagent (Roche

Diagnostics, Mannheim), diluted in 990 µl detection buffer, for 15 minutes at 37°C. Finally,

light emission was detected using Amersham HyperfilmTM MP (GE Healthcare, Munich) or a

ChemidocTM XRS+ molecular imager (Biorad, Munich).

Similarly to the protocol of gel shift assays, competitive assays were carried out. For this

purpose, unlabeled, overlapping 50 or 25 bp DNA fragments were added to the DNA-protein

samples. So, their function as inhibitors of GlnR-binding to its target DNA was tested.

TEN buffer:

10 mM Tris, 1 mM EDTA, 0.1 M NaCl, pH (HCl) 8.0


1 l 10 x TBE buffer:

108 g Tris, 53 g boric acid, 40 ml 0.5 M EDTA, pH (HCl) 8.0

Loading dye:

0.2 % (w/v) bromphenol blue, 40 % (w/v) glycerin, 60 % (v/v) 0.25 x TBE buffer

10 x blocking solution:

10 g blocking reagent (Roche Diagnostics, Mannheim) were suspended in 100 ml maleic

acid buffer and solved by heating to 60°C. After autoclavation for 20 minutes at 121°C, the

solution was further diluted if required.

Maleic acid buffer:

0.1 M maleic acid, 0.15 M NaCl, pH (NaOH) 7.5

Washing buffer:

Maleic acid buffer with 0.3 % Tween20

Detection buffer:

0.1 M Tris, 0.1 M NaCl, pH (NaOH) 9.0

CSPD solution:

1:100 dilution of CSPD reagent (Roche Diagnostics, Mannheim) in detection buffer

3.3.1.12 Electrophoretic mobility shift assay (EMSA)

EMSAs were also used to detect binding of GlnR to its target DNA. In principal, the binding

reaction was performed as described in section 3.3.1.11, but the DNA was not labeled with

any marker. Consequently, higher amounts of DNA fragment and purified protein were used.

After incubation of the DNA-protein samples, 2 µl loading dye were added and gel

electrophoresis was performed in 2 % agarose gels at 8 V/cm of gel length. Again, binding of

the protein should slow down passing of the DNA through the gel. After electrophoresis, DNA

was stained with ethidium bromide solution and detected with the Doc Print 1000 Gel

documentation (peqlab Biotechnologie GmbH, Erlangen).

1 x TAE buffer:



6 x loading dye:


3.3.1.13 Southern blot analysis

For detection of insertion or deletion mutants of M. smegmatis (see section 3.4.6), Southern

blot analyses were performed. For this purpose, the DIG High Prime DNA Labeling and

Detection Starter kit I (Roche Diagnostics, Mannheim) was used. First, an appropriate 300-

500 bp DNA probe was chosen, amplified by PCR (section 3.3.1.5) and isolated by gel

electrophoresis and extraction from the agarose block (section 3.3.1.3). This DNA fragment

was diluted to a final amount of 1 µg in 16 µl H2O and incubated for 10 minutes at 95°C. 4 µl

DIG High Prime (Roche Diagnostics, Mannheim) were added. After incubation of up to 20

hours at 37°C, 2 µl 0.2 M EDTA were added to stop the reaction. Finally, the samples were

incubated for 10 minutes at 65°C. To test the labeling efficiency, 1 µl of each sample was

applied to a positively charged nylon membrane (Roche Diagnostics, Mannheim) and treated

as described in section 3.3.1.11. Signals were detected by adding 40 µl NBT/BCIP (Roche

Diagnostics, Mannheim) and 1.96 ml detection buffer and incubation in the dark. TE buffer

was added to stop the reaction.

For the actual Southern blot analysis, 5 µg chromosomal DNA of M. smegmatis (see section

3.3.1.4) were digested overnight with appropriate restriction enzymes in a total volume of 20

µl (see section 3.3.1.8). The DNA samples were separated by gel electrophoresis in a 1 %

agarose gel (see section 3.3.1.3). If required, a digoxigenin-labeled DNA marker was added

to the gel (DNA Molecular Weight Marker VII, Roche Diagnostics, Mannheim). After that, the

gel was incubated for 15 minutes in depurination solution, for 2 x 15 minutes in denaturation

solution and for 2 x 10 minutes in neutralization solution. The DNA was subsequently

transferred to a positively charged nylon membrane (Roche Diagnostics, Mannheim) by a

capillary blot using a VacuGene vacuum pump (GE Healthcare, Munich) at 50 mbar. The gel

was covered with 20 x SSC. After three hours, the DNA was linked to the membrane by UV

radiation (2 x Autocrosslink, UV-Stratalinker® 1800, Stratagene, Heidelberg). The agarose

gel was stained with ethidium bromide to control successful blotting. The membrane was

incubated in 10 ml DIG Easy Hyb (Roche Diagnostics, Mannheim) for at least 30 minutes at

42°C. After addition of 3 µl DNA probe, which was denatured for five minutes at 95°C before,

the membrane was incubated overnight at 50°C rotating in a hybridization oven (HYBAIDTM,

Biometra, Göttingen). To remove unspecifically bound probe, the membrane was washed on

the next day for 2 x five minutes with washing buffer 2 at room temperature and for 2 x 15

minutes with washing buffer 3 at 68°C. The membrane was thereupon washed for three


minutes in washing buffer and incubated for one hour in 1 x blocking solution. Then, alkaline

phosphatase conjugated anti-digoxigenin Fab fragments (Roche Diagnostics, Mannheim)

were added in a 1:10.000 dilution. After 30 minutes of incubation, the membrane was

washed for 2 x 15 minutes in washing buffer. Accordingly, the membrane was equilibrated for

three minutes in detection buffer. Signals were detected by addition of 10 ml detection buffer

with 200 µl NBT/BCIP (Roche Diagnostics, Mannheim) and incubation in the dark. The

reaction was stopped by adding TE buffer.

1 x TAE buffer:


6 x loading dye:


Depurination solution:

0.25 M HCl (36.44 ml 25 % (v/v) HCl, H2O to 1 l)

Denaturation solution:

1.5 M NaCl, 0.5 M NaOH

Neutralization solution:

0.5 M Tris, 1.5 M NaCl, pH (HCl) 7.5

20 x SSC:

3 M NaCl, 0.3 M sodium citrate, pH (HCl) 7.0 Washing buffer 2:

2 x SSC, 0.1 % (w/v) SDS

Washing buffer 3:

0.2 x SSC, 0.1 % (w/v) SDS






Maleic acid buffer:


Washing buffer:


Detection buffer:


TE buffer:

100 mM Tris, 0.1 mM EDTA, pH (HCl) 8.0

3.3.2 Procedures to work with RNA

3.3.2.1 Isolation of total RNA from M. smegmatis

For isolation of total RNA from M. smegmatis, cells were grown as described in section 3.2.3.

7 ml samples were taken and centrifuged for two minutes at 8,000 x g and 4°C.

Subsequently, the cell pellet was frozen in liquid nitrogen and stored at -80°C. RNA isolation

was performed using the NucleoSpin® RNA II kit (Macherey-Nagel, Düren). For this purpose,

cells were thawed on ice, resuspended in RA1 buffer containing 1 % (v/v) 2-mercaptoethanol

and transferred to Cryo tubes (Thermo Scientific, Denmark) containing glass beads. Cells

were disrupted by vigorous shaking at 6.5 m s-1 for 30 seconds in a FastPrep FP120

instrument (Q-BIOgene, Heidelberg). After five minutes of incubation on ice, the disruption

step was repeated. After that, the samples were centrifuged for three minutes at 13,000 x g

and 4°C. While cell debris and glass beads were discarded, the supernatant was added to

350 µl 70 % (v/v) ethanol and mixed by vortexing. After that, the solution was transferred to

RNA purification columns from the NucleoSpin® RNA II kit (Macherey-Nagel, Düren). Total

RNA was isolated using the kit as recommended by the supplier, whereat the RNA was

routinely treated with DNAse (peqlab Biotechnologie GmbH, Erlangen) to avoid

contamination with chromosomal DNA. This guaranteed a sufficient purity of total RNA for

Dot blot experiments (see section 3.3.2.4). Finally, the RNA was eluted in 60 µl RNAse-free

H2O. Furthermore, for quantitative real time RT PCR (section 3.3.2.5) or microarray

experiments (section 3.3.2.6), total RNA was treated with TURBO-DNase (Ambion, Austin,

USA) and again purified according to the NucleoSpin® RNA II kit (Macherey-Nagel, Düren)


protocol. Otherwise, the RNeasy kit (Qiagen, Hilden) was used as recommended by the

supplier.

RNA concentrations were analyzed at a wavelength of 260 nm (ε = 5 cm2/mg) in the

Nanodrop® spectrometer ND-100 (peqlab Biotechnologie GmbH, Erlangen). Purity was

determined by the quotient of A260 / A280, which is 2.0 for pure RNA. Samples were stored at

-80°C until further usage.

3.3.2.2 RNA gel electrophoresis

Gel electrophoresis was used to test the quality of isolated RNA (see section 3.3.2.1). For

this purpose, 5 µl RNA loading buffer were added to 3 µl purified RNA. The mixture was

incubated for 10 minutes at 65°C. After cooling of the samples for five minutes on ice, they

were analyzed by gel electrophoresis according to the evaluation of DNA (refer to section

3.3.1.3).

RNA loading buffer:

0.2 % (w/v) bromphenol blue, 0.2 % (w/v) xylene cyanol, 65 % (v/v) formamide, 12 % (v/v)

formaldehyde, 2 x MOPS buffer, 2 % (w/v) sucrose

10 x MOPS buffer:

200 mM MOPS, 50 mM sodium acetate, 10 mM EDTA, pH (NaOH) 7.0

3.3.2.3 Synthesis of digoxigenin-labeled RNA probes

For specific detection of certain mRNA species in total RNA samples, digoxigenin-labeled

RNA probes were used. The probes were synthesized by in vitro transcription. Initially, an

approximately 500 bp DNA fragment was amplified by PCR (see section 3.3.1.5) using a

reverse primer with a specific T7 promoter sequence at its 5’-end. Digoxigenin-11-dUTPs

instead of dUTPs were added to the labeling reaction. The composition for an in vitro

transcription sample was as follows:

14 µl purified PCR product

2 µl DIG RNA Labeling Mix (Roche Diagnostics, Mannheim)

2 µl 10 x RNA polymerase buffer (NEB, Schwalbach)

1 µl T7 RNA Polymerase (NEB, Schwalbach)


Incubation for two hours at 37°C ensured the generation of a digoxigenin-labeled RNA probe

by T7 RNA polymerase. Then, 1 µl RNase-free peqGOLD DNaseI (peqlab Biotechnologie

GmbH, Erlangen) was added. The sample was again incubated for 25 minutes at 37°C to

ensure digestion of template DNA. After that, the functionality of the generated probe was

tested (see test of gel shift fragments, section 3.3.1.11). All probes were stored at -80°C until

further usage.

3.3.2.4 RNA hybridization analysis (Dot blot)

To analyze gene transcript levels, RNA hybridization experiments were carried out. For this

purpose, 1 µg total RNA of M. smegmatis (see section 3.3.2.1) was mixed with 100 µl 10 x

SSC marked with bromphenol blue and transferred onto a positively charged nylon

membrane (Roche Diagnostics, Mannheim). Therefore, the Minifold I Dot Blotter (Schleicher

& Schuell, Dassel) and a VacuGene vacuum pump at 20 mbar (GE Healthcare, Munich)

were used. Before that, the membrane had been equilibrated in 10 x SSC. When transfer of

the RNA samples was completed, the membrane was dried for five minutes at 100 mbar.

After that, RNA was crosslinked by UV radiation (2 x Autocrosslink, UV-Stratalinker® 1800,

Stratagene, Heidelberg). The membrane was subsequently incubated for one hour in 12.5 ml

prehybridization solution at 50°C in the hybridization oven HYBAIDTM (Biometra, Göttingen).

After 1.2 µl digoxigenin-labeled RNA probe (see section 3.3.2.3) were added, the reaction

temperature was set to 68°C and hybridization occurred overnight. On the next day, the

membrane was washed for 2 x 15 minutes at room temperature with washing buffer 2. Then,

the membrane was washed for 2 x 25 minutes at 68°C with washing buffer 3 and for five

minutes at room temperature with washing buffer. The membrane was incubated for one

hour in 25 ml 1 x blocking solution. For further treatment and detection see section 3.3.1.11.

Prehybridization solution:

50 ml formamide, 20 ml 10 x blocking buffer, 25 ml 20 x SSC, 1 ml 10 % (w/v) sodium lauroyl

sarcosinate, 0.2 ml 10 % (w/v) SDS, 3.8 ml H2O

20 x SSC:

3 M NaCl, 0.3 M sodium citrate, pH (HCl) 7.0

Washing buffer 2:

2 x SSC, 0.1 % (w/v) SDS


Washing buffer 3:

0.2 x SSC, 0.1 % (w/v) SDS





Maleic acid buffer:


Washing buffer:


Detection buffer:


3.3.2.5 Quantitative real time RT PCR

Quantitative real time RT PCR was performed to identify differences in the quantity of a

specific mRNA. To achieve this, cells were incubated under nitrogen starvation (see section

3.2.3) and total RNA was prepared as described before (see section 3.3.2.1). RT PCR is

based on quantification of mRNA levels by transcription of cDNA followed by a common PCR

resulting in the amplification of a specific DNA fragment. Total RNA was used as a template

for reverse transcription. Primers were generated by MWG (Ebersberg) and dissolved in H2O

to a final concentration of 100 pmol/µl. To guarantee constant polymerase efficiency, all

amplified DNA fragments were of 100 bp size. The iScript One-Step RT-PCR kit with SYBR

Green (Biorad, Munich) was used for setting up the reactions.

Composition of a 25 µl real time RT PCR sample:

12.5 µl SYBR mix (Biorad, Munich)

0.5 µl primer forward

0.5 µl primer reverse

0.5 µl reverse transcriptase (Biorad, Munich)

1 µl H2O

10 µl total RNA


Real time RT PCR program:

10 min 50°C

5 min 95°C

10 sec 95°C

45 x 15 sec 57°C

10 sec 72°C

1 min 95°C

1 min 55°C

81 x 10 sec 55-95°C

For all real time RT PCR experiments, the MyiQ Single-Color Real Time PCR Detection

system (Biorad, Munich) was used. In the first 10 minutes at 50°C, mRNA was transcribed by

the reverse transcriptase into cDNA. Incubation for five minutes at 95°C ensured

denaturation of the DNA/RNA hybrid. During the next 45 cycles, a specific cDNA product was

amplified. The amount of PCR product was determined after each cycle. Therefore, the

increase of fluorescence was monitored, as the fluorescent dye SYBR Green 1 is able to

bind double stranded DNA. Cq values were calculated when the sample fluorescence

crossed the threshold line, which was set automatically by the analysis software to separate

background fluorescence. In 81 cycles, where the temperature was risen from 55°C to 95°C,

melt curves were generated to analyze the purity and specificity of the amplified PCR

products. To guarantee the quality of the experiment, a no template control (H2O instead of

RNA) was carried along for each tested mRNA. Also to control the specificity of the PCR

product, each sample was analyzed in a 4 % agarose gel, where only the specific 100 bp

band should be visible.

Primarily, RNA dilution series were tested in duplicates. 100, 10, 1 and 0.1 ng RNA were

used. From the determined Cq values, a standard curve was calculated for every dilution

series. The slope of the curve was used to calculate the efficiency of the PCR reaction. All

primer pairs with an efficiency of 90-105 % were used for further experiments. To analyze

differences in gene expression, 10 ng RNA of the M. smegmatis wild type and the glnR

deletion strain were used for each reaction. Of each sample, at least 3 replicates were used

to determine the relative transcript level of various target genes compared to a reference

gene, whose transcript level did not differ significantly under the tested conditions. The MyiQ

Optical Software provided by the supplier (Biorad, Munich) was used to analyze the data.

Standard curves were analyzed using the applications “Gene Expression” and “Melt Curves”,

differences in transcript levels were quantified using the application “Gene Expression

Analysis”. Relative fold expression ratios were calculated according to Pfaffl et al. (2001)

using the following formula:


E target Relative expression ratio = E reference

3.3.2.6 cDNA microarrays

To compare mRNA levels in the total genome of different M. smegmatis strains, cDNA

microarrays were carried out. For this purpose, total RNA of M. smegmatis was prepared

(see section 3.3.2.1). The RNA was transcribed into cDNA, labeled with cy3 and cy5

fluorescence markers and bound to 60 bp oligonucleotides, which represented the whole

M. smegmatis genome and were fixed to a microarray chip. These steps were performed at

the Department of Biochemistry, Friedrich-Alexander University Erlangen-Nuremberg, using

the Two-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technologies,

Waldbronn). The microarray chip was also designed by Agilent Technologies. For data

analyses, the program GeneSpring GX 11 (Agilent Technologies, Waldbronn) was used.

3.3.3 Procedures to work with proteins

3.3.3.1 Preparation of total cell extract

For preparation of total cell extract, 500 ml C. glutamicum or M. smegmatis cells were grown

as described in section 3.2.3 and harvested by centrifugation (10 min, 4,000 x g, 4°C). After

the pellet was resuspended in 20 ml basic buffer, 500 µl protease inhibitor Complete (Roche

Diagnostics, Mannheim) and a spot of lysozyme (Merck, Darmstadt) were added. The cells

were subsequently disrupted using the sonication instrument SonoPlus (BANDELIN

electronic, Berlin) for 5 x 30 seconds at 60 % capacity. Between the intervals, samples were

incubated for five minutes on ice. Cell debris was removed by centrifugation (30 min, 14,000

x g, 4°C). The supernatant was transferred into a new reaction tube and stored at 4°C until

further usage.

Basic buffer:

100 mM Tris, 150 mM NaCl, 1 mM EDTA, pH (HCl) 8.0

Cq target (wt - ΔglnR)

Cq reference (wt - ΔglnR)


3.3.3.2 Protein purification via affinity chromatography

All tagged M. smegmatis proteins were overexpressed in E. coli Rosetta2. For that, cells

were grown as described in section 3.2.3. To induce overexpression of proteins tagged with

6 x His or MBP (maltose binding protein), 1 mM IPTG was added, whereas overexpression

of proteins with Strep-tag was induced by adding 1 µg/ml doxycycline. After four hours of

incubation, cells were harvested by centrifugation (10 min, 4,000 x g, 4°C). The cell pellet

was stored at -80°C until further usage or directly solved in 20 ml basic buffer (His, Strep or

MBP). 500 µl protease inhibitor Complete (Roche Diagnostics, Mannheim) and a spot of

lysozyme (Merck, Darmstadt) were added. Cells were subsequently disrupted using the

sonication instrument SonoPlus (BANDELIN electronic, Berlin) for 3 x 30 seconds at 50 %

capacity. Between the intervals, the samples were incubated for five minutes on ice. Cell

debris was removed by centrifugation (30 min, 14,000 x g, 4°C). The resulting cell extract

was filtered through Filtropur S 0.2 filter (Sarstedt, Nümbrecht) to remove remaining

granules.

His-tagged proteins were purified using an Äkta prime system with a 1 ml HighTrap affinity

column (GE Healthcare, Munich) following the protocol of the supplier. After loading the cell

extract onto the column and washing it with His basic buffer, His-tagged proteins were eluted

by adding rising concentrations of imidazole. MBP-tagged proteins were purified using the

same system with a 10 ml amylose resin affinity column (GE Healthcare, Munich) according

to the supplier’s protocol. MBP-tagged proteins were eluted from the column by adding rising

concentrations of maltose. Strep-tagged proteins were purified using a 1 ml Strep-TactinR-

SepharoseR matrix (IBA BioTAGnology, Göttingen) in a Poly-Prep chromatography column

(Biorad, Munich) following the description of the supplier. In order to elute Strep-tagged

proteins, D-desthiobiotin was added. Columns were regenerated following the

manufacturers’ protocols. All elution fractions were collected and stored at 4°C until further

usage.

For further purification or to separate proteins of different size, gel filtration was carried out.

Therefore, an Äkta system with a Superdex G250 column was used as recommended by the

supplier (GE Healthcare, Munich). After 1-2 column volumes of gel filtration buffer, the

protein sample was manually loaded onto the Äkta system.

His basic buffer:

300 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, pH (NaOH) 8.0

His elution buffer:

300 mM NaCl, 50 mM NaH2PO4, 500 mM imidazole, pH (NaOH) 8.0


Strep basic buffer:


Strep elution buffer:

100 mM Tris, 500 mM NaCl, 1 mM EDTA, 2.5 mM D-desthiobiotin, pH (HCl) 8.0

Strep regeneration buffer:

100 mM Tris, 500 mM NaCl, 1 mM EDTA, 1 mM HABA, pH (HCl) 8.0

MBP basic buffer:


MBP elution buffer:

20 mM Tris, 200 mM NaCl, 1 mM EDTA, 20 mM maltose, pH (HCl) 7.4

Gel filtration buffer:

20 mM Tris, 200 mM NaCl, pH (HCl) 8.0

All buffers were sterilized by filtration.

3.3.3.3 Quantification and enrichment of proteins

Protein concentrations were measured according to Bradford (1976). 0, 2, 4, 6, 8 and 10 µg

bovine serum albumine (BSA) were used to generate a standard curve. All samples were

filled with H2O to a final volume of 800 µl. Subsequently, 200 µl Biorad Protein Assay

(Biorad, Munich) were added. After vortexing, the samples were incubated for 20 minutes at

room temperature and vortexed again. Measurements were carried out in plastic cuvettes

(bio one, Greiner, Essen) in the photometer Ultraspec 2100 pro (Amersham Biosciences,

USA) at a wavelength of 595 nm against a reference consisting of 800 µl H2O and 200 µl

Biorad Protein Assay (Biorad, Munich).

The Nanodrop® spectrometer ND-100 (peqlab Biotechnologie GmbH, Erlangen) was also

used to determine protein concentrations. With the application “Protein A280”, the protein

concentration was measured at 280 nm considering the molecular weight and the extinction

coefficient of the purified protein.


To increase the amount of protein in a sample, it was concentrated using Centriprep

centrifugal filters with a pore size of 10 kDa (Millipore, Massachusetts, USA). First, the filter

was equilibrated with elution buffer of the particular protein (see section 3.3.3.2) by

centrifugation for five minutes at 3,000 x g and 4°C. After that, the buffer was completely

removed, the protein sample was added to the filter and also centrifuged at 3,000 x g and

4°C for 30 minutes. Subsequently, the flow through was removed. This procedure was

repeated until the desired volume of protein sample remained.

3.3.3.4 SDS polyacrylamide gel electrophoresis

14 % SDS polyacrylamide gels were used for electrophoretic separation of protein samples

(according to Schägger and von Jagow, 1987). The gels contained urea and SDS, leading to

denaturation of the proteins.

Composition of a separation gel:

3.75 ml acrylamide/bisacrylamide (37.5 %/1 %)

3.75 ml Schägger gel buffer

4.05 g urea

3.75 µl TEMED

37.5 µl 10 % (w/v) APS

Composition of a stacking gel:

250 µl acrylamide/bisacrylamide (37.5 %/1 %)

775 µl Schägger gel buffer

2 ml H2O

2.5 µl TEMED

25 µl 10 % (w/v) APS

Loading buffer was added to the protein samples. After incubation for five minutes at 95°C,

the samples were loaded onto the gel. For gel electrophoresis, a BlueVertical 101 apparatus

(Serva Electrophoresis GmbH, Heidelberg) was used. First, the voltage was set to 50 V. It

was risen to 120 V as soon as the protein samples had crossed the stacking gel. As running

buffers cathode and anode buffers were used. To determine the size of the investigated

proteins, protein markers (peqGOLD Protein Marker II or peqGOLD Prestained Protein-

Marker IV, peqlab Biotechnologie GmbH, Erlangen) were added to the gel.

http://de.wikipedia.org/wiki/Massachusetts


Schägger gel buffer:

3 M Tris, 1 M HCl, 0.3 % (w/v) SDS

5 x loading buffer:

20 % (w/v) SDS, 60 % (w/v) glycerol, 250 mM Tris, 10 % (v/v) 2-mercaptoethanol, 0.01 %

(w/v) serva blue G-250, pH (HCl) 6.8

Cathode buffer:

0.1 M Tris, 0.1 M tricine, 0.1 % (w/v) SDS

Anode buffer:

0.2 M Tris, pH (HCl) 8.9

3.3.3.5 Native polyacrylamide gel electrophoresis

Electrophoretic separation of proteins was also performed using native polyacrylamide gels,

where no denaturation of the proteins occurred.

Composition of a native gel:

2.5 ml acrylamide/bisacrylamide (19 %/1 %)

1 ml 10 x TBE buffer

6.195 ml H2O

5 µl TEMED

300 µl 10 % (w/v) APS

After native loading buffer was added to the protein samples, the mixture was loaded onto

the gel. Gel electrophoresis was performed in a BlueVertical 101 apparatus (Serva

Electrophoresis GmbH, Heidelberg) at 100 V. 1 x TBE buffer was used as running buffer. To

determine the size of the investigated proteins, protein markers (peqGOLD Protein Marker II

or peqGOLD Prestained Protein-Marker IV, peqlab Biotechnologie GmbH, Erlangen) were

added to the gel.

1 l 10 x TBE buffer :

108 g Tris, 53 g boric acid, 40 ml 0.5 M EDTA, pH (HCl) 8.0


4 x native loading buffer:

15 % (w/v) glycerin, 125 mM Tris, 1 mM EDTA, bromphenol blue, pH (HCl) 6.8

3.3.3.6 Staining with Coomassie Brilliant Blue

Proteins separated in SDS or native polyacriylamide gels (see sections 3.3.3.4 and 3.3.3.5)

were stained with Coomassie Brilliant Blue (Sambrook et al., 1989). For this purpose, the

polyacrylamide gel was incubated in staining solution at room temperature for 15 minutes.

Subsequently, it was decolorized for 1-5 hours in 10 % (v/v) acetic acid.

Staining solution:

45 % (v/v) methanol, 10 % (v/v) acetic acid, 0.1 % (w/v) Coomassie Brilliant Blue G-250

3.3.3.7 Western blot analysis

To detect proteins with specific antibodies, Western blot analyses were carried out. After

protein samples had been separated in SDS or native polyacrylamide gels, they were

dispatched to a polyvinylidene difluoride membrane (Millipore Immobilon P, Roth, Karlsruhe)

by electroblotting. For this purpose, the membrane, equilibrated in 60 % (v/v) methanol and

transfer buffer, was put on filter paper which was also equilibrated in transfer buffer. The

protein gel was put on top and covered with filter paper again deposed in transfer buffer.

Blotting was carried out for one hour at 0.8 mA per cm2 of membrane. After that, the

membrane was incubated in 1 x TBS buffer for five minutes and subsequently in blocking

solution for one hour. Then, a specific antibody (His, MBP or GlnR, each generated in

rabbits) was added in a 1:10.000 dilution and the membrane was incubated overnight. On

the next day, the membrane was washed for 3 x five minutes in TBS-T buffer and after that,

an anti-antibody with alkaline phosphatase (Anti-Mouse or Anti-Rabbit IgG, Sigma-Aldrich,

Steinheim) was added in a 1:10.000 dilution in blocking solution. The membrane was again

washed for 3 x five minutes in TBS-T buffer.

For detection of anti-GlnR or anti-MBP, the BCIP/NBT alkaline phosphatase substrate (Roth,

Karlsruhe) was used. For this, 65 µl BCIP and NBT stock solutions were diluted in 10 ml

detection buffer. The reaction was stopped by adding H2O. For detection of anti-His, the ECL

Western blot plus kit (GE Healthcare, Munich) was used as recommended by the supplier.

The signal was detected on Amersham HyperfilmTM MP (GE Healthcare, Munich).


Transfer buffer:

25 mM Tris, 0.2 M glycine, 20 % (v/v) methanol, pH (HCl) 8.5

1 l 10 x TBS buffer:

24.2 g Tris, 80 g NaCl, pH (HCl) 7.6

TBS-T buffer:

1 x TBS buffer, 0.1 % (v/v) Tween20

Blocking solution:

15 ml 10 x TBS buffer, 135 ml H2O, 7.5 g dry milk, 150 µl Tween20

NBT stock solution:

0.5 g 4-nitro blue tetrazolium chloride (NBT) were dissolved in 10 ml 70 % (v/v)

dimethylformamide and stored at -20°C.

BCIP stock solution:

0.5 g 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (BCIP) were dissolved in 20 ml

100 % dimethylformamide and stored at -20 °C.

Detection buffer:

100 mM NaCl, 5 mM MgCl2, 100 mM Tris, pH (NaOH) 9.5

3.3.3.8 Pull down assays

To identify GlnR interaction partners in pull down assays, GlnR protein with Strep-tag

overexpressed in E. coli Rosetta2 was used (see sections 3.2.3 and 3.3.3.2). First, the Strep-

GlnR containing E. coli cell extract was loaded onto a 1 ml Strep-TactinR-SepharoseR matrix

(IBA BioTAGnology, Göttingen) in a Poly-Prep chromatography column (Biorad, Munich).

After three washing steps with 1.5 ml strep washing buffer, total cell extract of M. smegmatis

(see section 3.3.3.1) was added. The washing steps were repeated, followed by five elution

steps with 500 µl strep elution buffer each. Purified GlnR bound to the column as well as

possible GlnR interaction partners were analyzed in SDS polyacrylamide gel electrophoresis

(see section 3.3.3.4). Visible protein bands were excised from the gel and sent to ZMMK,

University of Cologne, for mass spectroscopic analysis (MALDI-ToF-MS).


3.3.3.9 Radioactive phosphorylation of proteins

Protein phosphorylation was investigated using radioactive [γ-32P] ATP supplied by Hartmann

Analytics, Braunschweig.

Composition of a 10 µl phosphorylation reaction:

1.5 µl [γ-32P] ATP (0.05 µM)

1 µl ATP (5 µM)

1 µl protein (here: sensor histidine kinase, 1µg)

6.5 µl TKMD buffer

The mixture was prepared on ice, whereas radioactive material was added at last. A sample

with H2O instead of ATP was used as a negative control. All samples were incubated at

30°C. After 10 minutes of incubation, 5 µl purified GlnR protein (10 x amount of kinase) were

added, followed by another 20 minutes of incubation. At different time points, 3 µl loading dye

were added and the samples were placed on ice. After that, the proteins were separated in

SDS PAGE (see section 3.3.3.4). To detect radioactivity, the gels were wrapped in plastic foil

and placed on a phosphor imaging plate for 90 minutes at -80°C. Signals were detected

using the BAP-IP MP 2025 reader (Fujifilm, Düsseldorf). To determine phosphorylation of

GlnR, some modifications were made to the protocol such as using higher amounts of

protein or adding 5 mM α-ketoglutarate or Na-glutamate or total cell extract of M. smegmatis.

The signal strengths detected on the phosphor imaging plate were analyzed using the

program ImageJ (http://rsbweb.nih.gov/ij/).

For phosphorylation of GlnR, acetyl [32P] phosphate was produced. For this purpose, 0.19 ml

pyridine, 0.3 ml 0.33 M K2HPO4 and 0.1 ml [32P] phosphoric acid were mixed on ice.

Subsequently, 22 µl acetic acid anhydride were slowly added. After three minutes of

incubation on ice, 4 M LiOH was added until pH 7.0 was reached. 4.5 ml 100 % ethanol were

added after another three minutes on ice and after that, the sample was incubated for one

more hour on ice. Accordingly, the sample was centrifuged for five minutes at 8,000 x g and

4°C and washed twice with 5 ml 100 % ethanol. After the pellet was dried for two hours, it

was solved in resuspension buffer.

The concentration of generated acetyl [32P] phosphate was measured in a Spectro-

Photometer (GE Healthcare, Munich) at 620 nm. Acetyl phosphate concentrations from 0.25

to 2.5 mM were used to generate a standard curve. Treatment of GlnR with acetyl [32P]

phosphate was performed according to Hiratsu et al. (1995) and Bouché et al. (1998). GlnR

was incubated with rising amounts of acetyl [32P] phosphate for 30 minutes at 30°C or with

50 mM acetyl [32P] phosphate, while samples were taken after 1, 5, 10, 15, 30 and 60

http://rsbweb.nih.gov/ij/


minutes. For detection of radioactive signals in SDS PAGE, the samples were treated as

described above.

TKMD buffer:

50 mM Tris, 200 mM KCl, 5 mM MgCl2, 5 mM DTT, pH (HCl) 7.5, sterilization by filtration

5 x loading dye:

20 % (w/v) SDS, 60 % (w/v) glycerol, 250 mM Tris, 10 % (v/v) 2-mercaptoethanol,

0.01 % (w/v) serva blue G-250, pH (HCl) 6.8

Resuspension buffer:

50 mM Tris, 0.1 mM EDTA, 1 mM DTT, 5 % (w/v) glycerol, pH (HCl) 7.5

3.4 Genetic manipulation of bacteria

3.4.1 Preparation of competent E. coli cells

To generate chemically competent E. coli, the cells were cultivated in 4 ml LB medium

overnight. With this overnight culture, 100 ml LB medium were inoculated to an oD600 of 0.1

and cultivated to an oD600 of 0.4. The cells were subsequently centrifuged for 10 minutes at

4,000 x g and 4°C, before the cell pellet was resuspended in 40 ml cold, 50 mM CaCl2 and

incubated for 30 minutes on ice. After that, the cells were again centrifuged for 10 minutes at

4,000 x g and 4°C. The cell pellet was solved in 5 ml cold, 50 mM CaCl2. After 10 minutes

incubation on ice, 1.3 ml cold, 87 % glycerin were added and 100 µl aliquots were frozen in

liquid nitrogen. The competent cells were stored at -80°C until further usage.

3.4.2 Transformation of competent E. coli cells

Transformation of chemically competent E. coli cells (section 3.4.1) was performed using

heat shock process. After a 100 µl aliquot of cells was thawed on ice, 1-2 µl plasmid DNA

(section 3.3.1.1) or 10-20 µl ligation (section 3.3.1.9) were added. The mixture was incubated

for 30 minutes on ice. Absorption of plasmid DNA into the cells took place when a heat shock

was performed for 90 seconds at 42°C. Cells were subsequently incubated for another 30

minutes on ice, before 1 ml LB or SOC medium (see table 3, section 3.2.1) was added. After

that, the cell suspension was cultivated for one hour at 37°C and 700 rpm using a TS 100


Thermoshaker (peqlab Biotechnologie GmbH, Erlangen). Finally, 200 µl of the cell

suspension were plated on LB plates containing the appropriate antibiotic (retransformation

with plasmids). For transformation with a ligation sample, the complete cell suspension was

centrifuged (2 min, 5,000 x g), resuspended in the backflow of medium and completely plated

on LB plates containing the appropriate antibiotic. The plates were incubated for 24 hours at

37°C.

3.4.3 Preparation of competent E. coli Rosetta2 cells and TSS transformation

Overexpression of tagged M. smegmatis proteins was carried out in E. coli Rosetta2 cells

which had been transformed using the TSS method according to Chung et al. (1989). For

preparation of competent cells, these were cultivated in 4 ml LB medium to an oD600 of 0.5.

2 ml of this cell suspension were centrifuged for one minute at 10,000 x g and 4°C, before

the cell pellet was solved in 200 µl cold TSS solution. 0.5-1 µg plasmid DNA was added

subsequently and the mixture was incubated for 30 minutes on ice. After addition of 800 µl

LB medium, the cells were regenerated for one hour at 37°C and 400 rpm using a TS 100

Thermoshaker (peqlab Biotechnologie GmbH, Erlangen). The complete sample was plated

on LB plates containing 25 µg/ml chloramphenicol and a second, appropriate antibiotic.

100 ml TSS solution:

10 g polyethylene glycol, 5 ml 100 % (v/v) DMSO, 5 ml 1M MgCl2, 50 ml 2 x LB medium,

sterilization by filtration

3.4.4 Preparation of electrocompetent M. smegmatis cells

To prepare electrocompetent M. smegmatis cells, 100 ml 7H9 medium were inoculated and

cultivated to an oD600 of 0.8. After that, cells were centrifuged (10 min, 4,000 x g, 4°C) and

the cell pellet was solved in 25 ml cold, 10 % (v/v) glycerin with 0.05 % (v/v) Tween80. After

recentrifugation, the remaining cell pellet was resuspended in 10 ml cold, 10 % (v/v) glycerin

with 0.05 % (v/v) Tween80 and centrifuged once more. Finally, the cell pellet was solved in

5 ml cold, 10 % (v/v) glycerin with 0.05 % (v/v) Tween80. 100 µl aliquots were immediately

frozen in liquid nitrogen and stored at -80°C until further usage.


3.4.5 Transformation of electrocompetent M. smegmatis cells

Transformation of electrocompetent M. smegmatis cells was performed by electroporation.

After 100 µl competent cells (section 3.4.4) were thawed on ice, 1 µg plasmid DNA was

added. The mixture was incubated for further 10 minutes on ice and then transferred to a

cold electroporation cuvette with 2 mm electrode clearance (peqlab Biotechnologie GmbH,

Erlangen). Electroporation was carried out using a Gene-Pulser (Biorad, Munich) at 2.5 kV,

1000 Ω and 25 µF. Subsequently, cells were supplied with 1 ml SOC medium (see table 3,

section 3.2.1) and recovered for three hours at 37°C and 700 rpm using a TS 100

Thermoshaker (peqlab Biotechnologie GmbH, Erlangen). The complete cell suspension was

centrifuged (3 min, 5,000 x g), resuspended in the backflow of medium and completely plated

on 7H10 plates containing an appropriate antibiotic. The plates were incubated for 2-5 days

at 37°C.

3.4.6 Generation of genomic insertion and deletion mutants of M. smegmatis

To generate insertions or deletions in the genome of M. smegmatis, cells were transformed

(see section 3.4.5) with the plasmid pML814 carrying homologous sequences of the target

genes. After incubation of 4-7 days on 7H10 agar plates containing 50 µg/ml hygromycin B,

the arisen colonies were transferred to new 7H10-hygromycin plates to exclude

unspecifically grown colonies. From these cultures, the chromosomal DNA was prepared

(see section 3.3.1.4) and tested in Southern blot analyses (section 3.3.1.13). This procedure

was sufficient to determine mutants with insertion of the plasmid into target genes. While

generating deletion mutants with two homologous sequences upstream and downstream of

target genes, single and double cross overs occurred. This was also investigated in Southern

blot analyses (section 3.3.1.13). Cells showing single cross over were further cultivated

overnight at 37°C in 4 ml 7H9 medium containing 50 µg/ml hygromycin B. After that, cells

were filtered using a Filtropur S 5 µm filter (Sarstedt, Nümbrecht) and again cultivated as

described before. Afterwards, the oD600 was set to 0.05 and different dilutions were plated on

7H10 plates containing 50 µg/ml hygromycin B.

Using this procedure, cells were scattered and chances for a second single cross over were

increased. This was again examined in Southern blot analyses (section 3.3.1.13). After a

chromosomal double cross over, the target gene was exchanged against a hygromycin

resistance gene. To remove this, cells were transformed (section 3.4.5) with another plasmid,

pMN234, carrying the gene for Flpe recombinase for recombination of FRT sites, and plated

on 7H10 plates containing 25 µg/ml kanamycin. The resulting colonies were also cultivated

overnight at 37°C in 4 ml 7H9 medium containing 25 µg/ml kanamycin, filtered and plated on


7H10 plates containing 25 µg/ml kanamycin as described above. Finally, colonies were

plated on 7H10 plates containing 50 µg/ml hygromycin B and 400 µg/ml streptomycin,

respectively. In a last Southern blot analysis (section 3.3.1.13), the removal of the

hygromycin resistance gene was monitored.

3.4.7 Fluorescence measurements

To determine GlnR-dependent promoter activities, the green fluorescent protein (GFP) was

used as a reporter. For this purpose, different promoter fragments were cloned into vectors

carrying the gfpuv gene. M. smegmatis cells were transformed with this constructs by

electroporation (see section 3.4.5) and cultivated as described in section 3.2.3. When cells

reached the exponential growth phase, a 10 ml sample was harvested by centrifugation

(10 min, 4,000 x g, 4°C) and resuspended in 1 x PBS buffer. To analyze the influence of

nitrogen starvation on the promoter activities, the rest of the cultures was centrifuged

(10 min, 4,000 x g, 4°C), resuspended in 7H9-N medium and incubated for further

30 minutes. Finally, another 10 ml sample was taken as described above.

After the samples were excited at a wavelength of 395 nm, the emission between 490 and

520 nm was detected in the fluorimeter Fluorolog 3 Double Spectrometer (Spex, Edison,

USA). Promoter strength was calculated depending on the fluorescence at 508 nm and the

dry weight of the cells per ml. An oD600 of 1 corresponds to 0.36 mg dry weight per ml

(Weinand, 2004).

1 l 1 x PBS buffer:

8 g NaCl, 3.58 g Na2HPO4 x 12H2O, 0.2 g KCl, 0.24 g K2HPO4, pH (HCl) 7.4

3.4.8 Fluorescence microscopy

M. smegmatis cells were grown, harvested and resuspended in 1 x PBS buffer as described

in section 3.4.7. 5 µl of the solution were transferred to an object slide and covered with a

cover slip. Fluorescence was detected using the LSM reverse laser microscope (Zeiss,

Göttingen) at the Institute for Clinical Microbiology, Friedrich-Alexander University Erlangen-

Nuremberg.


3.4.9 β-galactosidase assays

To generate a more sensitive reporter system for measuring promoter activities, the lacZ

gene coding for β-galactosidase was used. M. smegmatis strains carrying plasmids

expressing the lacZ gene under the control of different promoters were grown as described in

section 3.4.7. When cells reached the exponential growth phase, the oD600 was exactly

determined and 1 ml sample was centrifuged (2 min, 4,000 x g, 4°C), subsequently frozen in

liquid nitrogen and stored at -80°C.

On the next day, the cell pellets were thawed on ice and resuspended in 1 ml 1 x Z buffer

before they were transferred to Cryo tubes (Thermo Scientific, Denmark) containing glass

beads. Cells were disrupted as described in section 3.3.2.1. Cell debris and glass beads

were removed by centrifugation (3 min, 13,000 x g, 4°C). The supernatant was transferred to

a new reaction tube. 300 µl 1 x Z buffer were mixed with 25 µl 0.1 % SDS, 50 µl CHCl3 and

200 µl 5 x Z buffer with 2-mercaptoethanol. 500 µl of the cell supernatant were added at last.

After 20 minutes of incubation at 28°C, 200 µl fresh ONPG solution were added under

vortexing. Samples were further incubated until a strong yellow coloring was visible. Then the

reaction was stopped by adding 500 µl 1M Na2CO3. Finally, the absorption at 420 and 550

nm was measured using the photometer Ultraspec 2100 pro (Amersham Biosciences, USA).

β-galactosidase activity was calculated in Miller Units according to the following formula:

MU =

5 x Z buffer:

300 mM Na2HPO4 x 12 H2O, 200 mM NaH2PO4 x H2O, 50 mM KCl, 5 mM MgSO4 x H2O

5 x Z buffer with 2-mercaptoethanol:

50 ml 5x Z buffer, 850 µl 2-mercaptoethanol

ONPG solution:

4 mg/ml ONPG in 1 x Z buffer with 2-mercaptoethanol

A420 - 1.75 x A550

A600 x V x t


3.4.10 Determination of urease activity

For determination of urease activity in M. smegmatis, an indophenol test was performed

according to Jahns et al. (1988), in which ammonium can be detected. Cells were grown as

described in section 3.2.3. When cells reached exponential growth, 20 ml were harvested by

centrifugation (10 min, 4,000 x g, 4°C), washed in 20 ml KH2PO4 buffer and resuspended in

500 µl KH2PO4 buffer. The rest of the cells was also centrifuged (10 min, 4,000 x g, 4°C),

washed and incubated for 30 minutes in 7H9-N medium, before another sample was taken

as described above.

Cells were transferred to Cryo tubes (Thermo Scientific, Denmark) containing glass beads

and disrupted by vigorous shaking at 6.5 m s-1 for 30 seconds in a FastPrep FP120

instrument (Q-BIOgene, Heidelberg). After five minutes of incubation on ice, the disruption

step was repeated twice. After that, samples were centrifuged for 20 minutes at 13,000 x g

and 4°C to remove cell debris and glass beads. Protein concentrations of all samples were

measured using the application “Protein A280” of the Nanodrop® spectrometer ND-100

(peqlab Biotechnologie GmbH, Erlangen). 50 and 200 µl cell extract were filled with KH2PO4

buffer to a final volume of 1 ml and incubated for 10 minutes at 37°C. After that, 50 µl of a

200 mM urea solution were added, followed by further incubation at 37°C for 15 minutes. To

start the indophenol reaction, 1 ml solution 1 and 1 ml solution 2 were added to each sample,

followed by another incubation at 50°C for 15 minutes. “Blind samples” (addition of urea after

solution 1 and 2) used as references to the actual samples were carried along, as well as

rising concentrations of ammonium (100 µM to 5 mM) to generate a calibration curve. The

absorption of all samples at a wavelength of 546 nm was measured in plastic cuvettes (bio

one, Greiner, Essen) in the photometer Ultraspec 2100 pro (Amersham Biosciences, USA).

Urease activity was calculated as units per mg protein according to the following formula:

Urease activity =

KH2PO4 buffer:

50 mM KH2PO4, pH (KOH) 7.0

Solution 1:

4.72 ml phenol, 0.025 g sodium nitroprusside in 100 ml H2O

Solution 2:

6.25 g NaOH, 13.125 ml sodium hypochlorite in 250 ml H2O

µmol generated ammonium

1 min x mg protein

Results 53

4 Results

4.1 General analyses of nitrogen metabolism and control in M. smegmatis

4.1.1 Utilization of different nitrogen sources

At the beginning of this study, only few experimental data were available about the ability of

M. smegmatis to use different nitrogen sources and especially about the role of GlnR in the

corresponding processes. In a bioinformatic approach, various genes coding for uptake and

assimilation systems for ammonium, nitrate, nitrite, urea and others were identified,

suggesting the ability of M. smegmatis to utilize a variety of different substrates as nitrogen

sources (Amon et al., 2009). Furthermore, ammonium, alanine, asparagine, glutamic acid,

glutamine, nitrate, nitrite and urea have already been proven to suffice as single nitrogen

source for the M. smegmatis wild type (Iwainsky and Sehrt, 1967; Ahmad et al., 1986;

Sritharan et al., 1987).

In this study, all amino acids, bases and different inorganic substrates were tested. Growth of

the M. smegmatis wild type SMR5 and the glnR deletion strain MH1 was observed over 72

hours in 7H9-N minimal medium, to which either 10 or 100 mM of the corresponding

substrate were added as putative nitrogen source. Substances such as tryptophan, tyrosine,

adenine, guanine, thymine, uracil and uric acid were only added in 10 mM, as higher

amounts were not dissolved in the medium. Moreover, nitrite concentrations higher than

10 mM were toxic for the cells. Significant changes in growth behavior were only inspected

within the first 32 hours. All growth experiments were carried out in triplicates, while one

representative growth curve for each putative nitrogen source is shown in figure 6 and 7. A

sample with 7H9 minimal medium was always carried along as a positive control, where the

generation time of SMR5 as well as of MH1 was 4-5 hours and a maximum oD600 of 4 was

reached (see figure 8 and 9). 7H9-N lacking any nitrogen source was used as negative

control; as expected, no growth was detected and cells reached a maximum oD600 of 0.7,

which was less than two doublings. To distinguish between growth behavior of the wild type

and the glnR deletion strain, a 7H9 sample to which MSX was added was carried along.

MSX is a glutamate analog that blocks GS activity (Berlicki, 2008). It has already been

shown before, that due to artificially caused nitrogen starvation the glnR deletion strain MH1

was no longer able to grow in this medium compared to the wild type (Amon et al., 2008).

Independently from the concentration, no growth was detected when glycine, phenylalanine,

adenine, thymine, creatinine, glucosamine or N-acetylglucosamine were used as nitrogen

Results 54

Results 55

Fig. 6: Growth of M. smegmatis with different substrates used as single nitrogen source. Substrates were added in a final concentration of 10 mM. □: SMR5 (wild type strain); ■: MH1 (glnR deletion strain); ○: wild type strain DSM 43756.

Results 56

Results 57

Fig. 7: Growth of M. smegmatis with different substrates used as single nitrogen source. Substrates were added in a final concentration of 100 mM. □: SMR5 (wild type strain); ■: MH1 (glnR deletion strain); Δ: SMR5 (addition of trace element solution); ○: wild type strain DSM 43756.

sources (see figure 6 and 7). Surprisingly, although also classified as nitrogen source for

M. smegmatis before (Iwainsky and Sehrt, 1967), no growth was visible with urea. As a

result, the same growth experiment was carried out adding a trace element solution required

for growth of C. glutamicum (see table 3, section 3.2.1) and moreover, with an alternative

M. smegmatis wild type strain, namely DSM 43756, which corresponds to type strain NCTC

8159. But even these cells were not able to utilize urea as single nitrogen source under the

tested conditions (see figure 6 and 7).

However, M. smegmatis SMR5 grew poorly in media where isoleucine, leucine, lysine,

methionine, tryptophan, tyrosine or methylamine was used as nitrogen source (figure 6 and

7). The cells showed a generation time of up to 30 hours which was slightly improved at

higher substrate concentrations (figure 8A) and reached an oD600 of fairly higher than 1

(figure 9A). Nevertheless, cells were able to grow on 7H10 agar plates containing these

substances, indicating that they could still be used as nitrogen sources (figure 10).

Detectable growth of the wild type close to 7H9 minimal medium existed in media with

aspartic acid, cysteine, glutamic acid, histidine, threonine and valine as well as guanine,

uracil, ammonium chloride, ammonium sulfate, ethanolamine, ferric ammonium citrate,

potassium nitrate, sodium nitrite and uric acid as single nitrogen source (figure 6 and 7). In

some cases, growth rates were even improved by increasing the substrate concentration, but

never reached 7H9 level (figure 8A), whereas the maximum oD600 in 7H9 medium was partly

outrun (figure 9A). Maximum growth of the wild type SMR5 was achieved using the amino

acids alanine, arginine, asparagine, glutamine, serine and proline as single nitrogen source

(figure 6 and 7). While growth rates such as in 7H9 medium were not quite reached (figure

8A), the maximum oD600 was easily exceeded (figure 9A).

Recapitulating these data, it was found that M. smegmatis can indeed utilize a variety of

organic and inorganic substances as single nitrogen source to guarantee growth. Regarding

Results 58

GlnR as the major regulatory protein of nitrogen metabolism in M. smegmatis, the question

arose if it is also involved in uptake and/or assimilation of these newly found nitrogen

sources. For this purpose, all growth experiments were also carried out with the glnR

deletion strain MH1. While growth in some media was unaffected by the absence of GlnR,

there was a distinct difference in media with alanine, histidine, proline, serine, guanine,

uracil, ethanolamine, potassium nitrate, sodium nitrite and uric acid, where the glnR deletion

strain showed extremely reduced or no growth compared to the wild type. This was observed

with either 10 or 100 mM substrate concentration (figure 6 and 7). The generation times of

MH1 were intensely increased compared to the wild type (figure 8B), while the maximum

oD600 was reduced (figure 9B). Reduced growth of the glnR deletion strain was also

observed when aspartic acid, isoleucine, leucine, lysine and methionine were used as single

nitrogen source, even though the wild type showed very slow and poor growth (see figure 6,

7, 8B and 9B). These results indicated that GlnR is indeed involved in uptake and/or

assimilation of a multitude of different substances that can be used as nitrogen sources.

Growth was not only tested in liquid cultures, but also on 7H10 agar plates. 10 mM of every

substrate that has been tested before were added to 7H10-N agar plates as single nitrogen

source. Plates were incubated for 48 hours, confirming the data obtained before: the

M. smegmatis wild type or the glnR deletion strain did not grow on plates containing glycine,

phenylalanine, adenine, thymine, creatinine, glucosamine, N-acetylglucosamine or urea

(figure 10). On plates, it could no longer be distinguished between poor and strong growth of

the wild type, as large colonies were visible on plates containing all other tested substrates

(figure 10). Furthermore, the slight differences in growth between wild type and glnR deletion

strain in media containing aspartic acid, isoleucine, leucine, lysine and methionine as well as

the strong difference in media containing histidine and ethanolamine monitored before were

no longer detectable due to the long incubation time of the plates. Nevertheless, growth

differences in media containing alanine, proline, serine, guanine, uracil, potassium nitrate,

sodium nitrite and uric acid were clearly visible (figure 10).

Results 59

A

B

Fig. 8: Generation times calculated from growth curves of M. smegmatis. A. Comparison of generation times of the wild type SMR5 in all media where growth was observed (see figure 6 and 7). B. Comparison of the generation times of wild type and glnR deletion strain in media where growth of the wild type was observed before. Generation times higher than 35 h are represented by open bars (only MH1, indicating no growth in these media). Turquoise: generation times in 7H9 medium as a control; light blue: 10 mM substrate; dark blue: 100 mM substrate; filled bars: wild type SMR5; patterned bars: glnR deletion strain MH1.

Results 60

A

B

Fig. 9: Maximum oD600 reached during growth of M. smegmatis. A. Comparison of the maximum oD600 of the wild type SMR5 in all media where growth was observed (see figure 6 and 7). B. Comparison of the maximum oD600 of wild type and glnR deletion strain in media where growth of the wild type was observed before. Turquoise: oD600 in 7H9 medium as control; light blue: 10 mM substrate; dark blue: 100 mM substrate; filled bars: wild type SMR5; patterned bars: glnR deletion strain MH1.

Results 61

His Ile Leu Lys Met

Phe Pro Ser Thr Trp

Sodium nitrite Urea Uric acid

7H10 7H10-N Ala Arg Asn

Asp Cys Gln Glu Gly

Tyr Val Adenine Guanine Thymine

Uracil Ammonium chloride Ammonium sulfate Creatinine Ethanolamine

Ferric ammonium citrate Glucosamine Methylamine N-acetylglucosamine Potassium nitrate

Fig. 10: Growth of M. smegmatis on 7H10 agar plates. Each plate contained 10 mM of a putative nitrogen source. 7H10 and 7H10-N minimal medium were carried along, each as positive and negative control. Amino acids, bases and inorganic substances were tested as single nitrogen source for the wild type SMR5 (left) and the glnR deletion strain MH1 (right). Plates were incubated for 48 hours.

4.1.2 Relation between growth behavior and initiation of nitrogen response

It was of special interest for this study not only to identify substrates which can be utilized as

nitrogen sources by M. smegmatis, but also to identify the regulatory network behind these

processes. For that reason, it was tested which substrate caused nitrogen response in the

wild type SMR5 (see figure 11). Nitrogen response is defined as the upregulation of genes

involved in central nitrogen metabolism to guarantee sufficient nitrogen (ammonium) supply

under limiting conditions. For M. smegmatis, it has already been shown that the transcript

levels of the genes glnA (encoding glutamine synthetase I), amtB and amt1 (encoding

ammonium transporters) were enhanced when cells were transferred from nitrogen-rich

medium to a medium lacking any nitrogen source (Amon et al., 2008).

Results 62

No growth Poor growth Growth

7H9

7H9-N

Gly

Phe

Adenine

Thymine

Creatinine

Glucosamine

N-acetylglucosamine

Urea

7H9

7H9-N

Ile

Leu

Lys

Met

Trp

Tyr

Methylamine

7H9

7H9-N

Ala

Arg

Asn

Asp

Cys

Gln

Glu

His

Pro

Ser

amtB amtB amtB

Thr

Val

Guanine

Uracil

Ammonium chloride

Ammonium sulfate

Ethanolamine

Ferric ammonium citrate

Potassium nitrate

Sodium nitrite

Uric acid

amtB

Fig. 11: Transcript level of amtB in M. smegmatis SMR5 incubated with different substrates as single nitrogen source. After 20 minutes growth in the different media, total RNA was prepared from the cells and hybridized with an amtB probe. Samples were divided corresponding to the growth ability observed before. A 7H9 sample was carried along as negative control (weak signal), a 7H9-N sample as positive control (strong signal of amtB). An enhanced amtB transcript level was seen in media with phenylalanine, adenine, thymine, N-acetylglucosamine, tyrosine, methylamine, arginine, glutamic acid, histidine, proline, threonine, ethanolamine, potassium nitrate and sodium nitrite.

In this study, the genes amtA and amtB (encoding ammonium transporters) as well as urtA

(encoding a urea transporter) were tested in the wild type SMR5 depending on different

nitrogen sources. As the results for these three genes were similar, only amtB is shown in

figure 11. Surprisingly, a clear correlation between growth ability and nitrogen response was

not detected. An increase of transcript was monitored in media with phenylalanine, adenine,

thymine, N-acetylglucosamine, tyrosine, methylamine, arginine, glutamic acid, histidine,

proline, threonine, ethanolamine, potassium nitrate and sodium nitrite, while the cells were

only able to utilize half of these substrates as nitrogen sources.

Only a very small amount or no transcript was detected in the other 21 tested media (see

figure 11), again independently from the ability of the bacteria to use them as nitrogen

sources. This led to the conclusion that uptake and assimilation of these substrates is not

subject to nitrogen control in M. smegmatis.

Results 63

amtB

Controls Addition of ammonium Addition of glutamine

0 5 10 15 30 60 0 5 10 15 30 60 min

Special attention was paid to the samples containing ammonium, glutamine and glutamate.

Regarding nitrogen metabolism, a very important question is which molecule indicates the

nitrogen status of the cells and is thus responsible for activation of nitrogen response.

Considering the GDH/GS/GOGAT-model (see section 2.2.1), these mechanisms are used to

provide the cells with sufficient amounts of nitrogen in form of glutamine and glutamate. It

has already been shown in this study that nitrogen response of M. smegmatis is activated

when glutamate is supplied as the only nitrogen source, and not activated or even inhibited in

the presence of ammonium or glutamine (figure 11). A second RNA hybridization experiment

revealed the correlation of these observations. Cells were grown with 100 mM glutamate as

single nitrogen source, before 100 mM ammonium chloride or glutamine were added. The

mRNA levels of amtA, amtB and urtA were monitored at different time points. As the results

for these three genes were similar, only amtB is shown in figure 12.

Fig. 12: Transcript level of amtB, depending on the substrate used as nitrogen source. Cells were incubated in medium containing 100 mM glutamate (control 1, positive) and in media containing 100 mM glutamate and 100 mM ammonium chloride or 100 mM glutamine, respectively (control 2 and 3, negative). When cells reached exponential growth phase, 100 mM ammonium chloride or glutamine were added to control 1, respectively, and samples were taken after 0, 5, 10, 15, 30 and 60 minutes.

As observed before, the transcript level of amtB was high when glutamate was used as

single nitrogen source. As a control, samples with glutamate and glutamine, and glutamate

and ammonium were carried along, showing no amtB signal. This indicated a negative and

stronger influence of glutamine and ammonium to the activation of nitrogen response

compared to glutamate. When 100 mM ammonium or glutamine were added to cells already

growing in medium with glutamate, the amtB signal was intensely decreased after five

minutes and completely disappeared after 10 minutes. Thus, it can be concluded that

glutamate is not the indicator of the nitrogen status of M. smegmatis; it is rather possible that

the intracellular glutamine level, which is increased by GS using ammonium, signalizes the

cells’ nitrogen surplus or limitation.

Results 64

0

0.5

1

1.5

2

SMR5 +N DSM43756 +N SMR5 -N DSM43756 -N

µm

olN

H4

+m

in-1

(mg

pro

tein

)-1

Urease activity in M. smegmatisUrease activity in M. smegmatis

µm

olN

H4

+m

in-1

(mg

pro

tein

)-1

SMR5 +N DSM 43756 +N SMR5 -N DSM 43756 -N

4.1.3 Uptake and assimilation of urea

Surprisingly, performing growth experiments with M. smegmatis using different substances

as single nitrogen source, no growth was detected upon the addition of urea (see section

4.1.1). Nevertheless, various genes important for uptake and assimilation of urea exist in

M. smegmatis (Amon et al., 2009) and growth with urea as nitrogen source has already been

described (Iwainsky and Sehrt, 1967). To exclude mutations in the laboratory strain SMR5

used in this study, growth experiments were repeated with an alternative wild type strain,

DSM 43756, also considered as urease-positive (www.dsmz.de). But even this strain was not

able to utilize urea as nitrogen source under the tested conditions (see section 4.1.1).

Consequently, the ability of these two M. smegmatis strains to degrade urea was

determined. As ureases catalyze hydrolysis of urea to CO2 and ammonia, the production of

ammonium was measured using an indophenol reaction. Urease activities of SMR5 and

DSM 43756, prior incubated under nitrogen surplus and starvation, were calculated as µM

produced ammonium per minute per mg protein (figure 13).

Fig. 13: Urease activity measurement in M. smegmatis strains SMR5 and DSM 43756. Cells were previously incubated under nitrogen surplus (+N) and starvation (-N). Urease activity was calculated as µM produced ammonium per minute per mg protein.

As shown in figure 13, both M. smegmatis strains had similar urease activities of 1 µmol

produced ammonium per minute per mg protein, which were barely increased under nitrogen

starvation. This indicated that a functional urease converting urea into ammonium existed in

both of the tested strains, even though no growth was observed with urea as single nitrogen

source under the tested conditions. Thus, the reason for this still remains unknown.

Results 65

4.1.4 Generation of an in vivo reporter system for nitrogen response

So far, this study revealed the response of M. smegmatis on gene transcript level to varying

nitrogen sources and suggested an important role of GlnR in these processes. To further

investigate this, a functional system, which can be used to characterize nitrogen- and

especially GlnR- dependent response, should be developed. In C. glutamicum, a closely

related actinomycete, a reporter system based on GFP, the green fluorescent protein, has

already been established (Jeßberger, 2008). With this system, it was possible to monitor

changes in promoter activities of gdh, amtA and amtB (and others), according to the nitrogen

status of the cells or the presence or absence of AmtR, the global regulatory protein of

nitrogen metabolism in this species (see section 2.2.3). Since GlnR has a great influence on

the transcript levels and thus promoter activities of amtB and glnA in M. smegmatis (Amon et

al., 2008), their corresponding promoter sequences were cloned upstream of the gfpuv gene

into an appropriate M. smegmatis vector and cells of the wild type and the glnR deletion

strain were transformed with these constructs. Additionally, also promoter sequences of

amtA, gdhA (msmeg_5442, further described as gdh) and glnR were used, as well as a

promoterless construct as negative control and a construct with psmyc, a strong

mycobacterial promoter, as positive control.

Cells were incubated in 7H9 medium until exponential growth phase and then transferred

into nitrogen-free medium, followed by another 30 minutes of incubation. Promoter activities

of all samples were calculated depending on the fluorescence and the dry weight of the cells

per ml. Contrary to expectations, fluorescence and thus promoter activity decreased under

nitrogen starvation (see figure 14). Detectable promoter activity was only seen in cells

transformed with psmyc-gfp, a strong and nitrogen-independent mycobacterial promoter;

whereas signals were significantly lower than in similar measurements with C. glutamicum

(Jeßberger, 2008). None of the remaining promoters showed increased activity under

nitrogen starvation, although this was expected for amtA, amtB and glnA promoter in the wild

type and should only be reversed in the absence of GlnR (MH1). In fact, promoter activities

of all tested constructs decreased under nitrogen starvation in the wild type as well as in the

glnR deletion strain (figure 14). Even when experimental conditions were varied from using

higher amounts of cells to longer or shorter incubation times in nitrogen-free medium or

adding MSX to induce nitrogen starvation, no stronger fluorescence signal could be detected

in any of the tested constructs (data not shown). This led to the conclusion that this GFP-

dependent system for measuring changes in nitrogen response was not functional in M.

smegmatis. This was also confirmed by fluorescence microscopy, where only cells

transformed with the psmyc-gfp construct were detected (figure 15).

Results 66

SMR5 MH1

Promoter activities M. smegmatis SMR5 vs. MH1

- psmyc amtAp amtBp glnAp gdhp glnRp - psmyc amtAp amtBp glnAp gdhp glnRp

SMR5 MH1

Pro

mote

r activity

900000

800000

700000

600000

500000

400000

300000

200000

100000

0

Fig. 14: Promoter activities of M. smegmatis strains SMR5 and MH1. Both strains, carrying gfpuv constructs with no promoter, psmyc, amtAp, amtBp, glnAp, gdhp and glnRp, were grown under nitrogen surplus and limitation. After fluorescence was measured at 508 nm, promoter activities were calculated as fluorescence per oD600 x 0.36 g dry weight per ml. Filled bars: wild type SMR5; striped bars: glnR deletion strain MH1; light blue: 7H9 medium, nitrogen surplus; dark blue: 7H9-N medium, nitrogen starvation.

Fig. 15: Fluorescence microscopy of M. smegmatis strains SMR5 and MH1. Only cells

transformed with the construct psmyc-gfpuv showed fluorescence.

Results 67

β-galactosidase activities SMR5 vs. MH1

Activity

(MU

)

Activity

(MU

)

psmyc - amtAp amtBp glnAp gdhp glnRp - amtAp amtBp glnAp gdhp glnRp

SMR5 MH1 SMR5 MH1

180

160

140

120

100

80

60

40

20

0

30

25

20

15

10

5

0

Due to the fact that a reporter system for detection of changes in nitrogen response based on

GFP was not functional in M. smegmatis, another reporter system based on β-galactosidase

activity measurements was tested. For this purpose, the gfpuv gene was replaced by the

lacZ gene (from E. coli, derived from pWH948, see table 2, section 3.1) in the existing

constructs (see above). These were again used to transform cells of the M. smegmatis

strains SMR5 and MH1. Cells were grown as described above. β-galactosidase activities

were calculated in Miller units from optical density of the samples at 600, 550 and 420 nm

and are shown in figure 16. Again, the strongest activity was obtained with the psmyc

construct, independently from nitrogen supply. Different from the GFP-system, an increase of

promoter activity for the amtA, amtB and glnA promoter was observed in the wild type under

nitrogen starvation, not for gdh and glnR promoter. In the strain MH1 lacking GlnR, no

increase of activity for any tested promoter was detected under nitrogen starvation. These

results confirmed the expectation that the presence of GlnR is necessary for an enhanced

activity of the promoters of amtA, amtB and glnA, even though the calculated

β-galactosidase activities were very low and the differences between nitrogen surplus and

starvation were not distinctly demonstrated.

Fig. 16: β-galactosidase activities of M. smegmatis strains SMR5 and MH1. Cells were transformed with plasmids carrying the lacZ gene under the control of psmyc, amtAp, amtBp, glnAp, gdhp and glnRp. After cells were grown in 7H9 medium until exponential growth phase and incubated for further 30 minutes under nitrogen starvation, β-galactosidase activities were calculated depending on optical density of the samples at 420, 550 and 600 nm. Filled bars: wild type SMR5; striped bars: glnR deletion strain MH1; light blue: 7H9 medium, nitrogen surplus; dark blue: 7H9-N medium, nitrogen starvation.

Results 68

4.1.5 Purification of GlnR

Considering the data obtained in this study so far, it is clear that the regulatory protein GlnR

plays an important role in M. smegmatis, generally in uptake and assimilation of different

nitrogen sources and particularly in the regulation of genes involved in nitrogen metabolism,

such as amtA, amtB or glnA. No data of the GlnR protein itself were available at the

beginning of this study. Therefore, another important aim of this study was to develop

functional overexpression and purification methods to supply high amounts of pure GlnR

protein for further experiments such as DNA binding assays or the generation of a GlnR-

specific antibody. Efforts to purify GlnR with different approaches are described below.

A very common approach to isolate a specific protein from total cell extract is to add a tag

consisting of six histidine residues, which binds specifically to a Ni2+ matrix in affinity

chromatography. First, an E. coli Rosetta2 strain carrying a pUC19-his-glnR construct (see

table 2, section 3.1) was used. This strain also carried a second plasmid, pRARE, encoding

rarely used tRNA species in E. coli. When cells reached exponential growth, overexpression

of the protein was induced by adding IPTG. After four hours, cells were harvested and

disrupted. After that, the His-GlnR protein was separated from total cell extract by binding of

the tag to a Ni2+ NTA matrix in an affinity column (see figure 17A). Binding was released by

adding rising concentrations of imidazole. The size of the purified protein was tested in SDS

PAGE and in Western blot analysis using a His-specific antibody. Size of M. smegmatis GlnR

combined with six additional histidine residues was 30 kDa. Weak bands of this size were

detected both in SDS PAGE and Western blot (figure 17A), but another band of

approximately 13 kDa was dominant. This appearance in Western blot analysis, where a His-

specific antibody was used, led to the conclusion that His-GlnR was either dismantled during

purification or not even correctly synthesized in E. coli.

Therefore, the his-glnR construct was cloned into the vector pMN016 suitable for

M. smegmatis SMR5. This strain was grown for 10 hours to mid-exponential growth phase

and harvested subsequently. The his-glnR construct was constitutively expressed from the

non-inducible, strong mycobacterial promoter psmyc. His-tagged proteins were again

separated from total cell extract as described above and purification was controlled in SDS

PAGE and Western blot using a His-specific antibody (figure 17B). Only a single band at size

of approximately 60 kDa was visible and identified via mass spectroscopic analysis as

GroEL1, the bigger subunit of a mycobacterial chaperone, naturally equipped with seven

histidine residues at its C-terminal end and thus blocking the binding sites at the Ni2+ NTA

matrix. As it was not possible to purify His-GlnR from M. smegmatis SMR5, another strain

was used, namely M. smegmatis ML371. In this strain, DNA of the mycobacteriophage BxbI

is integrated at the 3’-end of the groEL1 gene changing the ability of the GroEL1 protein to

bind to the purification column. This new strain was transformed with the pMN016his-glnR

Results 69

construct and treated as described above. SDS PAGE and Western blot analysis using a

His-specific antibody revealed that only one specific protein with a size of 30 kDa was

purified (figure 17C), most likely His-GlnR. Figure 17A-C shows all results for purification of

GlnR bound to an N-terminal His-tag. These results were also obtained when the six histidine

residues were attached to the C-terminus of GlnR (data not shown).

In parallel, a different approach using MBP (maltose binding protein) as affinity tag was

carried out. Here, GlnR was fused to the C-terminus of MBP by cloning the glnR gene into

the vector pMal-c2. E. coli Rosetta2 cells were transformed with this construct and treated as

described above. The gene fusion was overexpressed for four hours in the presence of

IPTG. After cells were harvested and disrupted, the MBP-GlnR fusion protein was separated

from total cell extract by binding of MBP to an amylose resin matrix in affinity

chromatography (figure 17D). The pure fusion protein was released by adding rising

concentrations of maltose. Purity was tested once more in SDS PAGE and Western blot

analysis using an MBP-specific antibody. As MBP is a large protein of a size of 44 kDa, the

MBP-GlnR fusion protein was detected at a size of approximately 71 kDa (figure 17D). After

the fusion protein was cleaved by protease factor Xa at its cleavage site, the resulting

proteins were separated by gel filtration. While two peaks indicating two protein fractions of

different size were identified during gel filtration, a single 30 kDa band representing GlnR

could not be detected in SDS PAGE (figure 17D), indicating that it was not possible to cleave

the MBP-GlnR fusion protein without the loss of GlnR. Nevertheless, the fusion protein was

used in a variety of following experiments, as MBP did not affect e.g. DNA binding manners

of GlnR (see section 4.1.7).

Furthermore, GlnR was also attached to an N-terminal Strep-tag by cloning the glnR gene

into the vector pASK-IBA5plus. The construct was used to transform E. coli Rosetta2 cells as

described above. Cells were again incubated until exponential growth phase, before

overexpression of the strep-glnR fusion was carried out for four hours in the dark in the

presence of doxycycline. After cells were harvested and disrupted, Strep-tagged proteins

were purified using a Strep-TactinR-SepharoseR matrix in a hand column. The Strep-tagged

GlnR protein was released by adding D-desthiobiotin and could be detected in SDS PAGE

and Western blot analysis using a Strep-specific antibody (figure 17E). Later in this study,

other M. smegmatis proteins such as Msmeg_1918 and Msmeg_5241 (see section 4.3.1), as

well as M. tuberculosis GlnR (see section 4.5) were purified via MBP- or Strep-tag. Strep-

tagged GlnR was also used in following experiments such as DNA binding assays,

phosphorylation experiments, pull down assays or the generation of a GlnR-specific

antibody.

Results 70

85 kDa

70 kDa

30 kDa

15 kDa

10 kDa

Purification protocol SDS PAGE Western blot

72 kDa

34 kDa

17 kDa

10 kDa

A

72 kDa

55 kDa

60 kDa

B

30 kDa

72 kDa

34 kDa

C

D

80 kDa

70 kDa72 kDa

55 kDa

E

85 kDa

70 kDa

50 kDa

30 kDa

15 kDa

1 2

55 kDa

34 kDa

50 kDa

30 kDa

SDS PAGE Western blot

Gel filtration SDS PAGE

1

2

Fig. 17: Purification of M. smegmatis GlnR. Purification protocols (Äkta apparatus) are shown as far as available, as well as detection of purified proteins in SDS PAGE and Western blot analyses. A. Purification of His-GlnR from E. coli Rosetta2. Samples indicated by a peak in the purification protocol did not show sufficient amounts of protein at GlnR size of 30 kDa, neither in SDS PAGE nor in Western blot using a His-specific antibody. B. Purification of His-GlnR from M. smegmatis SMR5. SDS PAGE, Western blot with His-specific antibody and mass spectroscopic analyses showed the 60 kDa protein GroEL1. C. Purification of His-GlnR from M. smegmatis ML371. Due to changes in GroEL1, only the 30 kDa His-GlnR protein was detected in SDS PAGE and Western blot. D. Purification of MBP-GlnR from E. coli Rosetta2. The 71 kDa fusion protein was detected both in SDS PAGE and Western blot using an MBP-specific antibody. Cleavage of the fusion and purification of the single GlnR protein via gel filtration did not result in a protein of correct size of 30 kDa. E. Purification of Strep-GlnR from E. coli Rosetta2 in a hand column. The Strep-GlnR protein was detected at a size of 30 kDa both in SDS PAGE and Western blot analysis using a Strep-specific antibody.

Results 71

72 kDa

55 kDa

36 kDa

4.1.6 Generation of a GlnR-specific antibody

To detect even small amounts of GlnR in different experimental samples or cell extracts, it is

greatly beneficial to have an antibody that specifically binds GlnR. For that purpose, high

amounts of GlnR were purified using the Strep-system as described in section 4.1.5. After

testing of rabbit pre-immune serums, 2 mg Strep-GlnR were sent to Pineda AK Service,

Berlin, where an appropriate rabbit was immunized with the protein sample. Every 30 days,

the specificity of the originating antibody was tested in Western blot analysis using purified

GlnR and total cell extract of the M. smegmatis wild type strain SMR5 as targets. Figure 18

shows the results of the Western blot analysis after 150 days.

Fig. 18: Ability of the GlnR-specific antibody to distinctly bind GlnR. Western blot analysis was carried out after 150 days. The antibody was used in a 1:50.000 dilution. Targets were purified Strep-GlnR, MBP-GlnR, Strep-Msmeg_1918, purified AmtR protein of C. glutamicum and total cell extract of M. smegmatis strains SMR5 and MH1.

As expected, the antibody was able to detect purified Strep-GlnR (31 kDa) which had been

used to immunize the rabbit before. Furthermore, also GlnR protein purified with MBP-tag

(71 kDa) was bound by the antibody. Another M. smegmatis protein, Msmeg_1918, also

attached to a Strep-tag, was visible at size of 54 kDa, indicating that the antibody recognized

not only GlnR, but also the Strep-tag, no matter which protein was attached. But the

specificity of the antibody was still guaranteed, as purified AmtR protein from C. glutamicum

was not detected. Moreover, the antibody was specific enough not only to detect purified

GlnR, but also GlnR protein in total cell extract of M. smegmatis SMR5 at a size of 28 kDa.

No signal was detected when total cell extract of the glnR deletion strain MH1 was used.

Consequently, the generation of a GlnR-specific antibody was successful and the functional

antibody could be used for further experiments such as detecting GlnR mutants (see section

4.3.2).

Results 72

4.1.7 DNA binding ability of purified GlnR

At the beginning of this study, it was already clear that GlnR has a great influence on the

transcript levels of genes involved in nitrogen metabolism in M. smegmatis, such as amtB or

glnA (Amon et al., 2008). Due to the fact that the mRNA signals of these genes were

dramatically decreased in the glnR deletion strain compared to the wild type under nitrogen

starvation, it was assumed that GlnR works as transcriptional activator of its target genes.

Such a protein activates transcription by RNA polymerase by binding to specific sites at the

promoter region of its target gene. In a previous study where no purified GlnR was available,

DNA binding assays were performed using a 220 bp DNA fragment directly upstream of the

amtB start codon and total cell extract of M. smegmatis strains SMR5 and MH1 (Bräu, 2008).

Indeed, a slight shift of the DNA on an electrophoretic gel was detected in the SMR5 sample,

which was even increased when total cell extract of this strain incubated under addition of

MSX was used, and which was not visible using total cell extract of the glnR deletion strain

MH1. MSX is a glutamate analog that blocks GS activity (Berlicki, 2008) and thus, induces

nitrogen response in the wild type strain (Amon et al., 2008). These results led to the

conclusion that GlnR actually binds to the promoter region of amtB and that this is even

enhanced under nitrogen limitation.

To investigate these processes in detail, purified GlnR (see section 4.1.5) was necessary.

While a detailed characterization of DNA binding manners of GlnR is presented later in this

study (see section 4.2.4), it is shown in figure 19 that every GlnR protein purified with a

different approach (see section 4.1.5) was able to bind to the 220 bp amtB promoter

fragment described above. The digoxigenin-labeled DNA fragments were separated via gel

electrophoresis and later visualized using an anti-digoxigenin-alkaline-phosphatase

conjugate. When rising amounts of purified GlnR were added, a distinct shift of DNA was

visible, as the DNA-protein complex of bigger mass passed slower through the gel than pure

DNA. Binding of His-GlnR to the amtB promoter fragment was obtained by adding protein

amounts of 200 ng or higher. In figure 19, GlnR attached to an N-terminal His-tag purified

from M. smegmatis ML371 is shown, but the same results were seen when a C-terminal His-

tag fusion or even the wild type strain SMR5 expressing nearly exclusively groEL1 (see

section 4.1.5) was used (data not shown). Due to the large size of the maltose binding

protein (44 kDa) compared to a small His- or Strep-tag, binding of the MBP-GlnR fusion

protein to the DNA was first detected at a protein amount of 400 ng. A sample in which only

800 ng of purified MBP were used was added as a control showing that pure MBP had no

effect on DNA binding (figure 19). Furthermore, binding of purified Strep-GlnR to the

digoxigenin-labeled amtB promoter fragment was observed when protein amounts of 100 ng

or higher were used (figure 19). It was monitored that the more purified GlnR was added, the

higher the shift of the DNA was, indicating that GlnR indeed binds to its target DNA in a

Results 73

His-GlnR MBP-GlnR Strep-GlnR

Binding of purified GlnR to target DNA

“galloping” manner as it was presumed before (Yoshida et al., 2006; Amon et al., 2008). Shift

of DNA attached to MBP-GlnR was largest due to the large size of the fusion protein. This

experiment gave a first hint that DNA binding of GlnR in vitro is independent from further

activation mechanisms of the protein, such as phosphorylation.

Fig. 19: Binding of GlnR protein to its target DNA. A 220 bp digoxigenin-labeled DNA fragment directly upstream of the amtB start codon was used as target. A sample of free DNA was carried along as a control in every approach. The following amounts of His-GlnR (here: N-terminal His-tag, purified from M. smegmatis ML371) were added: 200, 400, 600, 800 and 1000 ng. After a sample with free DNA and one with 800 ng pure maltose binding protein as controls, MBP-GlnR was added in amounts of 50, 100, 200, 400, 800, 1000, 1500 and 2000 ng. Strep-GlnR was added after a control sample of free DNA in amounts of 100, 200, 400, 800, 1600 and 3200 ng.

4.2 Characterization of the GlnR regulon

4.2.1 Global approach using DNA microarray analyses

Little was known about the role of GlnR in nitrogen metabolism in M. smegmatis at the

beginning of this study. First experiments revealed enhanced transcript levels of the genes

amtB and amt1, encoding ammonium transporters, and glnA, encoding GS I, under nitrogen

limitation when GlnR was present. These data were obtained performing RNA hybridization

experiments and real time RT PCR (Amon et al., 2008), indicating that GlnR is the

transcriptional activator of genes involved in nitrogen metabolism. A multitude of further

nitrogen-related genes was identified in M. smegmatis in a bioinformatic approach (Amon et

al., 2009). Among these were various glnA-like genes encoding different types of glutamine

synthetases, gltBD encoding glutamate synthase, glnE encoding ATase putatively involved in

posttranslational regulation of GSI, various genes involved in uptake and hydrolysis of urea,

nirBD and the nar operon encoding assimilatory nitrate and nitrite reductases, genes

encoding various nitrate/nitrite transporters, glnK and glnD encoding signal transduction

Results 74

proteins of nitrogen control well understood in the related actinomycete C. glutamicum (see

section 2.2.3), and also amtR encoding a putative second regulatory protein of nitrogen

metabolism (for an overview, see Amon et al., 2009). Moreover, first experiments of this

study (see section 4.1.1) revealed that GlnR has great influence on uptake and assimilation

of different substrates such as guanine, uracil, nitrate, nitrite and uric acid or the amino acids

alanine, proline and serine which can be used by M. smegmatis as nitrogen sources.

Therefore, more putative and so far unknown permeases or transport proteins as well as

different metabolic enzymes were assumed to be controlled by GlnR. This is supported by a

closer look at nitrogen control in actinomycetes related to M. smegmatis. Whereas in

C. glutamicum at least 40 genes involved in nitrogen metabolism are controlled by the global

regulatory protein AmtR (see section 2.2.3), influence of the transcriptional regulator GlnR on

at least 15 target genes was reported for S. coelicolor (Fink et al., 2002; Tiffert et al., 2008).

All these data indicate that in M. smegmatis a multitude of further GlnR target genes might as

well exist.

To verify this, a global analysis of total gene transcripts was carried out in a DNA microarray

experiment. Therefore, the M. smegmatis wild type SMR5 and the glnR deletion strain MH1

were incubated in 7H9 medium until exponential growth and then transferred into nitrogen-

free medium. After another 30 minutes, cells were harvested and total RNA was prepared.

Transcription of the RNA with labeling of the resulting cDNA with cy3 and cy5 fluorescent

markers, as well as hybridization of the samples with oligonucleotides representing the whole

M. smegmatis genome on a microarray chip was performed at the Department of

Biochemistry, Friedrich-Alexander University Erlangen-Nuremberg. In a first experiment, total

transcript of the wild type SMR5 grown under nitrogen surplus was compared to that

obtained under nitrogen starvation. A second assay revealed differences in total transcript

between the wild type and the glnR deletion strain, both cultivated under nitrogen starvation.

Data were exposed to different analysis procedures until all M. smegmatis genes whose

transcript levels differed between +N and -N or between wild type and glnR deletion strain by

a factor higher than 3 were shown. Results of these analyses are summarized in figure

20-23. Here, all genes identified in the transcriptome analysis are represented in %

according to their COG (Clusters of Orthologous Groups) numbers. Dividing proteins and

their corresponding genes into COGs based on orthologous relationships (Tatusov et al.,

2003) makes it easier to understand their individual structure and function. The complete lists

of genes with changing transcript levels between nitrogen surplus and starvation or between

wild type and glnR deletion strain can be viewed in section 7.2.

Results 75

When total transcript of the M. smegmatis wild type was compared under nitrogen surplus

and starvation, mRNA levels of 231 genes were found to be decreased under nitrogen

starvation, but only by a maximum factor of 15, indicating that these genes were not involved

in nitrogen stress response. Classification of these genes into COGs is shown in figure 20.

50 % of these genes were allocated to the major group metabolism, including genes for lipid

(COG_I), carbohydrate (COG_G), inorganic ion (COG_P) and amino acid (COG_E) transport

and metabolism, as well as genes for secondary metabolites biosynthesis, transport and

catabolism (COG_Q), energy production and conversion (COG_C), coenzyme (COG_H) and

nucleotide (COG_F) transport and metabolism. Another 27 % were allocated to the major

category poorly characterized with genes of predicted (COG_R) or unknown (COG_S)

function or no COG classification (X). 16 % of the genes with decreased transcripts in the

wild type under nitrogen starvation belonged to the group information storage and

processing, with genes involved in translation, ribosomal structure and biogenesis (COG_J),

transcription (COG_K) and replication, recombination and repair (COG_L). The remaining

7 % correlated with the group cellular processes and signaling, with genes involved in

posttranslational modification, protein turnover, chaperones (COG_O), cell

wall/membrane/envelope biogenesis (COG_M), cell cycle control, cell division, chromosome

partitioning (COG_D), signal transduction mechanisms (COG_T), cell motility (COG_N) and

intracellular trafficking, secretion and vesicular transport (COG_U). Only 4 % of these 231

genes are described as putatively involved in nitrogen metabolism.

In contrast, the amount of transcripts of 284 genes was enhanced in the wild type under

nitrogen starvation compared to nitrogen surplus by factors of up to 99. These data indicated

that the transcripts of a variety of genes in M. smegmatis are increased as soon as the

organism is exposed to nitrogen starvation. Contrary to the genes with decreased mRNA

levels (see above), 12.5 % of these 284 genes are associated with nitrogen metabolism.

Figure 21 shows the classification of these genes into COGs. 49 % of these genes were

poorly characterized, either not in COG (X), or of predicted (COG_R) or unknown (COG_S)

function. 37 % belonged to the major category metabolism, with genes involved in transport

and metabolism of amino acids (COG_E) and inorganic ions (COG_P), as well as in energy

production and conversion (COG_C) and biosynthesis, transport and catabolism of

secondary metabolites (COG_Q). Furthermore, genes involved in transport and metabolism

of coenzymes (COG_H), carbohydrates (COG_G), lipids (COG_I) and nucleotides (COG_F)

were found. 8 % of the genes with enhanced transcripts in the wild type under nitrogen

starvation were allocated to information storage and processing, with genes involved in

transcription (COG_K), translation, ribosomal structure and biogenesis (COG_J), replication,

recombination and repair (COG_L) and chromatin structure and dynamics (COG_B).

Results 76

M. smegmatis SMR5: decreased transcript levels of 231 genes under nitrogen starvation

Metabolism

Poorly characterized

Information storage and processing

Cellular processes and signaling

Metabolism

I: Lipid transport and metabolism

G: Carbohydrate transport and metabolism

P: Inorganic ion transport and metabolism

Q: Secondary metabolites biosynthesis, transport and catabolism

E: Amino acid transport and metabolism

C: Energy production and conversion

H: Coenzyme transport and metabolism

F: Nucleotide transport and metabolism


X: Not in COG

R: General function prediction only

S: Function unknown


J: Translation, ribosomal structure and biogenesis

K: Transcription

L: Replication, recombination and repair


O: Posttranslational modification, protein turnover, chaperones

M: Cell wall/membrane/envelope biogenesis

D: Cell cycle control, cell division, chromosome partitioning

T: Signal transduction mechanisms

N: Cell motility

U: Intracellular trafficking, secretion and vesicular transport

Fig. 20: Allocation of 231 M. smegmatis SMR5 genes with decreased transcript levels under nitrogen starvation into COGs.

Results 77

M. smegmatis SMR5: increased transcript levels of 284 genes under nitrogen starvation


Metabolism




Metabolism

X: Not in COG


S: Function unknown






D: Cell cycle control, cell division, chromosome partitioning

V: Defense mechanisms

U: Intracellular trafficking, secretion and vesicular transport









K: Transcription


L: Replication, recombination and repair

B: Chromatin structure and dynamics

Fig. 21: Allocation of 284 M. smegmatis SMR5 genes with increased transcript levels under nitrogen starvation into COGs.

Results 78

SMR5 vs. MH1 -N: increased transcript levels of 6 genes in the glnR deletion strain

Poorly characterized; X: not in COG

Cellular processes and signaling; O: Posttranslational

modification, protein turnover, chaperones

The remaining 6 % belonged to cellular processes and signaling, namely to posttranslational

modification, protein turnover, chaperones (COG_O), signal transduction mechanisms

(COG_T), cell wall/membrane/envelope biogenesis (COG_M), cell cycle control, cell division,

chromosome partitioning (COG_D), defense mechanisms (COG_V) and intracellular

trafficking, secretion and vesicular transport (COG_U).

These data indicated that M. smegmatis indeed changes its pattern of transcripts due to

sudden nitrogen starvation. While the mRNA levels of genes involved in carbohydrate, lipid

or energy metabolism were decreased, those of genes involved in nitrogen metabolism were

enhanced to maintain a nitrogen status sufficient for survival of the cells.

Most interesting for this study was the comparison of transcript levels between the wild type

SMR5 and the glnR deletion strain MH1 under nitrogen starvation. This experiment resulted

in a change of transcripts of 131 genes. Of these, the mRNA amounts of only 6 genes were

increased in the glnR deletion strain compared to the wild type (figure 22). While four of

these genes were not characterized in COGs, the remaining two belonged to cellular

processes and signaling as part of COG_O (posttranslational modification, protein turnover,

chaperones) and were not associated with nitrogen metabolism.

Fig. 22: Allocation of 6 M. smegmatis genes into COGs. These genes showed increased transcript levels under nitrogen starvation in the glnR deletion strain MH1 compared to the wild type SMR5.

In contrary, transcripts of 125 genes were decreased in the glnR deletion strain compared to

the wild type up to a factor of 136, leading to the first conclusion that GlnR might be involved

in expression of these genes under nitrogen limitation. This was also supported by the fact

that more than 33 % of these genes are described as putatively involved in nitrogen

metabolism. Allocating these genes into COGs (see figure 23), it was apparent that 52 %

belonged to the metabolism group, with more than half of these genes associated to amino

acid transport and metabolism (COG_E). This corroborated the thesis that GlnR is indeed

responsible for expression of a variety of genes involved in amino acid metabolism. Further

genes were designated to energy production and conversion (COG_C), transport and

Results 79

SMR5 vs. MH1 -N: decreased transcript levels of 125 genes in the glnR deletion strain

Metabolism


X: Not in COG


S: Function unknown





V: Defense mechanisms










K: Transcription


Metabolism




Fig. 23: Allocation of 125 M. smegmatis genes into COGs. These genes showed decreased transcript levels under nitrogen starvation in the glnR deletion strain MH1 compared to the wild type SMR5.

Results 80

metabolism of inorganic ions (COG_P), nucleotides (COG_F), carbohydrates (COG_G),

lipids (COG_I) and coenzymes (COG_H), as well as to secondary metabolites biosynthesis,

transport and catabolism (COG_Q). The next often detected group of genes (34 %) was

poorly characterized, either not in the COG-system (X) or of predicted (COG_R) or unknown

(COG_S) function. Furthermore, 9 % of the genes with decreased transcript levels under

nitrogen starvation in the glnR deletion strain were putatively involved in information storage

and processing, namely in transcription (COG_K) and translation, ribosomal structure and

biogenesis (COG_J). The remaining 5 % were allocated to the cellular processes and

signaling group, namely to cell wall/membrane/envelope biogenesis (COG_M),

posttranslational modification, protein turnover, chaperones (COG_O), defense mechanisms

(COG_V) and signal transduction mechanisms (COG_T).

A closer look was taken on the 125 genes which showed decreased mRNA levels in the glnR

deletion strain MH1 compared to the wild type SMR5 under nitrogen starvation. The

classification into COGs gave a first hint that GlnR is indeed responsible for transcriptional

activation of these genes, especially as many genes involved in uptake and assimilation of

amino acids or other possible nitrogen sources were detected. As this classification does not

show the factor of difference of transcript levels between presence and absence of GlnR, the

125 genes of interest are again listed in table 5, including the msmeg_ gene number, the fold

change of transcripts, the annotation of the gene and the corresponding COG number.

Tab. 5: List of all 125 M. smegmatis genes obtained from a DNA microarray experiment that showed decreased transcript levels by a factor higher than 3 in the glnR deletion strain MH1 compared to the wild type SMR5 under nitrogen starvation. The msmeg_ gene numbers as well as the fold change of transcripts, an annotational description and the corresponding COG numbers are given. Data are sorted by the strength of fold change in an ascending manner. Genes chosen for further analyses are highlighted in blue.

Locus tag Fold change

Description COG number

msmeg_3358 3.02 YaeQ protein COG4681S

msmeg_4965 3.07 hypothetical protein X

msmeg_6507 3.07 glycogen debranching enzyme GlgX (glgX) COG1523G

msmeg_1792 3.12 conserved hypothetical protein X

msmeg_4567 3.14 conserved hypothetical protein COG1305E

msmeg_4382 3.15 dehydrogenase-reductase SDR family member 10 COG1028IQR

msmeg_4011 3.26 putative pyrimidine permease RutG COG2233F

msmeg_2116 3.33 PTS system, glucose-specific IIBC component COG1263G

msmeg_4570 3.34 conserved hypothetical protein COG2308S

msmeg_2187 3.36 urea amidolyase COG1984E


msmeg_1292 3.48 FAD binding domain in molybdopterin dehydrogenase protein COG1319C

msmeg_4171 3.58 ribose transport system permease protein RbsC COG1172G

msmeg_3994 3.64 short chain dehydrogenase COG0300R


msmeg_2189 3.68 allophanate hydrolase (atF) COG0154J

msmeg_0393 3.73 Fmt protein COG2226H

Results 81

msmeg_1153 3.73 FAD dependent oxidoreductase X


msmeg_6332 3.79 amino acid ABC transporter, permease protein COG1174E

msmeg_3626 3.89 urease, beta subunit (ureB) COG0832E


msmeg_1152 4.01 citrate-proton symporter COG2814G

msmeg_5331 4.04 UDP-glucoronosyl and UDP-glucosyl transferase family X

msmeg_1151 4.08 DNA-binding protein COG1396K

msmeg_5783 4.10 acetyltransferase, GNAT family X

msmeg_1155 4.16 carnitinyl-CoA dehydratase COG1024I


msmeg_3912 4.20 acetoacetyl-CoA reductase COG1028IQR

msmeg_0565 4.33 putative glycosyl transferases group 1 COG0438M

msmeg_1184 4.40 serine esterase, cutinase family X

msmeg_1156 4.44 dihydrodipicolinate synthetase COG0329EM

msmeg_1185 4.46 transcriptional regulator, AsnC family COG1522K


msmeg_1088 4.63 glutamyl-tRNA(Gln)-aspartyl-tRNA(Asn) amidotransferase COG0154J

msmeg_0505 4.72 probable sugar ABC transporter, substrate-binding protein COG1653G

msmeg_6880 4.92 hydrophobic amino acid ABC transporter, putative COG0683E

msmeg_6879 4.97 Nat permease for neutral amino acids NatD COG0559E

msmeg_3722 5.04 bifunctional coenyme PQQ synthesis protein C-D X

msmeg_1596 5.05 transcriptional regulator COG1309K

msmeg_6264 5.06 putative oxidoreductase COG0665E

msmeg_2523 5.11 efflux ABC transporter, permease protein, putative X

msmeg_4381 5.14 amidase COG2421C

msmeg_1090 5.24 amidase COG0154J

msmeg_1508 5.54 amino acid permease-associated region COG0531E

msmeg_5729 5.75 hydantoin racemase COG4126E

msmeg_6733 6.32 hydrolase, carbon-nitrogen family COG0388R

msmeg_2748 6.36 soluble pyridine nucleotide transhydrogenase (sthA) COG1249C

msmeg_1085 6.41 dipeptide transport system permease protein DppB COG0601EP

msmeg_2569 6.61 oxidoreductase, 2OG-Fe(II) oxygenase family COG3491R

msmeg_0429 6.64 putative ferric uptake regulator X

msmeg_6878 6.97 inner-membrane translocator COG4177E

msmeg_6263 7.01 glutamate synthase family protein COG0069E

msmeg_6262 7.40 FwdC-FmdC family protein COG0070E

msmeg_1295 7.42 transthyretin COG2351R

msmeg_3402 7.82 cytosine permease, putative COG1457F


msmeg_1296 8.73 uricase COG3648Q

msmeg_0780 8.79 phosphotransferase enzyme family protein COG2334R

msmeg_6877 8.90 branched-chain amino acid transport system ATP-binding protein COG0411E

msmeg_0566 8.94 aliphatic amidase COG0388R

msmeg_3403 9.26 formamidase COG0388R


msmeg_1086 9.94 ABC transporter permease protein COG1173EP

msmeg_0778 10.22 putative transcriptional regulator COG2188K

msmeg_6261 10.36 glutamine amidotransferase, class II COG0067E

msmeg_6660 10.75 permease, cytosine-purines, uracil, thiamine, allantoin family COG1457F

msmeg_6817 11.34 RNA polymerase sigma factor, sigma-70 family COG1595K

msmeg_1052 11.63 amino acid carrier protein COG1115E

msmeg_6259 11.67 ammonium transporter (amt1) COG0004P

msmeg_6881 11.75 transcriptional regulator, GntR family COG1802K


Results 82

msmeg_2525 14.19 amino acid permease superfamily COG0531E

msmeg_1293 14.73 xanthine-uracil permeases family protein COG2233F

msmeg_2978 15.78 ABC transporter ATP-binding protein COG0410E

msmeg_6260 16.11 glutamine synthetase, type III (glnT) COG0174E

msmeg_4206 16.44 molybdopterin oxidoreductase X

msmeg_3400 17.66 glutamyl-tRNA(Gln) amidotransferase subunit A COG0154J


msmeg_2427 18.22 protein-P-II uridylyltransferase (glnD) COG2844O


msmeg_3401 18.68 LamB-YcsF family protein COG1540R


msmeg_1597 18.80 Transcription factor WhiB X

msmeg_2979 18.88 ABC transporter ATP-binding protein COG4674R

msmeg_2981 19.17 branched-chain amino acid ABC-type transport system COG0559E

msmeg_6115 19.21 phosphoglycerate dehydrogenase COG0111HE

msmeg_6735 19.26 amino acid permease, putative COG0531E

msmeg_1087 19.33 oligopeptide ABC transporter ATP-binding protein X


msmeg_5084 20.59 glycosyl transferase, group 2 family protein COG0463M

msmeg_5359 21.35 cyanate hydratase (cynS) COG1513P

msmeg_5360 22.01 formate-nitrate transporter COG2116P

msmeg_4501 22.33 sodium:dicarboxylate symporter COG1301C

msmeg_4635 22.90 ammonium transporter family protein (amtA) COG0004P

msmeg_2980 23.33 putative membrane protein COG4177E

msmeg_2524 24.34 ABC transporter, ATP-binding protein COG1136V

msmeg_2522 24.38 efflux ABC transporter, permease protein X

msmeg_5358 25.10 acetamidase-Formamidase family COG2421C

msmeg_4294 26.01 glutamine synthetase, type I (glnA2) COG0174E

msmeg_5765 29.55 globin COG2346R

msmeg_4638 30.14 vanillate O-demethylase oxidoreductase COG1018C




msmeg_6816 33.03 molybdopterin oxidoreductase COG0243C

msmeg_0432 34.03 uroporphyrinogen-III synthetase COG1587H

msmeg_4290 34.13 glutamine synthetase, type I (glnA) COG0174E

msmeg_0569 34.95 flavoprotein involved in K+ transport COG2072P

msmeg_0779 35.16 short-chain dehydrogenase-reductase SDR COG1028IQR

msmeg_0781 36.20 amino acid permease COG0531E


msmeg_5730 45.31 permease for cytosine-purines, uracil, thiamine, allantoin COG1953FH

msmeg_1082 47.68 putative response regulator COG2197TK



msmeg_0428 48.97 nitrite reductase [NAD(P)H] small subunit (nirD) COG2146PR

msmeg_2426 51.56 nitrogen regulatory protein P-II (glnK) COG0347E

msmeg_1084 51.81 peptide-opine-nickel uptake family ABC transporter COG0747E

msmeg_0427 52.06 nitrite reductase [NAD(P)H], large subunit (nirB) COG1251C

msmeg_0433 54.53 nitrite extrusion protein (narK3) COG2223P

msmeg_2425 56.21 ammonium transporter (amtB) COG0004P

msmeg_6734 62.84 dibenothiophene desulfuriation enzyme A COG2141C

msmeg_2526 66.62 copper methylamine oxidase COG3733Q

msmeg_2982 136.17 putative periplasmic binding protein (urtA) COG0683E

Results 83

This list of putative GlnR target genes includes genes already described to be involved in

nitrogen metabolism in M. smegmatis, such as msmeg_2425 (amtB; 56.21 fold change),

msmeg_4290 (glnA; 34.13 fold change) and msmeg_6259 (amt1; 11.67 fold change; see

also Amon et al., 2008). Furthermore, genes of known function in nitrogen metabolism of

related species such as C. glutamicum or S. coelicolor (see section 2.2.3) were detected.

These were msmeg_2426/2427 (glnK and glnD; 51.56 and 18.22 fold change) or

msmeg_0427/0428 (nirBD; 52.06 and 48.97 fold change). These genes were chosen for

further analyses to experimentally verify the microarray data. Moreover, a variety of other

interesting genes was tested. These included genes with a very high fold change of transcript

levels and genes putatively involved in uptake and assimilation of different nitrogen sources

to test correlation of the data with growth experiments (see section 4.1.1). These were

msmeg_0781, msmeg_1052, msmeg_2184 and msmeg_6735 (different permeases and

transporters for amino acids; 36.2, 11.63, 48.27 and 19.26 fold change), as well as

msmeg_1293, msmeg_2748, msmeg_4011, msmeg_5730 and msmeg_6660 (transporters

for purines and pyrimidines; 14.73, 6.36, 3.26, 45.31 and 10.75 fold change). Also urea

transport and assimilation systems were found: msmeg_2187 (urea amidolyase; 3.36 fold

change), msmeg_2982 (urtA; 136.17 fold change), msmeg_2981 (urtB; 19.17 fold change)

and msmeg_3626 (urease; 3.89 fold change). In addition to that, msmeg_0566 and

msmeg_1090 (amidases; 8.94 and 5.24 fold change), msmeg_0433 and msmeg_5360

(nitrate/nitrite-associated genes; 54.53 and 22.01 fold change), msmeg_0571 (hydrolase;

48.04 fold change) and msmeg_2526 (copper methylamine oxidase; 66.62 fold change) were

focus of attention. The operon msmeg_6259-6264 (including amt1; 5.06-16.11 fold change)

was of special interest, as well as msmeg_4635 (amtA; 22.9 fold change), msmeg_4294

(glnA2; 26.01 fold change), msmeg_6816 (molybdopterin oxidoreductase; 33.03 fold change)

and msmeg_5765 (29.55 fold change), a globin gene not yet known to be involved in

nitrogen assimilation. Every gene used for further analyses such as RNA hybridization, real

time RT PCR or gel retardation experiments is highlighted blue in table 5.

4.2.2 Verification of the microarray data

In a global approach comparing total transcript of the M. smegmatis wild type SMR5 and the

glnR deletion strain MH1 incubated under nitrogen starvation, 125 putative target genes of

GlnR have been identified (see table 5, section 4.2.1). From these, several genes of either

very high changes in transcript levels between wild type and glnR deletion strain or putative

function in nitrogen metabolism were chosen for further investigations. Results of an RNA

hybridization experiment carried out for verification of the microarray data are shown in

Results 84

figures 24 and 25. Additionally, a putative connection between transcript levels and length of

nitrogen starvation in minutes can be seen in this approach. For this experiment,

M. smegmatis SMR5 and MH1 were grown under nitrogen surplus until exponential growth

phase and then transferred into nitrogen-free medium. After samples were taken at different

time points, total RNA was prepared and 1 µg was used in each case for hybridization with

specific RNA probes representing the genes of interest. Figure 24 demonstrates nitrogen-

and GlnR-dependent mRNA levels of 32 genes. While in the wild type (upper lane) a rising

mRNA signal was detected depending on the length of nitrogen starvation, this was not

observed in the glnR deletion strain (lower lane), confirming that GlnR plays an essential role

in transcriptional activation of these genes.

Interestingly, comparing the signal strength of various genes at the same time points, it was

apparent that they differed significantly. The maximum signal strength of nirB and narK3 e.g.

was reached after 60 minutes of nitrogen starvation, whereas this was observed for amtB,

glnA or amt1 after 30 minutes or even earlier regarding genes likes urtA or msmeg_2184.

These differences might result from variable activation abilities of GlnR, caused by deviating

binding sites in the promoter regions of the tested genes. Binding of GlnR to its target genes

is investigated in detail later in this study (see section 4.2.4). A connection between signal

strength in this experiment and fold change of transcripts obtained from the microarray

analysis can hardly be postulated, as processing of the two approaches is very different.

Furthermore, for some genes nitrogen- or GlnR-dependent transcript levels could not be

detected in RNA hybridization experiments (see figure 25). These were genes with low fold

change of mRNA levels obtained from the microarray analysis, such as msmeg_3626

(urease, 3.89 fold change) or msmeg_4011 (putative permease, 3.26 fold change).

Surprisingly, the other tested genes (msmeg_1084, 51.81 x; msmeg_5360, 22.01 x and

msmeg_6735, 19.26 x) showed a rather high fold change in the microarray analysis, but no

signal in the RNA hybridization experiment. No increase of signal strengths was also

obtained when the genes ureC1, ureC2, gltB, gltD and gdh, described as generally involved

in nitrogen metabolism (marked with *), were tested.

Results 85

Fig. 24: RNA hybridization experiment. 1 µg total RNA was used for hybridization with specific RNA probes against genes of interest. RNA was prepared from wild type (upper lane) and glnR deletion strain (lower lane), both incubated under nitrogen surplus (+N) and starvation (-N). Samples were taken at different time points (directly and 5, 10, 15, 30 and 60 minutes after induction of nitrogen starvation). 32 genes with increased transcript levels under nitrogen starvation in the wild type, but with no or weak signal in the glnR deletion strain, are shown.

+N -N0 5 10 15 30 60 +N -N0 5 10 15 30 60

msmeg_0427 (nirB)

msmeg_0433 (narK3)

msmeg_0571

msmeg_0779

msmeg_0781

msmeg_1052

msmeg_1090

msmeg_1293

msmeg_2184

msmeg_2425 (amtB)

msmeg_2426 (glnK)

msmeg_2427 (glnD)

msmeg_2522

msmeg_2526

msmeg_2748

msmeg_2981

msmeg_2982 (urtA)

msmeg_3400

msmeg_4290 (glnA)

msmeg_4294 (glnA2)

msmeg_4381

msmeg_4635 (amtA)

msmeg_4638

msmeg_5358

msmeg_5730

msmeg_5765 (glbN)

msmeg_6259 (amt1)

msmeg_6260 (glnT)

msmeg_6261

msmeg_6660

msmeg_6734

msmeg_6816

Results 86

+N -N0 5 10 15 30 60 +N -N0 5 10 15 30 60

msmeg_0566

msmeg_1084

msmeg_1094 (ureC1)*

msmeg_3225 (gltB)*

msmeg_3226 (gltD)*

msmeg_3625 (ureC2)*

msmeg_3626

msmeg_4011

msmeg_5360

msmeg_5442 (gdh)*

msmeg_6735

Fig. 25: RNA hybridization experiment. 1 µg total RNA was used for hybridization with specific RNA probes against genes of interest. RNA was prepared from wild type (upper lane) and glnR deletion strain (lower lane), both incubated under nitrogen surplus (+N) and starvation (-N). Samples were taken at different time points (directly and 5, 10, 15, 30 and 60 minutes after induction of nitrogen starvation). For 11 genes, no enhanced signal was found in the wild type under nitrogen starvation. No influence of GlnR was detected. *: genes described as involved in nitrogen metabolism, not found in microarray analysis.

Additionally, some genes of interest were chosen for further verification of the data obtained

in DNA microarray and RNA hybridization analyses by real time RT PCR. With this method,

changes in the amounts of transcript of a specific target gene can be detected precisely. For

this experiment, total RNA was prepared from M. smegmatis SMR5 and MH1, again grown

until exponential growth phase and then incubated for 30 minutes under nitrogen starvation.

Purity of the RNA was guaranteed by performing additional DNAse digest, followed by

control PCR that gave no results when RNA was used as template. In a real time RT PCR

experiment, RNA is transcribed into cDNA which serves as template for further amplification

of a gene of interest. In this study, a 100 bp DNA fragment of every target gene was

amplified. Amplification rates were monitored and efficiencies of the PCR reactions were

determined. An example for a standard curve with resulting PCR efficiency of 95 % for a

100 bp fragment of msmeg_2427 (glnD) is shown in figure 26A. Additional controls such as

melt curves and detection of the 100 bp fragments on 4 % agarose gels guaranteed the

quality of the experiment (see figure 26B). All 21 genes used for further experiments showed

a PCR efficiency of 90-105 %.

Results 87

PCR efficiency (%) R squared slope y-intercept

95.0 0.998 -3.447 26.823

A

B

1 2 3 4 5 6 7 8 9

Fig. 26: Real time RT PCR experiments determinating PCR efficiency of a 100 bp fragment of glnD. A. Amplification chart and determination of PCR efficiency from a standard curve using RNA amounts of 100, 10, 1 and 0.1 ng per reaction, carried out in duplicates. B. Quality control of PCR reaction by generation of a melt curve and by detecting the 100 bp fragments on agarose gel. 1-8: RNA dilution series in duplicates. 9: non-template control.

In a second run, amounts of specific transcripts were compared in the wild type and the glnR

deletion strain. Therefore, relative quantification was chosen, i.e. the amount of a specific

RNA template in the wild type was determined in relation to the amount in the glnR deletion

strain, which was set one. Additionally, a reference gene was needed for this calculation. The

gene msmeg_3084 (encoding G3PDH) was used, as its change of transcript level between

wild type and glnR deletion strain was less than 10 % (see figure 27A and B). So, after

experiments were carried out at least in triplicates, the relative fold expression of every target

gene in the wild type was calculated according to Pfaffl et al. (2001), considering PCR

efficiencies and Cq values of target and reference gene in the wild type and in the ΔglnR

sample (for experimental setup and calculations refer to section 3.3.2.5). Results are

summarized in figure 27B showing the GlnR-dependent increase of transcript of 20 genes in

the wild type under nitrogen starvation. Very high differences in transcript levels were

obtained for amtB, glnK, urtA, glnA, amtA and msmeg_6816. Significant changes were also

Results 88

400

350

300

250

200

150

100

50

03084 3084 0428 0779 1090 1293 2184 2425 2426 2427 2522 2526 2982 3400 4290 4294 4635 5730 5765 6259 6261 6816

nirD amtB glnK glnD urtA glnA glnA2 amtA glbN amt1

SMR5 0.9 3.4 12.4 2.6 8.4 18.0 340.4 47.7 19.0 6.7 10.5 169.8 23.5 59.6 21.2 103.6 8.5 11.5 6.0 10.9 188.5

A B

1.2

1.0

0.8

0.6

0.4

0.2

0

rela

tive e

xp

ressio

nra

tio

rela

tive e

xp

ressio

nra

tio

MH1 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

monitored for msmeg_0779, msmeg_2184, glnD, msmeg_2526, msmeg_3400, glnA2,

msmeg_5765 (glbN) and msmeg_6261. The genes msmeg_1293, msmeg_2522,

msmeg_5730 and amt1 showed lower, but still detectable changes in this approach. In most

cases, the calculated transcript ratios deviated from those obtained in the previously shown

microarray analysis (section 4.2.1), which might be a result of different experimental

procedures. Nevertheless, the influence of GlnR on the transcripts of the tested genes was

again confirmed in this independent approach.

Fig. 27: Results of real time RT PCR analyses comparing transcript ratios of target genes in the M. smegmatis wild type and the glnR deletion strain. For this, total RNA of the strains SMR5 and MH1 incubated for 30 minutes under nitrogen starvation was prepared and used as template for reverse transcription and PCR reaction. Specific primers were used for amplification of 100 bp fragments of the target genes. A. The gene msmeg_3084 was carried along as a control; transcripts of this housekeeping gene (G3PDH) were not significantly different in wild type (blue bar) and glnR deletion strain (blue patterned bar). B. Relative fold transcript levels of 20 target genes in the wild type SMR5 were calculated (blue bars), while the amounts of transcripts in the glnR deletion strain were set one. Relative fold transcription levels were calculated in normalization to the reference gene msmeg_3084. Genes are sorted according to their msmeg_ numbers and the calculated transcript ratios in the wild type strain SMR5 are shown.

Recapitulating recent results, it can be stated that GlnR plays an important role in increasing

the amount of transcripts of various target genes under nitrogen starvation. This was proven

in three independent experimental approaches. First, in a global approach analyzing the

whole transcriptome of M. smegmatis wild type SMR5 and the glnR deletion strain MH1, 125

putative GlnR target genes were identified. These genes showed decreased mRNA levels

under nitrogen starvation in the absence of GlnR. More than 33 % of these genes are

putatively associated with nitrogen metabolism. A selection of these genes was further tested

in RNA hybridization experiments (see table 5, section 4.2.1). With this method, nitrogen-

Results 89

and GlnR-dependent transcript levels were confirmed for 32 genes (see figure 24). Using

real time RT PCR as another independent approach, GlnR-dependent transcripts of 20 target

genes found in the microarray analysis were monitored. Obviously, many new GlnR target

genes have been found, resulting in the question if the increase of their transcript levels is

due to binding of GlnR to the corresponding promoter sequences and thus activation of RNA

polymerase, or if it might be a secondary effect due to the absence of GlnR in the organism.

4.2.3 Binding of GlnR to promoter sequences of its target genes

Previous experiments in this study revealed that the increase of transcript levels of a

multitude of genes mainly involved in nitrogen metabolism depends on OmpR-type regulator

GlnR. It has already been shown that transcripts of amtB, amt1 and glnA are enhanced by

GlnR in response to nitrogen starvation (Amon et al., 2008). This was proven in this study for

more than 30 additional genes (see section 4.2.2). Furthermore, binding of purified GlnR

protein to a 220 bp promoter sequence upstream of amtB could be shown earlier in this

study (see section 4.1.7), supporting the idea that GlnR activates gene transcription by RNA

polymerase by binding to specific sequences in the promoter regions of its target genes. In

the following, binding of GlnR to upstream sequences of target genes identified before (see

section 4.2.1 and 4.2.2) is investigated.

Gel retardation experiments were performed as described in section 3.3.1.11 and 4.1.7. For

these, 200-300 bp fragments directly upstream of the target genes’ or operons’ start (refer to

figure 56, section 5.1.2) were amplified and labeled with digoxigenin for further detection.

After incubation with 400 ng purified MBP-GlnR, the samples were separated via gel

electrophoresis. The results of this experiment are shown in figure 28. Free DNA was

detected as the band proceeding furthest in the gel (lane 1), while a distinct shift was

monitored when DNA-GlnR complexes were formed (lane 2). When 3 µg (1000 fold surplus)

of competitor DNA polyd[I-C] were added (lane 3), a differentiation of specific and unspecific

binding was possible.

Results 90

0572 0781 2184 2425amtB 2526 2982urtA 3400

4290glnA 4294glnA2 4635 amtA 6259amt1 6660 6735

0427nirB 1052 1084 1090 1293 2748 2981

A

B

4638 5141narK 5734 5765glbN 6258 6734 6816

Fig. 28: Gel retardation experiments. 200-300 bp DNA fragments upstream of GlnR target genes labeled with digoxigenin were used. For each gene free DNA (lane 1), DNA plus 400 ng MBP-GlnR (lane 2) and DNA plus 400 ng MBP-GlnR plus 3 µg competitor DNA polyd[I-C] (lane 3) were tested. A. Target sequences with specific binding. B. Unspecific or no binding. Numbers indicate msmeg_ numbers.

Results 91

For 13 promoter fragments, specific binding of GlnR was spotted (figure 28A), as binding

was not inhibited by addition of surplus of competitor DNA. These were fragments upstream

of msmeg_0572, msmeg_0781, msmeg_2184, msmeg_2425 (amtB), msmeg_2526,

msmeg_2982 (urtA), msmeg_3400, msmeg_4290 (glnA), msmeg_4294 (glnA2),

msmeg_4635 (amtA), msmeg_6259 (amt1), msmeg_6660 and msmeg_6735. On the

contrary, 14 promoter regions with no or only unspecific binding of GlnR were found (figure

28B), as binding was not detected or was inhibited by addition of competitor DNA. These

fragments were located upstream of msmeg_0427 (nirB), msmeg_1052, msmeg_1084,

msmeg_1090, msmeg_1293, msmeg_2748, msmeg_2981, msmeg_4638, msmeg_5141

(narK), msmeg_5734, msmeg_5765 (glbN), msmeg_6258, msmeg_6734 and msmeg_6816.

Furthermore, promoter fragments upstream of msmeg_1091 and msmeg_3627 (ureE1 and

ureA2, encoding putative urea assimilating genes located in operons; not found in the

microarray analysis), but binding of GlnR could not be detected (Amon et al., 2008). These

data indicated that direct binding of GlnR to the promoter sequences of at least 13 target

genes and operons is indeed responsible for enhanced levels of transcripts, but also that

transcription of at least the same amount of genes must be regulated differently, presumably

by a secondary effect which is yet unknown or by influence of further transcriptional

regulatory proteins.

4.2.4 Determination of binding properties of GlnR

It has already been demonstrated in this study, that binding of GlnR to DNA promoter

sequences plays an important role for enhanced transcript levels of the corresponding target

genes. For approximately half of all tested promoter sequences, specific binding of GlnR was

demonstrated (see section 4.2.3) leading to the question which factors (e.g. binding site,

nitrogen status of the cells or activation of GlnR) determine whether GlnR binds or not. To

answer this, the definition of the DNA binding motif of GlnR was essential. This had already

been done in a previous study where upstream sequences of known GlnR target genes such

as amtB, glnA and amt1 of different mycobacteria were compared in a bioinformatic

approach resulting in a putative 44 bp DNA binding motif shown in figure 29 (Amon et al.,

2009). Four highly conserved domains were found indicating indeed at least two putative

binding sites for GlnR within these sequences.

Results 92

Fig. 29: DNA binding motif of GlnR determined in bioinformatic analysis of promoter regions of different target genes. Putative GlnR cis elements in M. smegmatis (msmeg), M. tuberculosis (H37rv), M. bovis (bovis) and M. avium (avium) upstream of the genes glnA, amtB and amt1 (only smeg) were compared (Amon et al., 2009).

To verify this in gel retardation experiments, the 220 bp region upstream of amtB was

chosen, as strong and specific binding of GlnR has been detected here before (see figure 28,

section 4.2.3). To localize the GlnR binding sites in detail, competitive gel retardation assays

were performed. Therefore, samples were set up as described before (see section 3.3.1.11

and 4.2.3). Each sample of 220 bp digoxigenin-labeled amtB promoter was mixed with 1 µg

purified MBP-GlnR (also GlnR-His, not shown) and additionally with one of eight non-labeled,

50 bp overlapping DNA fragments covering the whole promoter sequence. One of these

fragments containing the GlnR binding site would immediately inhibit the shift of labeled

DNA, as the protein would much likelier bind to the unlabeled DNA which was supplied in

1000 fold surplus. A model and the results of the first competitive gel retardation experiment

are shown in figure 30. Addition of fragments 1, 3, 7 and 8 did not lead to any inhibition of

the DNA shift caused by GlnR binding. Fragments 4 and 6 led to the strongest inhibition,

indicating that the GlnR binding site is located in between this 100 bp fragment. Interestingly,

when fragment 5 was added, the inhibition of the shift was almost abolished. Another weak

binding site might exist in fragment 2, as a very slight inhibition was also spotted here.

Results 93

- + 1 2 3 4 5 6 7 8

220 bp upstream region amtB

1

2

3

4

5

6

7

8

A

B

Fig. 30: Competitive gel retardation experiment. A. Model of eight overlapping 50 bp DNA fragments covering the 220 bp upstream region of amtB. B. When each of these fragments was added in 1000 fold surplus to gel retardation samples consisting of 220 bp digoxigenin-labeled amtB promoter fragment and purified GlnR, an inhibition of the shift was spotted for fragments 4, 6 and weakly 2. -: free DNA as negative control. +: DNA plus 600 ng MBP-GlnR as positive control.

Subsequently, new gel retardation experiments were performed to further specify the

localization. Therefore, the 50 bp fragments 4 and 6 which caused inhibition of GlnR-binding

to the 220 bp promoter fragment before were used as digoxigenin-labeled DNA probes. 25

and 15 bp DNA fragments were used as competitor DNA (figure 31A and C). Fragment 6.1

caused indeed a further inhibition of GlnR-binding to fragment 6, indicating that the binding

site is located within this 25 bp sequence (figure 31A and B). Further localization of the

binding site spotted in fragment 4 was not possible, as addition of 25 bp fragments 4.1, 4.2

and 4.3 did not lead to inhibition of the shift (figure 31C and D).

These results verified the supposed existence of more than one GlnR binding sites in the

promoter regions of its target genes. For amtB, two binding sites were experimentally

verified, one located 75-100 bp upstream (fragment 6.1), and the other 100-150 bp upstream

of the gene’s start codon (fragment 4). Sequence analyses revealed a putative third binding

site 50-75 bp upstream of the start codon (putatively fragment 6.3). An additional, fourth

binding site might exist 150-200 bp upstream of the start codon (fragment 2). These data

supported the “galloping” model which was predicted for GlnR binding before (Yoshida et al.,

2006; Amon et al., 2008).

Results 94

50 bp fragment 6 amtB

6a

6.2

6b

6.3

6c

6.1

6d

A

B

TGCGTTCACTTTCCGGAAACGCAACGGCAGCACCGGCGAAACGCCGGCAA

D

C

50 bp fragment 4 amtB

4.2

4.34.1

ACGACCTGTGCTTTGTCGGCGGGAAACATGAGCGTAACAGTGATCGGGAT

- + 6

- + 4

Fig. 31: Competitive gel retardation experiments. A. Model of three overlapping 25 bp DNA fragments (6.1-6.3) and four 15 bp fragments (6a-d) covering the 50 bp fragment 6 in the upstream region of amtB. B. Addition of these fragments in 1000 fold surplus to gel retardation samples containing 50 bp digoxigenin-labeled fragment 6 and 600 ng MBP-GlnR each. An inhibition of the shift was only detected for fragment 6.1. C. Model of three overlapping 25 bp DNA fragments (4.1-4.3) covering the 50 bp fragment 4 in the upstream region of amtB. D. When these fragments were added to gel retardation samples containing 50 bp digoxigenin-labeled fragment 4 and 600 ng MBP-GlnR each, no inhibition of the shifts was spotted. -: free DNA as negative control. +: DNA plus 600 ng MBP-GlnR as positive control.

Results 95

-GlnR-His+N GlnR-His-N

Another interesting point, next to the question where GlnR exactly binds, was if binding

depends on the nitrogen status of the cells. So far, GlnR-dependent increase of transcripts of

various target genes under nitrogen starvation has been identified in this study (see figure 24

and 27, section 4.2.2). It was also shown that this enhanced transcript levels partially

correlate with DNA binding of GlnR (see figure 28A, section 4.2.3). To analyze a putative

connection between binding affinity of the regulator protein to its target DNA and nitrogen

status of the cells, another gel retardation experiment was carried out. Therefore, two

samples of the M. smegmatis wild type SMR5 carrying a GlnR-His expression vector were

grown until exponential phase. While one was directly used for purification of the tagged

protein, the other was transferred into nitrogen-free medium and incubated for further

60 minutes before protein purification. Purification was carried out as described in section

4.1.5. Protein samples were used for binding to a 220 bp promoter fragment upstream of

amtB. Binding of GlnR to this sequence has already been shown (figure 19, section 4.1.7;

figure 28A, section 4.2.3; figure 30 and 31, see above).

When rising amounts of GlnR-His+N or GlnR-His-N were added to the amtB promoter

fragment, an increased shift of the DNA was spotted (figure 32). Considering previous results

obtained for purification of GlnR, this was surprising in two different ways. First, purification of

proteins from M. smegmatis SMR5 via His-tag results in loss of protein of interest (GlnR) in

favor of GroEL1 (see section 4.1.5). This gel retardation experiment confirmed the high DNA

binding affinity of GlnR, as only very small amounts of this protein in samples contaminated

with GroEL1 led to a shift of DNA (figure 32). Furthermore, as addition of both GlnR-His

proteins led to shift of DNA, it can be said that binding of GlnR in vitro is independent from

the nitrogen status of the cells the protein was purified from. Thus, the signal making GlnR

activate transcription of its target genes remained so far unclear. Further investigations

concerning putative modifications of GlnR are carried out later in this study (refer to section

4.3.2).

Fig. 32: Gel retardation experiment with 220 bp digoxigenin-labeled fragment upstream of amtB and purified GlnR. GlnR was purified via N-terminal His-tag from M. smegmatis SMR5 grown under nitrogen surplus (+N) and starvation (-N). Rising amounts of protein were added (380, 760, 1140, 1520 and 1900 ng).

Results 96

+N -N0 5 10 15 30 60 min

A B

4.2.5 Autoregulation of GlnR

It has already been demonstrated in this study that an increase of transcripts of more than 30

target genes depends on OmpR-type regulator GlnR (refer to section 4.2.2). Although

binding of GlnR to promoter sequences of its target genes has been investigated in detail,

the signal leading to activation of GlnR has not yet been identified and no correlation

between nitrogen status of the cells and DNA binding behavior of GlnR in vitro could be seen

(see section 4.2.4). Thus, another interesting point is the transcription of the glnR gene itself.

It is reported for the closely related actinomycete S. coelicolor, that the transcript of the glnR

gene is enhanced due to nitrogen limitation (Tiffert et al., 2008), which was not observed in

transcriptome analyses of M. smegmatis in this study. When total transcript of the wild type

SMR5 incubated under nitrogen surplus and starvation was compared, changes of transcript

levels of more than 500 genes were observed (see table 9 and 10, section 7.2), but

msmeg_5784 (glnR) was not among those genes. In a second approach, changes in

transcript levels (higher than 3) of 131 genes were revealed between wild type and glnR

deletion strain incubated under nitrogen starvation; again, the glnR gene was not detected

(see table 11 and 12, section 7.2). Nevertheless, additional experiments were carried out to

verify this. Nitrogen- and GlnR-dependence of the glnR gene was tested in an RNA

hybridization experiment (as described in section 4.2.2), but no increase of the transcript

level was detected at different time points under nitrogen starvation (figure 33A).

Furthermore, binding of purified MBP-GlnR protein to a 250 bp sequence upstream of the

glnR start codon was investigated (as described in section 4.2.3), but specific binding was

not detected (figure 33B).

Fig. 33: Investigation of regulation of the glnR gene. A. RNA hybridization experiment. 1 µg total RNA was used for hybridization with a specific glnR RNA probe. RNA was prepared from wild type (upper lane) and glnR deletion strain (lower lane), both incubated under nitrogen surplus (+N) and starvation (-N). Samples were taken at different time points (directly and 5, 10, 15, 30 and 60 minutes after induction of nitrogen starvation). B. Gel retardation experiment showing unspecific binding. A 250 bp digoxigenin-labeled glnR promoter fragment was used. Lane 1: free DNA, lane 2: DNA plus 400 ng MBP-GlnR, lane 3: DNA plus 400 ng MBP-GlnR plus 3 µg competitor DNA polyd[I-C].

Results 97

All data obtained from DNA microarray, RNA hybridization and gel retardation analyses

indicated that the glnR gene in M. smegmatis is obviously only weakly and constitutively

expressed. Contrary to observations in S. coelicolor, the glnR transcript level was not

enhanced due to nitrogen starvation. An autoregulation by GlnR was not monitored either.

4.3 Activation of GlnR

It has already been proven in this study that transcriptional activator GlnR is responsible for

enhanced transcript levels of at least 30 target genes under nitrogen starvation in

M. smegmatis (see section 4.2.2). Until now, nothing was known about the mechanism or the

putative interaction partner in M. smegmatis that signalizes GlnR to activate gene

transcription under nitrogen starvation. This mechanism is well understood in C. glutamicum

(refer to section 2.2.3). Under nitrogen starvation, the signal transduction protein GlnK is

adenylylated by GlnD and thus able to interact with transcriptional repressor AmtR. As a

consequence, the repressor is released from its target DNA and gene expression can take

place. Another system is described for E. coli (refer to section 2.2.2). Here, the PII signal

transduction protein is uridylylated by GlnD under nitrogen starvation and thus, no longer

able to interact with sensor histidine kinase NtrB which is subsequently autophosphorylated

and as a consequence able to phosphorylate NtrC. NtrC is a transcriptional regulator that

modulates expression of its target genes due to its phosphorylation state. Due to sequence

homologies, M. smegmatis GlnR is classified as OmpR-type transcriptional regulator (Amon

et al., 2008), for which a mechanism similar to the NtrB/NtrC-system applies. OmpR also

modulates transcription of its target genes after it has been phosphorylated by sensor

histidine kinase EnvZ. Because of these sequence homologies, it was assumed at the

beginning of this study that M. smegmatis GlnR might also be regulated depending on

nitrogen status of the cell via (de)phosphorylation by a sensor histidine kinase (Amon, 2010).

In the following, the search for a GlnR interaction partner (putative sensor histidine kinase)

and for evidence of phosphorylation of GlnR is described.

Results 98

A B

1

2

3

4

R W1 W2 W3 M W4 W5 W6 E1 E2 E3 E4 E5 W7 E2

70 kDa

60 kDa

50kDa

40 kDa

30 kDa

4.3.1 Search for GlnR interaction partner

Due to sequence homologies, M. smegmatis GlnR is characterized as a response regulator

belonging to a typical bacterial two-component system (Yoshida et al., 2006; Amon et al.,

2008). Again typical for such a system is that genes encoding response regulator and

corresponding sensor kinase are located in close vicinity to each other on the genome. This

is the case e.g. for NtrB/NtrC (genes located in operon glnAntrBC) and OmpR/EnvZ (genes

located in operon ompRenvZ) in E. coli. In contrast, M. smegmatis GlnR is characterized as

orphan response regulator (Amon, 2010), as the gene encoding its corresponding kinase is

not situated nearby, which makes its identification rather difficult. Due to this fact, different

approaches were used to find putative interaction partners of GlnR.

4.3.1.1 Global pull down analysis

First, a pull down experiment coupled with MALDI-ToF-MS was carried out. Therefore, the

Strep-GlnR protein was overexpressed in E. coli Rosetta2 and purified via affinity

chromatography as described in section 3.3.3.2 and 4.1.5. Before elution of the protein, total

cell extract of M. smegmatis SMR5 incubated for 60 minutes under nitrogen starvation was

added to the column leading to a mixture of Strep-GlnR and putative interaction partners in

the elution fractions. These fractions were separated in SDS PAGE (figure 34) and four

distinct bands at size of 55-75 kDa were analyzed via mass spectroscopy (University of

Cologne).

Fig. 34: Pull down experiment. A. Documentation of the experimental process in SDS PAGE. R: total cell extract of E. coli Rosetta2 with overexpressed Strep-GlnR. W1-W3: washing fractions. M: total cell extract of M. smegmatis SMR5 incubated for 60 minutes under nitrogen starvation. W4-W6: washing fractions. E1-E5: elution fractions. W7: washing fraction. B. Enlargement of elution fraction 2 (after enrichment of proteins) with purified Strep-GlnR at 31 kDa and four distinct protein bands at 55-75 kDa (see arrows), which were further investigated in MALDI-ToF-MS.

Results 99

A mass spectroscopic analysis revealed for protein 1 (approximately 75 kDa) closest relation

to a hypothetical protein CAT7_10475 from Carnobacterium sp. AT7 and for protein 4

(approximately 55 kDa) closest relation to a TetR-type transcriptional regulator from

Paenibacillus sp. JDR-2, indicating that these proteins were caught unspecifically. Protein 2

(approximately 70 kDa) was classified as glutamine synthetase I from E. coli, probably left

over from total Rosetta2 cell extract. Protein 3 (approximately 60 kDa) was the only

M. smegmatis protein found and classified as propionyl-CoA decarboxylase beta chain.

Propionyl-CoA decarboxylase is involved in fatty acid metabolism and thus probably no

interaction partner of GlnR.

4.3.1.2 Analyses of deletion and insertion mutants

As no GlnR interaction partner was found in a global pull down experiment (see section

4.3.1.1), another, bioinformatic approach was chosen. Genomic screening of various

actinomycete genomes was carried out (Amon, 2010). Here, the focus was on finding

putative sensor histidine kinases that appeared in every genome with a GlnR homolog and

were orphan kinases corresponding to the orphan location of glnR in the M. smegmatis

genome. Another condition was that these kinases should not appear in corynebacterial

genomes, as nitrogen metabolism in corynebacteria is regulated by AmtR. The screening

resulted in 22 putative sensor histidine kinases spotted in the genome of M. smegmatis that

met more or less accurate the criteria described above (for full list of candidates, see table

14, section 7.3, adapted from Amon, 2010). Four of these candidates were chosen for

further, experimental investigations: on the one hand, these were Msmeg_1918 and

Msmeg_5241, characterized as the best candidates that fulfilled all criteria according to

Amon (2010). On the other hand, Msmeg_4989, described as PhoR, was chosen. Although

homologs were also found in corynebacterial genomes (Amon, 2010), this kinase was of

special interest as it appeared also in transcriptome analyses carried out in this study. When

total transcript of the M. smegmatis wild type SMR5 was compared under nitrogen surplus

and starvation, it was found that the amount of transcript of msmeg_4989 was enhanced

under nitrogen starvation by a factor of 5.94 (refer to table 10, section 7.2).

Msmeg_0936, characterized as SenX3, was also a very interesting target for further

investigations, although it was also found in corynebacterial genomes and is not described

as orphan (Amon, 2010). Moreover, it is described as sensor histidine kinase of the

SenX3/RegX3-system controlling phosphate metabolism in M. smegmatis (Glover et al.,

2007), but in a recent publication (Rodríguez-García et al., 2009), a correlation between

phosphate and nitrogen metabolism was found in the related species S. coelicolor.

Results 100

A

B

D

C

1918

StuI BsaI

4122 bp

hph

StuI BsaI

2546 bp

BsaI

wt

d

wt (sco) d

1918StuI BsaI

4122 bp

StuI BsaI

3266 bp

BsaI

wt

d

Kin

hph

wt d

5241

BclI XmnI

4455 bp

hph

BclI XmnI

2149 bp

BclI

wt

d

wt d

4989

StuI StuI

6605 bp

pML814

StuI StuI

3813 bp

StuI

wt

i

wt i

Transcriptional control of nitrogen-involved genes such as glnR, glnA or amtB by PhoP

(RegX3 in M. smegmatis) was actually shown postulating “phosphate control over nitrogen

metabolism” in S. coelicolor.

Fig. 35: Southern blot analyses showing genomic situations in M. smegmatis wild type (wt) and deletion or insertion strains (d or i). A. Replacement of msmeg_1918 by hph. (sco): single cross over. B. Replacement of msmeg_1918 kinase domain by hph. C. Replacement of msmeg_5241 by hph. D. Insertion of plasmid pML814 into msmeg_4989.

Results 101

As a first experiment, deletion or insertion strains of three of the genes of interest were

generated as described in section 3.4.6. The plasmid pML814 (see table 2, section 3.1) was

inserted into msmeg_4989, leading to a disruption of the gene. The genes msmeg_1918 and

msmeg_5241 were completely replaced by a hygromycin resistance cassette, which was

subsequently removed by Flpe recombination. Additionally, only the putative kinase domain

of msmeg_1918 was deleted and a double deletion strain Δmsmeg_1918Δmsmeg_5241 was

generated. All genomic modifications were verified via Southern blot analysis showing DNA

fragments of distinct size for wild type and deletion or insertion strains (figure 35).

Putative effects of the chromosomal deletion of msmeg_1918 and msmeg_5241 or the

disruption of msmeg_4989 on nitrogen-dependent growth behavior and gene transcripts

were monitored. Growth experiments were carried out with all generated strains as described

in section 4.1.1. M. smegmatis wild type SMR5 and glnR deletion strain MH1 were carried

along as controls. As it was already described before (see section 4.1.1 and Amon et al.,

2008), no difference in growth of the wild type was observed when MSX was added to 7H9

minimal medium, whereas the glnR deletion strain MH1 was no longer able to grow after

addition of MSX (figure 36A). Furthermore, a deletion of the genes msmeg_1918 and

msmeg_5241 or a deletion of the kinase domain of msmeg_1918 as well as a disruption of

msmeg_4989 did not result in a change of growth behavior compared to the wild type. Figure

36A shows that this was detected for growth in 7H9 minimal medium and also for growth

under addition of MSX, which is supposed to cause nitrogen starvation to the cells by

interacting with glutamine synthetase I (Berlicki, 2008). Growth of the double deletion strain

Δmsmeg_1918Δmsmeg_5241 was also independent from MSX, but slightly reduced

compared to all other tested strains (figure 36A). The same growth experiments were also

performed using precursor strains where the hygromycin resistance gene hph has not yet

been removed. At this, no differences emerged to the strains without hph (data not shown),

except in the Δmsmeg_1918Δmsmeg_5241 strain, which showed even more reduced growth

(figure 36A). These data were clarified by comparing generation times and maximum oD600 of

the tested strains (figure 36B and C). While most of the strains showed a generation time of

about 4-5 hours and a maximum oD600 of 3-4, independently from presence or absence of

MSX, only for the glnR deletion strain MH1 a significantly enlarged generation time

(approximately 10 hours) and a reduced maximum oD600 (0.9) were detected. Moreover, also

a slightly enlarged generation time (6-8 hours) and a lower maximum oD600 (1.5-2) were

spotted for the strain Δmsmeg_1918Δmsmeg_5241, also independently from presence or

absence of MSX.

Results 102

Fig. 36: Growth of various M. smegmatis strains. A. Growth of the wild type SMR5, the glnR deletion strain MH1 and the strains Δmsmeg_1918, Δmsmeg_1918 kin, Δmsmeg_5241, Δmsmeg_1918Δmsmeg_5241 and Imsmeg_4989 was tested in 7H9 minimal medium (Δ), under addition of MSX (▲) and in 7H9-N (x). B. From these growth experiments, generation times were calculated in hours. C. The maximum oD600 of every tested strain is given. Light blue: 7H9-MSX. Dark blue: 7H9+MSX.

C

B

t [h

]

Maximum oD600

oD

600

Generation times

Ao

D6

00

t [h]

oD

60

0

t [h]

oD

60

0

t [h]

oD

60

0

t [h]

oD

60

0

t [h]

oD

60

0

t [h]

oD

60

0

t [h]

oD

60

0t [h]

Results 103

These data indicated that all tested M. smegmatis strains were still able to respond properly

to artificially caused nitrogen starvation. As MSX binds and thus blocks GS activity, further

growth of the cells can only be guaranteed by providing higher levels of GS and thus glnA

transcripts, which was proven to depend on GlnR (Amon et al., 2008). Consequently, GlnR

was still active in the tested strains and the putative activator of this response regulator was

not disabled. This gave a first hint that the corresponding GlnR interaction partner has not yet

been found.

Another interesting point obtained from these data was that a simultaneous deletion of

msmeg_1918 and msmeg_5241 obviously led to reduced growth even under nitrogen

surplus. As this was not seen for the single deletion mutants, it can be assumed that these

two kinases fulfill some function in growth of M. smegmatis in general and that a loss of one

of these kinases can be compensated by the other. Furthermore, a deletion of both of these

kinases was observed to lead to an even more reduced growth in the presence of

hygromycin.

In addition to the growth experiments, nitrogen-dependent transcript levels of genes involved

in nitrogen metabolism were investigated in every strain. As described in section 4.2.2, cells

were grown under nitrogen surplus until exponential growth phase and subsequently

transferred into nitrogen-free medium. Samples were taken at different time points. Signals of

the genes amtB (figure 37A), glnA (similar to amtB, not shown) and glnR (figure 37B) were

analyzed. As it has already been shown in this study, transcripts of GlnR target genes are

enhanced under nitrogen starvation in the wild type, whereas this is not the case in the

absence of GlnR (see figure 24, section 4.2.2). For that reason, total RNA samples of the

wild type SMR5 and the glnR deletion strain MH1 were carried along as controls. Transcript

levels were tested in the strains Δmsmeg_1918, Δmsmeg_1918 kin, Δmsmeg_5241,

Δmsmeg_1918Δmsmeg_5241 and Imsmeg_4989. As seen in figure 37, no significant

changes in transcript levels of amtB or glnR appeared in any of the tested mutant strains

compared to the wild type, indicating that nitrogen- and GlnR-dependent gene expression

was still completely functional in these strains.

Results 104

SMR5

MH1

Δmsmeg_1918

Δmsmeg_1918 kin

Δmsmeg_5241

Δmsmeg_1918Δmsmeg_5241

Imsmeg_4989

+N -N0 5 10 15 30 60 min +N -N0 5 10 15 30 60 min

amtB glnR

Fig. 37: RNA hybridization experiment. Total RNA from M. smegmatis strains SMR5, MH1, Δmsmeg_1918, Δmsmeg_1918 kin, Δmsmeg_5241, Δmsmeg_1918Δmsmeg_5241 and Imsmeg_4989, grown under nitrogen surplus (+N) and subsequently incubated under nitrogen starvation (-N) for one hour (time points 0, 5, 10, 15, 30 and 60 minutes), was used for hybridization with specific probes against the genes amtB and glnR.

4.3.1.3 Correlation between phosphate and nitrogen metabolism

Besides Msmeg_1918, Msmeg_5241 and Msmeg_4989, the putative involvement in GlnR

activation of another histidine kinase, namely SenX3, was postulated (see section 4.3.1.2).

Although this kinase is part of the two-component system SenX3/RegX3 regulating

phosphate metabolism in M. smegmatis (Glover et al., 2007), it might also be involved in

regulation of nitrogen-related genes, as described for S. coelicolor (Rodríguez-García et al.,

2009).

As a preliminary step to investigate a putative correlation between nitrogen and phosphate

metabolism in M. smegmatis, growth experiments were carried out. For this purpose, ST

medium was used according to Gebhard and Cook (2008). In this medium, L-asparagine and

ammonium chloride served as nitrogen sources, while K2HPO4 was used as phosphate

source (refer to table 3, section 3.2.1). As it can be seen in figure 38A, similar growth of the

M. smegmatis wild type SMR5 was monitored in both, 7H9 and ST medium. Cells were no

longer able to grow when ST-P medium was used, i.e. when no phosphate source was

available. Starting from that, further RNA hybridization experiments were performed. For this

purpose, cells were grown until exponential phase in ST or 7H9 medium, respectively. Then

they were transferred to either phosphate-free ST-P or nitrogen-free 7H9-N medium.

Samples were taken at different time points. After that, phosphate- or nitrogen-dependent

mRNA signals of different genes involved in phosphate (msmeg_0936 senX3, msmeg_0937

Results 105

A

oD

60

0

t [h]

A CB

+P -P10 1h 2h 4h 24h

senX3

regX3

pstS

amtB

glnR

senX3

regX3

pstS

+N -N0 5 10 15 30 60 min

SMR5

MH1

SMR5

MH1

SMR5

MH1

regX3 and msmeg_5782 pstS, encoding a phosphate ABC transporter) and nitrogen (amtB

and glnR) metabolism were monitored. However, no changes in senX3, regX3 or pstS

transcripts were spotted either due to phosphate or to nitrogen starvation (see figure 38B and

C). Transcript levels of nitrogen related genes amtB and glnR were also detected to be

independent from the phosphate status of the cells (figure 38B). These data gave a first

indication that the correlation between phosphate and nitrogen metabolism, which was

described for S. coelicolor (see above), does not apply for M. smegmatis.

Fig. 38: Analyses of growth and gene expression of M. smegmatis due to nitrogen and phosphate starvation. A. Growth in 7H9 (Δ), ST (♦) and ST-P (lacking phosphate, ◊) minimal medium. B. mRNA signals of senX3, regX3, pstS, amtB and glnR under phosphate surplus (+P) and 10 minutes and 1, 2, 4 and 24 hours after induction of phosphate starvation (-P). C. mRNA signals of senX3, regX3 and pstS under nitrogen surplus (+N) and 0, 5, 10, 15 and 60 minutes after induction of nitrogen starvation, tested in the strains SMR5 and MH1.

4.3.1.4 Analyses of protein-protein interactions and phosphorylation of GlnR

As deletion or disruption of genes encoding putative GlnR phosphorylating kinases did not

result in changes of growth or gene transcripts compared to the wild type, in vitro interaction

studies of purified proteins were carried out. For this purpose, GlnR was purified via Strep- or

MBP-tag as described in section 4.1.5. The sensor histidine kinases Msmeg_1918 and

Msmeg_5241 were purified using the same methods, resulting in MBP-1918 (97 kDa), MBP-

5241 (104 kDa), Strep-1918 (56 kDa) and Strep-5241 (64 kDa) shown in figure 39. The

phosphate metabolism-associated kinase SenX3* was purified via Strep-tag according to

Glover et al. (2007) without its pristine 131 amino acids N-terminal transmembrane domain

and was thus detected at 29 kDa (figure 39).

Results 106

100 kDa

85 kDa

70 kDa

60 kDa

50 kDa

40 kDa

30 kDa

- + 1918

Fig. 39: Purification of different kinases with Strep- or MBP-tag. All fusion proteins were overexpressed in E. coli Rosetta2 and purified as described in section 4.1.5. Specific bands were detected in SDS PAGE at 97 kDa (MBP-1918), 104 kDa (MBP-5241), 56 kDa (Strep-1918), 64 kDa (Strep-5241) and 29 kDa (Strep-SenX3*). In the last lane a mixture of Strep-SenX3* (29 kDa) and Strep-GlnR (31 kDa) is shown, indicating that these two proteins could be separated in SDS PAGE despite their similar size.

As a first test of interaction between Msmeg_1918 and GlnR, rising amounts of the purified

kinase were added to gel retardation samples including purified GlnR and the 220 bp

digoxigenin-labeled DNA fragment upstream of amtB. A putative interaction was supposed to

result in a large DNA-GlnR-kinase complex leading to an increased DNA shift. To exclude

interference of the 55 kDa maltose binding protein, only Strep-tagged GlnR and kinase were

used. Although elevation of the DNA seemed to vary a bit in the different samples, addition of

Strep-1918 did not result in an increased DNA shift, indicating that no protein-protein

interaction took place during this experiment (see figure 40).

Fig. 40: Gel retardation experiment. A 220 bp digoxigenin-labeled amtB promoter fragment was used as negative (-) control. 100 ng purified Strep-GlnR were added (+). To that, rising amounts of Strep-1918 were given (100, 200, 1000 and 2600 ng).

Results 107

To investigate putative phosphorylation of the response regulator GlnR by one of the chosen

sensor histidine kinases, phosphorylation assays with [γ-32P]-labeled ATP were carried out

as described in section 3.3.3.9. In a first experiment, autophosphorylation of the kinases was

tested. While this has already been proven for phosphate metabolism-associated kinase

SenX3 in different mycobacteria such as M. bovis (Himpens et al., 2000) and M. smegmatis

(Glover et al., 2007), nothing was known so far about (auto)phosphorylation qualities of

Msmeg_1918 and Msmeg_5241.

For this experiment, 1 µg kinase was mixed with 5 µM ATP and 0.05 µM [γ-32P] ATP and

incubated at 30°C. Autophosphorylation of Msmeg_1918, Msmeg_5241 and SenX3* already

started after one minute, while the strongest signals were detected after 15 minutes of

incubation (see figure 41A). In a second approach, purified GlnR protein was added in 10

fold surplus compared to the kinases, but no signal was detected at 31 (Strep-GlnR) or

71 (MBP-GlnR) kDa, respectively (figure 41B). This led to the conclusion that either these

kinases were not able to phosphorylate GlnR or that in this in vitro experiment a

phosphorylation stimulating factor was missing, which would be present in vivo in the cells.

For that reason, 5 mM Na-glutamate or 2-oxoglutarate were added to the samples. These

substances are known to function as indicators of nitrogen starvation in some bacteria (refer

to section 2.2) and might thus activate phosphorylation of the response regulator GlnR by its

corresponding kinase. However, in this experiment, Na-glutamate or 2-oxoglutarate did not

stimulate phosphorylation of GlnR by one of the tested kinases (data not shown).

Still proceeding on the assumption that a small metabolite was missing in this experiment to

activate GlnR phosphorylation, total cell extract of the M. smegmatis wild type SMR5,

previously incubated for one hour under nitrogen starvation, was added to the samples

described above. In these samples, a weak signal of radioactive labeled phosphate was

detected at a size of approximately 30 kDa, independently from presence or absence of

Msmeg_1918, Msmeg_5241, SenX3* or even purified GlnR (figure 41C). When total cell

extract was filtered and a fraction of proteins larger than 10 kDa was used for

phosphorylation assays, the 30 kDa band was still visible, but when the fraction smaller than

10 kDa was used, no signal could be detected (figure 41C). This indicated that some protein

at approximately 30 kDa size was phosphorylated during the experiment. To identify this

protein, different cell extracts were used. Addition of total cell extract of the C. glutamicum

wild type ATCC 13032, incubated for one hour under nitrogen starvation, resulted in loss of

phosphorylation of the 30 kDa protein, suggesting that it might be GlnR, as this does not

exist in C. glutamicum (figure 41D). An involvement of Msmeg_1918 and Msmeg_5241 in

phosphorylation of this protein was definitely excluded by using total cell extract of

M. smegmatis Δmsmeg_1918Δmsmeg_5241. A signal at 30 kDa was still detected (figure

41D). More evidence against the assumption that the phosphorylated 30 kDa protein was

Results 108

GlnR was given by using purified Strep-GlnR and total M. smegmatis cell extract smaller

than 10 kDa. This experiment showed that no additional metabolite in M. smegmatis

activated phosphorylation of purified GlnR (figure 41D). Furthermore, when purified MBP-

GlnR was added to total M. smegmatis cell extract in the experiment, only the 30 kDa band

was visible and no band at 71 kDa (figure 41D). The ultimate evidence against GlnR as the

phosphorylated 30 kDa protein was given by the use of total cell extract of the M. smegmatis

glnR deletion strain MH1, previously incubated for one hour under nitrogen starvation. Even

in this approach, the 30 kDa band was detected (figure 41D).

Summarizing these results, it can be said that sensor histidine kinases Msmeg_1918,

Msmeg_5241 and SenX3* were able to phosphorylate themselves in in vitro experiments

using [γ-32P]-labeled ATP, but no phosphorylation of purified GlnR protein was detected.

When total cell extract of M. smegmatis was used, phosphorylation of a 30 kDa protein was

monitored. This protein was not detected when the experiment was carried out with

C. glutamicum cell extract, pointing out that it might be GlnR. This was clearly disproved by

using total cell extract of M. smegmatis ΔglnR, where the phosphorylated 30 kDa protein still

appeared. The identity of the phosphorylated protein remained unclear.

Results 109

Fig. 41: In vitro phosphorylation experiments with [γ-

32P] ATP. Radioactive signals detected on a

phosphor imaging plate (right) and detection of protein bands in SDS PAGE (left) are shown. A. Autophosphorylation of kinases Msmeg_1918 (56 kDa with Strep-tag), Msmeg_5241 (64 kDa with Strep-tag) and SenX3* (29 kDa with Strep-tag). B. When 10 fold surplus of Strep-GlnR (31 kDa) or

A Autophosphorylation of kinases

56 kDa

64 kDa

29 kDa

1918 5241 SenX3*

B Addition of GlnR

C Addition of total cell extract SMR5

D Addition of different cell extracts

56 kDa

31 kDa

64 kDa

31 kDa

71 kDa

31 kDa

29 kDa

1918 + GlnR 5241 + GlnR SenX3* + GlnR

1918 + GlnR + ce 5241 + GlnR + ce SenX3* + GlnR + ce ce >10 <10

56 kDa

31 kDa

64 kDa

31 kDa

71 kDa

31 kDa

29 kDa

GlnR + ce Cgl ce Δ1918Δ5241 GlnR + ce SMR5<10 MBP-GlnR + ce SMR5 ce MH1 ΔglnR

71 kDa

31 kDa

Results 110

MBP-GlnR (71 kDa) was added to each sample (see A), only signals of the phosphorylated kinases were detected. C. Addition of total cell extract (ce) from M. smegmatis SMR5, previously incubated for one hour under nitrogen starvation. When ce was added to a sample including Strep-1918 and Strep-GlnR, two radioactive bands at 56 and approximately 30 kDa were detected. When ce was added to a sample including Strep-5241 and Strep-GlnR, two radioactive bands at 64 and approximately 30 kDa were detected. When ce was added to two samples including Strep-SenX3* and Strep-GlnR (left) or MBP-GlnR (right), respectively, one radioactive band at 29 kDa was detected probably hiding the weaker 30 kDa band. The 30 kDa band was also seen when only ce was used without addition of any purified protein. Cell extract was filtered; the 30 kDa band was also detected in ce bigger that 10 kDa, not in ce smaller than 10 kDa. D. When purified MBP-GlnR (left) or Strep-GlnR (right) were incubated with total cell extract of C. glutamicum (Cgl), no radioactive signal was detected. Usage of only cell extract from msmeg_1918msmeg_5241 double deletion strain resulted again in appearance of the 30 kDa phosphorylated band. This band disappeared in a sample where purified Strep-GlnR and M. smegmatis SMR5 cell extract (ce) smaller than 10 kDa were mixed. Again, the 30 kDa band was detected when total M. smegmatis SMR5 cell extract was added to purified MBP-GlnR. The 30 kDa phosphorylated protein was also seen when only total cell extract from the glnR deletion strain MH1, which has previously been incubated for one hour under nitrogen starvation, was used.

After all these experiments, it was still unclear if GlnR, which as a member of the OmpR-

family of transcriptional regulators (Amon et al., 2008) is supposed to be a response

regulator of a bacterial two-component system, is phosphorylated at all. Therefore, a new in

vitro phosphorylation assay was performed using acetyl [32P] phosphate. In this approach, no

additional kinase is required for phosphorylation. First, acetyl [32P] phosphate was produced

from [32P] phosphoric acid as described in section 3.3.3.9. Phosphorylation assays with GlnR

were carried out according to Hiratsu et al. (1995) and Bouché et al. (1998). For this

purpose, 10 µg purified Strep-GlnR protein were mixed with 50 mM acetyl [32P] phosphate

and incubated at 30°C, while samples were taken after 1, 5, 10, 15, 30 and 60 minutes

(figure 42A). In another approach, purified GlnR was mixed with rising concentrations of

acetyl [32P] phosphate (0, 1, 2, 5, 10, 50 and 100 mM) and incubated for 30 minutes at 30°C

(figure 42B). Samples were analyzed in SDS PAGE and on phosphor imaging plate as

described in section 3.3.3.9. As it can be seen in figure 42, no distinct signal at 31 kDa

(Strep-GlnR) was detected, indicating that GlnR was not phosphorylated in this approach.

Further modifications of this experiment, such as enlargement of reaction time, exchange of

protein against MBP-GlnR or the usage of different reaction buffers according to Hiratsu et

al. (1995), Bouché et al. (1998), Chamnongpol and Groisman (2000) and O’Hare et al.

(2008) did not improve the result (data not shown). So, phosphorylation of putative response

regulator protein GlnR has not yet been proven with this in vitro approach. As a

consequence, a new experiment was performed to analyze putative phosphorylation of

M. smegmatis GlnR in vivo.

Results 111

0 1 5 10 15 30 60 min 0 1 2 5 10 50 100 (50) mM ACP*

A B

Fig. 42: Phosphorylation assay. A. A mixture of 10 µg purified Strep-GlnR and 50 mM acetyl [32

P] phosphate (ACP*) was incubated at 30°C. Samples were taken after 0, 1, 5, 10, 15, 30 and 60 minutes and analyzed in SDS PAGE (above) and on phosphor imaging plate (below). B. 10 µg purified Strep-GlnR were mixed with 0, 1, 2, 5, 10, 50 and 100 mM acetyl [

32P] phosphate (ACP*) and

incubated for 30 minutes at 30°C. A sample without protein (50 mM ACP*) was carried along as control. Samples were analyzed in SDS PAGE (above) and on phosphor imaging plate (below).

For in vivo analyses of putative phosphorylation of M. smegmatis transcriptional regulator

GlnR, the wild type SMR5 and the glnR deletion strain MH1 were grown under nitrogen

surplus until exponential phase. After samples were taken, cells were transferred into

nitrogen-free medium and incubated for another hour, before new samples were taken. Total

cell extract of SMR5+N, SMR5-N, MH1+N and MH1-N was prepared and each analyzed in

SDS and native PAGE. Detection of GlnR was carried out in Western blot analyses using a

GlnR-specific antibody produced as described in section 4.1.6. As binding of PO43- increases

movement rates of proteins in a gel, a difference between phosphorylated and non-

phosphorylated proteins can be detected. This was not monitored in this experiment. Neither

in SDS (figure 43A) nor in native (figure 43B) PAGE an increased movement rate of GlnR in

the cell extract of the wild type SMR5 under nitrogen starvation (-N) was detected compared

to nitrogen surplus (+N). Total cell extract of the glnR deletion strain MH1 was carried along

as a control against putative unspecific binding of the antibody.

Results 112

30 kDa

A B

Fig. 43: Western blot analyses using a GlnR-specific antibody. A. Total cell extracts of the M. smegmatis wild type SMR5 and the glnR deletion strain MH1, incubated under nitrogen surplus (+N) and nitrogen starvation (-N), were analyzed in SDS PAGE. B. Total cell extracts (as described in A) were analyzed in native PAGE.

Summarizing these experiments, it can be said that putative phosphorylation of GlnR has not

yet been proven, neither in in vitro approaches using [32P]-labeled ATP or acetyl phosphate,

nor in in vivo studies with the M. smegmatis wild type SMR5 grown under nitrogen surplus

and starvation. Nevertheless, M. smegmatis GlnR is classified as OmpR-type transcriptional

regulator (Amon et al., 2008) and thus, most likely activated via phosphorylation by a

corresponding sensor histidine kinase.

4.3.2 Investigation of phosphorylation residues

Although phosphorylation of transcriptional regulator GlnR of M. smegmatis has not yet been

shown and a corresponding sensor histidine kinase could not yet be identified in this study

(see section 4.3.1), it is classified as OmpR-type regulator (Amon et al., 2008). These

proteins are response regulators of bacterial two-component systems, characterized by

interaction with corresponding kinases at specific amino acid residues (Itou and Tanaka,

2001; Yoshida et al., 2006). In an alignment of amino acid sequences of GlnR proteins from

different actinobacteria, three aspartic acid residues were identified as putative

phosphorylation sites (see figure 44 and Amon et al., 2008).

Results 113

43 48 52

Fig. 44: Alignment of GlnR homologs from different actinomycetes. MSMEG: M. smegmatis; MAP: Mycobacterium avium subsp. paratuberculosis; Mb: M. bovis; Rv: M. tuberculosis H37rv; SCO: S. coelicolor; SAV: S. avermitilis; nfa: N. farcinica; RHA1: Rhodococcus sp. strain RHA1; FRAAL: F. alni ACN14a; Francci3: Frankia sp. strain Cci3; Tfu: T. fusca; BL: B. longum; AAur: Arthrobacter aurescens; Lxx: Leifsonia xylii; ANA: A. naeslundii; AAN77733: A. mediterranei. Identical amino acids are indicated by black, similar amino acids by grey background. *: identical residues; :: conserved substitutions/residues; .: semi-conserved substitutions. Adapted from Amon et al. (2008). The N-terminal 67 amino acids are shown, including three highly conserved aspartic acid residues at position 43, 48 and 52 (highlighted in blue).

To investigate the role of these aspartic acid (Asp) residues in activation of GlnR, they were

each replaced by alanine (Ala) and by the structural more similar amino acids asparagine

(Asn) and glutamate (Glu). A triple exchange of Asp43, Asp48 and Asp52 to alanine was

also performed. Additionally, an exchange of Ala45 to serine was generated as a control.

Exchange of amino acids was attained by introducing specific point mutations into the glnR

gene. This was done by two-step PCR as described in section 3.3.1.5 and 3.3.1.6. The

obtained glnR* variants with specific base exchanges were cloned into the vector pMN016,

an E. coli/M.smegmatis shuttle vector (see table 2, section 3.1), and thus used for

transformation of the glnR deletion strain MH1. Growth and RNA hybridization experiments

were performed in order to investigate the ability of the constructed glnR* variants to

compensate the genomic glnR deletion. M. smegmatis MH1 transformed with the empty

vector pMN016control was carried along as negative control. MH1 transformed with the

construct pMN016glnR was used as a positive control, as complementation by this plasmid

encoded glnR gene has already been shown before (Amon et al., 2008; Bräu, 2008). First,

growth experiments were carried out. For all control and newly generated strains similar

growth with a generation time of approximately 4.5 hours and a maximum oD600 of

approximately 3.5 was monitored in 7H9 medium, whereas none of the strains grew in 7H9-N

medium (see figure 45). When 200 mM MSX, a glutamine synthetase binding and blocking

substrate (Berlicki, 2008), were added to the 7H9 medium, only the wild type SMR5 and the

MH1 strain transformed with pMN016glnR were able to grow, suggesting the existence of a

Results 114

oD

600

t [h]

oD

600

t [h]

oD

600

t [h]

oD

600

t [h]

oD

600

t [h]

oD

600

t [h]

oD

600

t [h]

oD

600

t [h]

oD

600

t [h]

oD

600

t [h]

oD

600

t [h]

oD

600

t [h]

oD

600

t [h]

oD

600

t [h]

oD

600

t [h]

functional GlnR protein in these strains. As expected, no growth under addition of MSX was

detected for the glnR deletion strain MH1 and the same transformed with empty vector

pMN016control (figure 45). Furthermore, no growth under MSX was detected when one of

the newly generated glnR* variants was used for complementation of the genomic glnR

deletion in MH1 (figure 45). This indicated that an exchange of either aspartic acid 43, 48 or

52 to either alanine, asparagine or glutamate resulted in a loss of functional GlnR protein. A

triple exchange of aspartic acid 43, 48 and 52 to alanine showed the same. Surprisingly, also

an exchange of adjacent alanine 45 to serine resulted in discontinuance of growth under

MSX (figure 45), suggesting that the loss of functional GlnR protein was not due to missing

phosphorylation sites, but rather due to loss of protein structure.

Fig. 45: Growth experiments with M. smegmatis wild type SMR5 and glnR deletion strain MH1, which was transformed with empty plasmid pMN016control, pMN016glnR and pMN016glnR*. glnR* are variants obtained from specific point mutations, leading to exchange of either aspartic acid (Asp) 43, 48 or 52 to either alanine (Ala), asparagine (Asn) or glutamate (Glu), to exchange of aspartic acid (Asp) 43, 48 and 52 to alanine (Ala) or to exchange of alanine (Ala) 45 to serine (Ser). Δ: growth in 7H9 minimal medium. ▲: growth under addition of 200 mM MSX. x: 7H9-N.

Results 115

SMR5

MH1

pMN016control

pMN016glnR

pMN016glnRAsp43Ala

pMN016glnRAsp48Ala

pMN016glnRAsp52Ala

pMN016glnRAsp43Asn

pMN016glnRAsp48Asn

pMN016glnRAsp52Asn

pMN016glnRAsp43Glu

pMN016glnRAsp48Glu

pMN016glnRAsp52Glu

pMN016glnR

Asp43/48/52Ala

pMN016glnRAla45Ser

+N -N0 15 30 60 +N -N0 15 30 60 +N -N0 15 30 60 min

amtB glnA glnR

The same results were obtained when RNA hybridization experiments were performed with

all strains mentioned above. Here, transcript levels of glnR and GlnR target genes amtB and

glnA were investigated under nitrogen surplus and after 0, 15, 30 and 60 minutes of nitrogen

starvation. As it had already been shown before (Amon et al., 2008), the M. smegmatis wild

type SMR5 was able to increase amtB and glnA mRNA levels under nitrogen starvation

(figure 46). In this experiment, this was only detected for MH1 transformed with

pMN016glnR, indicating a functional complementation of the genomic glnR deletion, whereas

any exchange of Asp43, 48 or 52 or even exchange of Ala45 led to a loss of GlnR function

(figure 46). On the contrary, a high amount of mRNA of the corresponding glnR* variants

encoded on vector pMN016 was detected (figure 46).

Fig. 46: RNA hybridization experiments. 1 µg total RNA of M. smegmatis wild type SMR5, glnR deletion strain MH1 and MH1 transformed with empty plasmid pMN016control, pMN016glnR and pMN016glnR* were used for hybridization with specific RNA probes against amtB, glnA and glnR. glnR* are variants with exchange of either aspartic acid (Asp) 43, 48 or 52 to either alanine (Ala), asparagine (Asn) or glutamate (Glu), with exchange of aspartic acids (Asp) 43, 48 and 52 to alanine (Ala) or with exchange of alanine (Ala) 45 to serine (Ser).

Results 116

30 kDa

To answer the question whether the observed loss of GlnR function was due to loss of

phosphorylation sites or rather due to loss of protein structure, Western blot analyses were

performed using the GlnR-specific antibody produced as described in section 4.1.6 in order

to detect GlnR protein in total cell extracts. For that purpose, total cell extract was prepared

from every strain used in growth and RNA hybridization experiments described above,

namely SMR5, MH1 and MH1 transformed with pMN016control, pMN016glnR and

pMN016glnRAsp43Ala, Asp43Asn, Asp43Glu, Asp48Ala, Asp48Asn, Asp48Glu, Asp52Ala,

Asp52Asn, Asp52Glu, Asp43/48/52Ala and Ala45Ser. As it is shown in figure 47, a protein

band at GlnR size of 28 kDa was only detected in total cell extract of the wild type SMR5 and

the glnR deletion strain MH1 transformed with pMN016glnR, which was the only construct

able to complement the genomic deletion (refer to figure 45 and 46). These results indicated

that the monitored loss of GlnR function by exchanging specific amino acids was rather due

to loss of protein structure and stability in M. smegmatis than to a phosphorylation problem.

Fig. 47: Western blot analysis. Total cell extract from M. smegmatis wild type SMR5, glnR deletion strain MH1 and MH1 transformed with pMN016control, pMN016glnR and pMN016glnRAsp43Ala, Asp43Asn, Asp43Glu, Asp48Ala, Asp48Asn, Asp48Glu, Asp52Ala, Asp52Asn, Asp52Glu, Asp43/48/52Ala and Ala45Ser was used for detection of GlnR at 28 kDa with a GlnR-specific antibody.

Although the generated GlnR* variants could not be detected in total cell extracts of

M. smegmatis, monitoring of the corresponding transcripts in E. coli Rosetta2 and purification

of the proteins via Strep-tag was possible. Figure 48A shows an RNA hybridization

experiment with a specific probe against glnR, indicating that high amounts of transcripts of

the glnR* variants were indeed present in E. coli. Furthermore, all GlnR* proteins were

detected in SDS PAGE (figure 48B) and Western blot analysis using the GlnR-specific

antibody (figure 48C). This indicated a successful purification of the single GlnR* variants

and thus the completeness of their structure in E. coli, even though single amino acids were

exchanged.

Results 117

B

31 kDa

C

31 kDa

A

Fig. 48: Analyses of purification of GlnR* variants in E. coli. A. Transcripts of the strep-glnR* variants were monitored by hybridization with a specific glnR probe. B. All purified GlnR* variants were detected in SDS PAGE at a size of 31 kDa. C. Detection of all purified GlnR* variants via Western blot analysis with a GlnR-specific antibody.

Purified GlnR* variants were supposed to be used in further experiments such as

phosphorylation assays. As phosphorylation of the wild type GlnR protein could not definitely

be shown (see section 4.3.1.4), they were only used for gel retardation studies to investigate

a putative effect of the N-terminal amino acid exchanges on DNA binding behavior of GlnR.

As it is shown in figure 49, every tested GlnR* variant was able to bind to a 220 bp

digoxigenin-labeled DNA fragment upstream of amtB in the same manner as wild type GlnR.

This demonstrated that the exchange of single amino acids in the N-terminal region of GlnR

had no influence on DNA binding of the protein, at least when it was purified from E. coli.

Fig. 49: Gel retardation experiment. Different purified GlnR* variants were used, as well as a 220 bp digoxigenin-labeled promoter fragment of amtB. Lane 1: free DNA, lane 2-6: addition of GlnR* variants (1 µg each) with exchange of aspartic acid 52 to alanine, asparagine and glutamate, of alanine 45 to serine and of aspartic acid 48 to asparagine.

Results 118

Summarizing the latest results, it can be stated that phosphorylation of M. smegmatis

transcriptional regulator GlnR at one or all of the predicted aspartic acid residues 43, 48 and

52 has not yet been proven. However, an exchange of one of these amino acids led to a

complete loss of GlnR function, which was shown in growth and RNA hybridization

experiments (see figure 45 and 46). Furthermore, GlnR function was also abolished when an

adjacent amino acid, alanine 45, was exchanged indicating that the loss of GlnR function

might rather be a problem of protein stability in M. smegmatis.

4.4 Nitrate and nitrite metabolism in M. smegmatis

Based on prior results obtained in this study, a closer investigation of nitrate and nitrite

metabolism in M. smegmatis was started. Extensive studies of nitrate and nitrite metabolism

in mycobacteria have just recently begun, mainly focusing on the importance for virulence of

M. bovis or M. tuberculosis, as these pathogens are often forced to survive under anaerobic

conditions after infiltrating host cells (see Weber et al., 2000; Sohaskey and Modesti, 2008).

A variety of different genes putatively involved in nitrate and nitrite transport or reduction

have been identified among the mycobacteria, while researchers disagree about their

assimilatory or dissimilatory function. While Malm et al. (2009) talk about the “mediation of

assimilatory nitrate and nitrite reduction” by NarGHJI and NirBD, others describe these

enzymes as involved in nitrate respiration under anaerobic conditions (Khan and Sarkar,

2006).

In M. smegmatis, a variety of genes and operons encoding enzymes putatively involved in

nitrate and nitrite metabolism exists. These were identified in a bioinformatic approach

(Amon et al., 2009) as assimilatory nitrite reductase (msmeg_0427/0428 nirBD), assimilatory

nitrate reductase (msmeg_5140-5137 narGHJI) and nitrate/nitrite transporters (msmeg_5141

narK and msmeg_0433 narK3). On the contrary, narGHJI has previously been described as

respiratory nitrate reductase in M. smegmatis (Khan and Sarkar, 2006; Khan et al., 2008).

Another, putative assimilatory nitrate reductase is encoded by msmeg_2837 (narB).

Interestingly, the homolog nasA in S. coelicolor is positively regulated by GlnR (Wang and

Zhao, 2009). Furthermore, it was shown in the related pathogen M. tuberculosis that a narG

deletion strain failed to grow on nitrate and a nirB deletion strain failed to grow on both,

nitrate and nitrite as single nitrogen source (Malm et al., 2009). The authors also showed

regulation of nirBD by GlnR. These results correlate with observation made earlier in this

study. Here, it was also seen that growth of M. smegmatis with nitrate or nitrite as single

nitrogen source was only possible in the presence of GlnR (refer to section 4.1.1).

Interestingly, nitrogen response was induced when nitrate or nitrite was used as single

Results 119

nitrogen source (refer to figure 11, section 4.1.2). Moreover, enhanced amounts of mRNA of

nirBD and narK3 were detected in total transcriptome analyses in the wild type under

nitrogen starvation, whereas this was not observed for narK, the narGHJI operon or narB

(section 4.2.1). When total transcript of the wild type SMR5 was compared to that of the glnR

deletion strain MH1, the same pattern was reported. Enhanced transcripts of nirBD and

narK3 were reported in the presence of GlnR, whereas this was not shown for narK, the

narGHJI operon or narB (section 4.2.1). GlnR-dependence was verified via RNA

hybridization experiments and/or real time RT PCR (see section 4.2.2). Subsequently

performed gel retardation experiment did not reveal binding of GlnR to promoter fragments of

nirBD or the nar operon (see figure 28, section 4.2.3).

Considering these results, the question arose if the genes encoding putative nitrate and

nitrite transporters and reductases are controlled by further regulatory proteins which are

indeed encoded by genes located nearby on the genome. The gene msmeg_0426, which is

located directly upstream of nirBD, encodes a GntR-family regulatory protein. A TetR-type

regulator is encoded by msmeg_2106, a gene whose homologs in Mycobacterium gilvum

and Mycobacterium vanbaalenii are located in close genomic neighborhood of nirBD (J.

Amon, personal communication). Furthermore, msmeg_5143 was identified as encoding

HTH-type transcriptional regulator DegA, a putative regulatory protein for the nar operon. To

investigate the role of these putative regulators and nitrate/nitrite reduction systems, insertion

mutagenesis was performed. The genes msmeg_0426 (regulator), msmeg_2106 (regulator),

msmeg_2837 (narB), msmeg_5140 (narG) and msmeg_5143 (regulator) were disrupted by

genomic insertion of the plasmid pML814 (see section 3.4.6 and 4.3.1.2) and insertion was

verified via Southern blot analyses (see figure 50).

Results 120

A

B

D

C

wt i

E

5143

SnaBI PacI

>13000 bp

pML814

SnaBI PacI

4809 bp

PacI

wt

i

wt i

wt i

2106

NruI NruI

3040 bp

pML814

NruI NruI

wt

i

wt i

0426

SphI SphI

9172 bp

pML814

SphI SphI

5402 bp

SspI

wt

i

wt i

6725 bp

2837 narB

SphI SphI

6885 bp

pML814

SphI SphI

wt

i

12814 bp

5140 narG

EcoRV NsiI

4996 bp

pML814

EcoRV NsiI

wt

i

10924 bp

Fig. 50: Southern blot analyses showing genomic situations in M. smegmatis wild type (wt) and insertion strains (i). A. Insertion of plasmid pML814 into msmeg_0426. B. Insertion of plasmid pML814 into msmeg_2106. C. Insertion of plasmid pML814 into msmeg_2837 (narB). D. Insertion of plasmid pML814 into msmeg_5140 (narG). E. Insertion of plasmid pML814 into msmeg_5143.

Results 121

To determine the importance of the tested genes, growth experiments were carried out. As

shown in figure 51A, no differences in growth in 7H9 minimal medium were observed for any

of the generated insertion strains compared to the wild type SMR5 or the glnR deletion strain

MH1, indicating that none of the disrupted genes was generally required for growth. When

10 mM potassium nitrate were used as single nitrogen source, the glnR deletion strain MH1

showed no growth at all and Imsmeg_5140 (narG) an intensely reduced growth. This effect

was abolished for Imsmeg_5140 (narG) when a higher concentration of nitrate (100 mM) was

used. Here, all insertion strains showed growth behavior very similar to the wild type SMR5

(figure 51A). Also when 10 mM sodium nitrite were used as single nitrogen source, none of

the newly generated insertion strains failed to grow (figure 51A). These observations were

elucidated by calculating generation times and showing the maximum oD600 reached by the

tested strains in this experiment. Generation times were lowest (approximately 4-5 hours) in

7H9 medium, independently from any genomic insertion, and comparatively high in media

with nitrate or nitrite as nitrogen source (approximately 8-15 hours, see figure 51B).

Generation times higher than 30 hours (open bars) indicated no growth. This was only the

case for MH1 in nitrate and nitrite and for Imsmeg_5140 (narG) in 10 mM nitrate. The highest

maximum oD600 of approximately 4 was again reached in 7H9 medium, whereas this was

lower in media with nitrate or nitrite (figure 51C). Only the glnR deletion strain MH1 did not

reach an oD600 of 1 in medium with nitrate and nitrite, indicating no growth in these media.

Summarizing these results, it can be said that a disruption of the genes msmeg_0426,

msmeg_2106, msmeg_2837 (narB), msmeg_5140 (narG) and msmeg_5143 did not lead to

differences in growth behavior compared to the wild type, neither in 7H9 minimal medium nor

with nitrate or nitrite as single nitrogen source. Only the disruption of narG seemed to reduce

growth with 10 mM nitrate, but this effect was abolished when higher nitrate concentrations

were used.

As it was assumed that regulatory proteins encoded by msmeg_0426, msmeg_2106 and

msmeg_5143 might control expression of genes involved in nitrate and nitrite reduction, RNA

hybridization experiments were performed to investigate transcription levels in the different

strains under varying nitrogen sources. For that, all insertion strains as well as SMR5 and

MH1 as controls were grown in 7H9 medium until exponential phase and then transferred

into nitrogen-free medium or medium with 10 mM potassium nitrate or sodium nitrite as

single nitrogen source. Samples were taken at different time points, from which total RNA

was prepared. Signals of the genes nirB (encoding nitrite reductase subunit), narG (encoding

nitrate reductase subunit) and amtB (encoding an ammonium transporter) were monitored

(figure 52).

Results 122

A

C

BGeneration times

Maximum oD600

oD

60

0t

[h]

7H9 10 mM nitrate 100 mM nitrate 10 mM nitrite

7H9 10 mM nitrate 100 mM nitrate 10 mM nitrite

Fig. 51: Growth of M. smegmatis wild type SMR5, glnR deletion strain MH1 and strains Imsmeg_0426, Imsmeg_2106, Imsmeg_2837 (narB), Imsmeg_5140 (narG) and Imsmeg_5143. A. Growth curves in 7H9 (Δ) and in 7H9-N medium with 10 mM potassium nitrate (○), 100 mM potassium nitrate (●) and 10 mM sodium nitrite (▲). B. Calculated generation times in hours. C. Maximum oD600.

Results 123

-N Sodium nitrite Potassium nitrate

SMR5

MH1

I0426

I2106

I2837 (narB)

I5140 (narG)

I5143

+N 5 15 30 60 5 15 30 60 5 15 30 60 min

nirB


SMR5

MH1

I0426

I2106

I2837 (narB)

I5140 (narG)

I5143

amtB

+N 5 15 30 60 5 15 30 60 5 15 30 60 min


SMR5

MH1

I0426

I2106

I2837 (narB)

I5140 (narG)

I5143

+N 5 15 30 60 5 15 30 60 5 15 30 60 min

narG

A

B

C

As shown in figure 52A, the transcript level of narG was weak and independent from the

nitrogen source used and also from the disruption of any of the genes encoding a putative

regulatory protein. It did also not depend on GlnR, which was observed before (see section

4.2.1). On the contrary, the transcript level of nirB was strictly depending on GlnR (see figure

52B and section 4.2.1 and 4.2.2). Furthermore, nirB transcripts were increased under

nitrogen starvation (-N) and when nitrate or nitrite were used as single nitrogen source. The

same pattern was monitored for amtB (see figure 52C), again independent from the

disruption of the tested genes.

Fig. 52: RNA hybridization experiments. mRNA levels were tested in the M. smegmatis strains SMR5, MH1, Imsmeg_0426, Imsmeg_2106, Imsmeg_2837 (narB), Imsmeg_5140 (narG) and Imsmeg_5143, incubated under nitrogen surplus (+N), nitrogen starvation (-N) and with 10 mM potassium nitrate or sodium nitrite as single nitrogen source. Samples were taken after 5, 15, 30 and 60 minutes. A. Transcripts of narG. B. Transcripts of nirB. C. Transcripts of amtB.

Results 124

As a summary of the latest results it can be said that the putative regulatory proteins

encoded by msmeg_0426, msmeg_2106 and msmeg_5143 were not involved in growth of

M. smegmatis on nitrate or nitrite. Transcript levels of narG or nirB, encoding nitrate/nitrite

reduction systems, as well as amtB did not change when one of these genes was disrupted.

A strain with disruption of msmeg_2837 (narB), putatively encoding a further nitrate

reductase, showed also no differences in growth or gene transcripts compared to the wild

type. Only when msmeg_5140 (narG) was disrupted, the resulting strain showed reduced

growth in medium with 10 mM nitrate as single nitrogen source. But this effect disappeared

when higher amounts of nitrate were used. mRNA levels of nirB or amtB were also not

changed in this strain compared to the wild type. All results obtained in this study so far

indicated that uptake, as well as assimilation of nitrate and nitrite in M. smegmatis is

mediated by nitrate/nitrite transporters NarK and NarK3, nitrate reductase NarGHJI and

nitrite reductase NirBD, whereas NarK3 and NirBD are controlled by GlnR.

4.5 Investigation of a conserved mode of action of GlnR in mycobacteria

Up to this point, this study revealed the global role of OmpR-type regulator GlnR for nitrogen

metabolism in M. smegmatis. Whereas the wild type SMR5 was able to use a variety of

different substrates as single nitrogen source, this ability was reduced in the absence of

GlnR (see section 4.1.1). Additionally, it was shown that GlnR has a great influence on

transcript levels of more than 30 target genes under nitrogen starvation (see section 4.2.1

and 4.2.2). Binding of GlnR to promoter sequences of about half of these genes was also

demonstrated (see section 4.2.3). These data indicated the great importance of GlnR for

survival of M. smegmatis under nitrogen limitation. A similar result was obtained in studies of

the related species S. coelicolor (Tiffert et al., 2008). Furthermore, the authors suggested a

highly conserved mode of action of GlnR among the actinomycetes, as binding of

S. coelicolor GlnR to glnA promoter fragments of different actinomycetes was reported.

Moreover, a genomic glnR deletion in S. coelicolor could be complemented by

A. mediterranei glnR (Yu et al., 2006; Tiffert et al., 2008).

The glnR genes of M. smegmatis and M. tuberculosis and their corresponding proteins show

very high sequence homology. Not only the putative binding motif upstream of target genes

such as amtB or glnA is almost identical (refer to figure 29, section 4.2.4), also the putative

phosphorylation site at the N-terminal end and the DNA binding motif at the C-terminal end of

the proteins are very similar (figure 53). This led to the question if a genomic glnR deletion in

M. smegmatis could also be complemented by M. tuberculosis glnR and if purified

M. tuberculosis GlnR was able to bind M. smegmatis target genes.

Results 125

10 20 30 40 50 60....|....|....|....|....|....|....|....|....|....|....|....|

MSMEG_5784 -MDLLLLTVDPHPESVLPSLSLLAHTVRTAPTEVSSLLETGSADVAIVDARTDLAAARGL

Rv0818 MLELLLLTSELYPDPVLPALSLLPHTVRTAPAEASSLLEAGNADAVLVDARNDLSSGRGL

Clustal Consensus ::***** : :*:.***:****.*******:*.*****:*.**..:****.**::.***

70 80 90 100 110 120....|....|....|....|....|....|....|....|....|....|....|....|

MSMEG_5784 CRLLGTTGTSVPVVAVINEGGLVAVNHEWGLDEILLPSTGPAEIDARLRLLVGRRGGNAN

Rv0818 CRLLSSTGRSIPVLAVVSEGGLVAVSADWGLDEILLLSTGPAEIDARLRLVVGRRGDLAD

Clustal Consensus ****.:** *:**:**:.*******. :******** *************:*****. *:

130 140 150 160 170 180....|....|....|....|....|....|....|....|....|....|....|....|

MSMEG_5784 QENVGKITLGELVIDEGTYTARLRGKPLDLTYKEFELLKYLAQHAGRVFTRAQLLQEVWG

Rv0818 QESLGKVSLGELVIDEGTYTARLRGRPLDLTYKEFELLKYLAQHAGRVFTRAQLLHEVWG

Clustal Consensus **.:**::*****************:*****************************:****

190 200 210 220 230 240....|....|....|....|....|....|....|....|....|....|....|....|

MSMEG_5784 YDFFGGTRTVDVHVRRLRAKLGPEYEALIGTVRNVGYKAVRPSRGKPPAADASGEDVPDG

Rv0818 YDFFGGTRTVDVHVRRLRAKLGPEHEALIGTVRNVGYKAVRPARGRPPAADPDDEDADPG

Clustal Consensus ************************:*****************:**:*****...**. *

250 260....|....|....|....|..

MSMEG_5784 PDEGFGDDVDVDGPLAGRLTSQ

Rv0818 RD-------GMQEPLVDPLRSQ

Clustal Consensus * .:: **.. * **

Fig. 53: Sequence alignment of M. smegmatis (msmeg_5784) and M. tuberculosis (Rv0818) GlnR. The conserved putative phosphorylation site is highlighted blue, the conserved DNA binding motif dark blue. *: identical residues. :: similar amino acids. .: related amino acids.

First, growth and RNA hybridization experiments were performed to investigate the ability of

M. tuberculosis glnR to complement a genomic glnR deletion in M. smegmatis. The construct

pMN016glnR M. tub. was used for transformation of the glnR deletion strain MH1. Growth of

the resulting strain was tested in 7H9, 7H9-N and 7H9 medium with 200 mM MSX, a

glutamate analog that blocks GS activity (Berlicki, 2008). MH1 strains transformed with

pMN016control and pMN016glnR were carried along as controls. As it has already been

shown before (see figure 45, section 4.3.2), the glnR deletion strain transformed with the

empty vector was not able to grow under addition of MSX, whereas the strain carrying

pMN016glnR was, indicating a successful homologous complementation. However, figure

54A shows that a heterologous complementation of the genomic glnR deletion in

M. smegmatis with M. tuberculosis glnR was not successful, as this strain failed to grow

under the addition of MSX. Nevertheless, a slight increase of oD600 was detected after

approximately 28 hours, indicating that MSX might be inactivated faster as in the

pMN016control sample. On the basis of that, RNA hybridization experiments were

performed. First, a glnR probe was used to ensure that M. tuberculosis glnR transcripts were

obtained from plasmid pMN016. Whereas a strong signal for glnR was detected, transcripts

of amtB or glnA were not monitored leading to the conclusion that M. tuberculosis GlnR was

not able to activate nitrogen response in M. smegmatis (see figure 54B).

Results 126

B

t [h]

oD

600

t [h]

oD

60

0

t [h]t [h]

oD

600

oD

600

A

Fig. 54: Investigation of heterologous complementation of M. smegmatis glnR deletion with M. tuberculosis glnR in vector pMN016. A. Growth experiments with M. smegmatis MH1 transformed with empty vector pMN016control, pMN016glnR and pMN016glnR M. tub. in 7H9 (Δ), 7H9-N (x) and 7H9 medium with 200 mM MSX (▲). B. Total RNA of the three strains was used for RNA hybridization experiments with probes against glnR, amtB and glnA. Samples were taken under nitrogen surplus (+N) and directly (-N0), 15, 30 and 60 minutes after induction of nitrogen starvation.

These first experiments already indicated that a genomic glnR deletion in M. smegmatis

could not be complemented with M. tuberculosis glnR. In another approach, M. tuberculosis

GlnR was purified via affinity chromatography with an MBP-tag (refer to section 3.3.3.2 and

4.1.5) by cloning the corresponding gene into the vector pMal-c2. Purification of the 70.5 kDa

MBP-GlnR M. tub. fusion protein is documented in figure 55A. Figure 55B shows a gel

retardation experiment, which was performed using the 220 bp promoter fragment of amtB.

Rising amounts of MBP-GlnR and MBP-GlnR M. tub. were added, respectively.

Results 127

70 kDa

Purification protocol SDS PAGE

- MBP MBP-GlnR - MBP MBP-GlnR M. tub.

A

B

Fig. 55: Studies on M. tuberculosis GlnR. A. Purification of the 70.5 kDa MBP-GlnR M. tub. fusion protein, verified in SDS PAGE. B. Gel retardation experiments with digoxigenin-labeled 220 bp promoter fragment of amtB. Free DNA was used as negative control (-). 800 ng pure maltose binding protein were added as control (MBP). Rising amounts of MBP-GlnR from M. smegmatis (left) and MBP-GlnR M. tub. (right) were added. These were 50, 100, 200, 400 and 800 ng and 1, 1.5, 2, 3, 5, 10, 20 and 30 (only M. tub.) µg. A shift of DNA was visible with 400 ng M. smegmatis MBP-GlnR and 10 µg M. tuberculosis MBP-GlnR.

Whereas addition of 400 ng MBP-GlnR from M. smegmatis led to a shift of DNA, which was

even increased with higher amounts, binding of MBP-GlnR from M. tuberculosis was only

monitored at amounts higher than 10 µg, which was about 25 fold. This led to the conclusion

that M. tuberculosis GlnR was indeed able to bind M. smegmatis DNA, but only in high

excess of protein, which is contradictory to the almost identical DNA binding motif of the two

GlnR proteins (refer to figure 53).

Discussion 128

5 Discussion

5.1 GlnR as the global regulator of nitrogen metabolism in M. smegmatis

5.1.1 GlnR-dependent utilization of different nitrogen sources

The main objective of this study was to discover the mechanisms of nitrogen metabolism and

especially response to nitrogen starvation in M. smegmatis. To converge to this topic from a

general point, the ability of the bacterium to utilize different substrates as single nitrogen

source was tested. First hints of that were given by a bioinformatic analysis of various uptake

and assimilation systems (Amon et al., 2009). Indeed, Iwainsky and Sehrt (1967) monitored

growth of M. smegmatis with the amides urea, acetamide, nicotinamide and isonicotinamide

as well as the amino acids asparagine, glutamate and glutamine and the inorganic

substrates nitrate and nitrite. Addition of asparagine to other nitrogen sources even

increased growth. While these authors were the only ones to focus on growth behavior of

M. smegmatis, different other nitrogen sources were investigated incidentally in studies

focusing on mycobacteriophages (Gadagkar and Gopinathan, 1980) or on the identification

of mycobacterial drug targets to inhibit cell wall biosynthesis (Milligan et al., 2007).

In this study, it was shown that the M. smegmatis wild type strain SMR5 was able to utilize

the amino acids alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,

glutamine, histidine, proline, serine, threonine and valine as well as the bases guanine and

uracil and the inorganic forms ammonium, ethanolamine, nitrate, nitrite and uric acid (see

section 4.1.1). Poor growth was detected in media with isoleucine, leucine, lysine,

methionine, tryptophan, tyrosine or methylamine. Cells did not grow when glycine,

phenylalanine, adenine, thymine, creatinine, glucosamine, N-acetylglucosamine or urea were

used as nitrogen sources. The glnR deletion strain MH1 showed no or extremely reduced

growth compared to the wild type in media with alanine, histidine, proline, serine, guanine,

uracil, ethanolamine, potassium nitrate, sodium nitrite and uric acid. Reduced growth was

also detected with aspartic acid, isoleucine, leucine, lysine and methionine, even though the

wild type showed only poor growth in these media (refer to section 4.1.1). Consequently,

M. smegmatis was no longer able to use these 15 substrates as single nitrogen source in the

absence of GlnR. Thus, the regulator must be involved in the expression of genes encoding

enzymes for uptake and/or assimilation of these substrates. Surprisingly, there was no

correlation between growth ability and initiation of nitrogen response (refer to figure 11,

section 4.1.2). Transcript of amtB was increased in media with phenylalanine, adenine,

thymine, N-acetylglucosamine, tyrosine, methylamine, arginine, glutamic acid, histidine,

Discussion 129

proline, threonine, ethanolamine, potassium nitrate and sodium nitrite, but not in media with

the remaining 21 substrates, independently from growth of M. smegmatis within these media.

Thus, the metabolites indicating the cells’ nitrogen status were not distinctly identified.

A closer look at the corresponding assimilation pathways suggests how the tested substrates

might be degraded in M. smegmatis and how GlnR might be involved in these processes. In

table 6 all growth analyses are summarized. Furthermore, the enzymes of the different

assimilation pathways are shown according to Kanehisa et al. (2010). The msmeg_ numbers

indicate genes encoding the corresponding enzymes in M. smegmatis if present, whereas

most of them are not experimentally verified.

Tab. 6: Summary of growth analyses. Displayed are the tested nitrogen sources, growth ability of the M. smegmatis wild type SMR5 and the glnR deletion strain MH1, induction of nitrogen response, the assimilating enzymes and the corresponding genes in M. smegmatis, as far as available, according to Kanehisa et al. (2010). Verified GlnR target genes (refer to section 4.2 and 5.1.2) are highlighted blue. Purple: enhanced transcript level under nitrogen starvation though no GlnR target (refer to table 10, section 7.2).

Nitrogen source

Growth SMR5

Growth MH1

Nitrogen response

Assimilation pathway Genes involved

Alanine

√

no

no

aminotransferase alanine dehydrogenase

msmeg_0688 (alaT) msmeg_2659 ald

Arginine

√

√

√

various NAD

+-GDH

msmeg_4699, msmeg_ 6272

Asparagine

√

√

no

asparagine synthase aspartate oxidase

msmeg_2594 (asnB),

msmeg_4269 msmeg_3200 (nadB)

Aspartic acid

√

poor

no

aspartate-ammonia lyase aspartate oxidase

msmeg_1677 (aspA) msmeg_3200 (nadB)

Cysteine

√

√

no

cystathionine-gamma-lyase

msmeg_2394

Glutamic acid

√

√

√

glutamate dehydrogenase NAD

+-GDH

msmeg_4699, msmeg_6272

Glutamine

√

√

no

glutaminase glutamate synthase NAD

+-GDH

msmeg_3818 msmeg_3225/3226 (gltBD),

msmeg_5594, msmeg_6458 msmeg_4699, msmeg_6272

Glycine

no

no

no

glycine/serine hydroxymethyltransferase serine dehydratase serine-ammonia lyase

msmeg_5249 (glyA)

msmeg_3183, msmeg_3532 msmeg_3440 (sdaA)

Histidine

√

no

√

histidine-ammonia lyase

msmeg_1183 (hutH)

Isoleucine

poor

no

no

aminotransferase

msmeg_4276 (ilvE)

Leucine

poor

no

no

aminotransferase

msmeg_4276 (ilvE)

Lysine

poor

no

no

aminotransferase

msmeg_4276 (ilvE)?

Methionine

poor

no

no

S-adenosylmethionine synthetase

msmeg_3055 (metK)?

Phenylalanine

no

no

√

various

not detected

Proline

√

no

√

various NAD

+-GDH


Serine

√

no

no

serine dehydratase serine-ammonia lyase

msmeg_3183, msmeg_3532 msmeg_3440 (sdaA)

Discussion 130

Threonine

√

√

√

threonine aldolase threonine dehydratase

msmeg_4454 (ltaE) msmeg_3183 (ilvA), msmeg_3532

Tryptophan

poor

poor

no

various

not detected

Tyrosine

poor

poor

√

various

not detected

Valine

√

√

no

aminotransferase

msmeg_4276 (ilvE)

Adenine

no

no

√

various

msmeg_1701, msmeg_2964, no nitrogen assimilation!

Guanine

√

no

no

guanine deaminase

msmeg_1298

Thymine

no

no

√

thymine phosphorylase msmeg_1675 (deoA),

no nitrogen assimilation!

Uracil

√

no

no

uracil phosphoribosyl transferase

msmeg_1694, msmeg_3042, msmeg_3473, no nitrogen assimilation?

Ammonium chloride

√

√

no

NADP+-GDH

glutamate synthase glutamine synthetase

msmeg_5442 msmeg_3225/3226 (gltBD), msmeg_5594, msmeg_6458 msmeg_4290 (glnA1)

Ammonium sulfate

√

√

no

NADP+-GDH



Creatinine

no

no

no

creatinine amidohydrolase

msmeg_1425, msmeg_6870?

Ethanolamine

√

no

√

ethanolamine-ammonium lyase

msmeg_1553/1554

Ferric ammonium citrate

√

√

no

NADP+-GDH



Glucosamine

no

no

no

various

not detected

Methylamine

poor

poor

√

various

not detected

N-acetyl glucosamine

no

no

√

PTS-system

msmeg_2116/2117?

Potassium nitrate

√

no

√

nitrate reductase nitrite reductase

msmeg_5140-5137 (narGHJI) msmeg_2837 (narB)?

msmeg_0427/0428 nirBD) Sodium nitrite

√

no

√

nitrite reductase ferredoxin-nitrite reductase

msmeg_0427/0428 nirBD) msmeg_4527

Urea

no

no

no

urease

msmeg_1093/1094, msmeg_3625/3626/3627

Uric acid

√

no

no

urate oxidase hydroxyisourate hydrolase allantoinase allantoicase urease

msmeg_1296 msmeg_1295 not detected msmeg_5727 (alc) msmeg_1093/1094, msmeg_3625/3626/3627

Formamide

not tested

not tested

not tested

formamidase

msmeg_0484, 3403 (amiF),


Discussion 131

Special attention was paid to urea and uric acid as single nitrogen sources. Uric acid or

urate, as a product of purine degradation, is assimilated by urate oxidase (msmeg_1296) and

5-hydroxyisourate hydrolase (msmeg_1295) to allantoine and in a following step by

allantoinase (not identified in M. smegmatis so far) to allantoate, which is further degraded by

allantoicase (msmeg_5727 alc) to urea. Urea is again degraded by urease (msmeg_1093,

msmeg_1094, msmeg_3625, msmeg_3626, msmeg_3627) to NH4+ and CO2. Surprisingly,

growth of M. smegmatis was detected in this study with uric acid, suggesting that a complete

degradation pathway must exist. Another surprising observation was that no growth was

obtained with urea as single nitrogen source, although a variety of degrading enzymes

(ureases) is present in M. smegmatis. Therefore, urease activity was determined (see

section 4.1.3). The tested M. smegmatis wild type strains SMR5 and DSM 43756 showed

similar urease activities of 1 µmol produced ammonium per minute per mg protein, which can

be compared to C. glutamicum, for which a urease activity of 0.9 µmol produced ammonium

per minute per mg protein was detected in complex medium (Nolden et al., 2000). Whereas

urease activity in C. glutamicum was enhanced to 7.8 µmol produced ammonium per minute

per mg protein under nitrogen starvation (Nolden et al., 2000), it was not increased in

M. smegmatis under the same conditions. This measurement and the fact that uric acid could

be used as nitrogen source indicated that a functional urea degrading system exists in

M. smegmatis. Thus, it remains unclear why the cells were not able to grow with urea as

single nitrogen source. A transport problem can also be excluded, as urea, especially as it

was used in a concentration of 100 mM in this study, passes the cell membrane by diffusion.

A possible explanation is that an intracellular shift of pH might have prevented the cells from

utilizing urea.

5.1.2 Characterization of the GlnR regulon

Total transcriptome analyses were carried out in search for different GlnR target genes.

Comparing mRNA levels of the M. smegmatis wild type SMR5 under nitrogen surplus and

starvation, 231 genes with decreased mRNA level under -N were found (see figure 20,

section 4.2.1 and table 9, section 7.2). This indicates that components which were not

necessary for survival under nitrogen limitation were reduced. Based on transcript levels it

was indicated that transport and metabolism of several lipids, carbohydrates, inorganic ions,

secondary metabolites, amino acids, coenzymes and nucleotides were decreased.

Furthermore, mRNA levels of genes associated with energy metabolism (acetyl-CoA

metabolism, as well as some ATPases) were extremely reduced. Decreased transcript levels

of many genes encoding ribosomal proteins and chaperones as well as sugar transport

Discussion 132

systems pointed to a reduction of growth to a minimum in order to survive nitrogen

starvation. Surprisingly, transcripts of genes encoding glutamate synthase (msmeg_6458),

ferredoxin-dependent glutamate synthase I (msmeg_6459) and a putative glutamine

transport system (msmeg_6307) were also reduced.

In contrast, 284 genes showed increased transcript levels under nitrogen starvation (see

figure 21, section 4.2.1 and table 10, section 7.2). Many of these genes were associated with

amino acid or inorganic ion transport and metabolism. These data indicated that uptake and

assimilation of alternative nitrogen sources was very important. Genes encoding amino acid

and ammonium transporters or permeases showed intensely high mRNA levels, as well as

genes encoding nitrogen assimilating enzymes such as amidases, dehydrogenases,

hydrolases, lyases and oxidoreductases. Furthermore, transcripts of various glutamine

synthetase, glutamate synthase and signal transduction genes were enhanced. As the

corresponding enzymes work under high energy demand, transcripts of some genes

encoding ATPases were again monitored. mRNA levels of genes encoding nitrate, nitrite and

urea uptake and assimilation systems were also intensely increased. Interestingly, a

comparison of table 10 (increased transcript level in the wild type under nitrogen starvation)

and table 12 (putative GlnR target genes, see section 7.2) revealed that some genes

involved in nitrogen metabolism showed apparently increased transcript levels under

nitrogen starvation in a GlnR-independent manner. These genes were e.g. msmeg_3561

(glutamine synthetase) and msmeg_5374 (glutamate-ammonia ligase).

Similar results of genes with enhanced mRNA levels under nitrogen (ammonium) limitation

were obtained for C. glutamicum in an earlier study performed by Silberbach et al. (2005a,

b). In that study, transcriptome analyses in response to ammonium starvation were carried

out. As in M. smegmatis, transcript levels of genes encoding enzymes involved in cell

division and growth as well as enzymes involved in biosynthesis of some amino acids were

decreased as a result of general starvation response. Genes with enhanced transcript levels

under nitrogen starvation encoded different transporters such as AmtA and AmtB, creatinine

permease, a urea uptake system and some amino acid transporters. Transcripts of genes for

metabolism of these alternative nitrogen sources were also increased. These were mainly

gltBD encoding GOGAT, glnA encoding GSI, creatinine deaminase and a urease operon. In

contrast to C. glutamicum, an enhanced mRNA concentration of glutamate synthase-

encoding genes was not observed in M. smegmatis. Moreover, genes for uptake and

complete assimilation of creatinine are not described in M. smegmatis. High amounts of

transcript of genes encoding signal transduction proteins GlnK and GlnD were found in both

organisms under nitrogen starvation. While the role of these proteins is well investigated in

C. glutamicum (see section 2.2.3), little is known about their function in M. smegmatis so far

(refer to section 5.3). Even some genes involved in energy production showed enhanced

Discussion 133

transcript levels due to the high energy demand of nitrogen-assimilating enzymes such as

GS. In C. glutamicum, genes encoding chaperones such as GroEL and GroES were also

found under nitrogen limitation. Due to this fact, Silberbach et al. (2005b) suggested a “trend

towards protein stabilization” under ammonium limitation. Interestingly, mRNA levels of

genes encoding these chaperones in M. smegmatis were reduced under nitrogen starvation

compared to nitrogen surplus (refer to table 9, section 7.2, this study).

Apart from that, most interesting for this study was the identification of GlnR target genes,

which were determined in a comparative transcriptome analysis of the M. smegmatis wild

type SMR5 and the glnR deletion strain MH1 under nitrogen starvation. Only two described

genes showed enhanced transcript levels in the absence of GlnR (see figure 22, section

4.2.1). These were msmeg_2274 (hypC encoding hydrogenase assembly chaperone HypC-

HupF) and msmeg_2275 (hypD encoding hydrogenase expression-formation protein HypD),

which are probably not involved in nitrogen metabolism.

In contrast to that, the amount of mRNA of 125 genes was decreased in the absence of GlnR

compared to the wild type (see figure 23, section 4.2.1). As more than 33 % of these genes

are putatively involved in nitrogen metabolism, the conclusion was drawn that these might be

target genes of GlnR. This demonstrated that M. smegmatis GlnR mainly works as

transcriptional activator, whereas different other transcriptional regulators work in a

bifunctional manner. NtrC, the global regulator of nitrogen metabolism in E. coli, regulates

transcription of glnA and ntrBC by activating or repressing gene expression from different

promoters (refer to section 2.2.2). On the contrary, AmtR in C. glutamicum functions

exclusively as repressor of its target genes under nitrogen surplus (see section 2.2.3).

Interestingly, a bifunctional manner is described for S. coelicolor GlnR. While it activates e.g.

glnA and nirB transcription under nitrogen starvation, gdh and ureA transcription are

repressed at the same time (Tiffert et al., 2008). For M. smegmatis GlnR a bifunctional mode

of action was not observed in experiments carried out in this study.

By performing further experiments such as RNA hybridization and real time RT PCR, 32

genes were indeed confirmed as GlnR-controlled (see figure 24 and 27, section 4.2.2).

Differences in transcript levels between nitrogen surplus and starvation varied significantly in

the individual experiments, which can be explained by considering the different experimental

procedures. In the microarray experiments cDNA was labeled with two different fluorescence

markers and bound to 60 bp oligonucleotides on a microarray chip, each representing one

gene or intergenic region in the M. smegmatis genome. The transcript of only one single

gene was monitored in one real time RT PCR sample, depending on a multitude of variables

such as primer design, reaction temperature, RNA purity, pipetting etc. Additionally, the

sample was normalized against a reference gene with comparable transcript levels under

changing nitrogen conditions. In this study, msmeg_3084 was chosen, encoding G3PDH.

Discussion 134

Further genes described as suitable references such as msmeg_4936 (atpD), msmeg_5489

(ribosomal protein L32), msmeg_5695 (glutathione-S-transferase) and msmeg_4931

encoding 16S rRNA (Mannhalter et al., 2000; Andersen et al., 2004; Cronin et al., 2004) did

not fulfill the criteria necessary for this study.

RNA hybridization experiments did not show quantitative results, but transcripts of the

M. smegmatis wild type and the glnR deletion strain under nitrogen surplus or starvation as a

function of time instead (see figure 24, section 4.2.2). As described in section 4.2.2,

maximum signals of different target genes were reached at different time points, from 15 to

60 minutes of nitrogen starvation. Due to different signal strengths and time it can be

assumed that not all GlnR target genes are transcribed in the same manner, which might

result from different promoter structures of the target genes leading to different binding

properties of the transcriptional activator GlnR (see also section 5.2). Binding of GlnR to 13

promoter fragments was verified in gel retardation experiments, whereas no binding was

detected for at least the same amount of genes, although GlnR-control was shown in

transcriptome analyses (see figure 28, section 4.2.3). This indicated that either the

corresponding promoter sequences were not correctly identified due to the unexplored state

of the genes or that these must be regulated by a yet unknown GlnR-dependent secondary

effect. Indirect regulation is described for vanABK encoding vanillate demethylase and

protokatechuate transporter by AmtR, the global regulator of nitrogen metabolism in

C. glutamicum (Merkens et al., 2005). Besides that, subordinated transcriptional regulators

might also be involved mediating gene transcription in M. smegmatis in association with

GlnR. This is the case e.g. for msmeg_2184-2187, for which additional regulation by AmtR

has been identified (see section 5.6 for a detailed description).

Organization of all GlnR target genes identified in this study, as well as the change of their

mRNA level due to presence or absence of GlnR, and binding of GlnR to the corresponding

promoter regions is summarized in figure 56.

Discussion 135

52x 49x ( 7x) ( 34x) 55x

A

0426 0427 0428 0429 0430 0431 0432 0433

nirB nirD narK3

no binding

B

( 35x) ( 33x) 48x ( 18x)

0569 0570 0571 0572

binding

C

( 10x) 35x ( 9x) ( 36x

0778 0779 0780 0781

binding

D

12x

1052 1054 1053

no binding

F

( 4x) 15x ( 7x) ( 9x)

1292 1293 1294 1295 1296

no binding

56x 52x 18x

H

2425 2426 2427

binding

amtB glnK glnD

24x ( 5x) ( 24x) ( 14x) 66x

I

2521 2522 2523 2524 2525 2526

binding

tynA

( 16x) ( 19x) ( 23x) 19x 136x

K

2978 2979 2980 2981 2982

binding

urtE urtD urtC urtB urtA

no binding

3400 3401 3402 3403

18x ( 19x) ( 8x) ( 9x)

L binding

34x 26x

M

4290 4291 4292 4293 4294

binding

glnA dtd glnE glnA2

binding

5x ( 3x)

N

binding not tested

4380 4381 4382

23x ( 32x) ( 18x) 30x

O

4635 4636 4637 4638

binding

amtA

no binding

( 6x) 45x

Q

5729 5730 5731 5732 5733 5734

no binding

12x 16x 10x ( 7x) ( 7x) ( 5x)

S

6259 6260 6261 6262 6263 6264

binding

amt1 glnT

T

11x

6660

binding

( 6x) 63x ( 19x)

U

6733 6734 6735

bindingno binding

6816 6817

33x ( 11x)

V no binding

( 48x) ( 54x) ( 6x) ( 10x) ( 19x) ( 4x) 5x

E

1082 1083 1084 1085 1086 1087 1088 1089 1090

no/unspecific binding no binding

gatA

48x ( 13x) ( 32x) ( 3x) ( 4x)

G

2184 2185 2186 2187 2188 2189

binding

atzF

J

6x

2748

unspecific/no binding

sthA

amiF

25x ( 21x) ( 22x)

P

5358 5359 5360

binding not testedcynS

R

30x

5765

no binding

glbN

Fig. 56: All GlnR target genes identified in this study. Genes are listed according to their msmeg_ numbers. Location as single genes or in operons in the genome is shown according to www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=246196. Arrow number x indicate fold change of transcript levels in the presence of GlnR according to microarray data, while ( ) represents data not verified in further experiments. Binding of GlnR protein to promoter sequences is shown in dark red, while light red ovals show predicted binding sites where no binding could be detected.

Discussion 136

Many genes involved in uptake and assimilation of various nitrogen sources were activated

by GlnR under nitrogen starvation. The most interesting GlnR targets, according to figure 56,

are described below.

GlnR controls nitrite reduction and transport systems (figure 56A), a flavoprotein involved in

K+ transport and a carbon-nitrogen family hydrolase (figure 56B), a putative transcriptional

regulator, short-chain dehydrogenase-reductase SDR, a phosphotransferase and an amino

acid permease (figure 56C). Additionally, an amino acid carrier protein depends on GlnR

(figure 56D). This is also the case for another putative response regulator, a peptide-opine-

nickel uptake family ABC transporter, dipeptide transport system permease DppB, an ABC

transporter permease, an oligopeptide ABC transporter, a glutamyl-tRNA(Gln)-aspartyl-

tRNA(Asn) amidotransferase and an amidase (figure 56E). Moreover, a FAD binding domain

in molybdopterin dehydrogenase was identified, as well as a xanthine-uracil permease,

transthyretin and a uricase (figure 56F). Very interesting targets were an amino acid

permease, two conserved hypothetical proteins and a urea amidolyase (figure 56G), which

were further controlled by transcriptional regulator AmtR (see section 5.6). Also an

allophanate hydrolase was spotted (figure 56G).

The best investigated GlnR target is the operon msmeg_2425-2427 (amtB-glnK-glnD)

encoding ammonium transporter, PII regulatory protein and uridylyltransferase (figure 56H;

see also Amon et al., 2008).

Further GlnR-controlled were different ABC transporters and permeases, another amino acid

permease and copper methylamine oxidase TynA (figure 56I). This was also monitored for

soluble pyridine nucleotide transhydrogenase SthA (figure 56J). Different further ABC

transporters as well as a periplasmic binding protein involved in urea uptake are part of the

GlnR regulon (figure 56K). This is also the case for glutamyl-tRNA(Gln) amidotransferase

subunit A, a LamB-YcsF-family protein, a cytosine permease and formamidase AmiF (figure

56L).

Additionally, two glutamine synthetases type I were identified (figure 56M; see also Amon et

al., 2008). Another amidase and a dehydrogenase-reductase SDR family protein were

spotted (figure 56N), as well as ammonium transporter AmtA and a vanillate O-demethylase

oxidoreductase (figure 56O). Moreover, a formate-nitrate transporter, cyanate hydratase

CynS and an acetamidase-formamidase family protein were found (figure 56P). GlnR also

controls a permease for cytosine-purines, uracil, thiamine and allantoine as well as a

hydantion racemase (figure 56Q). This was also observed for the globin GlbN (figure 56R)

and ammonium transporter Amt1, a glutamine synthetase type III, a glutamine

amidotransferase class II, a FwdC-FmdC-family protein, a glutamate synthase family protein

and a putative oxidoreductase (figure 56S). Moreover, another permease for cytosine-

Discussion 137

purines, uracil, thiamine, and allantoine was found (figure 56T). Additionally, another amino

acid permease, dibenzothiophene desulfurization enzyme A and a carbon-nitrogen family

hydrolase were identified (figure 56U), as well as a molybdopterin oxidoreductase and RNA

polymerase sigma factor, sigma-70 family (figure 56V).

During this study, it became obvious that GlnR is the global regulator of nitrogen metabolism

in M. smegmatis and that many genes involved in transport of amino acids, peptides and

inorganic nitrogen sources as well as nitrogen assimilating enzymes such as amidases,

amidotransferases and glutamine synthetases are GlnR-controlled.

Surprisingly, also genes not directly associated with nitrogen assimilation were identified as

GlnR targets. One example is msmeg_5765 (glbN) encoding a truncated globin (refer to

figure 56R). This globin has become very interesting for recent research, as it is able to bind

oxygen and is thus involved in “defense against NO toxicity and nitrosative stress” in

mycobacteria (Lama et al., 2006). Involvement of GlnR in these processes has not been

reported so far. Another interesting putative target gene, although not yet further

experimentally verified, is msmeg_6817 encoding a sigma factor, sigma-70 family protein

(figure 56V). Sigma 70 is described as standard sigma factor responsible for transcription of

housekeeping genes. A variety of different genes encoding sigma factors exists in

M. smegmatis, including sigA, sigB, sigD, sigE, sigF, sigG, sigH, sigJ and others (Manganelli

et al., 2004). While extensive research on sigma factors in mycobacteria has been practiced

(Gomez et al., 1998; Manganelli et al., 2004; Mukherjee and Chatterji, 2005), they were not

associated with GlnR so far. Considering these data, it can be assumed that the regulatory

function of GlnR might not be restricted to nitrogen metabolism. This has also been

suggested in a similar study on S. coelicolor GlnR (Tiffert et al., 2011), where also genes

involved in carbon metabolism, stress response and antibiotic biosynthesis were found to be

GlnR-controlled confirming the regulatory function of GlnR beyond nitrogen metabolism.

Discussion 138

5.2 GlnR as OmpR-type transcriptional regulator

According to sequence homologies, M. smegmatis GlnR is associated with the OmpR-family

of transcriptional regulators (Amon et al., 2008). The OmpR/EnvZ-system in E. coli is

thoroughly investigated and regulates specific response to changing osmolarity conditions.

Key elements for that are the two outer membrane porins OmpC and OmpF. While OmpF,

possessing a larger pore and a higher flow rate than OmpC, is mainly present at low

osmolarity, the level of OmpC porin dominates at high osmolarity (Huang et al., 1997). The

expression of the corresponding genes ompC and ompF is regulated by the transcriptional

regulator OmpR depending on osmolarity.

OmpR itself is part of a two-component system including EnvZ, its corresponding sensor

histidine kinase. EnvZ is located at the inner membrane and autophosphorylated at histidine

243 residue depending on changing osmolarity (Mattison et al., 2002). Subsequently, it is

able to (de)phosphorylate OmpR at aspartate 55 residue. Mattison et al. (2002) give an

overview on the extensive studies that were carried out on structure and function of OmpR:

with a size of 27 kDa, this regulatory protein consists of an N-terminal phosphorylation

domain including aspartate 55 and a C-terminal DNA binding domain carrying a

characteristic winged helix-turn-helix DNA binding motif. The two domains are connected by

a 15 amino acid flexible linker. Two additional aspartate 11 and 12 residues as well as lysine

105 and other important conserved amino acids are required to stabilize phosphorylation.

The protein attaches to binding sites upstream of its two target genes. Each of these sites,

namely F1, F2, F3, F4, C1, C2 and C3, consists of two 10 bp tandem subunits, an “a site”, to

which the protein has a lower affinity, and a “b site”, to which the affinity is higher (Yoshida et

al., 2006). At low osmolarity, few phosphorylated OmpR molecules bind cooperatively to F1,

F2 and F3 and thus activate ompF transcription, whereas only C1 is bound and ompC is not

expressed. However, high osmolarity leads to a higher amount of OmpR-P in the cells. Now

the protein can also bind C2 and C3, resulting in expression of ompC. Furthermore, OmpR-P

also occupies F4, supporting the formation of a DNA loop upstream of ompF, which can no

longer be transcribed (Yoshida et al., 2006). The authors suggest a hierarchic model of DNA

binding in a “discontinuous, galloping manner” (see figure 57), i.e. that OmpR-P first binds

with high affinity to F1b or C1b, respectively. Phosphorylation is responsible for protein-

protein interaction and therefore for recruiting new OmpR-P molecules to the F1a or C1a

site, and subsequently to the other F or C sites. Only OmpR dimers are able to accomplish

stable DNA binding.

Discussion 139

Fig. 57: Model of activation and functionality of E. coli transcriptional regulator OmpR. A. Activation by phosphorylation via sensor histidine kinase EnvZ. Phosphorylation leads to dimer formation. Only dimers are able to completely bind DNA. B. “Galloping” system of recruiting new OmpR-P molecules to its binding sites, leading to activation or repression of ompF and ompC expression. Adapted from Yoshida et al. (2006).

In the sequence of M. smegmatis GlnR, an N-terminal phosphorylation domain carrying

highly conserved aspartate residues and a C-terminal DNA binding domain were identified

(see figure 44, section 4.3.2 and Amon et al., 2008). Binding of GlnR to corresponding

promoter fragments of its target genes was proven in this study, indicating that additional

activation of the regulator such as phosphorylation might not be necessary for binding (see

figure 28, section 4.2.3). Moreover, the shift of the DNA became more intense with

increasing amounts of used purified GlnR (see figure 19, section 4.1.7). This might be a first

indication of a “galloping” DNA binding of GlnR. The varying expression strengths and times

of GlnR target genes also support the idea that GlnR binds with distinctive affinity to their

promoter regions (refer to figure 24, section 4.2.2), which might be a result of deviating

binding sites. This phenomenon is also well investigated in C. glutamicum, where binding

strength of the global nitrogen regulator AmtR to its target DNA depends on variations of the

consensus DNA sequence (Jakoby et al., 2000; Beckers et al., 2005; Hasselt et al., 2010).

According to sequence analyses, a consensus sequence for DNA binding of M. smegmatis

GlnR has also been identified (see figure 29, section 4.2.4 and Amon et al., 2009),

suggesting putative “a” and “b” binding sites following the OmpR-model described above.

Indeed, gel retardation experiments performed in this study revealed the existence of more

than one GlnR binding sites in the promoter region of amtB (refer to figure 30 and 31, section

Discussion 140

amtB glnA

GlnR GlnR

4.2.4). These are located 75-100 bp (1), 100-150 bp (2), 50-75 bp (3, putatively) and

150-200 bp (4) upstream of the gene’s start codon, supporting the “galloping” model for

GlnR. These data were confirmed in electrophoretic mobility shift assays, where at least two

distinct shifts of amtB and glnA promoter fragments were spotted (figure 58), leading to the

conclusion that initially a first binding site is attached by GlnR resulting in a low shift of DNA.

When this site is completely occupied, addition of higher amounts of GlnR leads to protein-

protein interaction and recruitment of new GlnR molecules to the DNA. Thus, the second

binding site is occupied, which can be seen in the experiment as a second, higher shift.

Fig. 58: Electrophoretic mobility shift assay (EMSA). A 220 bp amtB promoter fragment and a 250 bp glnA promoter fragment were incubated with rising amounts of GlnR (0, 0.1, 0.5, 1, 2, 4, 6 and 8 µg Strep-GlnR).

DNAse protection assays are required to characterize these binding sites in detail. These are

in progress in cooperation with Y. Lu, Chinese Academy of Sciences, Shanghai, People’s

Republic of China, and J. Milse, Center of Biotechnology, Bielefeld.

Although three aspartic acid residues have been identified as phosphorylation sites of

M. smegmatis GlnR, phosphorylation of the regulator could not be proven in this study (see

figure 41, 42 and 43, section 4.3.1.4). Exchange of one of the three aspartic acids led to loss

of GlnR function. The glnR* variants showed high amounts of transcripts in M. smegmatis

and E. coli. Furthermore, purification of intact GlnR* variants with a functional DNA binding

domain was possible in E. coli. These observations suggested rather a problem of protein

stability than phosphorylation in M. smegmatis (refer to section 4.3.2). Moreover, the search

for a corresponding, GlnR phosphorylating sensor histidine kinase was difficult, as its

encoding gene is not situated close to glnR in the genome, which is usually the case for

bacterial two-component systems such as NtrB/NtrC (genes located in operon glnAntrBC,

refer to section 2.2.2) and OmpR/EnvZ (genes located in operon ompRenvZ, see above) in

E. coli.

A closer look on the genomic region surrounding glnR provides no indication of a sensor

kinase encoding gene. Upstream of msmeg_5784 (glnR), msmeg_5785 encodes a

Discussion 141

( 4.1x)

5779 5780 5781 5782 5783 5784 5785 5786

pstB pstA pstC pstS glnR

hypothetical protein and msmeg_5786 thiredoxin (figure 59). Further upstream, various

hypothetical proteins are encoded. Downstream of glnR, several genes involved in

phosphate transport and metabolism are located (msmeg_5782-5779, see figure 59).

Directly downstream of glnR, the gene msmeg_5783 is located, encoding a GNAT-family

acetyltransferase (figure 59). Interestingly, a decreased mRNA level of this gene by a factor

of 4.1 was detected in a glnR deletion strain compared to the wild type under nitrogen

starvation (refer to table 12, section 7.2), suggesting that it might be GlnR-controlled in some

way. As msmeg_5783 did not appear in transcriptome analyses comparing total transcript of

the M. smegmatis wild type under nitrogen surplus and starvation, it is assumed that its

expression does not depend on nitrogen availability. The two genes msmeg_5784 (glnR) and

msmeg_5783 are putatively organized as operon, although glnR transcripts could not be

experimentally verified in this study.

Fig. 59: Genomic organization of the GlnR encoding region in M. smegmatis. The gene msmeg_5783, encoding an acetyltransferase showed an enhanced transcript level in the presence of GlnR.

Several sensor hisidine kinases identified in sequence or transcriptome analyses were

tested, but none of those was able to phosphorylate GlnR in vitro (refer to section 4.3.1.4).

Furthermore, a deletion or disruption of the corresponding genes did not result in loss of

GlnR function (see section 4.3.1.2). Investigation of other sensor histidine kinases (refer to

table 14, section 7.3 and Amon, 2010) is in progress.

All results obtained in this study led to the conclusion that the OmpR-model might be correct

for DNA binding manners of M. smegmatis GlnR, but not necessarily for activation of the

transcriptional regulator. Various two-component systems were identified as OmpR-family

members (see table 7 and Egger et al., 1997; Itou and Tanaka, 2001). For all of these

systems, activation of the transcriptional regulator is exclusively described as mediated by a

corresponding sensor histidine kinase via phosphorylation. Aspartic acid residues as

phosphorylation sites are highly conserved, as well as further amino acids stabilizing the

phosphorylation (Itou and Tanaka, 2001). The authors suggest a “conservative variety”

among the OmpR-family members, as large homologies of N-terminal receiver and

C-terminal DNA binding domains exist, but also differences in the remaining sequence and

thus protein structure and interaction with RNA polymerase.

Discussion 142

Tab. 7: OmpR/EnvZ two-component system family members. Response regulator and corresponding sensor histidine kinase are shown, as well as the adaptive system, effector genes and signal as far as available. Partly adapted from Egger et al. (1997), expanded according to Itou and Tanaka (2001) and Ansaldi et al. (2004).

Adaptive system Signal Sensor (kinase) Response regulator Effector(s)

Osmolarity changes ? EnvZ OmpR ompC, ompF

Multiple systems ? CpxA CpxR traJ, degP, etc.

Phosphate regulation phosphate PhoR PhoB phoA, phoE, pst

Stress situations ? PhoQ PhoP psiG,H,I,J,L,O

? ? BaeS BaeR ?

? ? BasS BasR ?

Catabolite repression ? CreC CreB pho regulon

Potassium transport K+, turgor KdpD KdpE kdpA,B,C,F

Respiratory control ? ArcB ArcA aceF, pps, cyoA-E

Chemotaxis chemotaxins CheA CheB, CheY flagellar motor

Nitrogen fixation ? FixL FixJ fix, nif

? Mg2+

RstB RstA ?

Anaerobic respiration TMAO TorS TorR torCAC

In summary, it can be concluded that every transcriptional regulator so far identified as a

member of the OmpR-family is controlled via phosphorylation by its corresponding sensor

histidine kinase and that, in the case of M. smegmatis GlnR, the correct kinase has not yet

been found. Consequently, conditions of further experiments might be varied to show GlnR

phosphorylation in vitro.

5.3 Activation of nitrogen response

It has been clearly demonstrated in this study that nitrogen response in M. smegmatis

depends on OmpR-type regulator GlnR. This is performed either by direct binding of the

regulator to promoter sequences of its target genes or by a so far unknown, indirect

mechanism (section 4.2.1, 4.2.2 and 4.2.3; see figure 56, section 5.1.2 for overview). In a gel

retardation experiment with GlnR protein purified via His-tag from M. smegmatis SMR5,

grown under nitrogen surplus and starvation, the high affinity of the regulator to its target

DNA was demonstrated (see figure 32, section 4.2.4). Furthermore, as binding of GlnR was

independent from the nitrogen status of the cells, the assumption that nitrogen starvation

induces binding of GlnR to its target DNA was disproved, at least in vitro.

These observations led to the conclusion that gene transcription by RNA polymerase in vivo

in M. smegmatis is not only activated due to binding of GlnR to the promoter sequence, but

rather due to involvement of a so far unknown cofactor or OmpR-like phosphorylation and

Discussion 143

dimerization of the regulator. Thus, the identification of the signal inducing GlnR binding and

gene transcription in the cells is a major objective of current research. It was detected in this

study, that the glnR transcript level is not autoregulated and also not enhanced due to

nitrogen limitation (see figure 33, section 4.2.5). In consequence, it is still assumed that GlnR

is activated by phosphorylation, which, however, could not be proven in this study (refer to

figure 41, 42 and 43, section 4.3.1.4 and section 5.2).

Moreover, the indicator signalizing nitrogen starvation to the cells and thus activating putative

sensor histidine kinase still needs to be identified. In E. coli, the best investigated model

organism for nitrogen metabolism, the ratio of intracellular concentrations of glutamine and

2-oxoglutarate indicates nitrogen availability and determines (de)uridylylation of signal

transduction protein PII, which is then responsible for (de)activation of nitrogen response via

NtrB and NtrC (refer to section 2.2.2). Furthermore, a model for detection of extracellular

ammonium concentrations by ammonium transporter AmtB exists (Javelle et al., 2004).

Indication of nitrogen response in the actinomycete C. glutamicum is much discussed. While

growth of the organism with glutamine and glutamate as single nitrogen source resulted in

activation of nitrogen response, this was not monitored in the presence of ammonium (Rehm,

2010). It was supposed that glutamine and glutamate do not indicate the cells’ nitrogen

status, as their intracellular concentrations did not vary under the tested experimental

conditions (Rehm, 2010; Rehm et al., 2010). It is rather assumed that in C. glutamium the

intracellular concentration of 2-oxoglutarate is responsible for indication of nitrogen limitation,

whereas binding of the molecule to the PII signal transduction protein GlnK still needs to be

investigated (Müller et al., 2006; Rehm et al., 2010). Next to 2-oxoglutarate, the intracellular

ammonium concentration is supposed to be the positive indicator of nitrogen status of the

cells (Nolden et al., 2001b; Müller et al., 2006; Rehm et al., 2010).

Evidence for putative indicators of the nitrogen status of M. smegmatis was given in

experiments performed in this study. While nitrogen response was induced when glutamate

was used as single nitrogen source, this was not observed in the presence of glutamine or

ammonium (see figure 11, section 4.1.2). Furthermore, glutamate-dependent nitrogen

response was completely suppressed after 5-10 minutes when glutamine or ammonium was

added, respectively (see figure 12, section 4.1.2). This led to the conclusion that glutamate is

probably not the indicator of nitrogen status in M. smegmatis, as nitrogen response was

induced under high concentrations of this product of GS/GOGAT-pathway (see figure 60).

On the contrary, the intracellular and/or extracellular ammonium concentration might function

as a nitrogen indicator, as its effect was superior to that of glutamate. The same conclusion

was drawn concerning glutamine as nitrogen indicator.

Discussion 144

NH3

glutamine glutamate

ATPADP + Pi + H2O

glutamate

GOGAT

NH3/NH4+

glutamate

2-oxogutarate

GS

GOGAT

GDH

NADPH + H +

NADP +

NADP+ + H2O NADPH + H+

NH4+

NH4+

Moreover, nitrogen response was detected in the presence of phenylalanine, adenine,

thymine, N-acetylglucosamine, tyrosine, methylamine, arginine, histidine, proline, threonine,

ethanolamine, nitrate and nitrite (figure 11, section 4.1.2), but there is no information about

the function of these substances as indicators of nitrogen metabolism in M. smegmatis.

Fig. 60: Occurrence of ammonium, glutamate and glutamine in central nitrogen metabolism. Ammonium is assimilated under nitrogen surplus by glutamate dehydrogenase (GDH) and under nitrogen limitation by glutamine synthetase (GS) and glutamate synthase (GOGAT). Figure modeled after Merrick and Edwards (1995) and Leigh and Dodsworth (2007); see also section 2.2.1.

Searching for indication of nitrogen response and activation of the global nitrogen regulator

GlnR, genes encoding several signal transduction proteins have been identified in

M. smegmatis (Amon et al., 2009). The glnE gene encoding adenylyltransferase GlnE,

known to be responsible for (de)activation of GSI via (de)adenylylation (see section 2.2.1), is

located in the genome between the GSI encoding genes glnA and glnA2 (see figure 56,

section 5.1.2 for overview). This is a situation similar to M. tuberculosis, a related

mycobacterium, in which GlnE function has already been intensely investigated. glnE was

described as an essential gene for M. tuberculosis, as it could not be deleted without another

functional copy in the cell (Parish and Stoker, 2000). Additionally, upregulation of glnE was

detected depending on the presence of ammonium or glutamine in the medium (Pashley et

al., 2006). The authors also detected co-transcription of glnA2 and glnE. In further studies,

the essential role of GlnE was attributed to the adenylylation domain, indicating that GlnE

indeed controls fine regulation of GSI activity in M. tuberculosis in response to varying

nitrogen conditions (Carroll et al., 2008). Interestingly, GlnE is not essential in other

actinomycetes such as S. coelicolor (Fink et al., 1999) leading to the conclusion that GlnE is

important for M. tuberculosis to maintain a balance of glutamate and glutamine levels, as

these are essential for survival of the organism in the host as major components of the cell

wall (Pashley et al., 2006).

Discussion 145

Up to now, no studies have been performed on M. smegmatis GlnE. As the organism is

closely related to M. tuberculosis, but considered non- or facultative pathogenic, it would be

interesting to investigate the putative necessity of GlnE in M. smegmatis. A (de)adenylylating

and thus GSI-controlling function of M. smegmatis GlnE is expected, as this is widely

conserved among bacteria (Fink et al., 1999; Nolden et al., 2001a; Ninfa and Jiang, 2005;

Carroll et al., 2008; see Amon et al., 2010 for overview).

Next to glnE, the genes glnK and glnD, encoding PII and uridylyl/adenylyltransferase were

also identified in M. smegmatis (Amon et al., 2009). The situation of these genes in the

operon amtB-glnK-glnD was monitored and co-transcription of these genes was proven

(Bräu, 2008). While the glnK and glnD mRNA levels were increased in the wild type under

nitrogen starvation by factors of 31.84 and 16.95, respectively, they were decreased in the

glnR deletion strain by factors of 52.56 and 18.22 (table 10 and 12, section 7.2, this study).

It is known that GlnD and PII mediate nitrogen response in E. coli via (de)uridylylation (Ninfa

and Atkinson, 2000; Javelle et al., 2004) and in C. glutamicum via (de)adenylylation

(Strösser et al., 2004; Beckers et al., 2005). (De)adenylylation of PII by GlnD is also

described for S. coelicolor (Hesketh et al., 2002), but further signal transduction by modified

PII protein could not be proven yet (Reuther and Wohlleben, 2007). Even less information is

provided concerning M. tuberculosis. In this organism, no evidence for a central role of GlnD

in nitrogen regulation has been found, although the authors monitored a slight effect of a

glnD deletion on GS activity (Read et al., 2007). For M. smegmatis, no data are published

about the role of GlnK and GlnD in signal transduction. First investigations of a glnK and a

glnD deletion strain revealed no differences in growth or transcript levels of GlnR and AmtR

target genes compared to the wild type under nitrogen surplus and starvation. Furthermore,

interaction studies with purified GlnK and GlnD proteins did not show any result (W. Zhou,

personal communication). Increased GS activity was measured in the wild type under

nitrogen starvation, which was reduced after addition of ammonium. Interestingly, the

increase of activity was slightly lower in the glnD deletion strain and clearly reduced in the

glnK deletion strain (W. Zhou, personal communication). Thus, an influence of at least GlnK

on (de)activation of adenylyltransferase GlnE, which controls GSI activity, is suggested. It

can be concluded that GlnK and GlnD are not essential for activation of nitrogen response in

M. smegmatis, but that they are probably involved in fine regulation of GSI via GlnE. GlnK-

GlnE interaction studies are in progress as well as the generation of a glnE deletion strain

(W. Zhou, personal communication). Finally it can be stated that the nitrogen regulatory

system in M. smegmatis differs significantly from the NtrB/NtrC-system described in E. coli.

Changing nitrogen conditions are probably sensed by the so far unknown sensor histidine

kinase that is predicted to activate GlnR under nitrogen limitation by phosphorylation.

Discussion 146

5.4 Ammonium assimilation in M. smegmatis

Concerning nitrogen metabolism, synthesis and processing of glutamine and glutamate plays

a very important role. While glutamine provides nitrogen for the synthesis of e.g. purines,

pyrimidines and some amino acids, glutamate is the main nitrogen donor for 85 % of nitrogen

containing cell compounds (Harper et al., 2010).

As described earlier in this study (see section 2.2.1), ammonium assimilation is mediated

under nitrogen surplus by NADPH-dependent aminating GDH resulting in L-glutamate. This

enzyme works with low ammonium affinity and under low energy demand. It is encoded in

M. smegmatis by msmeg_5442 (gdhA), which was first identified by Sarada et al. (1980).

It was shown in this study that gdhA transcript was altered neither in response to nitrogen

starvation nor the presence or absence of GlnR (refer to figure 25, section 4.2.2 and table 9-

12, section 7.2). These data correlated with the recent observation that aminating GDH

activity did not change in response to varying ammonium supply or nitrogen limitation

(Harper et al., 2010). Thus, it can be concluded that gdhA is not subject to nitrogen control in

M. smegmatis, a phenomenon which is different in the related actinomycete C. glutamicum.

There, the transcript level of gdh is regulated by AmtR, the global regulator of nitrogen

metabolism in this species (Beckers et al., 2005; Hänßler et al., 2009).

In addition to gdhA, the genes msmeg_4699 and msmeg_6272 encoding NAD+-dependent

deaminating GDH enzymes were identified in M. smegmatis (O’Hare et al., 2008; Harper et

al., 2010). These enzymes are described as involved in glutamine/glutamate catabolism, but

only very low and ammonium- or nitrogen-independent deaminating GDH activity was

detected (Harper et al., 2010). Surprisingly, further NAD+-dependent aminating activity was

monitored. As a consequence, the authors suggested an unusual aminating role for at least

one of the NAD+-dependent GDH enzymes, which has not been described before.

Furthermore, it was suspected that at least one of the corresponding genes is regulated

depending on nitrogen availability. Indeed, an enhanced transcript level of msmeg_4699 was

observed after two hours of nitrogen starvation (Harper et al., 2010). On the contrary,

changes in transcript levels of msmeg_4699 and msmeg_6272 were detected neither due to

nitrogen limitation nor due to presence or absence of GlnR in transcriptome analyses

performed in this study (see table 9-12, section 7.2).

Under low ammonium concentrations, the GS/GOGAT-pathway is used because of the low

ammonium affinity of GDH (refer to section 2.2.1). First, ammonium is assimilated via

glutamine synthetase GS to glutamine in an ATP-consuming way. Various genes encoding

different types of glutamine synthetases were identified in M. smegmatis (table 8).

Discussion 147

Tab. 8: List of all putative GS encoding genes in M. smegmatis. Gene name, occurrence in M. smegmatis and M. tuberculosis, predicted function and references or homologs are given as far as available. According to Amon et al. (2009) and www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd= Retrieve&dopt=Protein%20Table&list_uids=20087. * indicates glnA homologs of M. smegmatis that are missing in other mycobacterial genomes.

Function, GS type Gene name M. smegmatis M. tuberculosis Reference/nearest homologs

Unknown glnA* msmeg_1116 n/a -

Unknown glnA4 msmeg_2595 Rv2860c Harth et al., 2005

Unknown glnA3 msmeg_3561 Rv1878 Harth et al., 2005

Unknown glnA* msmeg_3827 n/a E. coli K12 putative GS

Unknown glnA* msmeg_3828 n/a -

NH4+ assimilation, GSI glnA1 msmeg_4290 Rv2220 Harth et al., 2005

Unknown, GSI glnA2 msmeg_4294 Rv2222c Harth et al., 2005

Unknown glnA* msmeg_5374 n/a Rhodopseudomonas palustris glnA

Unknown, GSII/III ? glnT, glxA msmeg_6260 n/a -

Unknown, GSII/III ? glnA* msmeg_6693 n/a Agrobacterium tumefaciens glnA

According to Merrick and Edwards (1995), four different types of glutamine synthetases exist

in bacteria: the universal form GSI consists of 12 identical 55 kDa subunits and is encoded

by glnA. GSI of most bacterial species is posttranslationally modified by adenylyltransferase

GlnE, with the exception of Bacillus and Clostridium strains. GSII is composed of eight

identical 36 kDa subunits. Its encoding gene, glnII, has been identified in Agrobacterium,

Rhizobium and Streptomyces strains. A eukaryotic origin of this GS type is suspected. GSIII

contains six 75 kDa subunits and has been found in Bacteroides, Butyrivibrio and

Synechocystis species. The fourth GS form is named GlnT and consists of eight 47 kDa

subunits. It has been identified in some Rhizobium strains. The GS encoding genes in

M. smegmatis cannot easily be allocated to the different groups.

Best investigated in mycobacteria is GSI, encoded by glnA. Especially glutamine

synthetases I of M. bovis and M. tuberculosis are very interesting targets of research, as they

are probably involved in virulence and pathogenicity of these organisms (Tullius et al., 2003;

Harth et al., 2005; Chandra et al., 2010). The authors believe that GSI is necessary for the

synthesis of a poly-L-glutamate-glutamine layer at the cell wall of these pathogens.

Interestingly, this specific layer is absent in saprophytic or non-pathogenic mycobacteria

(Chandra et al., 2010). Indeed, different tested GS inhibitors blocked the formation of the

poly-L-glutamate-glutamine layer, but no evidence for a direct involvement of GSI has been

shown so far (Harth et al., 2000; Odell et al., 2009).

Four glnA genes exist in M. tuberculosis: glnA1 which encodes the pathogenicity-associated

GSI, and glnA2-4 which show strong sequence similarity to glnA1. All of the corresponding

proteins showed GS activity and were inhibited by MSX (Harth et al., 2005). GlnA1, 3 and 4

Discussion 148

catalyze the synthesis of L-glutamine, while GlnA2 catalyzes the synthesis of D-glutamine

and isoglutamine. Furthermore, it is assumed that GlnA1 is essential for growth and

pathogenicity of M. tuberculosis, while GlnA2-4 are described as glutamine synthetases of

subordinate function (Harth et al., 2005).

Homologs of glnA1-4 were also identified in M. smegmatis (see table 8). As in

M. tuberculosis, glnA1 (msmeg_4290) encodes the main GSI protein. An enhanced transcript

level of glnA1 due to nitrogen limitation was reported (see Amon et al., 2008; this study,

figure 24 and 27, section 4.2.2 and table 10, section 7.2). Moreover, Harper et al. (2010)

revealed that less glnA1 transcript was present compared to M. tuberculosis. The authors

monitored glnA1 transcript level and GSI activity in response to changing nitrogen supply. It

was found that GSI activity started increasing after 30 minutes of nitrogen starvation, which

was at its maximum after four hours. When an ammonium pulse was given, GSI activity was

rapidly decreased. The transcript level of glnA1 was also increased after 30 minutes of

nitrogen starvation (as reported in this study) and highest after two hours. Interestingly, a

decrease of transcript after an ammonium pulse was barely detected leading to the

conclusion that a rapid response of glutamine synthetase activity to changing nitrogen

conditions is mediated by posttranslational modification of GSI by GlnE, rather than by gene

expression (Harper et al., 2010). This phenomenon was demonstrated for several related

actinomycetes before, and first hints for M. smegmatis were found (refer to section 5.3).

No data about M. smegmatis glnA2-4 are published so far. In this study, enhanced mRNA

levels of msmeg_4294 (glnA2, 3.75 fold) and msmeg_3561 (glnA3, 3.04 fold) were detected

in the wild type under nitrogen starvation (see figure 24 and 27, section 4.2.2 and table 10,

section 7.2). Furthermore, it was proven that glnA1 and glnA2 are controlled by GlnR, the

main regulator of nitrogen metabolism in M. smegmatis. The transcript of glnA1 was reduced

34.13 fold in a glnR deletion strain compared to the wild type under nitrogen starvation, the

transcript of glnA2 26.01 fold (figure 24 and 27, section 4.2.2 and table 12, section 7.2). It

was also shown that GlnR binds directly upstream of these two genes (see figure 28, section

4.2.3).

Additionally, several glnA-like genes were identified in M. smegmatis which do not appear in

related species such as M. tuberculosis (see table 8). The genes msmeg_1116 and

msmeg_3828 are described as glnA4 homologs with less homology than msmeg_2595,

while msmeg_3827 showed closest relation to an E. coli glutamine synthetase. The gene

msmeg_5374 is related to glnA of Rhodopseudomonas palustris, and msmeg_6693 to

Agrobacterium tumefaciens glnA (Amon et al., 2009). Interestingly, in transcriptome analyses

performed in this study, transcript of msmeg_5374 was increased by a factor of 4.66 in the

M. smegmatis wild type under nitrogen starvation (refer to table 10, section 7.2). The gene

Discussion 149

msmeg_6260 is described as glnT encoding GSII (www.ncbi.nlm.nih.gov/sites/entrez

?db=genome&cmd=Retrieve&dopt=Protein%20Table &list_uids=20087) and also as glxA

encoding GSIII as part of a glx operon which is of Pseudomonas origin (Amon et al., 2009).

However, a 30.44 fold increased mRNA level of this gene was detected in the wild type

under nitrogen starvation and a 16.11 fold decreased mRNA level in the absence of GlnR

(table 10 and 12, section 7.2).

Summarizing recent insights, it can be said that M. smegmatis possesses at least 10 genes

that encode glutamine synthetases typical for various, not even closely related bacterial

species. The results of this study suggest that GS activity is not restricted to one of these

genes. Especially under nitrogen starvation genes encoding several glutamine synthetases

(msmeg_4290, msmeg_4294, msmeg_ 3561, msmeg_5374 and msmeg_6260) are activated

and the corresponding enzymes are probably used to maintain a glutamine level sufficient for

survival.

In the GS/GOGAT-system glutamine is assimilated together with 2-oxoglutarate to two

molecules glutamate by glutamate synthase GOGAT. The encoding genes for this enzyme

are msmeg_6458/6459 (gltBD), showing closest relation to C. glutamicum glutamate

synthase (Amon et al., 2009). The authors also identified further genes that resemble gltB

(msmeg_5594 and msmeg_6263) and gltD (msmeg_6262). Interestingly, transcriptome

analyses of this study revealed decreased transcript levels of msmeg_6458/6459 by a factor

of 3.23/4.53 in response to nitrogen starvation (table 9, section 7.2). On the contrary, the

transcripts of msmeg_6262 and msmeg_6263 were enhanced under nitrogen starvation by

factors of 7.6 and 10.48 (table 10, section 7.2). Furthermore, the amounts of their mRNA

were decreased in the absence of GlnR by a factor of 7.4 and 7.01, respectively (table 12,

section 7.2). Thus it can be concluded that nitrogen- and GlnR-dependent GOGAT activity is

proceeded from msmeg_6262 and msmeg_6263, which are located in the operon

msmeg_6259-6264 (see figure 56, section 5.1.2) and described as glxC and glxD (Amon et

al., 2009).

5.5 Nitrate and nitrite metabolism in M. smegmatis

A variety of different studies on assimilation and catabolism of nitrate and nitrite in bacteria

can be found, whereas concerning mycobacteria, such studies have only recently begun.

The first step of nitrate assimilation is the reduction to nitrite by nitrate reductase encoded by

narGHJI. NarJ assembles the subunits NarG, NarH and NarI to the functional nitrate

reductase (Malm et al., 2009). The corresponding genes msmeg_5140-5137 were identified

Discussion 150

52x 49x ( 7x) ( 34x) 55x

A

0426 0427 0428 0429 0430 0431 0432 0433

nirB nirD narK3

no binding

B

2107 2106 2108

C

hypothetical proteins and tRNAs 2837 2838

narB

D

5137 5138 5139 5140 5141 5142 5143

no binding

narI narJ narH narG narK degA

in M. smegmatis (Amon et al., 2009). In a second step, nitrite is reduced to ammonium by

nitrite reductase. This sirohaem-dependent enzyme is present in a variety of bacteria and

fungi (Malm et al., 2009) and is encoded in M. smegmatis by msmeg_0427/0428 (nirBD).

Additionally, nitrate/nitrite transporters (msmeg_5141 narK and msmeg_0433 narK3) were

identified, as well as another putative assimilatory nitrate reductase (msmeg_2837 narB). All

genes putatively involved in nitrate and nitrite metabolism that were investigated in this study

are shown in figure 61.

Fig. 61: Genomic situation of genes putatively involved in nitrate and nitrite metabolism in M. smegmatis. According to http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=246196. Arrow number x indicates fold change of transcript levels in the presence of GlnR according to transcriptome analyses, while ( ) represents data not verified in further experiments. Light red ovals show predicted binding sites where no binding could be detected.

Nitrogen- and GlnR-dependent increase of nirBD and narK3 transcript levels was monitored

in this study, whereas this was not shown for narK or the narGHJI orperon (see section 4.2.1

and 4.2.2 and figure 61 for overview). In addition to that, a glnR deletion strain was not able

to grow with nitrate or nitrite as single nitrogen source (refer to section 4.1.1). Interestingly,

nitrogen response was activated in the wild type with nitrate or nitrite as single nitrogen

source (figure 11, section 4.1.2). Binding of GlnR to promoter fragments of nirBD or the nar

operon was not detected (see figure 28, section 4.2.3 and figure 61 for overview). These

data correlated with earlier observations made in M. tuberculosis, where an influence of GlnR

to nirBD expression was also reported (Malm et al., 2009). The authors proved furthermore

that narG was essential for growth of M. tuberculosis on nitrate and that nirB was essential

Discussion 151

for growth on both, nitrate and nitrite, pointing to an assimilatory function of these enzymes.

In contrast, disruption of narG in M. smegmatis did not lead to a significant change of growth

or gene transcript levels compared to the wild type (see figure 51 and 52, section 4.4),

leading to the conclusion that this gene is not essential for survival of M. smegmatis with

nitrate as single nitrogen source and that its malfunction may be complemented by another

nitrate reduction system. Within the time frame of this study, it was not possible to generate a

nirB mutant in M. smegmatis.

The mRNA level of narB, which encodes another putative nitrate reductase, was not

regulated due to nitrogen availability in M. smegmatis. Thus, it can be concluded that it fulfills

a different task than in the related actinomycete S. coelicolor, where the transcript level of the

narB homolog nasA was reported to be GlnR-controlled (Wang and Zhao, 2009). The

authors demonstrated the importance of nasA for growth of the organism in nitrate-containing

medium and moreover, specific binding of GlnR to a cis-element in the promoter region of

the gene. Experiments performed in this study with an M. smegmatis strain lacking a

functional copy of narB did not reveal differences in growth or gene transcript levels with

nitrate or nitrite compared to the wild type (see figure 51 and 52, section 4.4).

In addition to GlnR, the involvement of three transcriptional regulators in expression of nirBD

or narGHJI was investigated. These were msmeg_0426, encoding a GntR-family regulator

located directly upstream of nirBD, msmeg_2106, encoding a TetR-type regulatory protein

which is associated with nirBD, and msmeg_5143, encoding HTH-type transcriptional

regulator DegA, a putative regulator of the nar operon (for overview see also figure 61).

Contrary to expectations, these regulatory proteins seem not to be involved in nitrate or

nitrite metabolism in M. smegmatis (see figure 51 and 52, section 4.4). All results pointed

towards a mediation of uptake and assimilation of nitrate and nitrite in M. smegmatis by

nitrate/nitrite transporters NarK and NarK3, nitrate reductase NarGHJI and nitrite reductase

NirBD, whereas NarK3 and NirBD are activated by GlnR.

Assimilatory or dissimilatory function of NirBD and NarGHJI has been intensely discussed.

First, nitrate and nitrite metabolism was investigated in the pathogenic species M. bovis and

M. tuberculosis, focusing on the pathogenicity aspect. Although mycobacteria are considered

obligate aerobes, in particular the pathogenic ones must be able to adapt to limiting oxygen

conditions in granulomas or abscesses that are formed due to infection (Weber et al., 2000).

In this environment, M. tuberculosis switches to a state of persistence, in which it is not

replicating and increases several enzyme activities such as nitrate reductase activity

(Sohaskey and Modesti, 2008). Thus, NarGHJI and NirBD were classified as respiratory

enzymes, even in the non-pathogenic species M. smegmatis (Khan and Sarkar, 2006).

Discussion 152

On the contrary, Malm et al. (2009) stated that NarGHJI mediates nitrate reduction under

both aerobic and anaerobic conditions. Only in E. coli and related enterobacteria NirBD

would have no assimilatory function. By investigating nirB and narG deletion mutants of

M. tuberculosis, the assimilatory function of the corresponding enzymes was proven (Malm

et al., 2009). Thus, it can be concluded that nitrate reductase NarGHJI and nitrite reductase

NirBD fulfill both, assimilatory and respiratory functions in mycobacteria, depending on the

environmental conditions.

Interestingly, M. smegmatis is able to survive anaerobic conditions in a dormancy state

similar to its pathogenic relatives (Khan and Sarkar, 2006). This was first investigated by

Dick et al. (1998). Although the organism died when suddenly exposed to oxygen limitation, it

was able to adapt to slowly decreasing oxygen concentrations by entering a persistent state.

In this state, the bacteria did not synthesize DNA or replicate. They were even resistant

against different antimycobacterial drugs. Effective growth of M. smegmatis under anaerobic

conditions has not been reported so far.

A variety of further genes putatively involved in nitrate and nitrite metabolism were identified

in M. smegmatis (according to www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=

Retrieve&dopt=Protein%20Table&list_uids=20087). These are nitrate/sulfonate/bicarbonate

ABC transporters (msmeg_0551, msmeg_1160, msmeg_2501, msmeg_4586, msmeg_4588,

msmeg_6494, msmeg_6495 and msmeg_6496), nitrate/sulfonate ABC transporter

(msmeg_2099), nitrate transporter NrtD (msmeg_2102), a formate/nitrate transporter

(msmeg_5360), a nitrate/nitrite response regulator protein (msmeg_2251) and a ferredoxin-

sulfit/nitrit-reductase also described as ammonia:ferredoxin oxidoreductase (msmeg_4527).

Only formate/nitrate transporter (msmeg_5360) was identified in transcriptome analyses

performed in this study as a putative GlnR target (see table 12, section 7.2 and figure 56,

section 5.1.2 for overview). Msmeg_2099 and msmeg_2102 were also tested as they are

located close to the putative TetR-type transcriptional regulator encoding gene

msmeg_2106, but nitrogen- or GlnR-dependent changes of their transcript levels were not

detected (data not shown).

5.6 Role of AmtR in M. smegmatis

Within the order Actinomycetales two major regulatory proteins of nitrogen metabolism exist,

namely GlnR and AmtR. While AmtR mediates nitrogen-dependent gene expression in

corynebacteria, the majority of the actinomycetes uses GlnR as global nitrogen regulator.

Interestingly, M. smegmatis is part of a small group of actinomycetes that possesses genes

encoding both of these regulatory proteins. Further species are C. michiganensis, N. farcinia

Discussion 153

4x 2x

48x ( 13x) ( 32x) ( 3x)

2184 2185 2186 2187 2188

AmtRGlnR

+ -

amino acid hyp. hyp. urea integral membrane

permease amidolyase protein

or S. avermitilis (refer to figure 5, section 2.2.3 and Amon, 2010). It is experimentally proven

that GlnR is the global regulator of nitrogen metabolism in M. smegmatis (see Amon et al.,

2008 and section 5.1, this study).

Following this study, total transcriptome analysis of an amtR deletion strain compared to the

wild type was carried out, in which two putative AmtR target genes were identified. These

were msmeg_2187 encoding urea amidolyase and msmeg_2188 encoding an integral

membrane protein (see also table 13, section 7.2). An AmtR-dependent change of transcripts

of the operon msmeg_2184-2188, as well as binding of purified AmtR protein to a promoter

sequence upstream of msmeg_2184 was indeed verified in further experiments.

Furthermore, negative autoregulation of amtR and binding of AmtR to a promoter fragment of

msmeg_4301, a gene which is co-transcribed with amtR, were detected (Y. Lu, Chinese

Academy of Sciences, Shanghai, People’s Republic of China, personal communication). A

decreased mRNA level of msmeg_4301 in the wild type under nitrogen starvation was also

monitored in transcriptome analyses carried out in this study (4.37 fold, refer to table 9,

section 7.2). A similar pattern was monitored by Y. Lu in the related actinomycete

S. avermitilis.

Interestingly, the genes msmeg_2184-2188 are also described as GlnR targets in

M. smegmatis (refer to table 12, section 7.2 and section 5.1.2), suggesting a dual regulation

by GlnR and AmtR (see figure 62). Studies on a glnR amtR double deletion strain revealed

GlnR as the superior regulator (Y. Lu, personal communication).

Fig. 62: Model for dual regulation of the operon msmeg_2184-2187 by GlnR and AmtR. While transcript levels are enhanced by GlnR under nitrogen starvation (red arrows), they are decreased by AmtR under nitrogen surplus (orange arrows). Numbers indicate fold change of mRNA levels obtained from transcriptome analyses.

It is supposed that AmtR is bound to its corresponding target sequence and thus represses

expression of the operon under nitrogen surplus. Meanwhile, GlnR is not able to activate

transcription due to its own non-activated (non-phosphorylated) state. When nitrogen

becomes limited, AmtR is released, whereas at the same time activated GlnR binds to its

corresponding DNA sequence and induces gene expression by RNA polymerase. So far, no

Discussion 154

data are available about (de)activation of AmtR. In C. glutamicum, AmtR binding is released

after protein-protein interaction with adenylylated PII protein GlnK, a process which is

mediated by adenylyltransferase GlnD (see section 2.2.3 for overview). Until now, interaction

of AmtR, GlnK and GlnD has not been analyzed in M. smegmatis.

The subordinate function of AmtR in M. smegmatis was also shown in growth experiments

performed similar to those described in section 4.1.1. Here, an amtR deletion strain showed

no differences in growth compared to the wild type when various substances were tested as

single nitrogen source, in which the absence of GlnR led to reduced or no growth (data not

shown). Considering these results, it can be concluded that AmtR in M. smegmatis is not

important for general nitrogen response. Thus, the function of GlnR as global nitrogen

regulator is once more confirmed.

5.7 Nitrogen response in M. smegmatis as a possible model for M. tuberculosis

The aim of this study was to investigate the mechanisms of nitrogen response in

M. smegmatis, while the main focus was set on the global nitrogen regulatory protein GlnR.

M. smegmatis is able to utilize a variety of different organic and inorganic nitrogen sources,

while some of them induce nitrogen response (see section 5.1.1). Various transport and

assimilation systems for amino acids and inorganic nitrogen sources were identified.

Investigating indication and sensing of nitrogen limitation was more difficult. It is believed that

the extracellular nitrogen concentration is mediated by a yet unknown indicator, probably

ammonium, and sensed by a membrane-bound sensor histidine kinase (see section 5.3 and

5.2). The kinase is predicted to activate nitrogen response by phoshorylating GlnR. Although

phosphorylation of GlnR has not yet been proven, typical aspartic acid residues were

identified as phosphorylation sites (see section 5.2). Furthermore, the corresponding sensor

histidine kinase has not yet been found (section 5.2). Nevertheless, GlnR has a positive

influence on transcript levels of at least 32 target genes under nitrogen starvation. These

genes are involved in uptake and assimilation of ammonium, nitrate, nitrite, amino acids,

peptides and others. Different amidases, aminotransferases, hydrolases, as well as signal

transduction proteins are controlled by GlnR (see section 5.1.2 for overview). Moreover,

involvement of transcriptional regulator AmtR in changing the transcript level of the operon

msmeg_2184-2188 was proven (see section 5.6), whereas interaction of signal transduction

proteins GlnK and GlnD with either GlnR or AmtR has not yet been shown (see section 5.3).

Ammonium assimilation is mediated via GDH or the GS/GOGAT-system. While influence of

GlnR on glnA transcript was proven, this was not shown for gdhA or gltBD (see section

Discussion 155

NH3/NH4+ supply

NH3

GlnK

GlnD

NH3

glutamine glutamate

ATPADP + Pi + H2O

glutamate

GOGAT

NH3/NH4+

glutamate

2-oxogutarate

GS

GOGAT

GDH

Nitrogen sources

Amino acids, nitrate,

nitrite, ethanolamine, uric

acid, guanine, uracil…

Ammonium assimilation

Signal transduction

Sensing

Sensor hisitidine

kinase

Transport

systems

Assimilation

Ammonium

transporter

Phosphorylation

GlnR

Gene expression

>32 target genes:

glnA, amtB…

AmtR

Gene expression

indicators?

GlnE

?

?

?

?

?

??

regulation

5.1.2). Further regulation of GSI activity by adenylyltransferase GlnE has not yet been

investigated in M. smegmatis. Figure 63 gives an overview about the current model of

nitrogen sensing and assimilation in M. smegmatis.

Fig. 63: Model of nitrogen assimilation and response in M. smegmatis according to results obtained in this study (refer to text).

Beyond this model, other enzymes also seem to be involved in nitrogen/ammonium

assimilation in M. smegmatis. These are further ammonium transporters amtA and amt1,

further glnA genes encoding GSI enzymes, a type III GS, two additional GDH enzymes and

others (refer to section 5.4).

A highly conserved mode of action of GlnR among the actinomycetes was suggested by

Tiffert et al. (2008). In that study, binding of S. coelicolor GlnR to glnA promoter fragments of

different actinomycetes was observed. Additionally, a glnR deletion in S. coelicolor was

complemented by A. mediterranei glnR (Yu et al., 2006; Tiffert et al., 2008). After

investigating the relation of M. tuberculosis and M. smegmatis GlnR in this study, it was

obvious that these two proteins cannot be replaced by each other. First experiments

indicated that a genomic glnR deletion in M. smegmatis could not be complemented with

M. tuberculosis glnR (refer to figure 54, section 4.5). Furthermore, binding of purified

M. tuberculosis GlnR to M. smegmatis target genes was detected, but only under very high

Discussion 156

protein surplus (figure 55, section 4.5). This result was not expected based on earlier studies

of the amino acid sequence of the two proteins, which was shown to be almost identical

(refer to figure 53, section 4.5 and Amon et al., 2008). Nevertheless, a conserved function of

GlnR as global regulator of nitrogen response in actinomycetes is obvious.

Interestingly, in a recent study dual regulation such as GlnR-AmtR-regulation of the operon

msmeg_2184-2188 in M. smegmatis was also predicted for some genes of nitrogen

metabolism in M. tuberculosis. In silico analyses revealed the existence of a putative

C. glutamicum AmtR-related protein encoded by Rv3160c, which might influence expression

of ureABCG, glnA1, glnD, glnB and further genes (Krawczyk et al., 2009). However, no

experimental data are available so far to support this thesis.

Considering recent insights it can be concluded that the model of nitrogen control in

M. smegmatis resulting from observations made in this study can indeed be used as a

pattern for similar investigations in M. tuberculosis, provided that differences between the

saprophyte M. smegmatis and the human pathogen M. tuberculosis are considered.

References 157

6 References

Abe, S., Takayama, K. & Kinoshita, S. (1967). Taxonomical studies on glutamic acid producing bacteria. J. Gen. Microbiol. 13, 279-301.

Ahmad, S., Bhatnagar, R. K. & Venkitasubramanian, T. A. (1986). Changes in the enzyme activities involved in nitrogen assimilation in Mycobacterium smegmatis under various growth conditions. Annales de l'Institut Pasteur. Microbiol. 137, 231-237.

Amon, J., Bräu, T., Grimrath, A., Hasselt, K., Hänßler, E., Höller, M., Jeßberger, N., Ott, L., Szököl, J., Titgemeyer, F. & Burkovski, A. (2008). Nitrogen control in Mycobacterium smegmatis: nitrogen-dependent expression of ammonium transport and assimilation proteins depends on OmpR-type regulator GlnR. J. Bacteriol. 21, 7108-7116.

Amon, J., Titgemeyer, F. & Burkovski, A. (2009). A genomic view on nitrogen metabolism and nitrogen control in mycobacteria. J. Mol. Microbiol. Biotechnol. 17, 20-29.

Amon, J. (2010). Mining the genomes of actinomycetes: identification of metabolic pathways and regulatory networks. PhD thesis, Friedrich-Alexander University Erlangen-Nuremberg.

Amon, J., Titgemeyer, F. & Burkovski, A. (2010). Common patterns - unique features: nitrogen metabolism and regulation in Gram-positive bacteria. FEMS Microbiol. Rev. 34, 588-605.

Andersen, C. L., Jensen, J. L. & Ørntoft, T. F. (2004). Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 64, 5245-5250.

Ansaldi, M., Théraulaz, L. & Méjean, V. (2004). TorI, a response regulator inhibitor of phage origin in Escherichia coli. Proc. Natl. Acad. Sci. USA 101, 9423-9428.

Aronson, J. D. (1926). Spontaneous tuberculosis in salt water fish. J. Infect. Dis. 39, 314-320.

Barksdale, L. & Kim, K. S. (1977). Mycobacterium. Bacteriol. Rev. 41, 217-372.

Barry III, C. E., Lee, R. E., Mdluli, K., Sampson, A. E., Schroeder, B. G., Slayden, R. A. & Yuan, Y. (1998). Mycolic acids: structure, biosynthesis and physiological functions. Prog. Lipid Res. 37, 143-179.

References 158

Beckers, G., Nolden, L. & Burkovski, A. (2001). Glutamate synthase of Corynebacterium glutamicum is not essential for glutamate synthesis and is regulated by the nitrogen status. Microbiology 147, 2961-2970.

Beckers, G., Bendt, A. K., Krämer, R. & Burkovski, A. (2004). Molecular identification of the urea uptake system and transcriptional analysis of urea transporter- and urease-encoding genes in Corynebacterium glutamicum. J. Bacteriol. 186, 7645-7652.

Beckers, G., Stösser, J., Hildebrandt, U., Kalinowski, J., Farwick, M., Krämer, R. & Burkovski, A. (2005). Regulation of AmtR-controlled gene expression in Corynebacterium glutamicum: mechanism and characterization of the AmtR regulon. Mol. Microbiol. 58, 580-595.

Belitsky, B. R. & Sonenshein, A. L. (1998). Role and regulation of Bacillus subtilis glutamate dehydrogenase genes. J. Bacteriol. 180, 6298-6305.

Bendt, A. K., Beckers, G., Silberbach, M., Wittmann, A. & Burkovski, A. (2004). Utilization of creatinine as an alternative nitrogen source in Corynebacterium glutamicum. Arch. Microbiol. 181, 443-450.

Bérdy, J. (2005). Bioactive microbial metabolites. J. Antibiot. 58, 1-26.

Bergey, D. H., Harrison, F. C., Breed, R. S., Hammer, B. W. & Huntoon, F. M. (1923). Bergey's Manual of Determinative Bacteriology, 1st edition, The Williams & Wilkins Co, Baltimore, 1-442.

Berlicki, L. (2008). Inhibitors of glutamine synthetase and their potential application in medicine. Mini Rev. Med. Chem. 8, 869-878.

Bouché, S., Klauck, E., Fischer, D., Lucassen, M., Jung, K. & Hengge-Aronis, R. (1998). Regulation of RssB-dependent proteolysis in Escherichia coli: a role for acetyl phosphate in a response regulator-controlled process. Mol. Microbiol. 27, 787-795.

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

Bräu, T. (2008). Transkriptionskontrolle in Mycobakterien. Diploma thesis, Friedrich-Alexander University Erlangen-Nuremberg.

References 159

Burkovski, A. (2003). Ammonium assimilation and nitrogen control in Corynebacterium glutamicum and its relatives: an example for new regulatory mechanisms in actinomycetes. FEMS Microbiol. Rev. 27, 617-628.

Burkovski, A. (2005). Nitrogen metabolism and its regulation. In: Bott, M. & Eggeling, L. (eds.) Handbook of Corynebacterium glutamicum. CRC Press LLC, Boca Raton, FL, 333-349.

Burkovski, A. (2007). Nitrogen control in Corynebacterium glutamicum: proteins, mechanisms, signals. J. Microbiol. Biotechnol. 17, 187-194.

Carroll, P., Pashley, C. A. & Parish, T. (2008). Functional analysis of GlnE, an essential adenylyl transferase in Mycobacterium tuberculosis. J. Bacteriol. 190, 4894-4902.

Castets, M., Rist, N. & Boisvert, H. (1969). La variété africaine du bacille tuberculeux humain. Médecine d'Afrique Noire. 16, 321-322.

Chamnongpol, S. & Groisman, E. A. (2000). Acetyl phosphate-dependent activation of a mutant PhoP response regulator that functions independently of its cognate sensor kinase. J. Mol. Biol. 300, 291-305.

Chandra, H., Basir, S. F., Gupta, M. & Banerjee, N. (2010). Glutamine synthetase encoded by glnA-1 is necessary for cell wall resistance and pathogenicity of Mycobacterium bovis. Microbiology 156, 3669-3677.

Chaoui, I., Sabouni, R., Kourout, M., Jordaan, A. M., Lahlou, O., Elouad, R., Akrim, M., Victor, T. C. & El Mzibri, M. (2009). Analysis of isoniazid, streptomycin and ethambutol resistance in Mycobacterium tuberculosis isolates from Morocco. J. Infect. Dev. Ctries. 3, 278-284.

Chater, K. F. (2001). Regulation of sporulation in Streptomyces coelicolor A3(2): a checkpoint multiplex? Curr. Opinion Microbiol. 4, 667-673.

Chung, C. T., Niemela, S. L. & Miller, R. H. (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. USA 86, 2172-2175.

Coyle, M. B. & Lipsky, B. A. (1990). Coryneform bacteria in infectious diseases: clinical and laboratory aspects. Clin. Microbiol. Rev. 3, 227-246.

References 160

Cronin, M., Ghosh, K., Sistare, F., Quackenbush, J., Vilker, V. & O’Connell, C. (2004). Universal RNA reference materials for gene expression. Clin. Chem. 50, 1464-1471.

da Costa Cruz, J. (1938). Mycobacterium fortuitum um novo bacilo acido-resistente patogenico para o homen. Acta Med. (Rio de Janeiro) 1, 298-301.

Dick, T., Lee, B. H. & Murugasu-Oei, B. (1998). Oxygen depletion induced dormancy in Mycobacterium smegmatis. FEMS Microbiol. Lett. 163, 159-164.

Dye, C., Scheele, S., Dolin, P., Pathania, V. & Raviglione, M. C. (1999). Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO global surveillance and monitoring project. JAMA 282, 677-686.

Egger, L. A., Park, H. & Inouye, M. (1997). Signal transduction via the histidyl-aspartyl phosphorelay. Genes Cells 2, 167-184.

Embley, T. M. & Stackebrandt, E. (1994). The molecular phylogeny and systematics of the actinomycetes. Annu. Rev. Microbiol. 48, 257-289.

Engbaek, H. C., Larsen, S. O., Rasmussen, K. N., & Vergmann, B. (1973). Initial treatment of tuberculosis with streptomycin and isoniazid combined with either aminosalyl or rifampicin. Scand. J. Respir. Dis. 54, 83-91.

Engleman, E. G. & Francis, S. H. (1978). Cascade control of E. coli glutamine synthetase: II. Metabolite regulation of the enzymes in the cascade. Arch. Biochem. Biophys. 191, 602-612.

Faller, M., Niederweis, M. & Schulz, G. E. (2004). The structure of a mycobacterial outer-membrane channel. Science 303, 1189-1192.

Fink, D., Falke, D., Wohlleben, W. & Engels, A. (1999). Nitrogen metabolism in Streptomyces coelicolor A3(2): modification of glutamine synthetase I by an adenylyltransferase. Microbiology 145, 2313-2322.

Fink, D., Weisschuh, N., Reuther, J., Wohlleben, W. & Engels, A. (2002). Two transcriptional regulators GlnR and GlnRII are involved in regulation of nitrogen metabolism in Streptomyces coelicolor A3(2). Mol. Microbiol. 46, 331-347.

Flannagan, R. S., Cosío, G. & Grinstein, S. (2009). Antimicrobial mechanisms of phagocytes and bacterial evasion strategies. Nat. Rev. Microbiol. 7, 355-366.

References 161

Forchhammer, K. (2008). P(II) signal transducers: novel functional and structural insights. Trends Microbiol. 16, 65-72.

Gadagkar, R. & Gopinathan, K. P. (1980). Growth of Mycobacterium smegmatis in minimal and complete media. J. Biosci. 2, 337-348.

Gebhard, S., Tran, S. L. & Cook, G. M. (2006). The Phn system of Mycobacterium smegmatis: a second high-affinity ABC-transporter for phosphate. Microbiology 152, 3453-3465.

Gebhard, S. & Cook, G. M. (2008). Differential regulation of high-affinity phosphate transport systems of Mycobacterium smegmatis: identification of PhnF, a repressor of the phnDCE operon. J. Bacteriol. 190, 1335-1343.

Glover, R. T., Kriakov, J., Garforth, S. J., Baughn, A. D. & Jacobs, W. R. (2007). The two-component regulatory system senX3-regX3 regulates phosphate-dependent gene expression in Mycobacterium smegmatis. J. Bacteriol. 189, 5495-5503.

Gomez, M., Doukhan, L., Nair, G. & Smith, I. (1998). sigA is an essential gene in Mycobacterium smegmatis. Mol. Microbiol. 29, 617-628.

Goux, W. J., Strong, A. D. A., Schneider, B. L., Lee, W. N. P. & Reitzer, L. J. (1995). Utilization of aspartate as a nitrogen source for Escherichia coli. J. Biol. Chem. 270, 638-646.

Grant, S. G. N., Jessee, J., Bloom, F. R. & Hanahan, D. (1990). Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc. Natl. Acad. Sci. USA 87, 4645-4649.

Grooters, A. M., Couto, C. G., Andrews, J. M., Johnson, S. E., Kowalski, J. J. & Esplin, R. B. (1995). Systemic Mycobacterium smegmatis infection in a dog. J. Am. Vet. Med. Assoc. 206, 200-202.

Guan, C., Li, P., Riggs, P. D. & Inouye, H. (1987). Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene 67, 21-30.

Hadfield, T. L., McEvoy, P., Polotsky, Y., Tzinserling, V. A. & Yakovlev, A. A. (2000). The pathology of diphtheria. J. Infect. Dis. 181, 116-120.

References 162

Hänßler, E. & Burkovski, A. (2008). Molecular mechanisms of nitrogen control in corynebacteria. In: Burkovski, A. (ed.) Corynebacteria: genomics and molecular biology. Caister Academic Press, Norfolk, UK, 183-201.

Hänßler, E., Müller, T., Palumbo, K., Patek, M., Brocker, M., Krämer, R. & Burkovski, A. (2009). A game with many players: control of gdh transcription in Corynebacterium glutamicum. J. Biotechnol. 142, 114-122.

Harper, C. J., Hayward, D., Kidd, M., Wiid, I. & van Helden, P. (2010). Glutamate dehydrogenase and glutamine synthetase are regulated in response to nitrogen availability in Myocbacterium smegmatis. BMC Microbiol. 10, 138.

Harth, G., Clemens, D. L. & Horwitz, M. A. (1994). Glutamine synthetase of Mycobacterium tuberculosis: extracellular release and characterization of its enzymatic activity. Proc. Natl. Acad. Sci. USA 91, 9342-9346.

Harth, G., Zamecnik, P. C., Tang, J. Y., Tabatadze, D. & Horwitz, M. A. (2000). Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-L-glutamate/glutamine cell wall structure, and bacterial replication. Proc. Natl. Acad. Sci. USA 97, 418-423.

Harth, G., Maslesa-Galic, S., Tullius, M. V. & Horwitz, A. A. (2005). All four Mycobacterium tuberculosis glnA genes encode glutamine synthetase activities but only GlnA1 is abundantly expressed and essential for bacterial homeostasis. Mol. Microbiol. 58, 1157-1172.

Hartmans, S., De Bont, J. A. M. & Stackebrandt, E. (2006). The Genus Mycobacterium-nonmedical. The Prokaryotes 3, 889-918.

Hasselt, K., Rankl, S., Worsch, S. & Burkovski, A. (2010). Adaptation of AmtR-controlled gene expression by modulation of AmtR binding activity in Corynebacterium glutamicum. J. Biotechnol. doi:10.1016/j.jbiotec.201009.930.

Hauduroy, P. (1955). Derniers aspects du monde des mycobactéries. Masson et Cie, Paris.

Hesketh, A. R., Chandra, G., Shaw, A. D., Rowland, J. J., Kell, D. B., Bibb, M. J. & Chater, K. F. (2002). Primary and secondary metabolism, and post-translational protein modifications, as portrayed by proteomic analysis of Streptomyces coelicolor. Mol. Microbiol. 46, 917-932.

Hiery, E. (2009). Charakterisierung eines Stickstoffregulators aus Mycobacterium smegmatis. Diploma thesis, Friedrich-Alexander University Erlangen-Nuremberg.

References 163

Himpens, S., Locht, C. & Supply, P. (2000). Molecular characterization of the mycobacterial SenX3–RegX3 two-component system: evidence for autoregulation. Microbiology 146, 3091-3098.

Hiratsu, K., Nakata, A., Shinagawa, H. & Makino, K. (1995). Autophosphorylation and activation of transcriptional activator PhoB of Escherichia coli by acetyl phosphate in vitro. Gene 161, 7-10.

Huang, K.-J., Lan, C.-Y. & Igo, M. M. (1997). Phosphorylation stimulates the cooperative DNA-binding properties of the transcriptional factor OmpR. Proc. Natl. Acad. Sci. USA 94, 2828-2832.

Itou, H. & Tanaka, I. (2001). The OmpR-family of proteins: insight into the tertiary structure and functions of two-component regulator proteins. J. Biochem. 129, 343-350.

Iwainsky, H. & Sehrt, I. (1967). Über die Anpassungsfähigkeit von Mycobacterium smegmatis an verschieden Stickstoffquellen. In: Sehrt, Mikro- und biochemische Untersuchungen über den Einfluss stickstoffhaltiger Substanzen auf den Stoffwechsel von Mycobakterien. PhD thesis, Humboldt-University Berlin.

Jahns, T., Zobel, A., Kleiner, D. & Kaltwasser, H. (1998). Evidence for carrier-mediated, energy dependent uptake of urea in some bacteria. Arch. Microbiol. 149, 377-383.

Jakoby, M., Nolden, L., Meier-Wagner, J., Krämer, R. & Burkovski, A. (2000). AmtR, a global repressor in the nitrogen regulation system of Corynebacterium glutamicum. Mol. Microbiol. 37, 964-977.

Javelle, A., Severi, E., Thornton, J. & Merrick, M. (2004). Ammonium sensing in Escherichia coli. Role of the ammonium transporter AmtB and AmtB-GlnK complex formation. J. Biol. Chem. 279, 8530-8538.

Jeßberger, N. (2008). Corynebacterium glutamicum AmtR - Regulation der Repressionsstringenz. Diploma thesis, Friedrich-Alexander University Erlangen-Nuremberg.

Jiang, P., Peliska, J. A. & Ninfa, A. J. (1998). Reconstitution of the signal-transduction bicyclic cascade responsible for the regulation of Ntr gene transcription in Escherichia coli. Biochemistry 37, 12795-12801.

Kamala, T., Paramasivan, C. N., Herbert, D., Venkatesan, P. & Prabhakar, R. (1994). Evaluation of procedures for isolation of nontuberculous mycobacteria from soil and water. Appl. Environ. Microbiol. 60, 1021-1024.

References 164

Kamberov, E. S., Atkinson, M. R. & Ninfa, A. J. (1995). The Escherichia coli PII signal transduction protein is activated upon binding 2-ketoglutarate and ATP. J. Biol. Chem. 270, 17797-17807.

Kanehisa, M., Goto, S., Furumichi, M., Tanabe, M. & Hirakawa, M. (2010). KEGG for representation and analysis of molecular networks involving diseases and drugs. Nucleic Acids Res. 38, 355-360.

Karlsen, A. G. & Lessel, E. F. (1970). Mycobacterium bovis nom. nov. Int. J. Syst. Bacteriol. 20, 273-282.

Keener, J. & Kustu, S. (1988). Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NtrB and NtrC of enteric bacteria: roles of the conserved amino-terminal domain of NtrC. Biochemistry 85, 4976-4980.

Keilhauer, C., Eggeling, L. & Sahm, H. (1993). Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J. Bacteriol. 175, 5595-5603.

Khan, A. & Sarkar, D. (2006). Identification of a respiratory-type nitrate reductase and its role for survival of Mycobacterium smegmatis in Wayne model. Microb. Pathog. 41, 90-95.

Khan, A., Akhtar, S., Ahmad, J. & Sarkar, D. (2008). Presence of a functional nitrate pathway in Mycobacterium smegmatis. Microb. Pathog. 44, 71-77.

Kim, A. I., Ghosh, P., Aaron, M. A., Bibb, L. A., Jain, S. & Hatfull, G. H. (2003). Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene. Mol. Microbiol. 50, 463-473.

Kirschner, P., Teske, A., Schröder, K. H., Kroppenstedt, R. M., Wolters, J. & Böttger, E. C. (1992). Mycobacterium confluentis sp. nov. Int. J. Syst. Bacteriol. 42, 257-262.

Krawczyk, J., Kohl, T. A., Goesmann, A., Kalinowski, J. & Baumbach, J. (2009). From Corynebacterium glutamicum to Mycobacterium tuberculosis - towards transfers of gene regulatory networks and integrated data analyses with MycoRegNet. Nucleic Acids Res. 37, e97.

Kronemeyer, W., Peekhaus, N., Krämer, R., Sahm, H. & Eggeling, L. (1995). Structure of the gluABCD cluster encoding the glutamate uptake system of Corynebacterium glutamicum. J. Bacteriol. 177, 1152-1158.

References 165

Lama, A., Pawaria, S. & Dikshit, K. L. (2006). Oxygen binding and NO scavenging properties of truncated hemoglobin, HbN, of Mycobacterium smegmatis. FEBS Lett. 580, 4031-4041.

Lehmann, K. B. & Neumann, R. O. (1896). Atlas und Grundriss der Bakteriologie und Lehrbuch der speciellen bacteriologischen Diagnostik. Teil II. J. F. Lehmanns Verlag, München.

Lehmann, K. B. & Neumann, R. O. (1899). Bakteriologische Diagnostik. L. F. Lehmanns Verlag, München, 2. Aufl., II. Band, 408-413.

Leigh, J. A. & Dodsworth, J. A. (2007). Nitrogen regulation in bacteria and archaea. Annu. Rev. Microbiol. 61, 349-377.

Leuchtenberger, W., Huthmacher, K. & Drauz, K. (2005). Biotechnological production of amino acids and derivates: current status and prospects. Appl. Microbiol. Biotechnol. 69, 1-8.

Malm, S., Tiffert, Y., Micklinghoff, J., Schultze, S., Joost, I., Weber, I., Horst, S., Ackermann, B., Schmidt, M., Wohlleben, W., Ehlers, S., Geffers, R., Reuther, J. & Bange, F.-C. (2009). The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis. Microbiology 155, 1332-1339.

Manganelli, R., Proveddi, R., Rodrigue, S., Beaucher, J., Gaudreau, L. & Smith, I. (2004). Sigma factors and global gene regulation in Mycobacterium tuberculosis. J. Bacteriol. 186, 859-902.

Mannhalter, C., Koizar, D. & Mitterbauer, G. (2000). Evaluation of RNA isolation methods and reference genes for RT-PCR analyses of rare target RNA. Clin. Chem. Lab. Med. 38, 171-177.

Mattison, K., Oropeza, R., Byers, N. & Kenney, L. J. (2002). A phosphorylation site mutant of OmpR reveals different binding conformations at ompF and ompC. J. Mol. Biol. 315, 497-511.

Meier-Wagner, J., Nolden, L., Jakoby, M., Siewe, R., Krämer, R. & Burkovski, A. (2001). Multiplicity of ammonium uptake systems in Corynebacterium glutamicum: role of Amt and AmtB. Microbiology 147, 135-143.

Merkens, H., Beckers, G., Wirtz, A. & Burkovski, A. (2005). Vanillate metabolism in Corynebacterium glutamicum. Curr. Microbiol. 51, 59-65.

References 166

Merrick, M. J. & Edwards, R. A. (1995). Nitrogen control in bacteria. Microbiol. Rev. 59, 604-622.

Milligan, D. L., Tran, S. L., Strych, U., Cook, G. M. &. Krause, K. L. (2007). The alanine racemase of Mycobacterium smegmatis is essential for growth in the absence of D-alanine. J. Bacteriol. 189, 8381-8386.

Mukherjee, R. & Chatterji, D. (2005). Evaluation of the role of sigma B in Mycobacterium smegmatis. Biochem. Biophys. Res. Commun. 338, 964-972.

Müller, T., Strösser, J., Buchinger, S., Nolden, L., Wirtz, A., Krämer, R. & Burkovski, A. (2006). Mutation-induced metabolite pool alterations in Corynebacterium glutamicum: towards the identification of nitrogen control signals. J. Biotechnol. 126, 440-453.

Mullis, K. B., Farone, F. A., Schar, S., Saiki, R., Horn, G. & Ehrlich, H. (1986). Specific amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51, 263-273.

Newman, E. B., Batist, G., Fraser, J., Isenberg, S., Weyman, P. & Kapoor, V. (2003a). The use of glycine as nitrogen source by Escherichia coli K12. Biochim. Biophys. Acta 421, 97-105.

Newman, D. J., Cragg, G. M. & Snader, K. M. (2003b). Natural products as sources of new drugs over the period 1981-2002. J. Nat. Prod. 66, 1022-1037.

Niederweis, M., Danilchanka, O., Huff, J., Hoffmann, C. & Engelhardt, H. (2010). Mycobacterial outer membranes: in search of proteins. Trends Microbiol. 18, 109-116.

Ninfa, A. J. & Atkinson, M. R. (2000). PII signal transduction proteins. Trends Microbiol. 8, 172-179.

Ninfa, A. J. & Jiang, P. (2005). PII signal transduction proteins: sensors of α-ketoglutarate that regulate nitrogen metabolism. Curr. Opin. Microbiol. 8, 168-173.

Nolden, L., Beckers, G., Möckel, B., Pfefferle, W., Nampoothiri, K. M., Krämer, R. & Burkovski, A. (2000). Urease of Corynebacterium glutamicum: organization of corresponding genes and investigation of activity. FEMS Microbiol. Lett. 189, 305-310.

Nolden, L., Farwick, M., Krämer, R. & Burkovski, A. (2001a). Glutamine synthetases in Corynebacterium glutamicum: transcriptional control and regulation of activity. FEMS Microbiol. Lett. 201, 91-98.

References 167

Nolden, L., Ngouoto-Nkili, C.-E., Bendt, A. K., Krämer, R. & Burkovski, A. (2001b). Sensing nitrogen limitation in Corynebacterium glutamicum: the role of glnK and glnD. Mol. Microbiol. 42, 1281-1295.

Nolden, L., Beckers, G. & Burkovski, A. (2002). Nitrogen assimilation in Corynebacterium diphtheriae: pathways and regulatory cascades. FEMS Microbiol. Lett. 208, 287-293.

O'Hare, H. M., Durán, R., Cerveñansky, C., Bellinzoni, M., Wehenkel, A. M., Pritsch, O., Obal, G., Baumgartner, J., Vialaret, J., Johnsson, K. & Alzari, P. M. (2008). Regulation of glutamate metabolism by protein kinases in mycobacteria. Mol. Microbiol. 70, 1408-1423.

Odell, L. R., Nilsson, M. T., Gising, J., Lagerlund, O., Muthas, D., Nordqvist, A., Karlén, A. & Larhed, M. (2009). Functionalized 3-amino-imidazo[1,2-a]pyridines: a novel class of drug-like Mycobacterium tuberculosis glutamine synthetase inhibitors. Bioorg. Med. Chem. Lett. 19, 4790-4793.

Ojha, A. K., Mukherjee, T. K. & Chatterji, D. (2000). High intracellular level of guanosine tetraphosphate in Mycobacterium smegmatis changes the morphology of the bacterium. Infect. Immun. 68, 4084-4091.

Ortalo-Magne, A., Lemassu, A., Laneelle, M. A., Bardou, F., Silve, G., Gounon, P., Marchal, G. & Daffé, M. (1996). Identification of the surface-exposed lipids on the cell envelopes of Mycobacterium tuberculosis and other mycobacterial species. J. Bacteriol. 178, 456-461.

Ozturk, C. E., Sanic, A., Kaya, D. & Ceyhan, I. (2005). Molecular analysis of isoniazid, rifampin and streptomycin resistance in Mycobacterium tuberculosis isolates from patients with tuberculosis in Düzce, Turkey. Jpn. J. Infect. Dis. 58, 309-312.

Parish, T. & Stoker, N. G. (2000). glnE is an essential gene in Mycobacterium tuberculosis. J. Bacteriol. 182, 5715-5720.

Pashley, C. A., Brown, A. C., Robertson, D. & Parish, T. (2006). Identification of the Mycobacterium tuberculosis GlnE promoter and its response to nitrogen availability.

Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, 2002-2007.

Philip, R. (1932). Robert Koch’s discovery of the tubercle bacillus. Am. Ref. Tuberc. 26, 637-652.

References 168

Pimentel-Schmitt, E. F., Thomae, A. W., Amon, J., Klieber, M. A., Roth, H. M., Muller, Y. A., Jahreis, K., Burkovski, A. & Titgemeyer, F. (2007). A glucose kinase from Mycobacterium smegmatis. J. Mol. Microbiol. Biotechnol. 12, 75-81.

Pitulle, C., Dorsch, M., Kazda, J., Wolters, J. & Stackebrandt, E. (1992). Phylogeny of rapidly growing members of the genus Mycobacterium. Int. J. Syst. Bacteriol. 42, 337-343.

Rastogi, N., Legrand, E. & Sola, C. (2001). The mycobacteria: an introduction to nomenclature and pathogenesis. Rev. Sci. Tech. 20, 21-54.

Read, R., Pashley, C. A., Smith, D. & Parish, T. (2007). The role of GlnD in ammonium assimilation in Mycobacterium tuberculosis. Tuberculosis 87, 384-390.

Reed, G. B. (1957). Family I mycobacteriaceae Chester 1897. In: Bergey's Manual of Determinative Bacteriology, 7th edition (ed. by Breed, Murray & Smith), 695.

Rehm, N., Georgi, T., Hiery, E., Degner, U., Schmiedl, A., Burkovski, A. & Bott, M. (2010). L-glutamine as a nitrogen source for Corynebacterium glutamicum: derepression of the AmtR regulon and implications for nitrogen sensing. Microbiology 156, 3180-3193.

Rehm, N. (2010). Biochemische und molekularbiologische Untersuchungen zur Verstoffwechselung von Ammonium und Glutamin in Corynebacterium glutamicum. PhD thesis, Friedrich-Alexander University Erlangen-Nuremberg.

Reuther, J. & Wohlleben, W. (2007). Nitrogen metabolism in Streptomyces coelicolor: transcriptional and post-translational regulation. J. Mol. Microbiol. Biotechnol. 12, 139-146.

Rexer, H. U., Schäberle, T., Wohlleben, W. & Engels, A. (2006). Investigation of the functional properties and regulation of three glutamine synthetase-like genes in Streptomyces coelicolor A3(2). Arch. Microbiol. 186, 447-458.

Reyrat, J. M. & Kahn, D. (2001). Mycobacterium smegmatis: an absurd model for tuberculosis? Trends Microbiol. 9, 472-473.

Richardson, A. (1971). The experimental production of mastitis in sheep by Mycobacterium smegmatis and Mycobacterium fortuitum. Cornell. Veterinarian. 61, 640-646.

References 169

Rodríguez-García, A., Sola-Landa, A., Apel, K., Santos-Beneit, F. & and Martín, J. F. (2009). Phosphate control over nitrogen metabolism in Streptomyces coelicolor: direct and indirect negative control of glnR, glnA, glnII and amtB expression by the response regulator PhoP. Nucleic Acids Res. 37, 3230-3242.

Rogall, T., Wolters, J., Flohr, T. & Bottger, E. C. (1990). Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. Int. J. Syst. Bacteriol. 40, 323-330.

Runyon, E. H. (1959). Anonymous mycobacteria in pulmonary disease. Med. Clin. North. Am. 43, 273-290.

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Habor Laboratory Press, Cold Spring Harbor, N. Y.

Sander, P., Meier, A. & Böttger, E. C. (1995). rpsL+: a dominant selectable marker for gene replacement in mycobacteria. Mol. Microbiol. 16, 991-1000.

Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467.

Sarada, K. V., Appaji Rao, N. & Venkitasubramanian, T. A. (1980). Isolation and characterization of glutamate dehydrogenase from Mycobacterium smegmatis CDC 46. Biochim. Biophys. Acta 615, 299-308.

Schägger, H. & von Jagow, G. (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368-379.

Schreiber, J., Burkhardt, U., Rüsch-Gerdes, S., Amthor, M., Richter, E., Zugehör, M., Rosahl, W. & Ernst, M. (2001). Nichttuberkulöse Mykobakteriose der Lungen durch Mycobacterium smegmatis. Pneumologie 55, 238-243.

Silberbach, M., Hüser, A., Kalinowski, J., Pühler, A., Walter, B., Krämer, R. & Burkovski, A. (2005a). DNA microarray analysis of the nitrogen starvation response of Corynebacterium glutamicum. J. Biotechnol. 119, 357-367.

Silberbach, M., Schäfer, M., Hüser, A. T., Kalinowski, J., Pühler, A., Krämer, R. & Burkovski, A. (2005b). Adaptation of Corynebacterium glutamicum to ammonium limitation: a global analysis using transcriptome and proteome techniques. Appl. Environ. Microbiol. 71, 2391-2402.

References 170

Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T. & Jacobs Jr., W. R. (1990). Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol. Microbiol. 4, 1911-1919.

Sohaskey, C. D. & Modesti, L. (2008). Differences in nitrate reduction between Mycobacterium tuberculosis and Mycobacterium bovis are due to differential expression of both narGHJI and narK2. FEMS Microbiol. Lett. 290, 129-134.

Son, H. S. & Rhee, S. G. (1987). Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII protein and nucleotide sequence of its structural gene. J. Biol. Chem. 262, 8690-8695.

Springer, B., Böttger, E. C., Kirschner, P. & Wallace, R. J. (1995). Phylogeny of the Mycobacterium chelonae-like organism based on partial sequencing of the 16S rRNA gene and proposal of Mycobacterium mucogenicum sp. nov. Int. J. Syst. Bacteriol. 45, 262-267.

Sritharan, V., Ratledge, C. & Wheeler, P. R. (1987). Effect of homoserine on growth of Mycobacterium smegmatis: inhibition of glutamate transport by homoserine. J. Gen. Microbiol. 133, 2781-2785.

Stahl, D. A. & Urbance, J. W. (1990). The division between fast- and slow-growing species corresponds to natural relationships among the mycobacteria. J. Bacteriol. 172, 116-124.

Strösser, J., Lüdke, A., Schaffer, S., Krämer, R. & Burkovski, A. (2004). Regulation of GlnK activity: modification, membrane sequestration and proteolysis as regulatory principles in the network of nitrogen control in Corynebacterium glutamicum. Mol. Microbiol. 54, 132-147.

Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. (1990). Use of T7 polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60-89.

Talaata, A. M., Trucksisa, M., Kaneb, A. S. & Reimschuessel, R. (1999). Pathogenicity of Mycobacterium fortuitum and Mycobacterium smegmatis to goldfish, Carassius auratus. Veterin. Microbiol. 66, 151-164.

Tatusov, R. L., Fedorova, N. D., Jackson, J. D., Jacobs, A. R., Kiryutin, B., Koonin, E. V., Krylov, D. M., Mazumder, R., Mekhedov, S. L., Nikolskaya, A. N., Rao, B. S., Smirnov, S., Sverdlov, A. V., Vasudevan, S., Wolf, Y. I., Yin, J. J. & Natale, D. A. (2003). The COG database: an updated version includes eukaryotes. BMC Bioinformatics 4. doi:10.1186/1471-2105-4-41.

References 171

Tiffert, Y., Supra, P., Wurm, R., Wohlleben, W., Wagner, R. & Reuther, J. (2008). The Streptomyces coelicolor GlnR regulon: identification of new GlnR targets and evidence for a central role of GlnR in nitrogen metabolism in actinomycetes. Mol. Microbiol. 67, 861-880.

Tiffert, Y., Franz-Wachtel, M., Fladerer, C., Nordheim, A., Reuther, J., Wohlleben, W. & Mast, Y. (2011). Proteomic analysis of the GlnR-mediated response to nitrogen limitation in Streptomyces coelicolor M145. Appl. Microbiol. Biotechnol. 89, 1149-1159.

Traag, B. A., Driks, A., Stragier, P., Bitter, W., Broussard, G., Hatfull, G., Chu, F., Adams, K. N., Ramakrishnan, L. & Losick, R. (2010). Do mycobacteria produce endospores? Proc. Natl. Sci. USA 107, 878-881.

Tsukamura, M. (1966). Adansonian classification of mycobacteria. J. Gen. Microbiol. 45, 253-273.

Tsukamura, M. (1981). Numerical analysis of rapidly growing, nonphotochromogenic mycobacteria, including Mycobacterium agri (Tsukamura 1972) Tsukamura sp. nov., nom. rev. Int. J. Syst. Bacteriol. 31, 247-258.

Tullius, M. V., Harth, G. & Horwitz, A. A. (2003). Glutamine synthetase GlnA1 is essential for growth of Mycobacterium tuberculosis in human THP-1 macrophages and guinea pigs. Infect. Immun. 71, 3927-3936.

van Crevel, R., Ottenhoff, T. H. M. & van der Meer, J. W. M. (2002). Innate immunity to Mycobacterium tuberculosis. Clin. Microbiol. Rev. 15, 294-309.

van Heeswijk, W. C., Rabenberg, M., Westerhoff, H. V. & Kahn, D. (1993). The genes of the glutamine synthetase adenylylation cascade are not regulated by nitrogen in Escherichia coli. Mol. Microbiol. 9, 443-457.

Wallace Jr., R. J., Nash, D. R., Tsukamura, M., Blacklock, Z. M. & Silcox, V. A. (1988). Human disease due to Mycobacterium smegmatis. J. Infect. Dis. 158, 52-59.

Walter, B. (2007). Funktion der Ammoniumtransporter AmtA und AmtB in Corynebacterium glutamicum. PhD thesis, Friedrich-Alexander University Erlangen-Nuremberg.

Walter, B., Küspert, M., Ansorge, D., Krämer, R. & Burkovski, A. (2008). Dissection of ammonium uptake systems in Corynebacterium glutamicum: mechanism of action and energetics of AmtA and AmtB. J. Bacteriol. 190, 2611-2614.

References 172

Wang, R. & Marcotte, E. M. (2007). The proteomic response of Mycobacterium smegmatis to anti-tuberculosis drugs suggests targeted pathways. J. Proteome Res. 7, 855-865.

Wang, J. & Zhao, G.-P. (2009). GlnR positively regulates nasA transcription in Streptomyces coelicolor. Biochem. Biophys. Res. Commun. 386, 77-81.

Wayne, L. G. (1966). Classification and identification of mycobacteria. III. Species within Group III. Amer. Rev. Resp. Dis. 93, 919-928.

Weber, I., Fritz, C., Ruttkowski, S., Kreft, A. & Bange, F.-C. (2000). Anaerobic nitrate reductase (narGHJI) activity of Mycobacterium bovis BCG in vitro and its contribution to virulence in immunodeficient mice. Mol. Microbiol. 35, 1017-1025.

Weinand, M. (2004). Expressionsregulation von Transportern kompatibler Solute in Corynebacterium glutamicum. PhD thesis, University of Cologne.

Wray, L. V. & Fisher, S. H. (1993). The Streptomyces coelicolor glnR gene encodes a protein similar to other bacterial response regulators. Gene 130, 145-150.

Yoshida, T., Qin, L., Egger, L. A. & Inouye, M. (2006). Transcription regulation of ompF and ompC by a single transcription factor, OmpR. J. Biol. Chem. 281, 17114-17123.

Yu, H., Peng, W., Liu, Y., Wu, T., Yao, Y., Cui, M., Jiang, W. & Zhao, G. P. (2006). Identification and characterization of glnA promoter and its corresponding trans-regulating protein GlnR in the rifamycin SV producing actinomycete, Amycolatopsis mediterranei U32. Acta. Biochim. Biophys. Sin. 38, 831-843.

Appendix 173

7 Appendix

7.1 Plasmid constructions

Constructions of the plasmids listed in table 2, section 3.1, are described below. Restriction

sites or exchanged bases in the primer sequences are highlighted bold.

pASK-IBA5+glnR*

glnR* variants (generated as described below: pMN016glnR*) were each amplified via PCR

using each pMN016glnR* as template and the primers 5’-G CGC CTC GAG GGA TCT ACT

GCT ACT GAC C-3’ and 5’-G CGC AAG CTT TCA CTG ACT GGT CAA C-3’. The resulting

fragments were restricted with XhoI and HindIII and cloned to the XhoI-HindIII-restricted

vector pASK-IBA5plus (IBA BioTAGnology, Göttingen). After sequencing of the constructs,

the GlnR* proteins could be purified via N-terminal Strep-tag.

pASK-IBA5+senX3*

For purification of SenX3 without its 131 amino acids N-terminal transmembrane domain via

N-terminal Strep-tag, a truncated version of the corresponding gene msmeg_0936 (senX3)

was amplified using chromosomal DNA of M. smegmatis as template and the primers 5’-G

CGC GGC GCC GTC TAC ATC GAC GAC C-3’ and 5’-G CGC CCC GGG TCA TCG TTC

TCG TTG GTC CTC-3’. The resulting fragment was restricted with KasI and XmaI and

cloned to the KasI-XmaI-restricted vector pASK-IBA5plus (IBA BioTAGnology, Göttingen).

pMal-c2-1918

For purification of Msmeg_1918 via MBP-tag the corresponding gene msmeg_1918 (1497 bp

without start codon) was amplified via PCR using chromosomal DNA of M. smegmatis as

template and the primers 5’-G CGC GAA TTC TCG ACC CTC GGT GAT CTG-3’ and 5’-G

CGC AAG CTT CTA CTG CGC GCC CCG GCT GC-3’. It was subsequently restricted with

EcoRI and HindIII and cloned to the EcoRI-HindIII-restricted vector pMal-c2 (Guan et al.,

1987). The resulting construct was sequenced.

Appendix 174

pMal-c2-5241

For purification of Msmeg_5241 via MBP-tag the corresponding gene msmeg_5241 (1713 bp


template and the primers 5’-G CGC GAA TTC GGG GGC GTG AAC GGC CA-3’ and 5’-G

CGC AAG CTT TCA GTC GGG GAG CGG CGC GG-3’. It was subsequently restricted with

EcoRI and HindIII and cloned to the EcoRI-HindIII-restricted vector pMal-c2 (Guan et al.,


pMal-c2-glnR

For purification of M. smegmatis GlnR via MBP-tag the corresponding glnR gene (783 bp


template and the primers 5’-G CGC GGA TCC GAT CTA CTG CTA CTG ACC-3’ and 5’-G

CGC AAG CTT TCA CTG ACT GGT CAA C-3’. It was subsequently restricted with BamHI

and HindIII and cloned to the BamHI-HindIII-restricted vector pMal-c2 (Guan et al., 1987).

The resulting construct was sequenced.

pMal-c2glnR M.tub.

For purification of M. tuberculosis GlnR via MBP-tag the corresponding glnR gene (765 bp

without start codon) was amplified via PCR using chromosomal DNA of M. tuberculosis as

template and the primers 5’-G CGC GAA TTC TTG GAG TTA TTA CTG CTG ACC T-3’ and

5’-G CGC AAG CTT TCA CTG ACT GCG CAA CGG GT-3’. It was subsequently restricted

with EcoRI and HindIII and cloned to the EcoRI-HindIII-restricted vector pMal-c2 (Guan et al.,


pML814-D1918kin

A 1000 bp fragment upstream of the kinase domain encoding region of msmeg_1918 was

amplified via PCR using chromosomal DNA of M. smegmatis as template and the primers 5’-

GCG CAT TTA AAT GCA GCG GTG AAA AAG CCC CG-3’ and 5’-GCG CTT AAT TAA

GGG GCT GGT CAG CCG CCG CT-3’, restricted with SwaI and PacI and cloned to SwaI-

PacI-restricted vector pML814 (M. Niederweis, personal communication). A 1000 bp

fragment downstream of the kinase domain encoding region of msmeg_1918 was amplified

via PCR using chromosomal DNA of M. smegmatis as template and the primers 5’-G CGC

ACT AGT CTG CCG CTG GTC GCC GG-3’ and 5’-GCG CGT TTA AAC GCT GGG CTT

TGG AGT CGG G-3’, restricted with SpeI and PmeI and cloned to SpeI-PmeI-restricted

Appendix 175

vector pML814 (M. Niederweis, personal communication). Subsequently, the construct was

sequenced.

pML814-D5241

A 1000 bp fragment upstream of msmeg_5241 was amplified via PCR using chromosomal

DNA of M. smegmatis as template and the primers 5’-GCG CAT TTA AAT AGT CCG GAG

CCT TCC GTG CC-3’ and 5’-GCG CTT AAT TAA GCT GGC ATT TCC GGC GGC G-3’,

restricted with SwaI and PacI and cloned to SwaI-PacI-restricted vector pML814

(M. Niederweis, personal communication). A 1000 bp fragment downstream of msmeg_5241

was amplified via PCR using chromosomal DNA of M. smegmatis as template and the

primers 5’-G CGC ACT AGT AGT CGC GAG AGG AAG GTG ACC C-3’ and 5’-GCG CGT

TTA AAC GCG ACC CAC ATC GCG G-3’, restricted with SpeI and PmeI and cloned to

SpeI-PmeI-restricted vector pML814 (M. Niederweis, personal communication).

Subsequently, the construct was sequenced.

pML814-I0426

A 500 bp fragment of msmeg_0426 was amplified via PCR using chromosomal DNA of

M. smegmatis as template and the primers 5’-GCG CGT TTA AAC TCG AAA CAC GGC

CCG GTG TG-3’ and 5’-G CGC ACT AGT CAC CCC AGT AGG TGG GTG ACT C-3’. The

resulting fragment was restricted with PmeI and SpeI and cloned to the PmeI-SpeI-restricted

vector pML814 (M. Niederweis, personal communication).

pML814-I2106


M. smegmatis as template and the primers 5’-GCG CGT TTA AAC GGC CTC CGC GCG

CAT CGT-3’ and 5’-G CGC ACT AGT GGT TCC GAC AGC ACG ATG CCG-3’. The resulting

fragment was restricted with PmeI and SpeI and cloned to the PmeI-SpeI-restricted vector

pML814 (M. Niederweis, personal communication).

pML814-I2837 (narB)

A 500 bp fragment of msmeg_2837 (narB) was amplified via PCR using chromosomal DNA

of M. smegmatis as template and the primers 5’-GCG CGT TTA AAC GCC TCA CCG ACG

ACG ACG T-3’ and 5’-G CGC ACT AGT ATG GTC GAC GTC GGT GTA CGA G-3’. The

Appendix 176



pML814-I4989


M. smegmatis as template and the primers 5’-GCG CGT TTA AAC CCC AGG TCG CGC

TGC TCG CCG TG-3’ and 5’-G CGC ACT AGT CAC CGA GTT GGG CAG TCC GGT GAC

GA-3’. The resulting fragment was restricted with PmeI and SpeI and cloned to the PmeI-

SpeI-restricted vector pML814 (M. Niederweis, personal communication).

pML814-I5140 (narG)

A 500 bp fragment of msmeg_5140 (narG) was amplified via PCR using chromosomal DNA

of M. smegmatis as template and the primers 5’-GCG CGT TTA AAC GAC CGA CTA CCC

GTC CGT G-3’ and 5’-G CGC ACT AGT CGA CTC CGG GAC ATC GGT C-3’. The resulting

fragment was restricted with PmeI and SpeI and cloned to the PmeI-SpeI-restricted vector

pML814 (M. Niederweis, personal communication).

pML814-I5143


M. smegmatis as template and the primers 5’-GCG CGT TTA AAC CGA CGT GGC GCC

GGC CAC-3’ and 5’-G CGC ACT AGT GCC AGT CCA CCG GAG AAG TTG GTG-3’. The



pMN016glnR*

Point mutations were induced into the M. smegmatis glnR gene via two-step PCR. In a first

reaction, truncated glnR fragments were amplified using pMN016glnR (Bräu, 2008) as

template. As reverse primer 5’-G CGC AAG CTT TCA CTG ACT GGT CAA C-3’ was used.

Forward primers inducing specific point mutations were: 5’-GGT AGT GCC GCC GTC GCG

ATC-3’ (Asp43Ala), 5’-GGT AGT GCC AAC GTC GCG ATC G-3’ (Asp43Asn), 5’-GGT AGT

GCC GAG GTC GCG ATC G-3’ (Asp43Glu), 5’-G ATC GTC GCC GCT CGC ACA-3’

(Asp48Ala), 5’-GCG ATC GTC AAC GCT CGC ACA G-3’ (Asp48Asn), 5’-CG ATC GTC GAG

GCT CGC ACA G-3’ (Asp48Glu), 5’-GCT CGC ACA GCT CTG GCC GC-3’ (Asp52Ala), 5’-

GCT CGC ACA AAT CTG GCC GC-3’ (Asp52Asn), 5’-C GCT CGC ACA GAG CTG GCC

Appendix 177

GC-3’ (Asp52Glu), 5’-GGT AGT GCC GCC GTC GCG ATC GTC GCC GCT CGC ACA GCT

CTG GCC GCC-3’ (Asp43/48/52Ala) and 5’-CGA TCG TCG ACG CTC GCA CAG-3’ (no

mutation, but resulting in Ala45Ser). In a second reaction, the full-length glnR* genes (786

bp) were amplified using pMN016glnR (Bräu, 2008) as template, 5’-G CGC GCA TGC TTG

GAT CTA CTG CTA CT-3’ as forward primer and the products of PCR 1 as reverse primers.

The resulting fragments were restricted with SpeI and HindIII and cloned to the SpeI-HindIII-

restricted vector pMN016 (M. Niederweis, personal communication). Subsequently, the

constructs were sequenced.

pMN016glnR-his

His-tagged glnR of M. smegmatis was expressed from non-inducible, strong promoter psmyc

in this vector. The plasmid pQE70-glnR was restricted with HindIII and SphI, resulting in a

817 bp insert consisting of glnR and the sequence of a C-terminal His-tag. The insert was

ligated to the HindIII-SphI-restricted vector pMN016 (M. Niederweis, personal

communication).

pMN016his-glnR

From the plasmid pUC19-his-glnR (M. Höller, personal communication), a 817 bp fragment

consisting of the sequence of an N-terminal His-tag and M. smegmatis glnR was amplified

via PCR using the primers 5’-CGC GCG GCA TGC ATG CAC CAC CAC CAC CAC CA-3’

and 5’-GCG CGC AAG CTT TCA CTG ACT GGT CAA CCG CC-3’. The fragment was

restricted with HindIII and SphI and cloned to HindIII-SphI-restricted vector pMN016

(M. Niederweis, personal communication). The construct was subsequently sequenced.

pMN-amt1p-gfpuv

To measure nitrogen- and GlnR-dependent promoter activity via fluorescence, a 500 bp

promoter fragment of msmeg_6259 (amt1) was amplified via PCR using chromosomal DNA

of M. smegmatis as template and the primers 5’-G CGC TCT AGA CAC CAC ACT GGC

CGG G-3’ and 5’-G CGC GCA TGC CAA ACA TCT CCT CAC GG-3’. The resulting insert

was restricted with XbaI and SphI and cloned to the XbaI-SphI-restricted vector pMN016-

gfpuv (Y. Lu, personal communication). The construct was subsequently sequenced.

Appendix 178

pMN-amtAp-gfpuv


promoter fragment of msmeg_4635 (amtA) was amplified via PCR using chromosomal DNA

of M. smegmatis as template and the primers 5’-G CGC TCT AGA GCA GGC TCA GCA

TCT CGT GG-3’ and 5’-G CGC GCA TGC AAA AAC CTC CGC AGG GGG-3’. The resulting

insert was restricted with XbaI and SphI and cloned to the XbaI-SphI-restricted vector

pMN016-gfpuv (Y. Lu, personal communication). The construct was subsequently

sequenced.

pMN-amtBp-gfpuv


promoter fragment of msmeg_2425 (amtB) was amplified via PCR using chromosomal DNA

of M. smegmatis as template and the primers 5’-G CGC TCT AGA CGC GCC GCG GTC

GA-3’ and 5’-G CGC GCA TGC TTT GTG TGA ACC TCC TTG-3’. The resulting insert was

restricted with XbaI and SphI and cloned to the XbaI-SphI-restricted vector pMN016-gfpuv

(Y. Lu, personal communication). The construct was subsequently sequenced.

pMN-gdhp-gfpuv


promoter fragment of msmeg_5442 (gdhA) was amplified via PCR using chromosomal DNA

of M. smegmatis as template and the primers 5’-G CGC TCT AGA CGC CGA CCT CGC

CAA C-3’ and 5’-G CGC GCA TGC CTC AAT CGT TCT GAT AGC-3’. The resulting insert

was restricted with XbaI and SphI and cloned to the XbaI-SphI-restricted vector pMN016-

gfpuv (Y. Lu, personal communication). The construct was subsequently sequenced.

pMN-glnAp-gfpuv


promoter fragment of msmeg_4290 (glnA) was amplified via PCR using chromosomal DNA

of M. smegmatis as template and the primers 5’-G CGC TCT AGA GCT CTC CAG CCA

CTC ACC CG-3’ and 5’-G CGC GCA TGC TGA AGC TCC TAA ATG GAC TA-3’. The

resulting insert was restricted with XbaI and SphI and cloned to the XbaI-SphI-restricted

vector pMN016-gfpuv (Y. Lu, personal communication). The construct was subsequently

sequenced.

Appendix 179

pMN-glnRp-gfpuv


promoter fragment of msmeg_5784 (glnR) was amplified via PCR using chromosomal DNA

of M. smegmatis as template and the primers 5’-G CGC TCT AGA GCG TCC TCG CGG

ATC AGC CAC G-3’ and 5’-G CGC GCA TGC CAA GTC CTC CCG GCT CGT ACG-3’. The

resulting insert was restricted with XbaI and SphI and cloned to the XbaI-SphI-restricted

vector pMN016-gfpuv (Y. Lu, personal communication). The construct was subsequently

sequenced.

pMN-lacZ

To measure nitrogen- and GlnR-dependent promoter activity via β-galactosidase activity, the

gfp gene was exchanged by lacZ. For that purpose, the lacZ gene (3069 bp) was amplified

via PCR using the plasmid pWH948 (J. Bürger) as template and the primers 5’-G CGC ACT

AGT ATG AAA GGG AAT TCA CTG GCC GTC G-3’ and 5’-G CGC AAG CTT TTA TTT TTG

ACA CCA GAC CAA CTG G-3’. The resulting insert was restricted with SpeI and HindIII,

cloned to the SpeI-HindIII-restricted vector pMN-gfpuv (Y. Lu, personal communication) and

subsequently sequenced.

pMN016-lacZ

To measure nitrogen- and GlnR-dependent promoter activity via β-galactosidase activity, the

gfp gene was exchanged by lacZ. For that purpose, the lacZ gene (3069 bp) was amplified

via PCR using the plasmid pWH948 (J. Bürger) as template and the primers 5’- GCG CTT

AAT TAA ATG AAA GGG AAT TCA CTG GCC GTC G-3’ and 5’-G CGC AAG CTT TTA TTT

TTG ACA CCA GAC CAA CTG G-3’. The resulting insert was restricted with PacI and HindIII

and cloned to the PacI-HindIII-restricted vector pMN016-gfpuv (Y. Lu, personal

communication). The plasmid was subsequently sequenced.

The same method was used generating the constructs pMN-amtAp-lacZ, pMN-amtBp-lacZ,

pMN-amtRp-lacZ, pMN-gdhp-lacZ, pMN-glnAp-lacZ and pMN-glnRp-lacZ. Here, the lacZ

gene was cloned to PacI-HindIII-restricted vectors pMN-amtAp-gfpuv, pMN-amtBp-gfpuv,

pMN-amtRp-gfpuv, pMN-gdhp-gfpuv, pMN-glnAp-gfpuv and pMN-glnRp-gfpuv.

Appendix 180

pQE70-glnR

The glnR gene from M. smegmatis (786 bp) was amplified via PCR using chromosomal DNA

as template and the primers 5’-G CGC GCA TGC TTG GAT CTA CTG CTA CT-3’ and 5’-G

GCG CGC GGA TCC CTG ACT GGT CAA CCG CC-3’. The insert was restricted with NlaIII

and BamHI, cloned to the SphI-BamHI-restricted vector pQE70 (Qiagen, Hilden) and

subsequently sequenced. This resulted in a glnR-his construct for purification of GlnR from

E. coli via a C-terminal His-tag.

7.2 Complete data obtained from DNA microarray analyses

As described in section 4.2.1, a global approach to identify genes involved in nitrogen

metabolism of M. smegmatis was carried out. Additionally, genes putatively regulated by

GlnR were found in this analysis. In a first experiment, total transcript of the M. smegmatis

wild type strain SMR5, incubated under nitrogen surplus and starvation, was compared. After

analysis of the data, 231 genes with decreased transcript levels under nitrogen starvation

were found (minimum factor 3). These genes are listed in table 9. Furthermore, 284 genes

were identified showing enhanced mRNA levels in the wild type under nitrogen starvation

(minimum factor 3; shown in table 10). When total transcript of the wild type and the glnR

deletion strain was compared under nitrogen starvation, transcripts of only 6 genes were

increased by a minimum factor of 3 in the absence of GlnR. These can be seen in table 11.

In fact, the amount of mRNA of 125 genes was decreased in the glnR deletion strain

compared to the wild type under nitrogen starvation (minimum factor 3; listed in table 12).

Additionally, total transcriptome analysis was also performed with an amtR deletion strain

resulting in two putative AmtR target genes (see table 13).

Tab. 9: List of all 231 genes with decreased transcript levels in the M. smegmatis wild type strain SMR5 under nitrogen starvation. The msmeg_ gene numbers as well as the fold change of transcripts, an annotational description and the corresponding COG numbers are given. Data are sorted by the strength of fold change in an ascending manner.



msmeg_2536 3.01 3-oxoacyl-[acyl-carrier-protein] reductase COG1028IQR

msmeg_3509 3.01 conserved hypothetical protein COG0491R

msmeg_0016 3.01 conserved domain protein COG3251S

msmeg_3792 3.01 ribosomal protein L35 (rpmI) COG0291J

msmeg_0985 3.02 sugar transporter family protein COG2814G

msmeg_6238 3.02 putative two-component system sensor kinase COG4585T

msmeg_0531 3.03 putative acyl-CoA dehydrogenase COG1960I

msmeg_3363 3.03 regulatory protein, TetR COG1309K

msmeg_6313 3.04 queuine tRNA-ribosyltransferase (tgt) COG0343J

Appendix 181

msmeg_0401 3.04 putative non-ribosomal peptide synthase COG1020Q

msmeg_3364 3.05 RhtB family transporter COG1280E

msmeg_2349 3.05 glycosyl hydrolase, family 57 COG1543S

msmeg_5061 3.06 bacterial extracellular solute-binding protein COG1653G

msmeg_3710 3.08 cytochrome b561 family protein COG3038C

msmeg_4627 3.08 nucleoside diphosphate kinase COG0105F

msmeg_0788 3.10 putative conserved membrane protein X


msmeg_2963 3.12 bacterial extracellular solute-binding proteins, family 5 COG0747E

msmeg_2853 3.12 conserved hypothetical protein COG0767Q

msmeg_0313 3.13 phosphogluconate dehydratase (edd) COG0129EG

msmeg_6315 3.13 lipoprotein LpqH X

msmeg_5525 3.13 succinyl-CoA synthetase, beta subunit (sucC) COG0045C

msmeg_1019 3.14 ribonucleoside-diphosphate reductase, alpha subunit COG0209F

msmeg_5060 3.15 ABC transporter, permease protein SugA COG1175G

msmeg_2499 3.15 ABC transporter, membrane spanning protein COG0600P

msmeg_4644 3.16 molybdopterin-guanine dinucleotide biosynthesis protein A COG0746H



msmeg_1401 3.19 translation elongation factor Tu (tuf) COG0050J

msmeg_1870 3.19 conserved hypothetical protein COG0840NT

msmeg_5897 3.21 virulence factor mce family protein COG1463Q

msmeg_6458 3.23 glutamate synthase, small subunit COG0493ER

msmeg_1524 3.24 DNA-directed RNA polymerase, alpha subunit (rpoA) X

msmeg_1525 3.24 50S ribosomal protein L17 COG2030I

msmeg_4958 3.25 diaminopimelate decarboxylase (lysA) COG0019E

msmeg_4646 3.25 pyruvate synthase COG0674C

msmeg_6404 3.25 UDP-galactopyranose mutase (glf) COG0562M

msmeg_3159 3.26 methylmalonyl-CoA mutase large subunit COG1884I

msmeg_4656 3.27 sugar ABC transporter ATP-binding protein COG1129G

msmeg_1341 3.27 MaoC family protein COG0203J

msmeg_0082 3.27 conserved hypothetical protein COG0202K

msmeg_6402 3.28 PAP2 superfamily protein X

msmeg_4111 3.29 alpha-methylacyl-CoA racemase COG1804C

msmeg_1963 3.30 putative transcriptional regulatory protein COG3629T

msmeg_4118 3.31 acyl-CoA dehydrogenase COG0267J

msmeg_0140 3.32 probable conserved mce associated membrane protein X



msmeg_1339 3.33 ribosomal protein L33 (rpmG) COG1960I

msmeg_4114 3.35 naphthoate synthase (menB) COG0447H

msmeg_5432 3.35 peptidyl-tRNA hydrolase (pth) COG0193J


msmeg_2348 3.36 glycosyl transferase, group 1 family protein X

msmeg_1023 3.36 integral membrane transporter X


msmeg_4087 3.37 major facilitator superfamily COG2271G

msmeg_3962 3.38 lactate 2-monooxygenase COG1304C

msmeg_5222 3.39 GTP-binding protein YchF (ychF) COG0464O

msmeg_6050 3.40 solute-binding lipoprotein COG0803P

msmeg_5968 3.41 polysaccharide biosynthesis protein COG2244R

msmeg_6947 3.41 chromosomal replication initiator protein DnaA (dnaA) COG0593L

msmeg_0059 3.41 ATPase, AAA family COG0012J

msmeg_3051 3.43 guanylate kinase COG0194F

msmeg_6236 3.43 two-component system, regulatory protein COG2197TK

Appendix 182

msmeg_6021 3.43 xylose isomerase (xylA) COG2115G

msmeg_1398 3.43 ribosomal protein S12 (rpsL) X

msmeg_0061 3.44 ftsk-spoiiie family protein COG0048J

msmeg_1255 3.47 UvrD-Rep helicase X

msmeg_5211 3.48 aminotransferase class-III COG5012R

msmeg_6542 3.48 B12 binding domain protein COG1674D

msmeg_0078 3.48 hypothetical protein COG0210L

msmeg_6903 3.49 transcriptional regulator, PadR family protein COG0160E

msmeg_5901 3.49 TrnB2 protein COG1695K

msmeg_0787 3.51 bacterial extracellular solute-binding proteins, family 3 COG0183I

msmeg_5199 3.51 putative acyl-CoA dehydrogenase X

msmeg_2820 3.51 hypothetical protein COG0767Q

msmeg_0055 3.52 hypothetical protein COG0834ET

msmeg_5198 3.52 carnitinyl-CoA dehydratase X

msmeg_3708 3.53 catalase COG1463Q

msmeg_0238 3.53 O-acetylhomoserine-O-acetylserine sulfhydrylase COG1832R

msmeg_6758 3.56 transport integral membrane protein COG0753P

msmeg_5923 3.56 acetyl-CoA acetyltransferase COG0183I

msmeg_4645 3.56 alpha oxoglutarate ferredoxin oxidoreductase, beta subunit X

msmeg_4086 3.57 nitrilotriacetate monooxygenase component A (ssuD) COG1013C

msmeg_6400 3.57 probable conserved transmembrane protein COG0580G

msmeg_5896 3.58 virulence factor Mce family protein COG1024I

msmeg_5899 3.58 virulence factor Mce family protein COG2141C

msmeg_0410 3.60 MmpL protein COG1841J

msmeg_6638 3.61 methyltransferase (metE) X

msmeg_5895 3.63 virulence factor mce family protein COG0458EF

msmeg_3047 3.64 carbamoyl-phosphate synthase, large subunit (carB) COG2409R

msmeg_3161 3.64 lipoprotein, putative COG1463Q

msmeg_1473 3.64 ribosomal protein L30 (rpmD) COG1463Q


msmeg_0622 3.65 putative DNA-binding protein X

msmeg_0108 3.67 acyl-CoA dehydrogenase COG1960I

msmeg_6759 3.68 glycerol kinase (glpK) COG0554C

msmeg_0062 3.68 ftsk-spoiiie family protein COG1674D

msmeg_3599 3.69 sugar-binding transcriptional regulator, LacI family COG1879G

msmeg_4116 3.71 3-hydroxybutyryl-CoA dehydrogenase COG1250I

msmeg_4561 3.72 ABC Fe3+-siderophores transporter, periplasmic binding protein COG0614P

msmeg_3197 3.73 lipase X


msmeg_6904 3.74 myo-inositol-1-phosphate synthase COG1260I

msmeg_1474 3.75 ribosomal protein L15 (rplO) COG0200J

msmeg_5371 3.78 ectoine-hydroxyectoine ABC transporter, ATP-binding protein COG1126E

msmeg_6307 3.79 glutamine-binding periplasmic protein COG0765E

msmeg_3598 3.80 periplasmic sugar-binding proteins COG1879G

msmeg_6942 3.80 membrane protein OxaA COG0706U

msmeg_0132 3.82 conserved hypothetical protein COG1804C

msmeg_0620 3.82 pe family protein X

msmeg_4756 3.83 holo-(acyl-carrier-protein) synthase (acpS) COG0736I

msmeg_1704 3.83 ABC transporter COG4213G

msmeg_0138 3.84 virulence factor Mce family protein X

msmeg_2130 3.84 putative acyl-CoA dehydrogenase COG1960I

msmeg_1705 3.85 D-xylose transport ATP-binding protein XylG COG1129G

msmeg_2073 3.86 CAIB-BAIF family protein COG0767Q

msmeg_6761 3.87 glycerol-3-phosphate dehydrogenase 2 COG0578C

msmeg_3713 3.87 hypothetical protein COG1463Q

Appendix 183

msmeg_0380 3.88 MmpS4 protein X

msmeg_6585 3.89 acyl-CoA dehydrogenase COG1960I

msmeg_4115 3.90 3-hydroxybutyryl-CoA dehydrogenase COG1250I

msmeg_1349 3.92 dgpf domain family COG3795S

msmeg_4560 3.92 periplasmic binding protein COG0614P

msmeg_0239 3.92 O-acetylhomoserine-O-acetylserine sulfhydrylase COG2873E

msmeg_1342 3.92 conserved hypothetical protein COG2030I

msmeg_0786 3.99 serine-threonine protein kinase COG0515RTK

msmeg_0068 4.00 probable conserved transmembrane protein X



msmeg_3094 4.02 oxidoreductase, inc-binding dehydrogenase family COG1063ER

msmeg_4083 4.04 putative monooxygenase COG2141C

msmeg_4530 4.05 sulfate ABC transporter, ATP-binding protein (cysA) COG1118P

msmeg_4531 4.05 sulfate ABC transporter, permease protein CysW (cysW) COG4208P

msmeg_1448 4.06 integral membrane transporter COG0659P

msmeg_4557 4.09 ABC transporter, ATP-binding protein COG1120PH

msmeg_3602 4.10 ribose transport ATP-binding protein RbsA COG1129G

msmeg_3092 4.11 transcriptional regulator, sugar-binding family COG2390K

msmeg_3603 4.11 oxidoreductase, inc-binding dehydrogenase family COG1063ER

msmeg_5902 4.11 domain of unknown function superfamily COG0767Q

msmeg_1466 4.12 ribosomal protein L24 (rplX) COG0198J

msmeg_2503 4.14 lipoprotein, putative COG0715P


msmeg_6018 4.21 xylose transport system permease protein XylH COG4214G

msmeg_1467 4.23 50S ribosomal protein L5 COG0094J

msmeg_0520 4.23 porin X




msmeg_6229 4.31 glycerol kinase (glpK) X

msmeg_6057 4.32 MspD protein COG0554C

msmeg_0143 4.34 probable conserved mce associated membrane protein X

msmeg_1469 4.34 ribosomal protein S8 (rpsH) COG0096J

msmeg_0550 4.35 sulfonate binding protein COG0715P

msmeg_4301 4.37 acyl-CoA synthase COG2409R

msmeg_4532 4.38 sulfate ABC transporter, permease protein CysT (cysT) COG0555O

msmeg_0382 4.39 putative transport protein COG0318IQ

msmeg_6391 4.41 propionyl-CoA carboxylase beta chain COG0609P


msmeg_4559 4.42 ABC transporter, membrane spanning protein COG4799I

msmeg_0381 4.42 Mmp14a protein COG2409R


msmeg_1807 4.46 acetyl--propionyl-coenyme A carboxylase alpha chain COG4770I

msmeg_1443 4.47 ribosomal protein L16 (rplP) COG0197J



msmeg_6459 4.53 ferredoxin-dependent glutamate synthase 1 COG0069E

msmeg_0131 4.54 AMP-binding enzyme, putative COG0318IQ

msmeg_2534 4.56 putative carboxylesterase protein COG0596R

msmeg_5058 4.59 ABC transporter, ATP-binding protein SugC COG3839G

msmeg_2619 4.65 efflux protein COG2814G

msmeg_1741 4.65 TetR-family transcriptional regulator X

msmeg_3058 4.70 lipoprotein, nlpa family COG1464P

msmeg_1472 4.71 ribosomal protein S5 (rpsE) COG0098J

Appendix 184

msmeg_1442 4.72 ribosomal protein S3 (rpsC) COG0092J

msmeg_1436 4.74 ribosomal protein L3 (rplC) COG0087J

msmeg_3538 4.74 cyclopropane-fatty-acyl-phospholipid synthase 1 COG2230M

msmeg_1435 4.76 ribosomal protein S10 (rpsJ) COG0051J

msmeg_3095 4.77 D-ribose-binding periplasmic protein COG1879G

msmeg_0141 4.80 probable conserved mce associated transmembrane protein COG1714S


msmeg_2121 4.84 multiphosphoryl transfer protein (MTP) COG1080G


msmeg_1444 4.85 ribosomal protein L29 (rpmC) COG0255J

msmeg_4210 4.86 secreted protein COG3181S

msmeg_0549 4.87 ABC transporter, permease protein COG0600P


msmeg_1471 4.88 ribosomal protein L18 (rplR) COG0256J

msmeg_1350 4.91 cyclopropane-fatty-acyl-phospholipid synthase 1 COG2230M

msmeg_0134 4.97 virulence factor Mce family protein COG1463Q

msmeg_1465 5.03 ribosomal protein L14 (rplN) COG0093J


msmeg_0084 5.12 phosphocarrier protein hpr COG1925G

msmeg_1437 5.12 ribosomal protein L4-L1 family (rplD) COG0088J

msmeg_1445 5.15 30S ribosomal protein S17 COG0186J

msmeg_0135 5.18 virulence factor Mce family protein COG1463Q

msmeg_5059 5.21 ABC transporter, permease protein SugB COG0395G

msmeg_1439 5.21 ribosomal protein L2 (rplB) COG0090J

msmeg_1468 5.28 ribosomal protein S14p-S29e (rpsN) COG0199J

msmeg_1438 5.39 ribosomal protein L23 (rplW) COG0089J


msmeg_1440 5.64 ribosomal protein S19 (rpsS) COG0185J

msmeg_5418 5.95 iron permease FTR1 COG0672P

msmeg_0133 6.02 ABC-transporter integral membrane protein COG0767Q



msmeg_0137 6.09 virulence factor mce family protein COG1463Q

msmeg_5412 6.12 immunogenic protein MPT63 X

msmeg_6392 6.23 polyketide synthase COG3321Q

msmeg_0085 6.39 PTS system, Fru family, IIABC components COG1299G

msmeg_1583 6.58 chaperonin GroL (groL) COG0459O

msmeg_6242 6.65 alcohol dehydrogenase, iron-containing COG1454C


msmeg_0880 6.92 chaperonin GroL (groL) COG0459O

msmeg_0020 7.19 periplasmic binding protein COG0614P

msmeg_4326 7.50 acyl carrier protein (acpP) COG0236IQ

msmeg_5435 7.53 acyl-CoA synthase COG0318IQ

msmeg_4329 7.55 propionyl-CoA carboxylase beta chain COG4799I

msmeg_5420 7.77 Tat-translocated enzyme COG2837P

msmeg_1582 7.97 chaperonin GroS (groS) COG0234O

msmeg_4325 7.98 malonyl CoA-acyl carrier protein transacylase COG0331I


msmeg_4328 8.25 3-oxoacyl-[acyl-carrier-protein] synthase 2 COG0304IQ

msmeg_0530 8.35 short chain dehydrogenase COG1028IQR

msmeg_4757 9.08 fatty acid synthase COG4981I

msmeg_1813 9.18 propionyl-CoA carboxylase beta chain COG4799I

msmeg_4327 9.32 3-oxoacyl-[acyl-carrier-protein] synthase 1 COG0304IQ


msmeg_1811 11.24 septum formation protein Maf (maf) COG0424D

Appendix 185

msmeg_5589 12.60 manganese transport protein MntH COG1914P

msmeg_5419 14.63 lipoprotein X

Tab. 10: List of all 284 genes with increased transcript levels in the M. smegmatis wild type strain SMR5 under nitrogen starvation. The msmeg_ gene numbers as well as the fold change of transcripts, an annotational description and the corresponding COG numbers are given. Data are sorted by the strength of fold change in an ascending manner.



msmeg_6612 3.01 ATPase, MoxR family (moxR) COG0714R


msmeg_2416 3.01 conserved hypothetical protein COG3599D

msmeg_0451 3.02 oxidoreductase, FAD-linked X

msmeg_6506 3.02 nicotinamidase-pyrainamidase COG1335Q


msmeg_3561 3.04 glutamine synthetase, catalytic domain (glnA3) COG0174E

msmeg_3137 3.05 oxidoreductase COG1902C



msmeg_2916 3.06 DNA-binding response regulator, PhoP family COG0745TK




msmeg_0755 3.07 cobalt-inc-cadmium resistance protein COG1230P

msmeg_3254 3.07 RDD family, putative X

msmeg_2112 3.07 secreted protein X



msmeg_1301 3.12 NanT3 COG2197TK

msmeg_5014 3.12 copper-translocating P-type ATPase COG2217P

msmeg_1883 3.12 glycine betaine transporter OpuD COG1292M

msmeg_3816 3.13 excinuclease ABC, B subunit (uvrB) COG0556L

msmeg_0171 3.13 histone deacetylase superfamily X



msmeg_0267 3.20 esterase COG0657I

msmeg_2428 3.21 DNA-binding protein X


msmeg_0752 3.22 fructose-bisphosphate aldolase, class II (fbaA) COG0191G

msmeg_1552 3.22 ethanolamine permease (eat) COG1113E

msmeg_4546 3.23 oxidoreductase X

msmeg_4417 3.25 methionine-S-sulfoxide reductase (msrA) COG0225O



msmeg_3372 3.28 transcriptional regulator, ArsR family COG0640K

msmeg_2960 3.28 preprotein translocase, YajC subunit (yajC) COG1862U

msmeg_5935 3.29 ATP-dependent DNA helicase COG0514L

msmeg_1755 3.29 anti-sigma factor, ChrR COG1917S


msmeg_3138 3.29 thioredoxin (trx) COG3118O



msmeg_2696 3.31 putative conserved membrane alanine rich protein X


Appendix 186

msmeg_6212 3.33 hemerythrin HHE cation binding domain subfamily, putative X



msmeg_6477 3.38 methionine-S-sulfoxide reductase (msrA) COG0225O

msmeg_0172 3.38 probable conserved transmembrane protein, putative X






msmeg_1501 3.42 methyltransferase, putative, family COG3315Q

msmeg_6616 3.44 S-(hydroxymethyl)glutathione dehydrogenase COG1063ER


msmeg_1802 3.46 ChaB protein X



msmeg_6416 3.51 phosphoglycerate mutase family protein COG0406G

msmeg_0051 3.52 transcription factor WhiB family X

msmeg_3289 3.57 gp61 protein COG0596R


msmeg_6879 3.59 ABC-type Nat permease for neutral amino acids NatD COG0559E

msmeg_4465 3.59 cutinase X

msmeg_6876 3.60 branched chain amino acid transport ATP-binding protein COG0410E

msmeg_2913 3.65 hydrolase COG0596R

msmeg_1595 3.65 putative oxidoreductase COG4221R

msmeg_1769 3.68 UsfY protein X








msmeg_0637 3.78 iron-sulfur binding oxidoreductase COG0665E

msmeg_0585 3.79 L-carnitine dehydratase-bile acid-inducible protein F COG1804C

msmeg_1762 3.79 piperideine-6-carboxylic acid dehydrogenase COG1012C

msmeg_3022 3.80 transglycosylase associated protein X


msmeg_2925 3.84 permease membrane component COG1174E


msmeg_1605 3.85 phosphate transport system regulatory protein PhoU (phoU) COG0704P

msmeg_0074 3.88 IS1549, transposase COG0123BQ


msmeg_3862 3.91 FxsA cytoplasmic membrane protein X

msmeg_6213 3.91 Manganese containing catalase COG3546P

msmeg_6877 3.94 branched-chain amino acid transporter, ATP-binding protein COG0411E


msmeg_1698 3.96 putative ammonia monooxygenase superfamily COG3180R

msmeg_0911 3.97 isocitrate lyase (aceA) COG2224C

msmeg_3564 3.99 bacterioferritin (bfr) COG2193P



msmeg_2659 4.01 alanine dehydrogenase (ald) COG0686E


msmeg_5078 4.04 glucose-1-phosphate adenylyltransferase (glgC) COG0448G

Appendix 187




msmeg_0072 4.06 IS1549, transposase, interruption-C COG5421L

msmeg_1991 4.10 isovaleryl-CoA dehydrogenase COG1960I

msmeg_1088 4.13 glutamyl-tRNA(Gln)-aspartyl-tRNA (Asn) amidotransferase COG0154J

msmeg_1411 4.15 universal stress protein family COG0589T

msmeg_3902 4.16 ATPase, AAA family COG0464O


msmeg_2343 4.16 methylesterase COG4638PR


msmeg_1787 4.18 RsbW protein X

msmeg_5542 4.22 transcriptional regulator, HTH_3 family COG1476K

msmeg_6480 4.23 putative transcriptional regulatory protein COG1309K





msmeg_5016 4.29 conserved domain protein COG2608P

msmeg_5764 4.30 putative cyanamide hydratase X

msmeg_6354 4.33 serine esterase, cutinase family COG0400R



msmeg_4073 4.39 DNA-binding protein X


msmeg_3471 4.44 GTP cyclohydrolase COG0807H



msmeg_1530 4.51 integral membrane protein COG5006R


msmeg_6874 4.63 aldehyde dehydrogenase COG1012C

msmeg_5374 4.66 glutamate--ammonia ligase COG0174E






msmeg_4765 4.80 transcriptional regulator, MerR family COG0789K


msmeg_1771 4.82 methylase, putative COG2890J

msmeg_6467 4.85 starvation-induced DNA protecting protein COG0783P

msmeg_1791 4.90 UsfY protein X


msmeg_5402 4.92 dehydrogenase DhgA COG0300R

msmeg_4542 4.93 oligopeptide transport integral membrane protein COG1173EP

msmeg_3255 5.00 DoxX subfamily, putative COG2259S





msmeg_5617 5.24 immunogenic protein MPT63 X



msmeg_5375 5.32 GntR-family transcriptional regulator COG2186K


Appendix 188

msmeg_1134 5.38 putative protease HtpX COG0501O




msmeg_2695 5.52 35 kDa protein COG1842KT


msmeg_5341 5.56 dipeptidyl aminopeptidase-acylaminoacyl peptidase COG2267I


msmeg_1786 5.67 stas domain, putative COG1366T



msmeg_4989 5.94 sensor histidine kinase COG0642T

msmeg_2798 5.95 hypothetical protein COG1380R

msmeg_6223 5.97 TetR family transcriptional repressor LfrR X

msmeg_6478 6.02 putative cytochrome P450 135B1 COG2124Q



msmeg_5275 6.15 permease of the major facilitator superfamily COG4760S

msmeg_4298 6.16 3-methyl-2-oxobutanoate hydroxymethyltransferase (panB) COG0413H




msmeg_1234 6.26 taurine import ATP-binding protein TauB COG1116P

msmeg_1794 6.38 dehydrogenase COG2141C

msmeg_5072 6.40 extracytoplasmic function alternative sigma factor COG1595K


msmeg_3371 6.45 short-chain dehydrogenase-reductase SDR X

msmeg_0436 6.68 allophanate hydrolase subunit 1 COG2049E




msmeg_1131 6.94 tryptophan-rich sensory protein COG3476T





msmeg_1076 7.12 lipoprotein, putative X



msmeg_5485 7.50 molybdopterin biosynthesis protein COG0521H

msmeg_5015 7.51 secreted protein X


msmeg_0434 7.70 aminoglycoside 2-N-acetyltransferase (AAC(2)-Id) X


msmeg_6225 7.89 proton antiporter efflux pump COG2271G





msmeg_0435 8.36 allophanate hydrolase subunit 2 COG1984E


msmeg_2694 8.40 transcriptional regulator, XRE family X

msmeg_2752 8.63 sigma factor SigB COG0568K

msmeg_1951 8.68 conserved domain protein X


Appendix 189



msmeg_1030 9.06 monooxygenase COG2072P









msmeg_4990 10.73 DNA-binding response regulator COG0745TK




msmeg_1415 11.30 AsnC-family transcriptional regulator COG1522K



msmeg_1764 11.97 L-lysine-epsilon aminotransferase COG0160E


msmeg_3400 13.26 glutamyl-tRNA (Gln) amidotransferase subunit A COG0154J



msmeg_0586 14.47 stas domain, putative X

msmeg_5486 14.84 peptidase S1 and S6, chymotrypsin-Hap COG0265O

msmeg_4206 15.10 Molybdopterin oxidoreductase X








msmeg_2427 16.95 protein PII uridylyltransferase (glnD) COG2844O





msmeg_1413 21.40 ornithine--oxo-acid transaminase (rocD) COG4992E

msmeg_5358 21.65 acetamidase-formamidase family COG2421C
















Appendix 190





msmeg_2426 31.84 nitrogen regulatory protein PII (glnK) COG0347E



msmeg_2982 35.17 putative periplasmic binding protein COG0683E




msmeg_6734 44.58 dibenothiophene desulfuriation enyme A COG2141C







msmeg_1414 98.67 amidinotransferase COG1834E

Tab. 11: List of all 6 M. smegmatis genes that showed increased transcript levels (factor higher than 3) in the glnR deletion strain MH1 compared to the wild type SMR5 under nitrogen starvation. The msmeg_ gene numbers as well as the fold change of transcripts, an annotational description and the corresponding COG numbers are given. Data are sorted by the strength of fold change in an ascending manner.




msmeg_2274 4.24 hydrogenase assembly chaperone HypC-HupF (hypC) COG0298O

msmeg_2275 4.37 hydrogenase expression-formation protein HypD (hypD) COG0409O

msmeg_1738 4.46 probable conserved transmembrane protein X



Table 12: List of all 125 M. smegmatis genes that showed decreased transcript levels by a factor higher than 3 in the glnR deletion strain MH1 compared to the wild type SMR5 under nitrogen starvation. The msmeg_ gene numbers as well as the fold change of transcripts, an annotational description and the corresponding COG numbers are given. Data are sorted by the strength of fold change in an ascending manner.



msmeg_3358 3.02 YaeQ protein COG4681S






msmeg_4011 3.26 putative pyrimidine permease RutG COG2233F

msmeg_2116 3.33 PTS system, glucose-specific IIBC component COG1263G





Appendix 191




msmeg_2189 3.68 allophanate hydrolase (atF) COG0154J


msmeg_1153 3.73 FAD dependent oxidoreductase X



msmeg_3626 3.89 urease, beta subunit (ureB) COG0832E


msmeg_1152 4.01 citrate-proton symporter COG2814G



msmeg_5783 4.10 acetyltransferase, GNAT family X

msmeg_1155 4.16 carnitinyl-CoA dehydratase COG1024I



msmeg_0565 4.33 putative glycosyl transferases group 1 COG0438M


msmeg_1156 4.44 dihydrodipicolinate synthetase COG0329EM



msmeg_1088 4.63 glutamyl-tRNA(Gln)-aspartyl-tRNA(Asn) amidotransferase COG0154J

msmeg_0505 4.72 probable sugar ABC transporter, substrate-binding protein COG1653G


msmeg_6879 4.97 Nat permease for neutral amino acids NatD COG0559E






















msmeg_6877 8.90 branched-chain amino acid transporter, ATP-binding protein COG0411E








Appendix 192










msmeg_4206 16.44 molybdopterin oxidoreductase X

msmeg_3400 17.66 glutamyl-tRNA(Gln) amidotransferase subunit A COG0154J


msmeg_2427 18.22 protein PII uridylyltransferase (glnD) COG2844O













msmeg_5360 22.01 formate-nitrate transporter COG2116P






msmeg_5358 25.10 acetamidase-Formamidase family COG2421C



















msmeg_2426 51.56 nitrogen regulatory protein PII (glnK) COG0347E





Appendix 193

msmeg_6734 62.84 dibenothiophene desulfuriation enzyme A COG2141C


msmeg_2982 136.17 putative periplasmic binding protein (urtA) COG0683E

Tab. 13: List of two genes with increased transcript levels in M. smegmatis ΔamtR compared to the wild type under nitrogen starvation. The msmeg_ gene numbers as well as the fold change of transcripts, an annotational description and the corresponding COG numbers are given.



msmeg_2188 2.35 integral membrane protein X


7.3 Putative sensor histidine kinases activating GlnR

In a bioinformatic search for sensor histidine kinases able to phosphorylate and thus activate

GlnR, 22 putative candidates have been found (Amon, 2010). Criteria were appearance in

every genome with a GlnR homolog, no appearance in corynebacterial genomes and an

orphan location in the genome. Results of this approach are shown in table 14.

Tab. 14: List of all 22 sensor histidine kinases putatively interacting with GlnR found in a bioinformatic approach. Given are the predicted open reading frames with their corresponding annotation number for the respective genome and their identity in percentage according to BLASTP results. On the right, the putative role based on literature research and homology searches is given. n/a: not available. Adapted from Amon (2010).

M. smegmatis other mycobacteria

N. farcinica streptomycetes corynebacteria putative role

msmeg_5158 n/a NFA17980 (69%)

SCO1217 (49%)

SAV7118 (50%)

n/a LytS (regulation of cell autolysis)

msmeg_2793 yes (all) NFA6630 (46%)

n/a n/a PrrB

(mult. copies) msmeg_5663

msmeg_0246

msmeg_5304 n/a NFA46340 (54%)

SCO5435 (48%)

SAV2816 (47%)

CG0089 (33%) CitA regulating citrate/malate metabolism ?

msmeg_3239

(no orphan)

MAP_3274 (65%)

NFA54920 (48%)

SCO2121 (38%)

SAV6081 (39%)

n/a msmeg_3240 LuxR-type regulator, conserved across species…

Appendix 194

msmeg_5870 yes (all except ML)

NFA5450 (54%)

SAV4197 (46%) SCO4021 (49%)

CG2887 (44%) [JK0342 (48%), DIP1935 (45%), CE2493 (41%)]

PhoR ribonucleotide biosynthesis (mult. copies)

msmeg_4989

msmeg_0106

(no orphan)

yes (all except ML)

n/a SCO1369 (33%) CG2201 28% ?

msmeg_6864 n/a NFA12320 (32%)

SCO7562 (35%) CG3388 25% ?

msmeg_2248 n/a n/a n/a n/a ?


n/a CG2457 (49%) DIP1680 (53%), JK0667 (51%), CE2135 (50%)

?

msmeg_4211

no orphan, cit?

n/a n/a SCO1137 (43%) n/a similar msmeg_5304


SCO5239 (46%) SAV3017 (46%)

n/a ? (VERY good candidate)

msmeg_2915

(no orphan!)

yes (all except ML)

n/a … … similar to msmeg_4989 /5870…

msmeg_0980

(no orphan)

n/a n/a … … similar to msmeg_2248

msmeg_3448

(no orphan)

msmeg_2804

(no orphan)

msmeg_5241 yes (all except ML)

NFA28940 (63% BLASTN)

SCO0203 (61%)

SAV4257 (59%)

n/a “GAF family protein”; orphan, also very good candidate

msmeg_0854 n/a NFA7820 SCO6163/6424/5784 n/a

msmeg_1493 (no orphan)

n/a n/a n/a n/a

msmeg_4968 (no orphan)

n/a n/a SCO4597/4598 n/a

msmeg_0936

no orphan!

Rv0490 (all) NFA51870 SCO4229/sav3973 cg0483/ce0424 “SenX3”

Appendix 195

7.4 Abbreviations and units

A adenine

ACP* acetyl [32P] phosphate

ADP adenosine diphosphate

AMP adenosine monophosphate

AmpR resistance to ampicillin

APS ammonium peroxodisulfate

ATCC american type culture collection

ATP adenosine triphosphate

BCIP 5-bromo-4-chloro-3-indolyl phosphate toluidine salt

BSA bovine serum albumine

C cytosine

CamR resistance to chloramphenicol

CIP calf intestine phosphatase

CM cytoplasmic membrane

CMN corynebacteria, mycobacteria, nocardia

COG cluster of orthologous groups

CSPD disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2´-(5´-chloro)tricyclo

[3.3.1.13´7]decan}-4-yl)phenylphosphate

CTAB cetyl trimethyl ammonium bromide

ddUTP dideoxyuridine triphosphate

DIG digoxigenin

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

dNTP deoxynucleotide triphosphate

DSM(Z) Deutsche Sammlung von Mikroorganismen (und Zellkulturen)

DTT dithiothreitol

e.g. for example

EDTA ethylendiaminetetraacetic acid

Fig. figure

FRT Flpe recognition target

et al. et alii

G guanine

g 9.81 m s-2

G3PDH glyceraldehyde 3-phosphate dehydrogenase

GDH glutamate dehydrogenase

GFP green fluorescent protein

GOGAT glutamate synthase (glutamine:oxoglutarate-aminotransferase)

GS glutamine synthetase

HABA 2-(4-hydroxyphenylazo)benzoic acid

Appendix 196

i.e. that is

IPTG isopropyl β-D-1-thiogalactopyranoside

KanR resistance to kanamycin

MALDI-ToF-MS matrix-assisted laser desorption/ionisation time-of-flight mass

spectrometry

MBP maltose binding protein

MOM mycobacterial outer membrane

MOPS 3-morpholinopropane-1-sulfonic acid

MOTT mycobacteria other than tuberculosis

MSX L-methionine-DL-sulfoximine

+N nitrogen surplus

-N nitrogen starvation

NAD(P)H nicotinamide adenine (phosphate) dinucleotide

NBT 4-nitro blue tetrazolium chloride

NCTC national collection of type cultures

oD600 optical density at 600 nm

ONPG ortho-nitrophenyl-β-D-galactopyranoside

ORF open reading frame

PAA polyacrylamide

PAGE polyacrylamide gel electrophoresis

PBS phosphate buffered saline

PCR polymerase chain reaction

RNA ribonucleic acid

RT reverse transcriptase

SDS sodium dodecylsulphate

SSC sodium chloride/sodium citrate

StrR resistance to streptomycin

t time

T thymine

Tab. table

TAE tris acetate EDTA

TBE tris boric acid EDTA

TBS tris buffered saline

TE tris EDTA

TEN tris EDTA sodium chloride

TEMED N,N,N’,N’-tetramethyl-ethylendiamine

TKMD tris, potassium chloride, magnesium chloride, DTT

TMAO trimethylamine N-oxide

Tris 2-amino-hydroxymethylpropane-1,3-diol

TSS transformation and storage solution

UMP uridine monohosphate

UTP uridine triphosphate

Appendix 197

UV ultraviolet

v/v volume per volume

w/v Units

weight per volume

A Ampère

°C degree Celsius

bar unit of pressure

bp base pairs

Da Dalton

F Farad

g gram

kb kilo base pairs

l liter

m meter

M molar

min minutes

mol chemical amount

Ω Ohm

rpm rounds per minute

s seconds

U unit

V volt

Prefixes

k kilo

m milli

µ micro

n nano

p pico

Appendix 198

Amino acid nomenclature (IUPAC-IUB, 1969)

A Ala alanine

C Cys cysteine

D Asp aspartate

E Glu glutamate

F Phe phenylalanine

G Gly glycine

H His histidine

I Ile isoleucine

K Lys lysine

L Leu leucine

M Met methionine

N Asn asparagine

P Pro proline

Q Gln glutamine

R Arg arginine

S Ser serine

T Thr threonine

V Val valine

W Trp tryptophane

Y Typ tyrosine

Appendix 199

7.5 Publications

Amon, J., Bräu, T., Grimrath, A., Hasselt, K., Hänßler, E., Höller, M., Jeßberger, N., Ott, L., Szököl, J., Titgemeyer, F. & Burkovski, A. (2008). Nitrogen control in Mycobacterium smegmatis: nitrogen-dependent expression of ammonium transport and assimilation proteins depends on OmpR-type regulator GlnR. J. Bacteriol. 21, 7108-7116.

Muhl, D., Jeßberger, N., Hasselt, K., Jardin, C., Sticht, H. & Burkovski, A. (2009). DNA binding by Corynebacterium glutamicum TetR-type transcription regulator AmtR. BMC Mol. Biol. 10, 37.

Jeßberger, N., Lu, Y., Amon, J., Niederweis, M., Titgemeyer, F., Sonnewald, S., Reid, S. & Burkovski, A. (2011). The GlnR-dependent nitrogen regulatory network of Mycobacterium smegmatis. Mol. Microbiol. Submitted.

2 material und methoden - opus 4 · at least three glnr binding sites were identified in the...

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