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CHAPTER FOUR

Pleiomorphism in MycobacteriumLeif A. Kirsebom1, Santanu Dasgupta, Brännvall M. Fredrik PetterssonDepartment of Cell and Molecular Biology, Biomedical Centre, Uppsala, Sweden1Corresponding author: e-mail address: [email protected]

Contents

1.

AdvISShttp

Introduction

ances in Applied Microbiology, Volume 80 # 2012 Elsevier Inc.N 0065-2164 All rights reserved.://dx.doi.org/10.1016/B978-0-12-394381-1.00004-0

82
2. Morphological Variations in Bacteria 84
2.1
E. coli and some other Gram-negative bacteria 86 2.2 Gram-positive bacteria—Actinomycetes and Firmicutes 88
3.
Mycobacterium spp.—Morphology and Pleiomorphism 91 3.1 Overview of different morphologies among Mycobacterium spp. 91 3.2 Sporulation in Mycobacterium spp. 92
4.
Symmetric Versus Asymmetric Cell Division 94 4.1 Control of septum formation in the rod-shaped
bacteria E. coli and B. subtilis
94 4.2 Septum formation and cell division in Mycobacterium spp. 95 4.3 Expression and regulation of FtsZ 96
5.
Regulatory Genes and Spore Formation in the Firmicutes 97 6. Regulatory Genes Involved in the Sporulation Pathway—B. subtilis Versus
Mycobacterium spp.
100 6.1 Sensors, kinases, and phosphatases 100
7.
Domestication—“You Study What You Select For” 102 8. Concluding Remarks 103 Acknowledgments 104 References 104
Abstract

Morphological variants in mycobacterial cultures under different growth conditions, in-cluding aging of the culture, have been shown to include fibrous aggregates, biofilms,coccoids, and spores. Here we discuss the diversity in shape and size changes demon-strated by bacterial cells with special reference to pleiomorphism observed inMycobac-terium spp. in response to nutritional and other environmental stresses. Inherentasymmetry in cell division and compartmentalization of cell interior under differentgrowth conditions might contribute toward the observed pleiomorphism inmycobacteria. The regulatory genes comprising the bacterial signaling pathway res-ponsible for initiating morphogenesis are speculated upon from bioinformaticidentifications of genes for known sensors, kinases, and phosphatases existing in myco-bacterial genomes as well as on the basis of what is known in other bacteria.

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http://dx.doi.org/10.1016/B978-0-12-394381-1.00004-0

82 Leif A. Kirsebom et al.

1. INTRODUCTION

Bacteria of the genus Mycobacterium are acid-fast, hardy, and they in-

habit various environmental reservoirs, for example, ground and tap water,

soil, animals, and humans. TheMycobacterium genus includes nonpathogenic

environmental bacteria, opportunistic pathogens, and highly successful

pathogens such as Mycobacterium tuberculosis. While M. tuberculosis is human

specific, the closely related Mycobacterium bovis can infect both humans and

animals and lead to tuberculosis regardless of the host. Other diseases, such as

leprosy (Hansen’s disease) and Buruli ulcer are caused byMycobacterium leprae

and Mycobacterium ulcerans, respectively. In fact, M. leprae was the first Myco-

bacterium spp. that was identified (Hansen, 1880). Buruli ulcer is an emerging

disease in tropical areas, and although it is the third most common infection

caused by aMycobacterium spp., it is one of the most neglected. Another path-

ogenic Mycobacterium spp. is the multi-host, chronic enteric pathogen

M. avium subsp. paratuberculosis. It causes chronic diarrhea among ruminants

( Johne’s disease) and can be present in food products derived from cattle.

Some data suggest that M. avium subsp. paratuberculosis is the causative agent

of Crohn’s disease in humans; however, this is highly controversial. These

and some other Mycobacterium spp. are extremely slow growing under lab-

oratory conditions and require handling in special laboratories (M. leprae has

not yet been cultivated on plates or in liquid media). It is not uncommon

that the generation time for the slow-growing Mycobacterium spp. exceeds

20 h. Moreover, there are also some fast-growing Mycobacterium spp. The

environmental bacteria, M. smegmatis and M. phlei, have a generation time

of roughly 2 and 4–5 h, respectively, under optimal laboratory conditions.

These faster growing strains, in particular, M. smegmatis, are widely used

to study the molecular biology of Mycobacterium spp. in general. Moreover,

the genus ofMycobacterium spp. is highly diverse, and the number of known

Mycobacterium spp. is growing rapidly with more than 30 new species

identified between 2003 and 2006 (Tortoli, 2003, 2006). Many of these

are isolated from patients, in particular, from immune-compromised

patients.

Mycobacterium spp., like other bacteria, compete for nutrients and have

developed sophisticatedways to adapt to different environmental conditions.

These adaptations include transitions from planktonic growth to formation

of biofilms, cord-like aggregates, coccoids, and spores (Fig. 4.1). They

Figure 4.1 Micrographs showing different cell morphologies of derivatives of M. mar-inum (strain T CCUG 20998 corresponding to ATCC 927). (A) Characteristic growth andcording of M. marinum attB::rfp-HygR (RFP, red fluorescent protein linked to the KanR

gene integrated at the L5 attB site on the chromosome). The cells were grown in7H9 medium in the presence of 100 mg/ml hygromycin and harvested in late exponen-tial phase. (B) Planktonic growth of M. marinum attB::rfp-HygR. The cells were preparedas described in (A). (C) A spore observed in a 16-day-oldM. marinum attB::gfp-KanR (GFP,green fluorescent protein linked to the KanR gene integrated at the L5 attB site on thechromosome) culture grown on 7H10 medium in the presence of 25 mg/ml kanamycin.The left image shows phase contrast, while the right shows fluorescence of the samefield. The integrating plasmids carrying the RFP and GFP encoding genes linked togenes encoding for HygR and KanR, respectively, were kindly provided by Dr. D. G. Ennisand Ms. A. Mallick, University of Louisiana, Lafayette, USA).

83Cell Shape Morphology Variation in Mycobacterium spp.

respond to different stress situations such as nutrient starvation and oxidative

stress by making changes in their metabolism as well as in the cell shape,

which implies that the transcriptome has been altered. Many Mycobacterium

spp. are also invasive and can grow and multiply within cells such

as macrophages. Inside the macrophage, they reside within a membrane-

bound cytoplasmic vacuole, the mycobacteria phagosome. To achieve this,

pathogenic Mycobacterium spp. are equipped with an arsenal of tools to pre-

vent phagosomal degradation in this hostile environment. Ultimately, this

ensures survival and spread of the bacteria inside its host. Mycobacterium

spp. can also establish long-term infections that cause acute or chronic stages

of disease or a stage that is clinically asymptomatic. This asymptomatic in-

fection or latent stage is a long-term hidden threat to the host, since it

can result in active disease after long periods of dormancy. In the persistent

stage,Mycobacterium spp. such asM. tuberculosis reside in granulomas, which are

organized collections of differentiated macrophages and other cell types. It has

been hypothesized that within the granulomas, the bacteria are in a dormant,

nonreplicative state or in a state where the number of bacteria is controlled by

the immune system (Cosma, Sherman, & Ramakrishnan, 2003; Falkinham,


2011; Flynn & Chan, 2005; Koul, Herget, Klebl, & Ullrich, 2004; Nguyen &

Pieters, 2005; Primm, Lucero, & Falkinham, 2004; Russell, 2007;

Vaerewijick, Huys, Palomino, Swings, & Portaels, 2005).

In summary, the diversity of ecological niches inhabited byMycobacterium

spp. demands widely varied life styles with different growth patterns and

morphologies. Even the same bacterial strain responds to variations in the

environment, such as aging culture, oxygen deprivation, heat or cold shocks,

pH changes, and exposure to toxins/antibiotics or to the hostile immune

system of the host cell. This ability to switch to alternate life styles by global

shift in the transcriptome makesMycobacterium spp. into one of the most suc-

cessful human pathogens. However, very little is known about the signals

that induce the wide ranging pleiomorphism among Mycobacterium spp.

In fact, our knowledge about the diversity of the morphological variations

undertaken byMycobacterium spp. is rather limited and sporadic. At the same

time, an understanding of the biology and physiology of the members of the

genusMycobacterium is of utmost importance in medical care, food and water

quality, and the public health care in general. Here we will review our cur-

rent understanding of the pleiomorphism ofMycobacterium spp. compared to

other bacteria. In this context, we note that ever since M. tuberculosis was

isolated in 1882 (Koch, 1882), the existence of different cell morphologies

amongMycobacterium spp. has been discussed as well as whether the cell shape

can change dependent on growth conditions. Recent and previous data

clearly suggest that members of the genus Mycobacterium spp. indeed show

variation in cell morphology. We will discuss some of the morphological

variants thatMycobacterium spp. appear to demonstrate in response to known,

as well as unknown environmental signals.

2. MORPHOLOGICAL VARIATIONS IN BACTERIA

Biofilms. In their natural habitats,manybacteria have the capacity to form

biofilms, a complex structure that is mainly composed of bacteria held in a stra-

tum comprising extracellular polymeric substances, proteins, polysaccharides,

amyloid fibers, and extracellular DNA. Bacteria that grow in a protective bio-

film environment are more resistant to stress and tolerate exposure to higher

levels/concentrations of different antibiotics in comparison to bacteria in

planktonic states.Hence, understanding the underlyingmechanisms of biofilm

formation and its control is of significantmedical importance.Discussion about

bacterial biofilms is beyond the scope of the present review, and for further

reading, we refer to recent reviews on this topic (see, e.g., Fux, Costerton,


Stewart, & Stoodley, 2005; Hall-Stoodley & Stoodley, 2005; McDougald,

Rice, Barraud, Steinberg, & Kjelleberg, 2012).

Changes in shape. Most bacteria possess characteristic shapes that are

apparently maintained by their outer boundaries or walls. Here the synthesis

of the exocytoskeleton, the peptidoglycan layer, plays an essential role as do

the activities of the genes involved in this process. The shapes vary among

different bacteria, as exemplified by rod shapes for Escherichia coli and Bacillus

subtilis and by coccoids for Streptococcus pneumoniae. The recent discovery of

bacterial homologues of eukaryotic tubulin, actin, and intermediate filaments

(FtsZ, MreB, and Crescentin, respectively; Table 4.1) has revealed that in-

ternal dynamic cytoskeletal structures provide the subcellular architecture

responsible for giving the bacterial cell its characteristic shape. These cyto-

skeletal proteins appear to play an essential role in the localization of pepti-

doglycan synthetase and thereby contribute heavily in shape formation

(Osborn & Rothfield, 2007; Young, 2007). However, this seems to be a

feature unique to rod-shaped bacteria; coccoid bacteria in general lack an

MreB homologue and might have alternate mechanisms for the

maintenance of cell shape and polarity. For further reading on this

interesting topic, we refer to some excellent reviews (Cabeen & Jacobs-

Wagner, 2005; Harold, 2007; Koch, 2005; Typas, Banzhaf, Gross, &

Vollmer, 2011; Young, 2006, 2010). In addition to MreB, the tubulin-like

Table 4.1 Genes involved in cell division in different bacteria discussed in the main textGene and its function E. coli B. subtilis Mycobacterium

Slm/nucleoid occlusion Yes No No

Noc/nucleoid occlusion No Yes No

MinCDE/septum positioning Yes No No

MinCDDivIVA No Yes No

Wag31/DivIVA analog and growth marker

at the cell poles

No Yes Yes

FtsZ/tublin like protein that forms the septum ring Yes Yes Yes

MreB and Mbl/cytoskelatel protein Yes Yes No

RodA/control of peptidoglycan synthesis during

cellular elongation

Yes Yes Yes

PbpA/peptidoglycan synthetase,

penicillin-binding protein 2

Yes Yes Yes


protein FtsZ plays an essential role in cell division by guiding the divisome

assembly and eventual septum formation to the mid-cell region in

coordination with genome duplication and chromosome segregation. The

role of FtsZ will be discussed in more detail below.

Bacteria have a remarkable ability to adapt to environmental changes like

temperature, nutrient starvation, population density, and oxidative stress.

An important aspect is also survival in stationary phase compared to growth

in exponential phase. Response to environmental changes might also result

in altered shapes. Many bacteria also have the capacity to enter a dormant

state upon exposure to hostile conditions such as nutrient starvation and ex-

posure to heavy metals or toxins, and this too might be associated with a

change in shape. The best-known example of this is the formation of spores,

and here B. subtilis has been the paradigm for bacterial differentiation as

manifested through endosporulation (see, e.g., Hilbert & Piggot, 2004).

However, there are alternate developmental pathways triggered in other

bacteria in response to changes in the environment resulting in morpholog-

ical changes. Below we will provide a few examples of altered morphologies

in different bacterial strains. First, we will discuss morphological alterations

due to environmental and genetic changes in some Gram-negative bacteria

and Gram-positive bacteria. This will be followed by a discussion of the cell

morphology, including sporulation, in Mycobacterium spp.

2.1. E. coli and some other Gram-negative bacteria

2.1.1 E. coliThe best-studied Gram-negative bacterium, E. coli, changes its morphology

in response to environmental changes and as a result of mutations. It converts

into shorter rods of uniform length in the stationary phase compared to

the exponential phase; however, the ratio between length and diameter

remains unchanged (Koch, 2005). Interestingly, strains of uropathogenic

E. coli go through a developmental program that results in long filaments

during the infectious cycle. This formation of filaments is due to the

inhibition of cell division (Justice et al., 2004; Justice, Hunstad, Cegelski,

& Hultgren, 2008; Justice, Hunstad, Seed, & Hultgren, 2006).

Morphological changes and long filaments have also been observed when

E. coli is grown under starvation for a long time on different supports

(Wainwright, Canham, Al-Wajeeh, & Reeves, 1999). E. coli is also

capable of forming branched cells when the chromosome replication or

nucleoid segregation is disturbed (Akerlund, Nordstrom, & Bernander,


1993). Antibiotics that act on cell wall synthesis have a positive effect on

branching, and it appears that the physiological state of the cell induces

this branching (Gullbrand, Akerlund, & Nordstrom, 1999).

2.1.2 Legionella pneumophila and Helicobacter pyloriThe etiological agent of the Legionnaires’ disease, the rod-shaped

L. pneumophila, has been reported to grow in a “cyst-like” form with a

spore-like envelope composed of a multilayer outer boundary when grown

in HeLa cells (Faulkner & Garduno, 2002; Garduno, Garduno, Hiltz, &

Hoffman, 2002). This cyst-like L. pneumophila form is referred to as MIF

(mature intracellular form) and is highly resilient and infectious (Molofsky

& Swanson, 2004). Another Gram-negative bacterium of medical

importance is the spiral bacteria H. pylori, which changes its morphology

to a coccoid form as the culture ages, approaching stationary phase and

during other stress. This coccoid form of H. pylori appears to be

nonculturable but viable; the coccoid form has, however, been

controversial ever since it was described (Andersen & Rasmussen, 2009;

Worku, Sidebotham, Walker, Keshavarz, & Karim, 1999 and references

therein). These two Gram-negative bacteria illustrate that changes in cell

morphology can have an impact on growth and pathogenicity.

2.1.3 Myxococcus xanthus and Rhodobacter johriisp. nov.—Spore-forming Gram-negative bacteria

One example of a spore-forming Gram-negative bacterium is the

d-proteobacteria Myxobacteria among whichMyxococcus xanthus is the best

studied. These bacteria can form fruiting bodies during phosphate or carbon

starvation; within these fruiting bodies spores are produced (Kaiser, 2006;

Kroos, 2007). Another example of a spore-forming Gram-negative

bacterium is the a-proteobacterium R. johrii sp. nov. that produces spores

presumably via an endosporulating pathway. It should be noted that these

spores have increased concentrations of dipicolinic acid, as also seen in

the endospore-forming Firmicutes, for example, B. subtilis (see below;

Girija et al., 2010; Hilbert & Piggot, 2004).

2.1.4 Streptobacillus moniliformisThe Gram-negative S. moniliformis belongs to the phylum Fusobacteria.

Its common habitat is the ileal epithelium in rats and mice, and it displays

interesting life forms. When this filamentous endospore-forming bacterium

reproduces, it generates either an endospore with a clear septum or two new


cells. These newones are referred to as holdfast-bearing cells; they are released

by themother cell and can attach themselves to the epithelium. S.moniliformis

can also cause a human infectionknownas rat-bite fever (Angert, 2005;Chase

& Erlandsen, 1976; Gaastra, Boot, Ho, & Lipman, 2009).

The examples above show that Gram-negative bacteria undergo differ-

entiation that results in variation in cell shape and morphology including

endosporulation.

2.2. Gram-positive bacteria—Actinomycetes and Firmicutes

2.2.1 Corynebacterium spp.In 1884, Klebs and Loffler discovered Corynebacterium diphtheriae, the caus-

ative agent of diphtheria (Holmes, 2000). Early microscopy studies revealed

that cultures of this high GC-content Gram-positive bacteria (which be-

longs to the order actinomycetales, that is, the same order as Mycobacterium

spp. and Streptomyces spp., see below) showed different morphologies.

Apparently, C. diphtheriae can grow both as rods and as coccoids as well

as form long filaments. The shape and filamentation depend on the growth

conditions (Davis & Mudd, 1954; Denny, 1903; Hewitt, 1951). Moreover,

early isolates showed that the diphtheria bacillus yielded two types of

colonies, rough and smooth ones (Yu, 1930). Interestingly, in an electron

microscopy study, Kawata and Inoue (1965) observed remarkable bodies

inside aged C. diphtheria cells. As discussed above, MreB or its homologue

is a common cytoskeletal element for rod-shaped bacteria. However,

corynebacteria seem to lack MreB but nevertheless exist as rods. In other

rod-shaped bacterium such as E. coli and B. subtilis, the cell walls are

synthesized laterally. In contrast, the new cell walls in Corynebacterium spp.

are synthesized at the poles as revealed from studies of C. glutamicum (Letek

et al., 2008). Perhaps the lack of MreB homologues confers upon

Corynebacterium spp. the capability to change its shape dependent on growth

conditions. In this context, we note that overexpression of specific serine/

threonine protein kinases (PknA or PknB) in C. glutamicum results in

coccoid shape (Fiuza et al., 2008; see below). Hence, these kinases might be

part of a pathway involved in regulating the shapes of the bacteria.

Interestingly, the gene organization of pknA and pknB in the three

actinomycetes Streptomyces coelicolor, M. tuberculosis, and C. glutamicum is well

conserved (Molle & Kremer, 2010). Together, these observations might

indicate that switching into different shapes and growth depending on the

environmentmightbe ageneral characteristicof the actinomycetes (seebelow).


2.2.2 Streptomyces spp., Salinispora gen. nov., Nocardia spp.,Rhodococcus spp., and Micromonosporaceae

Another well-studied group of actinomycetes is Streptomyces spp., for exam-

ple, the soil-living S. coelicolor that produces several antibiotics (Bentley et al.,

2002; Hopwood, 1999 and references therein). S. coelicolor is a spore-

producing bacterium that undergoes distinct morphological changes

when it grows on surfaces. Hence, it has been a model organism to study

cell differentiation. Starting from a spore, it first grows out as vegetative

mycelia, characterized by branching cells that grow on and into solid

growth media. At a certain point, less branched and longer aerial mycelia

called aerial hyphae start to project from the surface. After several

synchronized cell divisions at the tip of the aerial mycelium, the cycle is

completed by production of spores (Fig. 4.2A; Wildermuth & Hopwood,

1970). Streptomyces spores are exospores and are less resistant to physical

and chemical stress than B. subtilis endospores (see below). However, it has

been reported that certain Streptomycetes, Streptomyces globisporus and

Streptomyces avermitilis, produce endospores (Filippova et al., 2005; Stastna

et al., 1992). For a discussion of the underlying genetic regulation of cell

differentiation in Streptomyces, we refer to the following reviews (Chater &

Chandra, 2006; Claesson, de Jong, Dijkhuizen, & Wosten, 2006; Flardh &

Buttner, 2009). There are also examples of actinomycetes that produce

nonmotile spore particles without forming aerial hyphae (Ara & Kudo,

Figure 4.2 Micrographs showing different cell shapes of Streptomyces coelicolor andNocardia asteroides. (A) S. coelicolor was grown for 32 h on a 45� slanted coverslip buriedinto Soya Flour-Mannitol (SFM) plate, and the cells were mounted with 50% glycerol on aglass slide and the phase contrast image was taken using a Zeiss Axoplan 2 microscope.The exospores appear as chains of round particles marked with arrows. This image waskindly provided by Dr. S. Bagchi (Uppsala University). (B) N. asteroides cells were grown inGYM Streptomyces liquidmedia for 13 days at 30 �C before theywere subjected tomicros-copy using the microscope mentioned above. (C) N. asteroides cells were grown on GYMStreptomyces plates for 13 days at 30 �C. A coverslip was pressed against the colonies andsubjected to microscopy using the microscope mentioned earlier.


2006; Asano &Kawamoto, 1986). The marine actinomycete Salinispora gen.

nov. produces substrate hyphae with a single spore or a cluster of spore

particles (Maldonado et al., 2005). Other actinomycetes, Rhodococcus spp.

and Nocardia spp., also show variations in cell morphology when grown

under different growth conditions (Fig. 4.2B and C). Nocardia spp. appears

to form spores while this has not yet been observed for any of the

Rhodococcus spp. (Bradley, 1959; Brown-Elliot, Brown, Conville, &

Wallace, 2006; Larkin, Kulakov, & Allen, 2006 and references therein).

Other actinomycetes that form spores are the bacteria of the family

Micromonosporaceae, for example, Micromonospora chalcea. Two types of

mycelia are generated where the spores are produced from branched

mycelium with frequent septation. These spores are not endospores, but

they are different from the spore of Streptomycetes (Luedemann & Casmer,

1973; Suarez & Hardisson, 1985). These examples show morphological

diversity among the actinomycetes, and this will be further accentuated

when we discussMycobacterium spp. (see below).

2.2.3 Firmicutes—Bacillus halodurans and Bacillus subtilisShifting pH can also affect bacterial shape as exemplified by B. halodurans,

a facultative alkaliphilic low GC-content Gram-positive bacterium belong-

ing to the phylum Firmicutes. It can grow at pH values ranging from 6.8 to

10.8; at pH above 7.5, it is rod shaped, whereas below this pH it has a coiled

morphology. The data suggest that the Mbl protein, an MreB homologue,

does not form a helical structure in the coil-shaped cells, but it does form

it in rod-shaped cells at pH 10. Hence, the level of Mbl protein and/or

its capacity to form helical structures appears to influence B. halodurans

morphology (Fujinami, Sato, & Ito, 2011).

Among the Firmicutes, the soil-living Gram-positive endosporulating

bacterium B. subtilis has been widely used as a model system to study gene

regulatory circuits, in particular,with respect to cell differentiation anddevel-

opment.Toendure different growth conditions,B. subtilishas developedvar-

ious survival strategies. Upon nutrient limitations, it expresses genes involved

in motility and chemotaxis, and when reaching stationary growth phase, it

starts to produce and secrete degradative enzymes. At this stage, cells also pro-

duce antibiotics, which gives it a competitive edge. Nutrient stress can also

lead to the development of competence as well as formation of endospores.

Entering the sporulation pathway is, however, irreversible,whereas the other

physiological states are reversiblewhen the cells again have access to nutrients,

for example, additionof freshmedia (Hamoen,Venema,&Kuipers, 2003 and


references therein). That spore formation is the ultimate resort to ensure sur-

vival of the population is further emphasized by the finding that before enter-

ing the sporulation pathway some cells produce toxins that kill neighboring

cells. This releases nutrients that allow the toxin-producing cells to continue

to grow and thereby delay initiation of the sporulation pathway (Ellermeier,

Hobbs, Gonzalez-Pastor, & Losick, 2006; Gonzalez-Pastor, Hobbs, &

Losick, 2003; see also Stragier, 2006). Expression of degrading enzymes,

development of competence, and entering the sporulation pathway are

dictated by intricate genetic regulatory circuits involving different

components. These include two-component signaling systems,

transcription factors, repressors, phosphorelay systems, small peptides,

proteases, and different s-factors. Interestingly, specific labeling of proteinsto mark cell status together with imaging studies reveals that in colonies of

B. subtilis, several distinct differentiated cell types are present. These motile

cells, matrix-producing cells, competent cells, and sporulating cells have

the same genotype but differ in phenotype and as such ensure that the

population can quickly respond to changes in growth conditions (Dubnau

& Losick, 2006; Hamoen et al., 2003; Lopez & Kolter, 2010; Lopez,

Vlamakis, & Kolter, 2008; Schultz, Wolynes, Jacob, & Onuchic, 2009;

Smits, Kuipers, & Veening, 2006; Veening & Kuipers, 2010; Veening,

Smits, Hamoen, & Kuipers, 2006; Veening, Smits, & Kuipers, 2008).

3. MYCOBACTERIUM SPP.—MORPHOLOGY ANDPLEIOMORPHISM

3.1. Overview of different morphologies amongMycobacterium spp.

Like the bacteriaCorynebacterium and Streptomyces,Mycobacterium spp. belongs

to the actinomycetes, and in cultures, these cells grow in “clumps”, referred

to as cording, which is different from planktonic growth (Fig. 4.1). Ever

since the identification ofM. tuberculosis as the causative agent of tuberculosis

(Koch, 1882), it has been discussed whetherMycobacterium spp. changes cell

shape during growth (Kahn, 1929 and references therein). It was early

reported that the timothy grass bacillus, Mycobacterium phlei, passes from

rod shape to coccoid upon aging (Fig. 4.3; Csillag, 1970; Juhasz, 1962;

Stewart-Tull, 1965; Wyckoff & Smithburn, 1933). Similar observations

were made and reported for the slow-growing M. tuberculosis (see, e.g.,

Csillag, 1961; Kahn, 1929; Lack & Tanner, 1953). Kahn followed the

growth of individual M. tuberculosis cells in single droplets for several

Figure 4.3 Cultures of M. phlei cells grown on Long's agar (for details, see Wyckoff andSmithburn, 1933). (A) M. phlei cells after 12 h of growth (B) and (C). The same field as in(A) but after prolonged incubation where the cells in (C) are the oldest. The arrows in (C)mark coccoid cells. This figure was reprinted with permission from the Oxford UniversityPress Publication.


generations, and he reported (1929) changes in cell shape, from rod to

coccoid, over time. Moreover, early work even suggested the presence of

spore-like structures in M. tuberculosis cultures (Brieger & Glaubert, 1956;

Csillag, 1961, 1963, 1964). However, Hilson questioned the coccoid “form

2 mycobacteria” (Csillag, 1961) and concluded that this was due to a

contamination (Hilson, 1965). More recent studies have shown that there

are Mycobacterium spp. that indeed can grow as coccoids or are pleomorphic

coccobacilli. This is exemplified by the environmental bacteriaMycobacterium

chlorophenolicum and the pathogens Mycobacterium septicum sp. nov. and

Mycobacterium elephantis. In comparison to M. tuberculosis, these Mycobacterium

spp. are considered to be fast growing (Haggblom et al., 1994; Schinsky

et al., 2000; Tortoli, 2003; Turenne et al., 2002). Microscopic images of

dormant forms of Mycobacterium smegmatis and M. tuberculosis also show cells

with more coccoid-like structures referred to as ovoid cells. Some spore-like

particles in these images are also observed (Anuchin et al., 2009; Shleeva

et al., 2011). Interestingly, images of extensive and extremely drug resistant

strains of M. tuberculosis reveal coccoid cells and it appears that these round

cells are formed inside the rod-shaped cells (Farnia et al., 2010) similar to

what has been observed in aging cultures of C. diphtheriae (Kawata & Inoue,

1965; see above). These observations clearly show that the cell shape and

morphology ofMycobacterium spp. depend on the growth conditions.

3.2. Sporulation in Mycobacterium spp.Contrary to common knowledge and description of Mycobacterium spp. as

nonsporulating bacteria in microbiology textbooks, we have provided

experimental evidence for sporulation in old cultures of the fish pathogen


Mycobacterium marinum and the M. bovis bacillus Calmette-Guerin bacillus

(BCG) strain (Ghosh et al., 2009; Figs. 4.1C and 4.4). A recent report also

showed that spores are present in old cultures of different strains ofM. avium

subsp. paratuberculosis (Lamont, Bannantine, Armien, Ariyakumar, &

Sreevatsan, 2012). Irrespective of the Mycobacterium spp., all transmission

electronmicrographs of spore particles show similar morphologies (Fig. 4.4).

In addition, old cultures of spore-forming Mycobacterium spp. contain an

increased concentration of dipicolinic acid, a biochemical hallmark for

endosporulating bacteria such as B. subtilis (Hilbert & Piggot, 2004). To rule

out the possibility of contamination, Sreevatasan and coworkers and we per-

formed rigorous control experiments (Ghosh et al., 2009; Lamont et al.,

2012). Furthermore, the observations reported by Lamont et al. (2012)

are also based on experiments done independently in two laboratories.

Moreover, we have introduced the genes encoding the green fluorescent

Figure 4.4 Transmission electron microscopy images of spore particles of different My-cobacterium spp. (A) M. marinum (see also Ghosh et al., 2009; Singh et al., 2010; imagetaken by Dr. J. Ghosh). (B) M. bovis BCG (see also Ghosh et al., 2009; image taken byDr. J. Ghosh). (C and D) Spores from two different derivatives of M. avium subsp.paratuberculosis, Ben and Linda as indicated. The image was taken from Fig. 4.4 inLamont et al. (2012), open license access, PLoS One.


protein and kanamycin resistance into the L5 attB site on the chromosome

of a M. marinum ATCC927 strain. This strain was grown in the presence

of kanamycin and we detected fluorescent, spore-like particles in old

cultures, confirming that they indeed were derived from M. marinum

ATCC927 (Fig. 4.1C; see also Singh, Ghosh, Islam, Dasgupta, & Kirsebom,

2010). Traag et al. (2010) could not repeat our original findings and

hence questioned whether Mycobacterium spp. could form spores via an

endosporulating pathway. One major argument here was the bioinformatics

(see below); Mycobacterium spp. lack many of the sporulation genes that

have been shown to be essential for endosporulating bacteria such as

B. subtilis (Hilbert & Piggot, 2004). However, elsewhere we have argued

that combining all our data provides a solid empirical foundation that

Mycobacterium spp. indeed can form spores (Singh et al., 2010). The recent

finding that also M. avium subsp. paratuberculosis sporulates supports our

observations and extends the number of Mycobacterium spp. known to form

spores (Lamont et al., 2012).

4. SYMMETRIC VERSUS ASYMMETRIC CELL DIVISION

Septum formation and cell division play important roles in cell

morphology. Below we summarize what is known about the placement

of septum and cellular growth in model systems such as E. coli and B. subtilis

and compare them with similar processes in Mycobacterium spp.

4.1. Control of septum formation in the rod-shapedbacteria E. coli and B. subtilis

Most of theMycobacterium spp. that havebeen identified and characterized form

rod-shapedcells.However, there are exceptions (see above).BothE. coli andB.

subtilis are rod shaped; they grow along their axis and divide by binary fission.

Cell division takes place after the formation of a division septum in themid-cell

region after near completionof replication and segregationof the twodaughter

chromosomes into opposite halves of the cell. The division site is placed pre-

cisely atmid-cell in coordinationwith cell growth, replication, and segregation

of the chromosome.This is of critical importance for propagation, and bacteria

suchasE. coliandB. subtilishaveevolvedelaboratedivisionsite selection systems

to ensuremid-cell division and equipartition.Thewell-conserved tubulin-like

protein FtsZ is responsible for mid-cell placement of the septum, which is the

nucleating site for the assembly of the division machinery referred to as the

divisome. Accumulated data show that MinCDE (MinCDDivIVA in


B. subtilis) prevents FtsZ-ring placement at the poles, and the nuclear occlusion

factors Slm (E. coli) and Noc (B. subtilis) have a role in preventing septum for-

mation over the nucleoids, thus ensuring mid-cell placement of the septum.

Cell growth in both E. coli and B. subtilis occurs by lateral synthesis of nascent

peptidoglycans along the helical MreB (Mbl) protein (Adams & Errington,

2009; Bernhardt & de Boer, 2005; Errington, Daniel, & Scheffers, 2003;

Margolin, 2001; Rothfield, Taghbalout, & Shih, 2005; Wu & Errington,

2004; Wu et al., 2009). Different factors involved in cell division are

summarized in Table 4.1.

4.2. Septum formation and cell division in Mycobacterium spp.In Mycobacterium spp., cell division also occurs after placement of the FtsZ

ring that leads to a typical V-shape of dividing cells (Dahl, 2004; Lack

and Tanner, 1953; Thanky, Young, & Robertson, 2007; B Singh et al.,

unpublished data). Interestingly, while septum formation in E. coli and

B. subtilis is symmetric, asymmetric placement of the FtsZ ring is frequently

observed in Mycobacterium spp. cell cultures growing in exponential

phase (Fig. 4.5; Aldridge et al., 2012; Singh et al., 2010; Thanky et al.,

2007). It should be noted that no MinCDE system, Slm, Noc, or MreB

have yet been identified in Mycobacterium spp. (Table 4.1). However, in

C. glutamicum that lacks MreB and MinCD, the FtsZ ring can assemble over

the nucleoids before segregation. This confirms the absence of a nucleoid

occlusion system in this group of bacteria (Ramos et al., 2005). Another

striking difference with respect to E. coli and B. subtilis is that Mycobacterium

Figure 4.5 Transmission electron microscopy images of exponentially growingM. phlei.(A) Field shows cells of different lengths where some contain more than one chromo-some. Note also the placement of septum that appears to be symmetric and asymmetric.(B) A cell with a likely asymmetric septum formation,markedwith a black arrow. The grayarrows mark the tight cell–cell contact points. The white round spheres inside the cellsmight be vacuoles; however, at present, we do not know what they represent. The cellswere grown in 7H9 medium at 37 �C and harvested in exponential phase.


spp. display apical growth where the peptidoglycan precursors are added at the

cell poles (Aldridge et al., 2012;Thanky et al., 2007;BSingh et al., unpublished

data). Endospore-forming bacteria, such as B. subtilis, undergo symmetric cell

division during vegetative growth. Formation of polar septa leading to

asymmetric cell division is the first morphological step in the

endosporulation pathway (Errington, 2003; Errington et al., 2003; Hilbert

& Piggot, 2004). Cell division in Mycobacterium spp. involves asymmetric

septal placement in growing cells, which need to be converted to binary

fission with equipartition of the chromosome to prevent loss of genomic

information. Evidently, this also includes the need for a robust control

system that ensures that the sporulation pathway is not triggered during

normal growth.

4.3. Expression and regulation of FtsZThe essential FtsZ protein is widely conserved among the bacteria (Adams &

Errington, 2009), and blocking septum formation leads to a filamentous phe-

notype (Goehring & Beckwith, 2005). Its level and activity are regulated in

response to growth conditions and environmental changes. A comparison

of different bacteria shows that the function of FtsZ appears to be similar al-

though the expression level and regulation of FtsZ can vary (Roy &

Ajitkumar, 2005; Roy, Anand, Vijay, Gupta, & Ajitkumar, 2011 and

references therein). The ftsZ gene is part of the dcw (division and cell wall)

cluster in different bacteria. The gene organization of this cluster is similar

in E. coli, B. subtilis, and M. tuberculosis (see, e.g., Tamames, Gonzalez-

Moreno, Mingorance, Valencia, & Vicente, 2001). In B. subtilis, two of

the three promoters involved in the transcription of ftsZ depend on sA

(the house keeping s-factor during vegetative growth), while the third is

driven by sH and is active during sporulation (Gonzy-Treboul, Karmazyn-

Campelli, & Stragier, 1992). In M. tuberculosis and M. smegmatis, ftsZ is also

transcribed from multiple promoters, and in M. tuberculosis, expression from

these promoters requires different s-factors: the house keeping sA and the

alternative s-factors sC, sE, sF, and sH. A comparison of B. subtilis and

M. tuberculosis suggests that none of the alternative s-factors in the latter

corresponds to sH in B. subtilis (Gruber & Gross, 2003; Rodrigue,

Provvedi, Jacques, Gaudreau, & Manganelli, 2006; Sachdeva, Misra, Tyagi,

& Singh, 2010). Additionally, it has been shown that M. tuberculosis FtsZ is

phosphorylated by PknA (Thakur & Chakraborti, 2006; see above), an

essential kinase in Mycobacterium spp. (Chao et al., 2010; Molle & Kremer,

2010; Wehenkel et al., 2008). Phosphorylation of FtsZ impairs the


polymerization and septum formation. These differences in the regulation and

activity of FtsZmight provide a key to our understanding of the cell shape and

different life forms of Mycobacterium spp. and hence warrant further studies.

Given the importance of FtsZ, it has also been explored as a drug target

(Kumar et al., 2010).

Recently a previously unidentified, septum site-determining protein,

Ssd (rv3660c), was described in M. tuberculosis H37Rv. The Ssd protein be-

longs to a group of FtsZ regulatory proteins, and it was identified in a search

for genes encoding putative minD- and ssd-like orthologs. However,

rv3660c shows less similarity to minD compared to ssd. The observation that

expression of ssd inM. tuberculosis andM. smegmatis promoted filamentation

is consistent with that rv3660c being involved in septum site placement

(England, Crew, & Slayden, 2011). The identification of ssd and character-

ization of its function may lead to a more detailed understanding of why

Mycobacterium spp. display both symmetric and asymmetric septum forma-

tion in growing cultures and how the cell division machinery copes with

such asymmetry. The molecular mechanisms of septum formation and reg-

ulation of cell division may explain the morphological pleiomorphism and

its control of Mycobacterium spp. For further reading and identification of

genes involved in regulation of cell division in Mycobacterium spp., we refer

the reader to recent reports (Dziedzic et al., 2010; Hett & Rubin, 2008;

Kiran et al., 2009; Maloney, Madiraju, & Rajagopalan, 2009; Slayden &

Belisle, 2009; Slayden, Knudson, & Belisle, 2006; Srinivasan, Rajeswari,

Bhatt, Indi, & Ajitkumar, 2007; Thanky et al., 2007; Vadrevu et al., 2011

and references therein).

5. REGULATORY GENES AND SPORE FORMATIONIN THE FIRMICUTES

As discussed above, sporulation has been extensively studied in the

Firmicutes, in particular, in B. subtilis, which produce one spore from one

mother cell (for reviews, see, e.g., Errington, 2003; Hilbert and Piggot,

2004; Paredes, Alsaker, & Papoutsakis, 2005). However, there are

significant variations in sporulation among the different Firmicutes. For

example, there are multiple-spore-forming bacteria such as Metabacterium

polyspora and Epulopiscium that go through a sporulation pathway as a

means of reproduction. Moreover, the phototrophic Heliobacterium forms

endospores with a low frequency in its ecological niche, but when

cultivated as a pure culture, it ceases to form spores (Kimble-Long &


Madigan, 2001). The hydrogenogen Carboxydothermus hydrogenoformans

Z-2901 forms spores despite its lack of many of the known sporulation

genes present in B. subtilis (Wu et al., 2005). A comparison of Clostridium

acetobutylicum and B. subtilis reveals that the former, which is considered to

belong to a more ancestral phylogenetic line than Bacillus, lacks Spo0F and

Spo0B. These phosphotransferases are essential for the phosphorylation of

Spo0A, the master regulator of the sporulation pathway in the Firmicutes

(Durre, 2011). Phosphorylation of Spo0A in C. acetobutylicum is performed

by two alternative, recently identified kinases (Steiner et al., 2011). The

data also suggest that there are differences in Clostridium spp. and B. subtilis

with respect to the regulation of expression of the spore-specific

s transcription factors such as the activation of sF by SpoIIE (Bi, Jones,

Hess, Tracy, & Papoutsakis, 2011; Jones, Hess, Tracy, & Papoutsakis, 2011).

In the future, it will be interesting to find if there are other differences in the

sporulation pathway among the Firmicutes.

In the case ofMycobacterium spp., bioinformatics reveals that many of the

genes specific for sporulation cannot be identified while some can be (de

Hoon, Eichenberger, & Vitkup, 2010; Ghosh et al., 2009; Lamont et al.,

2012; Traag et al., 2010). However, most of the orthologue spore genes so

far identified also have other functions in the cell; some such examples are

presented in Table 4.2. One of the key enzymes in endosporulating

bacteria is dipicolinic acid synthase. It is composed of two subunits

encoded by spoVFA and spoVFB (Hilbert & Piggot, 2004), both of which

are absent in Mycobacterium spp. genomes. Dipicolinc acid (DPA) is also

present in S. globisporus (Stastna et al., 1992); R. johrii sp. nov. (Girija et al.,

2010); and Clostridium perfringence, Clostridium botulinum, and Clostridium

tetani (Paredes et al., 2005). The genes encoding DPA synthase have not

been found in any of these bacteria. However, in C. perfringens, the

electron-transfer flavoprotein EtfA catalyzes the formation of DPA

(Osburn, Melville, & Popham, 2010), showing that there is more than one

pathway for the synthesis of DPA in bacteria. Hence, there has to be an

alternative DPA synthase, which in Mycobacterium spp. (and other bacteria

shown to have DPA, see above) is responsible for the synthesis of DPA.

The spore-specific penicillin-binding protein, PBP 5*, is suggested to be

involved in the assembly of the spore cortex in B. subtilis. Deletion of the

gene encoding PBP 5*, dacB, does not affect vegetative growth, but spores

produced from such a strain are extremely heat sensitive (Buchanan &

Gustafson, 1992). Interestingly, a homologue of dacB has been identified

in M. tuberculosis, and PknH phosphorylates the DacB protein. The role

Table 4.2 Examples of signature sporulation genes from known spore forming bacteria and their orthologue genes in Mycobacterium spp.along with their general conferred functionsOrthologs Sporulation function General function

SpoVK,a SpoIIIEa Maturation of spores and DNA translocase Widespread ATPases involved in many

other functions in nonsporulating bacteria

CotSAa Spore coat-associated protein Glycosyltransferase

SpoVEa and Soja Spore cortex synthesis centromere-like function Universal cell division proteins

SigEb s-factor controlling sporulation genes s-factor

SigFa,b s-factor controlling sporulation genes s-factor

SigGb s-factor controlling sporulation genes s-factor

SigKb s-factor controlling sporulation genes s-factor

KinAb Sporulation-specific kinase ATP-dependent kinase

KinBb Sporulation-specific kinase ATP-dependent kinase

KinCb Sporulation-specific kinase ATP-dependent kinase

KinEa Sporulation-specific kinase ATP-dependent kinase

Spo0Ab Master regulator of sporulation Regulator

Spo0Bb Sporulation initiation part of the phosphorelay Phosphotransferase

Spo0Fb Sporulation initiation part of the phosphorelay Phosphotransferase

WhiB2b and WhiB3b Septum formation

DacBc Sporulation-specific penicillin-binding protein

aTaken from Ghosh et al. (2009), see also Singh et al. (2010).bTaken from Lamont et al. (2012).cZheng, Papavinasasundaram, and Av-Gay (2007).


of DacB in M. tuberculosis is not known, but it is localized in the membrane

(Zheng et al., 2007). It will be interesting to understand if DacB inMycobac-

terium spp. has a similar role in the assembly process of the cortex as has been

described for B. subtilis spores.

6. REGULATORY GENES INVOLVED IN THESPORULATION PATHWAY—B. SUBTILIS VERSUS
MYCOBACTERIUM SPP.
Given that Mycobacterium spp. can form spores (Ghosh et al., 2009;

Lamont et al., 2012; see above), identification of the genes that control

the sporulation pathway is of utmost importance. Apart from the role of

the proteins discussed above, spore-specific s-factors (sE, sF, sG, and sK)

play essential roles in the expression of spore proteins in the Firmicutes, for

example, B. subtilis (Errington, 2003; Errington et al., 2003; Hilbert &

Piggot, 2004). Putative orthologues to sF, and possibly sG, have so far

been identified in Mycobacterium spp. (Table 4.1; Ghosh et al., 2009;

Lamont et al., 2012). Nothing is known about their role in spore

formation, but the expression level of sF increases in aging cultures of

M. marinum (Ghosh et al., 2009), which is consistent with a possible role in

spore formation. In Mycobacterium spp., there are a number of alternative

s-factors, most of them belonging to group 4. This group lacks the

s regions 1.2 and 3, which are present in, for example, the house

keeping s factor (Rodrigue et al., 2006). Clearly, the alternative s-factorsneed to be studied for their possible role in sporulation in Mycobacterium

spp. Another important question is to investigate the role (if any) of

different s factors with respect to pleiomorphic growth of Mycobacterium

spp. For example, are specific s factors involved in changing the cell shape

from rod to coccoid and vice versa. This might also be relevant with

respect to dormancy and latency. In the next section, we discuss proteins

involved in phosphorylation of regulators of gene expression with a focus

on sporulation and cell shape.

6.1. Sensors, kinases, and phosphatasesBacteria are subjected to variation in growth conditions. For example, soil

bacteria are exposed to variation in temperature, access to nutrients and

oxygen as well as to changes in pH. This is also true for Mycobacterium

spp., and in addition, the pathogenicMycobacterium spp. such asM. tuberculosis

can grow and reside inside macrophages where they are exposed to low pH,


oxygen and nitrogen radicals, as well as other immune response factors

(Huynh, Joshi, & Brown, 2011 and references therein; see above). To sur-

vive under these variable conditions, bacteria have sensors that affect the set

of genes that should be turned on and/or switched off. This is accomplished

through signal transduction systems where proteins are reversibly phosphor-

ylated. Here two-component systems consisting of a histidine kinase and

a response regulator have been shown to play important roles (Stock,

Robinson, & Goudreau, 2000). In addition, phosphorelay systems can also

involve kinases and response regulators, for example, the phosphorelay sys-

tem that leads to activation of the master regulator of sporulation in

Firmicutes, Spo0A (see, e.g., Hilbert & Piggot, 2004; see also above).

A comparison of the sequences of several different bacterial genomes

revealed that Mycobacterium spp. harbors relatively few two-component

signaling systems compared to other bacteria, for example, the Firmicutes

(Wehenkel et al., 2008). Moreover, of the 11 two-component signaling sys-

tems inM. tuberculosis, only 1, the MtrAB (Zahrt & Deretic, 2001), has been

reported to be essential for cell growth. Instead, theMycobacterium spp. have in

general a higher number of Serine/Threonine PhosphoKinases (STPK) rel-

ative to other bacteria including the Firmicutes.M. tuberculosis has 11 STPKs,

whileM.marinumhas 24 andM. lepraewith its degenerated genomehas only 4

(Chao et al., 2010;Molle&Kremer, 2010;Wehenkel et al., 2008). The genes

encoding the STPKs are referred to as pknA through pknL (M. tuberculosis).

Four STPKs (pknA, pknB, pknG, and pknL) have also been identified

in the actinomycete C. glutamicum. As discussed above, the gene

organization of pknA and pknB is highly conserved among the

actinomycetes, and they are colocalized with pbpA, rodA, and pstP where

the former are involved in peptidoglycan synthesis and cell shape,

respectively (see above; Henriques, Glaser, Piggot, & Moran, 1998; Molle

& Kremer, 2010). The PstP protein is a serine/threonine phosphatase that

dephosphorylates STPKs and their subtrates (Wehenkel et al., 2008).

STPKs are predicted to be transmembrane, receptor-like proteins and their

substrates are proteins involved in cell wall metabolism and cell division.

For example, FtsZ is a substrate of PknA, while both PknA and PknB

phosphorylate Wag31, the cell shape/cell division regulator DivIVA

homologue in Mycobacterium spp. In accordance with these findings,

overexpression or partial depletion of PknA and PknB influences cell

growth and shape (Kang et al., 2005). Overexpression of Wag31 also

affects cell shape by converting the rod-shaped cells into enlarged, bulging

bowling pins, an obvious defect in the regulation of growth and shape


(Nguyen et al., 2007). Moreover, the level of expression of PknA or PknB in

C. glutamicum influences growthandcell shape in that overexpressionof either

of these kinases leads to a more coccoid cell form (see above; Fiuza et al.,

2008). The role of STPKs in regulation of cell growth and septum

formation is further emphasized by the observation that the morphology of

M. tuberculosis changes when the level of PknF is reduced (Deol et al.,

2005). Moreover, the extracytoplasmatic domain of M. tuberculosis PknB

binds diaminopimelic (DAP) muropeptides (Mir et al., 2011). An STPK,

PrkC, in B. subtilis also binds DAP-type muropeptides, and this results in

triggering of germination of B. subtilis spores (Shah, Laaberki, Popham, &

Dworkin, 2008; see also Setlow, 2008). Although it appears that PknB

binding of DAP muropeptides only has a minor role in resuscitation of

M. tuberculosis (Mir et al., 2011), it might have a role in triggering spore

germination in Mycobacterium spp. (Ghosh et al., 2009). Taken together,

these data clearly suggest that Mycobacterium spp. are equipped with proteins

that can influence the cell shape when grown under various conditions.

Five kinases KinA-E can phosphorylate Spo0F, which is part of the pho-

sphorelay that activates the master regulator of sporulation Spo0A in

Firmicutes. Among these, KinA plays the major role (Piggot & Hilbert,

2004). Homologues to all kinases, except KinD, and Spo0A, Spo0B and

Spo0F candidates have been identified in M. avium subsp. paratuberculosis

(Lamont et al., 2012; see also Table 4.2). This raises the possibility that ini-

tiation of sporulation in Mycobacterium spp. involves these homologues.

Thus, there may be at least partially overlapping pathways in the Firmicutes

and Mycobacterium spp. Nevertheless, the roles of these proteins, together

with other regulatory proteins (see above), need to be studied with respect

to both cell shape and spore formation in Mycobacterium spp.

7. DOMESTICATION—“YOU STUDY WHAT YOUSELECT FOR”

Much of what is known about the bacterial life styles is based on do-

mesticated bacteria from the model systems E. coli and B. subtilis. Moreover,

expanding our knowledge of their life styles using natural isolates as well as

studies of other bacteria has revealed new information of how bacteria adapt

to their growth environment. For example, a natural isolate of B. subtilis

forms fruiting-body like structures with spores, whereas this has not yet been

seen in cultures of B. subtilis laboratory strains (Branda, Gonzalez-Pastor,

Ben-Yeduda, Losick, & Kolter, 2001). Another example that was discussed


above is that pure laboratory cultures of Heliobacterium cease to form spores

(Kimble-Long & Madigan, 2001). As the number of sequences of genomes

of domesticated and new isolates of bacteria increases, it has become appar-

ent that bacteria of the same species show large variation in genome size and

content. For example, a comparison of the genome size of E. coli K-12 and

E. coli 0157:H7 reveals an approximately �800,000 bp difference (Blattner

et al., 1997; Perna et al., 2001). Part of this difference is due to that E. coli

0157:H7 is equipped with more pathogenicity islands compared to E. coli

K-12; this increases its virulence (for further information, see Fux et al.,

2005). Loss of virulence and adaptation to growth conditions is also

evident in the case of Leptospira interrogans, a Gram-negative spirochete

and the causative agent of leptospirosis; it loses its virulence upon in vitro

cultivation (Ko, Goarant, & Picardeau, 2009).

ForMycobacterium spp., theM. bovis BCG strain provides a good example

of the evolution of an attenuated strain. It was evolved by Calmette in the

beginning of the 1900s. Genome sequence data show that the attenuation is

most likely due to the loss of genes involved in protein secretion, the ESX-1

secretion system (Brosch et al., 2007; Garnier et al., 2003). Moreover,

a comparison of different M. bovis BCG isolates used for vaccination

shows variation in the protective efficacy against pulmonary tuberculosis

(Behr, 2002). Genome sequence and transcriptome analysis show

differences in sequence as well as in protein expression for the different

M. bovis BCG strains (Berredo-Pinho et al., 2011; Brosch et al., 2007;

Gomes et al., 2011; Orduna et al., 2011; Pan et al., 2011; Seki et al., 2009).

In conclusion, it is evident that domestication constitutes a selection pres-

sure with the outcome that “you study what you select for”. Ultimately, this

emphasizes the importance of acquisition of knowledge and documentation

of the history of different bacterial strains in their diverse habitats. With

today’s ease of speedy acquisition of total genome sequence information, this

is now possible.

8. CONCLUDING REMARKS

To conclude, both early and more recent data clearly suggest that the

cell morphology of Mycobacterium spp. changes in response to growth con-

ditions. They also have the capacity to form spores under certain conditions.

This makes sense given that bacteria in general have evolved strategies to

pursue growth under various conditions. Studies using B. subtilis as a model

system have generated a deep understanding about how it handles different


situations where entering the sporulation pathway is the ultimate way to sur-

vive harsh conditions. With respect to Mycobacterium spp., we can build on

this knowledge to elucidate the molecular mechanisms that guide the

transitions between the different cell shapes including spore formation. In

particular, having this information might give us new tools to detect, trace,

and treat diseases caused byMycobacterium spp. such as tuberculosis with ap-

proximately 10 million new cases per year. Hence, we look forward to a

challenging and adventurous future. Finally, althoughwe searched for an an-

swer to the question why Mycobacterium spp. is considered to be a

nonsporulating bacterium, we have not been able to find any (or very

few, e.g., Hilson, 1965) experimental data confirming its inability to form

spores. Is it because the different Mycobacterium spp. we study today have

been domesticated or are there data that we have not been able to trace?Here

we do not mean to criticize; as the physicist Nils Bohr once said, “I do not

mean to criticize, I just want to understand” (taken from Segre, 2007).

ACKNOWLEDGMENTSWe thank our colleagues for discussions. Dr. S. Bagchi andMs.M.Ramesh are acknowledged

for providing Fig. 4.2A and assisting in generating Fig. 4.5, respectively, and Ms. T. Bergfors

for critical reading of the chapter. The ongoing work is supported by grants to L. A. K. from

the Soderberghs Foundation, the Swedish Research Council (M), Uppsala RNA Research

Center (Swedish Research Council, Linneaus Grant), and SIDA/SAREC.

REFERENCESAdams, D. W., & Errington, J. (2009). Bacterial cell division: Assembly, maintenance and

disassembly of the Z ring. Nature Reviews. Microbiology, 7, 642–653.Akerlund, T., Nordstrom, K., & Bernander, R. (1993). Branched Escherichia coli cells.

Molecular Microbiology, 10, 849–858.Aldridge, B. B., Fernandez-Suarez, M., Heller, D., Ambravaneswaran, V., Irimia, D.,

Toner, M., et al. (2012). Asymmetry and aging of mycobacterial cells lead to variablegrowth and antibiotic susceptibility. Science, 335, 100–104.

Andersen, L. P., & Rasmussen, L. (2009). Heliocobacter pylori-coccoid forms and biofilmformation. FEMS Immunology and Medical Microbiology, 56, 112–115.

Angert, E. R. (2005). Alternatives to binary fission in bacteria.Nature Reviews. Microbiology, 3,214–224.

Anuchin, A. M., Mulyukin, A. L., Suzina, N. E., Duda, V. I., El-Registan, G. I., &Kaprelyants, A. S. (2009). Dormant forms of Mycobacterium smegmatis with distinctmorphology. Microbiology, 155, 1071–1079.

Ara, I., & Kudo, T. (2006). Three novel species of the genus Catellatospora, Catellatosporachokoriensis sp. nov., Catellatospora coxensis sp. nov. and Catellatosporabangladeshensis sp. nov., and transfer of Catellatospora citrea subsp. methionotrophicaAsano and Kawamoto 1988 to Catellatospora methionotrophica sp. nov., comb. nov.International Journal of Systematic and Evolutionary Microbiology, 56, 393–400.


Asano, K., & Kawamoto, I. (1986). Catellatospora, a new genus of the Actinomycetales.International Journal of Systematic Bacteriology, 36, 512–517.

Behr, M. A. (2002). BCG-different strains, different vaccines?The Lancet Infectious Diseases, 2,86–92.

Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M., Challis, G. L., Thomson, N. R.,James, K. D., et al. (2002). Complete genome sequence of the model actinomyceteStreptomyces coelicolor A3(2). Nature, 417, 141–147.

Bernhardt, T. G., & de Boer, P. A. (2005). SlmA, a nucleoid-associated, FtsZ binding proteinrequired for blocking septal ring assembly over chromosomes in E. coli. Molecular Cell,18, 555–564.

Berredo-Pinho, M., Kalume, D. E., Correa, P. R., Gomes, L. H. F., Pereira, M. P.,da Silva, R. F., et al. (2011). Proteomic profile of culture filtrate from the Brazilianvaccine strain Mycobacterium bovis BCG moreau compared to M. bovis BCG Pasteur.BMC Microbiology, 11, 80.

Bi, C., Jones, S. W., Hess, D. R., Tracy, B. P., & Papoutsakis, E. T. (2011). SpoIIE isnecessary for asymmetric division, sporulation and expression of sF, sE, andsG but doesnot control solvent production in Clostridium acetobutylicum ATCC 824. Journal ofBacteriology, 193, 5130–5137.

Blattner, F. R., Plunkett, G., 3rd, Bloch, C. A., Perna, N. T., Burland, V., Riley, M., et al.(1997). The complete genome sequence of Escherichia coli K-12. Science, 277,1453–1462.

Bradley, S. G. (1959). Sporulation by some strains of Nocardiae and Streptomycetes. AppliedMicrobiology, 7, 89–93.

Branda, S. S., Gonzalez-Pastor, J. E., Ben-Yeduda, S., Losick, R., & Kolter, R. (2001).Fruiting body formation by Bacillus subtilis. Proceedings of the National Academy of Sciencesof the United States of America, 98, 11621–11626.

Brieger, E. M., & Glaubert, A. M. (1956). Spore-like structures in the tubercle bacillus.Nature, 178, 544.

Brosch, R., Gordon, S. V., Garnier, T., Eiglmeier, K., Frigui, W., Valenti, P., et al. (2007).Genome plasticity of BCG and impact on vaccine efficacy. Proceedings of the NationalAcademy of Sciences of the United States of America, 104, 5596–5601.

Brown-Elliot, B. A., Brown, J. M., Conville, P. S., &Wallace, R. J., Jr. (2006). Clinical andlaboratory features of the Nocardia spp. based on current molecular taxonomy. ClinicalMicrobiology Reviews, 19, 259–282.

Buchanan, C. E., & Gustafson, A. (1992). Mutagenesis and mapping of the gene for a spor-ulation specific penicilli-binding protein in Bacillus subtilis. Journal of Bacteriology, 174,5430–5435.

Cabeen, M. T., & Jacobs-Wagner, C. (2005). Bacterial cell shape. Nature Reviews. Microbi-ology, 3, 601–610.

Chao, J., Wong, D., Zheng, X., Poirier, V., Bach, H., Hmama, Z., et al. (2010). Proteinkinase and phosphatase signaling in Mycobacterium tuberculosis physiology and patho-genesis. Biochimica et Biophysica Acta, 1804, 620–627.

Chase, D. G., & Erlandsen, S. L. (1976). Evidence for a complex life cycle and endosporeformation in the attached, filamentous, segmented bacterium frommurine ileum. Journalof Bacteriology, 127, 572–583.

Chater, K. F., & Chandra, G. (2006). The evolution of development in Streptomycesanalysed by genome comparison. FEMS Microbiology Reviews, 30, 6512–6672.

Claesson, D., de Jong, W., Dijkhuizen, L., & Wosten, H. A. (2006). Regulation ofStreptomyces development: Reach for the sky!. Trends in Microbiology, 14, 313–319.

Cosma, C. L., Sherman, D. R., & Ramakrishnan, L. (2003). The secret lives of the patho-genic mycobacteria. Annual Review of Microbiology, 57, 641–676.


Csillag, A. (1961). Morphological and biochemical features of ’atypical’ mycobacteria. Journalof General Microbiology, 24, 261–272.

Csillag, A. (1963). Cellular morphology of form 2 mycobacteria in slide culture. Journal ofGeneral Microbiology, 30, 21–27.

Csillag, A. (1964). Growth of a form 2 mycobacterium and various bacillus species onLowenstein-Jensen medium. Journal of General Microbiology, 24, 261–272.

Csillag, A. (1970). A simple method to obtain the mycococcus form of Mycobacterium phlei.Journal of General Microbiology, 62, 251–259.

Dahl, J. L. (2004). Electron microscopy analysis of Mycobacterium tuberculosis cell division.FEMS Microbiology Letters, 240, 15–20.

Davis, J. C., & Mudd, S. (1954). The cytology of a strain of Corynebacterium diphtheriae.Journal of Bacteriology, 69, 372–386.

de Hoon, M. J. L., Eichenberger, P., & Vitkup, D. (2010). Hierarchical evolution of thebacterial sporulation network. Current Biology, 20, R735–R745.

Denny, F. P. (1903). Observations on the morphology of B. diphtheriae, B. pseudo-diphtheriae, and B. xerosis. Journal of Medical Research, 9, 117–136.

Deol, P., Vohra, R., Saini, A. K., Singh, A., Chandra, H., Chopra, P., et al. (2005). Role ofMycobacterium tuberculosis Ser/Thr kinase PknF: Implications in glucose transport andcell division. Journal of Bacteriology, 187, 3415–3420.

Dubnau, D., & Losick, R. (2006). Bistability in bacteria. Molecular Microbiology, 61,564–572.

Durre, P. (2011). Ancestral sporulation initiation. Molecular Microbiology, 80, 584–587.Dziedzic, R., Kiran, M., Plocinski, P., Ziolkiewicz, M., Brzostek, A., Moomey, M., et al.

(2010). Mycobacterium tuberculosis ClpX interacts with FtsZ and interferes with FtsZassembly. PLoS One, 5, e11058.

Ellermeier, C. D., Hobbs, E. C., Gonzalez-Pastor, J. E., & Losick, R. (2006). A three-proteinsignaling pathway governing immunity to a bacterial cannibalism toxin. Cell, 124,549–559.

England, K., Crew, R., & Slayden, R. A. (2011). Mycobacterium tuberculosis septum sitedetermining protein, Ssd encoded by rv3660c, promotes filamentation and elicits analternative metabolic and dormancy stress response. BMC Microbiology, 11, 79.

Errington, J. (2003). Regulation of endospore formation in Bacillus subtilis. Nature Reviews.Microbiology, 1, 117–126.

Errington, J., Daniel, R. A., & Scheffers, D. J. (2003). Cytokinesis in bacteria.Microbiology andMolecular Biology Reviews, 67, 52–65.

Falkinham, J. O., 3rd (2011). Surrounded bymycobacteria: Nontuberculosis mycobacteria inthe human environment. Journal of Applied Microbiology, 107, 356–367.

Farnia, P., Masjedi, M. R., Merza, M. A., Tabarsi, P., Zhavnerko, G. K., Ibrahim, T. A.,et al. (2010). Growth and cell-division in extensive (XDR) and extremely drug resistant(XXDR) tuberculosis strains: Transmission and atomic force observation. InternationalJournal of Clinical and Experimental Medicine, 3, 308–314.

Faulkner, G., & Garduno, R. A. (2002). Ultrastructural analysis of differentiation inLegionella pneumophila. Journal of Bacteriology, 184, 7025–7041.

Filippova, S. N., Gorbatiuk, E. V., Poglazova, M. N., Soina, V. S., Kuznetsov, V. D., &El’-Registan, G. I. (2005). Endospore formation by Streptomyces avermitilis in sub-merged culture. Mikrobiologiia, 74, 204–214.

Fiuza, M., Canova, M. J., Zanella-Cleon, I., Becchi, M., Cozzone, A. J., Mateos, L. M., et al.(2008). From the characterization of the four serine/threonine protein kinases (PknA/B/G/L) of Cornyebacterium glutamicum toward the role of PknA and PknB in cell divi-sion. The Journal of Biological Chemistry, 283, 18099–18112.

Flardh, K., & Buttner, M. J. (2009). Streptomyces morphogenetics: Dissecting differentiationin a filamentous bacterium. Nature Reviews. Microbiology, 7, 36–49.


Flynn, J. L., & Chan, J. (2005). What’s good for the host is good for the bug. Trends inMicrobiology, 13, 98–102.

Fujinami, S., Sato, T., & Ito, M. (2011). The relationship between a coiled morphology andMbl in alkaliphilic Bacillus halodurans C-125 at neutral pH values. Extremophiles, 15,587–596.

Fux, C. A., Costerton, J. W., Stewart, P. S., & Stoodley, P. (2005). Survival strategies ofinfectious biofilms. Trends in Microbiology, 13, 34–40.

Gaastra, W., Boot, R., Ho, H. T. K., & Lipman, L. J. A. (2009). Rat bite fever. VeterinaryMicrobiology, 133, 211–228.

Garduno, R. A., Garduno, E., Hiltz, M., & Hoffman, P. S. (2002). Intracellular growth ofLegionella pneumophila gives rise to a differentiated form dissimilar to stationary-phaseforms. Infection and Immunity, 70, 6273–6283.

Garnier, T., Eiglmeier, K., Camus, J. C., Medina, N., Mansoor, H., Pryor, M., et al. (2003).The complete genome sequence of Mycobacterium bovis. Proceedings of the NationalAcademy of Sciences of the United States of America, 100, 7877–7882.

Ghosh, J., Larsson, P., Singh, B., Pettersson, B. M. F., Islam, N. M., Sarkar, S. N., et al.(2009). Sporulation in mycobacteria. Proceedings of the National Academy of Sciences ofthe United States of America, 106, 10781–10786.

Girija, K. R., Sasikala, Ch., Ramana, Ch.V., Sproer, C., Takaichi, S., Thiel, V., et al. (2010).Rhodobacter johrii sp. nov., an endospore producing cryptic Rhodobacter speciesisolated from semi-arid tropical soils. International Journal of Systematic and EvolutionaryMicrobiology, 60, 2099–2107.

Goehring, N. W., & Beckwith, J. (2005). Diverse paths to midcell: Assembly of the cell di-vision machinery. Current Biology, 15, R514–R526.

Gomes, L. H. F., Otto, T. D., Vasconcellos, E. A., Ferrao, P. M., Maia, R. M.,Moreira, A. S., et al. (2011). Genome sequence of Mycobacterium bovis BCG moreau,the Brazilian vaccine strain against tuberculosis. Journal of Bacteriology, 193, 5600–5601.

Gonzalez-Pastor, J. E., Hobbs, E. C., & Losick, R. (2003). Cannibalism by sporulatingbacteria. Science, 301, 510–513.

Gonzy-Treboul, G., Karmazyn-Campelli, C., & Stragier, P. (1992). Developmental regula-tion of transcription of the Bacillus subtilis ftsAZ operon. Journal of Molecular Biology, 224,967–979.

Gruber, T. M., & Gross, C. A. (2003). Multiple sigma subunits and the partitioning ofbacterial transcription space. Annual Review of Microbiology, 57, 441–466.

Gullbrand, B., Akerlund, T., & Nordstrom, K. (1999). On the origin of branches inEscherichia coli. Journal of Bacteriology, 181, 6607–6614.

Haggblom, M. M., Nohynek, L. J., Palleroni, N. J., Kronqvist, K., Nurmiaho-Lassila, E.-L.,Salkinojasalonen, M. J., et al. (1994). Transfer of polychlorophenol-degradingRhodococcus chlorophenolicus (Apajalahti et al. 1986) to the genus Mycobacteriumas Mycobacterium chlorophenolicum comb. nov. International Journal of SystematicBacteriology, 44, 485–493.

Hall-Stoodley, L., & Stoodley, P. (2005). Biofilm formation and dispersal and the transmis-sion of human pathogens. Trends in Microbiology, 13, 7–10.

Hamoen, L. W., Venema, G., & Kuipers, O. P. (2003). Controlling competence in Bacillussubtilis: Shared use of regulators. Microbiology, 149, 9–17.

Hansen, G. A. (1880). Bacillus leprae. Virchows Archiv, 79, 32–42.Harold, F. M. (2007). Bacterial morphogenesis: Learning how cells make cells.Current Opin-

ion in Microbiology, 10, 591–595.Henriques, A. O., Glaser, P., Piggot, P. J., & Moran, C. P., Jr. (1998). Control of cell shape

and elongation by the rodA gene in Bacillus subtilis.Molecular Microbiology, 28, 235–247.Hett, E. C., & Rubin, E. J. (2008). Bacterial growth and cell division: A mycobacterial per-

spective. Microbiology and Molecular Biology Reviews, 72, 126–156.


Hewitt, L. F. (1951). Cell structure of Corynebacterium diphtheriae. Journal of GeneralMicrobiology, 5, 287–292.

Hilbert, D. W., & Piggot, P. J. (2004). Compartmentalization of gene expression duringBacillus subtilis spore formation.Microbiology and Molecular Biology Reviews, 68, 234–262.

Hilson, G. R. F. (1965). Taxonomic characteristics of so-called ‘form 2mycobacteria’. Journalof General Microbiology, 39, 407–421.

Holmes, R. K. (2000). Biology and molecular epidemiology of diphtheria toxin and the toxgene. Journal of Infectious Diseases, 181(Suppl. 1), S1156–S1167.

Hopwood, D. A. (1999). Forty years of genetics with Streptomyces: From in vivo throughin vitro to in silico. Microbiology, 145, 2183–2202.

Huynh, K. K., Joshi, S. A., & Brown, E. J. (2011). A delicate dance: Host response tomycobacteria. Current Opinion in Immunology, 23, 464–472.

Jones, S. W., Tracy, B. P., Gaida, S. M., & Papoutsakis, E. T. (2011). Inactivation of sF inClostridium acetobutylicum ATCC 824 blocks sporulation prior to asymmetric divisionand abolishes sE and sG protein expression but does not block solvent formation. Journalof Bacteriology, 193, 2429–2440.

Juhasz, S. E. (1962). Aberrant forms of Mycobacterium phlei produced by streptomycinand their multiplication on streptomycin-free media. Journal of General Microbiology,28, 9–13.

Justice, S. S., Hung, C., Theriot, J. A., Fletcher, D. A., Anderson, G. G., Footer, M. J., et al.(2004). Differentiation and developmental pathways of uropathogenic Escherichia coli inurinary tract pathogenesis. Proceedings of the National Academy of Sciences of the United Statesof America, 101, 1333–1338.

Justice, S. S., Hunstad, D. A., Cegelski, L., & Hultgren, S. J. (2008). Morphological plasticityas a bacterial survival strategy. Nature Reviews. Microbiology, 6, 162–168.

Justice, S. S., Hunstad, D. A., Seed, P. C., & Hultgren, S. J. (2006). Filamentation byEscherichia coli subverts innate defenses during urinary tract infection. Proceedings ofthe National Academy of Sciences of the United States of America, 103, 19884–19889.

Kahn, M. C. (1929). A developmental cycle of the tubercle bacillus as revealed by single-cellstudies. American Review of Tuberculosis, 20, 150–200.

Kaiser, D. (2006). A microbial genetic journey. Annual Review of Microbiology, 60, 1–25.Kang, C. M., Abbott, D. W., Park, S. T., Dascher, C. C., Cantley, L. C., & Husson, R. N.

(2005). The Mycobacterium tuberculosis serine/threonine kinases PknA and PknB,substrate identification and regulation of cell shape. Genes & Development, 19,1692–1704.

Kawata, T., & Inoue, T. (1965). Electron microscopic observations of a remarkable body inaged Corynebacterium diphtheriae. Journal of Bacteriology, 89, 1613–1614.

Kimble-Long, L. K., & Madigan, M. T. (2001). Molecular evidence that the capacity forendosporulation is universal among phototropic heliobacteria. FEMSMicrobiology Letters,199, 191–195.

Kiran, M., Chauhan, A., Dziedzic, R., Maloney, E., Mukherji, S. K., Madiraju, M., et al.(2009). Mycobacterium tuberculosis ftsH expression in response to stress and viability.Tuberculosis, 89, S70–S73.

Ko, A. I., Goarant, C., & Picardeau, M. (2009). Leptospira: The dawn of the molecular ge-netics era for an emerging zoonotic pathogen. Nature Reviews. Microbiology, 7, 736–747.

Koch, R. (1882). Die aetilogie der tuberculose. Berliner Klinische Wochenschrift, 19, 221–230.Koch, A. L. (2005). Shapes that Escherichia coli cells can achieve, as a paradigm for other

bacteria. Critical Reviews in Microbiology, 31, 183–190.Koul, A., Herget, T., Klebl, B., & Ullrich, A. (2004). Interplay between mycobacteria and

host signalling pathways. Nature Reviews. Microbiology, 2, 189–202.Kroos, L. (2007). The Bacillus andMyxococcus developmental networks and their transcrip-

tional regulators. Annual Review of Genetics, 41, 13–39.


Kumar, K., Awasthi, D., Berger, W. T., Tonge, P. J., Slayden, R. A., & Ojima, I. (2010).Discovery of anti-TB agents that target the cell-division protein FtsZ. Future MedicinalChemistry, 2, 1305–1323.

Lack, C. H., & Tanner, F. (1953). The significance of pleomorphism in Mycobacterium tu-berculosis var. hominis. Journal of General Microbiology, 8, 18–26.

Lamont, E. A., Bannantine, J. P., Armien, A., Ariyakumar, D. S., & Sreevatsan, S. (2012).Identification and characterization of a spore-like morphotype in chronically starvedMycobacterium avium subsp. paratuberculosis cultures. PLoS One, 7, e30648.

Larkin, M. J., Kulakov, L. A., & Allen, C. C. R. (2006). Biodegradation by members ofthe genus Rhodococcus: Biochemistry, physiology, and genetic adaptation. Advancesin Applied Microbiology, 59, 1–29.

Letek, M., Fiuza, M., Ordonez, E., Villadangos, A. F., Ramos, A., Mateos, L. M., et al.(2008). Cell growth and cell division in the rod-shaped actinomycete Corynebacteriumgluatmicum. Antoine Van Leeuwenhoek, 98, 165–177.

Lopez, D., & Kolter, R. (2010). Extracellular signals that define distinct and coexisting cellfates in Bacillus subtilis. FEMS Microbiology Reviews, 34, 134–149.

Lopez, D., Vlamakis, H., & Kolter, R. (2008). Generation of multiple cell types in Bacillussubtilis. FEMS Microbiology Reviews, 33, 152–163.

Luedemann, G.M., &Casmer, C. J. (1973). Electron microscope study of whole mounts andthin sections of Micromonospora chalcea ATCC 12452. International Journal of SystematicBacteriology, 23, 243–255.

Maldonado, L. A., Fenical, W., Jensen, P. R., Kauffman, C. A., Mincer, T. J., Ward, A. C.,et al. (2005). Salinispora arenicola gen. nov., sp. nov. and Salinispora tropica sp. nov.,obligate marine actinomycetes belonging to the family Micromonosporaceae. Interna-tional Journal of Systematic and Evolutionary Microbiology, 55, 1759–1766.

Maloney, E., Madiraju, M., & Rajagopalan, M. (2009). Overproduction and localization ofMycobacterium tuberculosis ParA and ParB proteins. Tuberculosis, 89, S65–S69.

Margolin, W. (2001). Spatial regulation of cytokinesis in bacteria. Current Opinion in Micro-biology, 4, 647–652.

McDougald, D., Rice, S. A., Barraud, N., Steinberg, P. D., & Kjelleberg, S. (2012). Shouldwe stay or should we go: Mechanisms and ecological consequences for biofilm dispersal.Nature Reviews. Microbiology, 10, 39–50.

Mir, M., Asong, J., Li, X., Cardot, J., Boons, G.-J., & Husson, R. N. (2011).The extracytoplasmic domain of the Mycobacterium tuberculosis Ser/Thr PknB bindsspecific muropeptides and is required for PknB localization. PLoS Pathogens, 7, e1002182.

Molle, V., & Kremer, L. (2010). Division and cell envelope regulation by Ser/Thr phosphor-ylation: Mycobacterium shows the way. Molecular Microbiology, 75, 1064–1077.

Molofsky, A. B., & Swanson, M. S. (2004). Differentiate to thrive: Lessons from theLegionella pneumophila life cycle. Molecular Microbiology, 53, 29–40.

Nguyen, L., & Pieters, J. (2005). The trojan horse: Survival tactics of pathogenicmycobacteria in macrophages. Trends in Cell Biology, 15, 269–276.

Nguyen, L., Scherr, N., Gatfield, J., Walburger, A., Pieters, J., & Thompson, C. J. (2007).Antigen 84, an effector of pleiomorphism in Mycobacterium smegmatis. Journal ofBacteriology, 189, 7896–7910.

Orduna, P., Cevallos, M. A., Ponce de Leon, S., Arvizu, A., Hernandez-Gonzalez, I. L.,Mendoza-Hernandez, G., et al. (2011). Genomic and proteomic analysis of Mycobac-terium bovis BCGMexico 1931 reveal a diverse immunogenic repertoire against tuber-culosis infection. BMC Microbiology, 12, 493.

Osborn, M. J., & Rothfield, L. (2007). Cell shape determination in Escherichia coli. CurrentOpinion in Microbiology, 10, 606–610.

Osburn, B. C., Melville, S. B., & Popham, D. L. (2010). EtfA catalyses the formation ofdipicolinic acid in Clostridium perfringens. Molecular Microbiology, 75, 178–186.


Pan, Y., Yang, X., Duan, J., Lu, N., Leung, A. S., Tran, V., et al. (2011). Whole-genomesequences of four Mycobacterium bovis BCG vaccine strains. Journal of Bacteriology, 193,3152–3153.

Paredes, C. J., Alsaker, K. V., & Papoutsakis, E. T. (2005). A comparative genomic view ofclostridial sporulation and physiology. Nature Reviews. Microbiology, 3, 969–978.

Perna, N. T., Plunkett, G. 3rd., Burland, V., Mau, G., Glasner, J. D., Rose, D. J., et al.(2001). Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature,409, 529–533.

Piggot, P. J., & Hilbert, D. W. (2004). Sporulation of Bacillus subtilis. Current Opinion inMicrobiology, 7, 579–586.

Primm, T. P., Lucero, C. A., & Falkinham, J. O., 3rd (2004). Health impacts of environmentalmycobacteria. Clinical Microbiology Reviews, 17, 98–106.

Ramos, A., Letek, M., Campelo, A. B., Vaquera, J., Mateos, L. M., & Gil, J. A. (2005).Altered morphology produced by ftsZ expression in Corynebacterium glutamicumATCC 13869. Microbiology, 151, 2563–2572.

Rodrigue, S., Provvedi, R., Jacques, P.-E., Gaudreau, L., & Manganelli, R. (2006). Thes factors of Mycobacterium tuberculosis. FEMS Microbiology Reviews, 30, 926–941.

Rothfield, L., Taghbalout, A., & Shih, Y. L. (2005). Spatial control of bacterial division-siteplacement. Nature Reviews. Microbiology, 3, 959–968.

Roy, S., & Ajitkumar, P. (2005). Transcriptional analysis of the principal cell division gene,ftsZ, of Mycobacterium tuberculosis. Journal of Bacteriology, 187, 2540–2550.

Roy, S., Anand, D., Vijay, S., Gupta, P., & Ajitkumar, P. (2011). The ftsZ gene of Myco-bacterium smegmatis is expressed through multiple transcripts.Open Microbiology Journal,5, 43–53.

Russell, D. G. (2007). Who puts the tubercle in tuberculosis?Nature Reviews. Microbiology, 5,39–47.

Sachdeva, P., Misra, R., Tyagi, A. K., & Singh, Y. (2010). The sigma factors of Mycobac-terium tuberculosis: Regulation of the regulators. The FEBS Journal, 277, 605–626.

Schinsky, M. F., McNeil, M. M., Whitney, A. M., Steigerwalt, A. G., Lasker, B. A.,Floyd, M. M., et al. (2000). Mycobacterium septicum sp. nov., a new rapidly growingspecies associated with catheter-related bacteraemia. International Journal of Systematic andEvolutionary Microbiology, 50, 575–581.

Schultz, D., Wolynes, P. G., Jacob, E. B., & Onuchic, J. N. (2009). Deciding fate in adversetime: Sporulation and competence in Bacillus subtilis. Proceedings of the National Academyof Sciences of the United States of America, 106, 21027–21034.

Segre, G. (2007). Faust in Copenhagen: A struggle for the soul of physics. New York: Penguin.Seki,M.,Honda, I., Fujita, I., Yano, I., Yamamoto, S., &Koyama, A. (2009).Whole genome

sequence analysis of Mycobacterium bovis bacillus Calmette-Guerin (BCG) Tokyo 172:A comparative study of BCG vaccine substrains. Vaccine, 27, 1710–1716.

Setlow, P. (2008). Dormant spores receive an unexpected wake-up call. Cell, 135, 410–411.Shah, I. M., Laaberki, M.-H., Popham, D. L., & Dworkin, J. (2008). A eukaryotic-like Ser/

Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments.Cell,135, 486–496.

Shleeva, M. O., Kudykina, Y. K., Vostroknutova, G. N., Suzina, N. E., Mulyukin, A. L., &Kaprelyants, A. S. (2011). Dormant ovoid cells of Mycobacterium tuberculosis areformed in response to gradual external acidification. Tuberculosis, 91, 146–154.

Singh, B., Ghosh, J., Islam, N. M., Dasgupta, S., & Kirsebom, L. A. (2010). Growth, celldivision and sporulation in mycobacteria. Antoine Van Leeuwenhoek, 98, 165–177.

Slayden, R. A., & Belisle, J. T. (2009). Morphological features and signature gene responseelicited by inactivation of FtsI in Mycobacterium tuberculosis. The Journal of AntimicrobialChemotherapy, 63, 451–457.


Slayden, R. A., Knudson, D. L., & Belisle, J. T. (2006). Identification of cell cycle regulatorsin Mycobacterium tuberculosis by inhibition of septum formation and global transcrip-tional analysis. Microbiology, 152, 1789–1797.

Smits,W. K., Kuipers, O. P., &Veening, J.-W. (2006). Phenotypic variation in bacteria: Therole of feedback regulation. Nature Reviews. Microbiology, 4, 259–271.

Srinivasan, R., Rajeswari, H., Bhatt, B. N., Indi, S., & Ajitkumar, P. (2007). GTP/GDPbinding stabilizes bacterial cell division protein FtsZ against degradation by FtsH proteasein vitro. Biochemical and Biophysical Research Communications, 357, 38–43.

Stastna, J., Goodfellow, M., Kristufek, V., Novotna, J., Jizba, J., Caslavska, J., et al. (1992).Characteristics of Streptomyces globisporus strain 0234A forming endospores in sub-merged cultures. Folia Microbiologica (Praha), 37, 111–116.

Steiner, E., Dago, A. E., Young, D. I., Heap, J. T., Minton, N. P., Hoch, J. A., et al. (2011).Multiple orphan histidine kinases interact directly with Spo0A to control the initiation ofendospore formation in Clostridium acetobutylicum. Molecular Microbiology, 80,641–654.

Stewart-Tull, D. E. S. (1965). Occurrence of dimorphic forms of Mycobacterium phlei.Nature, 208, 603–605.

Stock, A. M., Robinson, V. L., & Goudreau, P. N. (2000). Two-component signal trans-duction. Annual Review of Biochemistry, 69, 183–215.

Stragier, P. (2006). To kill but not be killed: A delicate balance. Cell, 124, 461–463.Suarez, J. E., & Hardisson, C. (1985). Morphological characteristics of colony development

in Micromonospora chalcea. Journal of Bacteriology, 162, 1342–1344.Tamames, J., Gonzalez-Moreno, M., Mingorance, J., Valencia, A., & Vicente, M. (2001).

Bringing order into bacterial shape. Trends in Genetics, 17, 124–126.Thakur, M., & Chakraborti, P. K. (2006). GTPase activity of mycobacterial FtsZ is impaired

due to its transphosphorylation by the eukaryotic-type Ser/Thr kinase, PknA.The Journalof Biological Chemistry, 281, 40107–40113.

Thanky, N. R., Young, D. B., & Robertson, B. D. (2007). Unusual features of the cell cyclein mycobacteria: Polar-restricted growth and the snapping-model of cell division. Tuber-culosis, 87, 231–236.

Tortoli, E. (2003). Impact of genotypic studies on mycobacterial taxonomy: The newmycobacteria of the 1990s. Clinical Microbiology Reviews, 16, 319–354.

Tortoli, E. (2006). The new mycobacteria: An update. FEMS Immunology and Medical Micro-biology, 48, 159–178.

Traag, B. A., Driks, A., Stragier, P., Bitter, W., Broussard, G., Hatfull, G., et al. (2010). Domycobacteria produce endospores? Proceedings of the National Academy of Sciences of theUnited States of America, 107, 878–881.

Turenne, C., Chedore, P.,Wolfe, J., Jamieson, F.,May, K., &Kabani, A. (2002). Phenotypicand molecular characterization of clinical isolates of Mycobacterium elephantis fromhuman specimens. Journal of Clinical Microbiology, 40, 1230–1236.

Typas, A., Banzhaf, M., Gross, C. A., & Vollmer, W. (2011). From the regulation of pep-tidoglycan synthesis to bacterial growth and morphology. Nature Reviews. Microbiology,10, 123–136.

Vadrevu, I. S., Lofton, H., Sarva, K., Blasczyk, E., Plocinska, R., Chinnaswamy, J., et al.(2011). ChiZ levels modulate cell division process in mycobacteria. Tuberculosis, 91,S128–S135.

Vaerewijick,M. J., Huys, G., Palomino, J. C., Swings, J., & Portaels, F. (2005). Mycobacteriain drinking water distribution systems: Ecology and significance for human health.FEMS Microbiology Reviews, 29, 911–934.

Veening, J.-W., & Kuipers, O. P. (2010). Gene position within a long transcript as a deter-minant for stochastic switching in bacteria. Molecular Microbiology, 76, 269–272.


Veening, J.-W., Smits, W. K., Hamoen, L. W., & Kuipers, O. P. (2006). Single cell analysisof gene expression patterns of competence development and initiation of sporulation inBacillus subtilis grown on chemically defined media. Journal of Applied Microbiology, 101,531–541.

Veening, J.-W., Smits, W. K., & Kuipers, O. P. (2008). Bistability, epigenetics, and bet-hedging in bacteria. Annual Review of Microbiology, 62, 193–210.

Wainwright, M., Canham, L. T., Al-Wajeeh, K., & Reeves, C. L. (1999). Morphologicalchanges (including filamentation) in Escherichia coli grown under starvation conditionson silicon wafers and other surfaces. Letters in Applied Microbiology, 29, 224–227.

Wehenkel, A., Bellinzoni, M., Grana, M., Duran, R., Villarino, A., Fernandez, P., et al.(2008). Mycobacterial Ser/Thr protein kinases and phosphatases: Physiological rolesand therapeutic potential. Biochimica et Biophysica Acta, 1784, 193–202.

Wildermuth, H., & Hopwood, D. A. (1970). Septation during sporulation in Streptomycescoelicolor. Journal of General Microbiology, 60, 51–59.

Worku, M. L., Sidebotham, R. L., Walker, M. M., Keshavarz, T., & Karim, Q. N. (1999).The relationship betweenHelicobacter pylorimotility,morphology and phase of growth:Implications for gastric colonization and pathology. Microbiology, 145, 2803–2811.

Wu, L. J., & Errington, J. (2004). Coordination of cell division and chromosome segregationby a nucleoid occlusion protein in Bacillus subtilis. Cell, 117, 915–925.

Wu, L. J., Ishikawa, S., Kawai, Y., Oshima, T., Ogasawara, N., & Errington, J. (2009). Nocprotein binds to specific DNA sequences to coordinate cell division with chromosomesegregation. The EMBO Journal, 28, 1940–1952.

Wu, M., Ren, Q., Durkin, A. S., Daugherty, S. C., Brinkac, L. M., Dodson, R. J., et al.(2005). Life in hot carbon monoxide: The complete genome sequence ofCarboxydothermus hydrogenoformans Z-2901. PLoS Genetics, 1, e65.

Wyckoff, R. W. G., & Smithburn, K. C. (1933). Micromotion pictures of the growth ofMycobacterium phlei. The Journal of Infectious Diseases, 53, 201–209.

Young, K. D. (2006). The selective value of bacterial shape.Microbiology and Molecular BiologyReviews, 70, 660–703.

Young, K. D. (2007). Bacterial morphology: Why have different shapes? Current Opinion inMicrobiology, 10, 596–600.

Young, K. D. (2010). Bacterial shape: Two-dimensional questions and possibilities. AnnualReview of Microbiology, 64, 223–240.

Yu, H. (1930). A study of the dissociation of diphtheria bacillus. Journal of Bacteriology, 20,107–120.

Zahrt, T. C., & Deretic, V. (2001). Mycobacterium tuberculosis signal transduction systemrequired for persistent infections. Proceedings of the National Academy of Sciences of the UnitedStates of America, 98, 12706–12711.

Zheng, X., Papavinasasundaram, K. G., & Av-Gay, Y. (2007). Novel substrates of Mycobac-terium tuberculosis PknH Ser/Thr kinase. Biochemical and Biophysical Research Communi-cation, 355, 162–168.

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