university of groningen bacteriocins of streptococcus ...virulence (132). cps prevents phagocytosis...
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University of Groningen
Bacteriocins of Streptococcus pneumoniae and its response to challenges by antimicrobialpeptidesMajchrzykiewicz, Joanna Agnieszka
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Bacteriocins of Streptococcus pneumoniae
and its response to challenges by
antimicrobial peptides
Joanna Majchrzykiewicz
Paranimfen
Bogusia Marciniak
Tomas Kloosterman
Front cover: ―Light in the tunnel‖, photo made in the Palácio Nacional de Sintra, Portugal.
Left corner: an example of a bacteriocin.
Back cover: Bacteriocin-like activity of the chosen samples/strains.
Printed by: JAKS (Wrocław, Poland)
The work described in this thesis was carried out in the Molecular Genetics Group
of the Groningen Biomolecular Sciences and Biotechnology Institute (Faculty of
Mathematics and Natural Sciences, University of Groningen, The Netherlands).
Printing of this thesis was financially supported by the Faculty of Mathematics and
Natural Sciences, University of Groningen.
RIJKSUNIVERSITEIT GRONINGEN
Bacteriocins of Streptococcus pneumoniae and
its response to challenges by antimicrobial
peptides
Proefschrift
ter verkrijging van het doctoraat in de
Wiskunde en Natuurwetenschappen
aan de Rijksuniversiteit Groningen
op gezag van de
Rector Magnificus, dr. F. Zwarts,
in het openbaar te verdedigen op
vrijdag 7 januari 2011
om 16.15 uur
door
Joanna Agnieszka Majchrzykiewicz
geboren op 1 juni 1980
te Kielce, Polen
Promotor: Prof. dr. O.P. Kuipers
Copromotor: Dr. J.J.E. Bijlsma
Beoordelingscommissie: Prof. dr. J.M. van Dijl
Prof. dr. A.J.M. Driessen
Prof. dr. P.W.M. Hermans
ISBN
978-90-367-4588-8 (printed version)
978-90-367-4589-5 (digital version)
“Spieszmy sie kochac ludzi, tak szybko odchodza…”
“Let’s hurry to love people, they leave so quickly…”
Jan Twardowski
Babci/For granny
―Bacteriocins are well described among the Gram-positive bacteria including a
variety of AMPs produced by the genus Streptococcus. Nevertheless, little is known about
bacteriocins produced by S. pneumoniae. Therefore, the main aim of the thesis was to find
and characterize bacteriocin(s) produced by S. pneumoniae. The thesis contributes to the
complex story of bacteriocins in S. pneumoniae. Moreover, it presents information about
three new clusters likely involved in nitrogen metabolism in the bacterium. It adds data on
the subject of S. pneumoniae resistance to selected AMPs. Additionally, it contributes to
development of novel lantibiotics that once might find use in food industry or in medicine‖.
Introduction; the scope of this thesis
Contents
CHAPTER 1 ......................................................................................................... 9
INTRODUCTION
CHAPTER 2 ...................................................................................................... 39
IDENTIFICATION AND COMPARATIVE ANALYSIS OF PUTATIVE BACTERIOCIN-GENE
CLUSTERS IN STREPTOCOCCUS PNEUMONIAE
CHAPTER 3 ...................................................................................................... 71
EXPLORING THE FUNCTION AND REGULATION OF A PUTATIVE PNEUMOCOCCAL
PEPTIDE AND ITS GENE CLUSTER IN STREPTOCOCCUS PNEUMONIAE
CHAPTER 4 ...................................................................................................... 93
PRODUCTION OF A CLASS IC TWO-COMPONENT LANTIBIOTIC OF STREPTOCOCCUS PNEUMONIAE USING THE CLASS IA NISIN SYNTHETIC
MACHINERY AND LEADER SEQUENCE
CHAPTER 5 .................................................................................................... 109
GENERIC AND SPECIFIC ADAPTATIVE RESPONSE OF STREPTOCOCCUS PNEUMONIAE
TO CHALLENGE WITH THREE DISTINCT ANTIMICROBIAL PEPTIDES: BACITRACIN, LL-
37 AND NISIN
CHAPTER 6 .................................................................................................... 131
GENERAL DISCUSSION
REFERENCE LIST ......................................................................................... 139
SAMENVATTING VOOR DE LEEK ............................................................... 165
ACKNOWLEDGEMENTS/DANKWOORD .................................................... 171
Chapter 1
Introduction
Chapter 1
10
Introduction
11
Streptococcus pneumoniae
S. pneumoniae, the pathogen
S. pneumoniae (the pneumococcus) was first identified in 1881 simultaneously by
L. Pasteur and G. M. Sternberg (540). The pneumococcus is a Gram-positive bacterium that
belongs to the genus Streptococcus. The term Streptococcus means literally, ―strepto‖-
twisted and ―coccus‖- from the Greek word ―kokkus‖ meaning berry or grain. The genus
consists of bacteria of round, spherical shape that occur single and/or in pairs, and/or in
short chains (Fig.1). The former name of S. pneumoniae was Diplococcus pneumoniae,
since it mostly grows in pairs. S. pneumoniae is an aerotolerant anaerobe, but some of fresh
clinical isolates are obligate anaerobes.
Figure 1. Scanning electron micrograph (SEM) of S.
pneumoniae cells. The image was obtained from Public
Health Image Library (PHIL; http://phil.cdc.gov/Phil/; image
credit: #263, Janice Haney Carr, CDC).
The main characteristics distinguishing S. pneumoniae from other streptococci are
the production of alpha hemolysis (a green zone) when grown in blood, bile solubility,
inulin hydrolysis and sensitivity to optochin (513). The genus Streptococcus represents part
of the nasopharyngeal microflora of human and some of the Streptococcus species can be
pathogenic. The upper respiratory tract can be colonized asymptomatically by the
pneumococcus but the colonization rate varies between individuals and depends on the
geographical region and population group. However, when conditions are favourable to S.
pneumoniae, such as in young children, elderly and people with immunodeficiency
disorders, the pneumococcus might relocate to other parts of the human body, e.g. lungs,
ears, sinusitis, blood or brain, which may eventually cause diseases such as pneumonia,
otitis media, sinusitis, bacteraemia or meningitis. More than one million people each year
suffer from S. pneumoniae infections, of which over 800 thousand children from
developing countries, younger than 5 years old, die annually (465).
Interactions between S. pneumoniae and other streptococcal species during the
nasopharyngeal colonization have not been studied extensively (11,54,97,317,318,402).
Nevertheless, it was shown that during otitis media, S. pneumoniae is able to cohabit in a
biofilm with Haemophilus influenzae and Moraxella catarrhalis (362). Sometimes, three
latter species and Staphylococcus aureus might together colonize asymptomatically the
Chapter 1
12
nasopharynx of young children (412), but upon the occurrence of unknown triggering
conditions, they will start to compete with each other. The factors of the competition
mechanism between microorganisms colonizing a human body are not exactly identified
but in vitro and in vivo data showed that bacteriocins, hydrogen peroxide, pili, host immune
responses and/or other (unknown) factors play a role in S. pneumoniae competition with
other respiratory pathogens such as Neisseria meningitidis, H. influenzae, M. catarrhalis
and S. aureus (97,317,318,402,434,435,437,503). Bacteriocins are antimicrobial peptides
(AMPs) produced by bacteria. The genus Streptococcus produces a great number and
diversity of known bacteriocins and one of the first published articles concerning AMPs in
this genus goes back to the 1960s, when the AMPs were described in D. pneumoniae and in
the group A streptococci (298,349).
A capsular polysaccharide (CPS), composed of carbohydrate polymers, encloses
the pneumococcus cell. CPS was the first factor shown to be important in a S. pneumoniae
virulence (132). CPS prevents phagocytosis and aggregation, affects colonization and
adhesion, helps the pneumococcus to survive in the lungs and spread to bloodstream, and
contributes to antibiotic tolerance (137,240,309,356,367,541). Based on the CPS
composition, S. pneumoniae strains are divided into 91 serotypes (399). Consequently,
virulence of the S. pneumoniae strains depends primarily on a type of the serotype.
Interestingly, some of the pneumococcus clones, e.g. (serotype indicated in a superscript) S.
pneumoniaeSpain23F
ATCC700669, are able to switch their capsule type (84,85,263). There
are a few completely assembled genome sequences of the pneumococcus available in NCBI
database, i.e. D392, TIGR4
4, G54
19F, CGSP
14, Hungary
19A-6, Taiwan
19F-14, P1031
1, JJA
14,
ATCC 70066923F
, 705855 and R6 unencapsulated strain derivative of D39, and many more
are in sequencing progress. These genome sequences have made it possible to compare
DNA sequences of not-and/or closely related species. Furthermore, the genome content
variability of S. pneumoniae strains of the same or different serotypes was demonstrated
(94,179,380). Notably, it was shown for eight S. pneumoniae clinical isolates of different
serotypes that 15.6% of the sequence was unique to the reference strain TIGR4 and 5.5%
was unknown for the sequenced pneumococcus strains (472). Additionally only 46% of the
homologous gene clusters were common between 17 S. pneumoniae strains of distinct
serotypes (205).
The S. pneumoniae serotypes dissemination among people differs and depends on
such factors as age and geographical area (189,190). Roughly 10% of the carriers that is
people colonized by the pneumococcus, can be colonized by more than one pneumococcus
strain at the same time if the strains are not particularly of the same serotype
(54,183,221,247). It has been shown that 95% of children below the age of two, in
Birmingham, Alabama, have been colonized by up to six various pneumococcal serotypes
(158). S. pneumoniae of serotype 3, 6A, 11A, 19F, 23F, and/or 14, depending on
geographical region, are commonly found at the same time in healthy carriers (52,53).
Nevertheless, isolates of these serotypes (and additionally the ones of 6B, 9V and 19A) are
Introduction
13
the most common cause of otitis media in children younger than 18 years old (442). In
Denmark serotypes 3, 10A, 11A, 15B, 16F, 17F, 19F, 31 and 35F are related to high
mortality among children older than 5 (182). In the Netherlands serotypes 1, 5, 7F, 15B, 20
and 33F are the ones with the lowest mortality, serotypes 4, 6A, 8, 9V, 10A, 11A, 12F, 14,
19A, 22A, 22F, 23F, and 24F have intermediate mortality rates, and serotypes 3, 6B, 9N,
16F, 18C, 19F, and 23A have the highest rates (234). In 2008 serotypes 1, 3, 7, 14, and 19
were the most common cause of the pneumococcal infectious diseases in Europe (133). All
together, the serotype distribution among countries may vary but those with a high invasive
diseases potential, e.g. of serotype 3, 7, 19 and 23, are the same around the world.
Penicillin, an antibiotic of the beta-lactam family, has been used to treat
pneumococcal infection diseases since 1940. The resistance of a S. pneumoniae clinical
isolate to penicillin was described as early as 1967 (181). Since then, the resistance of the
pneumococcus to commonly used antibiotics, i.e. beta-lactams, macrolides,
chloramphenicol, tetracyclines and fluoroquinolones, has increased worldwide. This
increase has varied yearly for each antibiotic and also varies by country (65,133). For
instance, the occurrence in European countries of S. pneumoniae strains resistant to
penicillin and macrolide varies between 5% and 50% depending on the country
(133,152,344). A multidrug-resistant, i.e. non-susceptible to two or more antibiotics, S.
pneumoniae clinical isolate was first reported in 1977. This isolate was located in South
Africa and it was resistant to penicillin, erythromycin, clindamycin, tetracycline and
chloramphenicol (264). Nowadays, pneumococci of serogroups 6, 9, 14, 19 and 23F are
commonly multidrug-resistant (88,138,262). Interestingly, these serotypes are more
prevalent among carriers.
S. pneumoniae, vaccines
Currently, two types of vaccine against S. pneumoniae are available on the market,
i.e. a non-conjugated pneumococcal polysaccharide vaccine (PPV23) and a pneumococcal
conjugate vaccine (PCV). The first vaccine with commercial name Pneumovax23, is
effective against 23 serotypes, i.e. 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B,
17F, 18C, 19F, 19A, 20, 22F, 23F and 33F. Nevertheless, it is useful only for older children
and adults because, in children under the age of two years, the PPV23 fails to mount an
adequate immunity response (547). The PCV7 vaccine (produced by Wyeth under the
commercial name Prevnar) consists of capsular poly- and/or oligosaccharide of serotypes 4,
6B, 9V, 14, 18C, 19F and 23F conjugated to a carrier protein (a nontoxic recombinant
variant of diphtheria toxin) and is safe for use by infants and elderly people (546). The
improved PCV7, Prevnar13, will be available on the market soon and it will give protection
against six additional serotypes, i.e. 1, 3, 5, 6A, 19A and 7F. The PCV10 produced by
GlaxoSmithKline, under the commercial name Synflorix, contains antigen from ten
serotypes of the pneumococcus, i.e. 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F, conjugated
to a carrier protein (a protein from non-typeable H. influenzae strains) (155,559).
Chapter 1
14
The number of the pneumococcal invasive diseases caused by penicillin,
erythromycin and cephalosporin non-susceptible strains, e.g. 6B, 9V, 19F and 23F, has
decreased since the introduction of PCV7. Nevertheless, the number of invasive diseases of
non-vaccine serotypes, i.e. 3, 15A, 33F, 22F, 35B and 19A, has increased. A similar
situation was reported for carriage serotypes, namely a reduction of vaccine serotypes and
an increase of non-vaccine serotypes in carriers (93). Nowadays, multidrug-resistant
serotypes, such as 19A, are the most common cause of the pneumococcal infectious
diseases (93,204). The PPV23 and PCV vaccines protect against a limited number of
serotypes, and thus an increased number of the pneumococcal invasive diseases, caused by
the non-vaccine serotypes, is observed. Hence, a new protein-based pneumococcal vaccine
is currently in development and/or being clinically trialed, and is likely to become a
universal vaccine that is effective against all serotypes for all age groups (10,266). The
protein-based vaccine would consist of numerous pneumococcal virulence factors and
surface proteins.
S. pneumoniae, virulence factors
Virulence factors are molecules contributing to the morbidity and mortality caused
by a pathogen. Generally, virulence factors facilitate pathogen colonization, proliferation,
escape from the host‘s immune response and person to person spread.
One of the most important virulence factors is the pneumococcal capsular
polysaccharide (CPS). Apart from CPS, S. pneumoniae has other important virulence
factors, described below. Pneumolysin (Ply) is a cytoplasmic cholesterol dependent toxin
that is released from a bacterial cell during lysis with help of autolysin (LytA). Once
released, Ply forms pores in the cellular membrane of eukaryotic cells causing discharge of
cytoplasm and consequently tissue damage (208). The non-cytotoxic activity of
pneumolysin involves inhibition of complement system (567). Ply is essential for S.
pneumoniae to survive in the upper and lower respiratory tracts and to disseminate from
lungs to blood in an in vivo model. Thus, Ply is an important cytotoxin in invasive diseases,
i.e. pneumonia, bacteraemia and meningitis (30,32,389). The major autolysin, LytA, a N-
acetyl-muramyl-L-alanine amidase, is involved in the prevention of phagocytosis and
production of cytokines (331) and, a lytA mutant is less virulent in pneumonia and
bacteraemia murine models (58). Nevertheless, it is thought that LytA only mediates
virulence by triggering the pneumococcal cell lysis and, as a consequence, release of
pneumolysin, inflammatory cell wall components and teichoic acids (331). Both the NanA
and NanB neuraminidases play a significant role during colonization and survival in the
lungs and the blood stream (313). PavA, pneumococcal adhesion and virulence A,
modulates adherence to immobilized fibronectin and plays an important role in virulence,
as it is attenuated in the sepsis and the meningitis model (213,419). Notably, this protein
stimulates adaptive immune response and production of cytokines, and it helps to avoid
phagocytosis (378). Another adhesin, namely the pneumococcal serine-rich repeat adhesin
Introduction
15
P (PsrP), is important in the development of pneumonia since it contributes to
pneumococcal adherence to lung epithelial cells (447,474). The pneumococcal pili encoded
either by the first pilus islet, PI-1 (also known as the rlrA islet) or by the second pilus islet,
PI-2, are not present in all pneumococcal clinical isolates. Those strains with either type of
the pili, PI-1 or PI-2, exhibit increased adhesion to human respiratory epithelial cells
(17,366). Additionally PI-1 triggers higher host inflammatory responses than strains
without pili, and they enhance the ability of S. pneumoniae to colonize, and to cause
invasive diseases (21,153). The choline-binding protein A, CbpA, also known as the
pneumococcal surface protein C, PspC, plays a role in colonization by facilitation of
adherence to epithelial cells, prevents phagocytosis and avoids complement activation
(178,235,383,448). Another choline-binding protein, i.e. pneumococcal surface protein A
(PspA), is a significant factor for the pneumococcus to colonize and to cause invasive
diseases as it prevents complement-mediate opsonisation and killing by lactoferrin
(383,471,519).
Metal-binding lipoproteins such as pneumococcal surface antigen A (PsaA),
pneumococcal iron acquisition A (PiaA) and pneumococcal iron uptake (PiuA) are
important for S. pneumoniae virulence since mutation of these transporters reduces
pneumococcal virulence in pneumonia, bacteraemia and additionally mutation of psaA
reduces colonization (31,50,320). PsaA is involved in the protection from oxidative stress
(517). The polyamine transporter (PotD) and immunoglobulin A protease (IgA) are
important virulence factors because IgA is the pneumococcal protease that reduces
efficiency of the immunity system by cleavage of a human immunoglobulin protease IgA1
(537) and a potD mutant is attenuated in pneumonia and the bacteraemia model (538).
Pneumococcal surface proteins such as enolase (Eno) and hyaluronate lysase (HylA)
contribute to S. pneumoniae virulence in invasive diseases (27,236). Another surface
virulence factors, i.e. β-galactosidase (BgaA) and β-N-acetylglucosaminidase (StrH),
mediate colonization of the human nasopharynx by facilitating pneumococcal adherence to
the epithelial cells (256). Surface anchored proteins PhtA, PhtB and PhtD (pneumococcal
histidine triad) protect against nasopharyngeal colonization, pneumonia and sepsis
(1,177,382,570).
Bacteriocins are peptides with an antimicrobial activity mainly against closely
related species and they are characterized in detail below. BlpM and BlpN (bacteriocin-like
peptides) are bacteriocins produced by S. pneumoniae strains (97,300). They are important
factors for S. pneumoniae strains that enable intra- and interspecies competition in the
nasopharynx (97) and consequently survival, colonization and transmission of the
pneumococcus to other parts of the human body.
Many of these virulence factors are highly immunogenic, which makes them good
candidates for a vaccine. In addition, this suggests that more than one virulence factor is
essential for S. pneumoniae virulence and therefore, for the protein vaccine. Virulence
factors such as PspA, PspC, PsaA, LytA, pneumolysin, PiuA, PiaA, PotD, NanA and PhtB,
Chapter 1
16
are good candidates for the vaccine because they trigger a protective immune response in
animal models and are likely to increase survival (381,499). However, the optimal
combination of the proteins mentioned above, which would give the best protection against
pneumococcal infection diseases, is still being investigated.
Antimicrobial peptides
Antimicrobial peptides, an introduction
Antimicrobial peptides (AMPs) are small proteins produced by the majority, if not
all, living organisms in order to eliminate environmental microorganisms such as viruses,
bacteria, fungi and/or protozoa, while they are immune. Thus, AMPs are defensive
weapons used widely by both bacteria and the human body. Regardless of their host
diversity, AMPs contain common features such as they are all natural products of living
organisms and they all have antimicrobial activity, and low molecular weight. AMPs are
grouped independently of their producer organism, but according to the way they are
synthesized and their structural characteristics. For instance, there are ribosomally and
nonribosomally (nonribosomal peptides, NRP) synthesized peptides, anionic or cationic
molecules, circular, linear, or globular ones and those with specific amino acids
compositions. One of the best characterized and most common group of AMPs is the class
of ribosomally synthesized and of cationic nature, named cationic antimicrobial peptides
(cAMP). Generally, cAMPs, of both human and bacteria, are produced as inactive peptides
consisting of an N-terminal leader sequence, which is cleaved off during release of the
peptide from the cell, and a C-terminal cationic sequence which forms the active peptide.
cAMPs become active once the N-terminus is cleaved off. Here, AMPs produced by
humans and Gram-positive bacteria will be discussed.
cAMPs of the human host
In the human body, the first defense barrier against pathogenic microorganisms is
the innate immune system. It is composed of multiple components: a mechanical barrier of
skin epithelium, tissues of epithelial cells producing mucus, lysozyme, neutrophils,
dendritic cells, macrophages, natural killer cells, phagocytes and/or effector molecules such
as antimicrobial peptides. In humans, the following distinct groups of cationic AMPs have
been identified: cathelicidin, defensins and histatins (19,109,345).
Cathelicidin
The cathelicidin family/group includes one member, the 18 kDa hCAP-18. The
hCAP-18 is produced by neutrophils, monocytes, NK cells, T and B cells and by epithelial
cells, as an inactive preproprotein (42,124). The serine proteinase cleaves the precursor
protein, cathelin, from the carboxyterminal peptide, which is then named LL-37 (486). The
Introduction
17
11 kDa cathelin displays antimicrobial activity against bacterial species that are resistant to
LL-37. However, the lack of a net-positive charge and structural similarity to the cystatin
family disqualifies cathelin from the cAMP group. LL-37 is a linear 37 amino acid cationic
peptide with activity against both Gram-positive and Gram-negative bacteria (20,124). It
has been shown that bactericidical action of LL-37 is due to immobilization of the peptide
into the membrane and as a consequence destabilization of the bacterial membrane
(388,522). In addition to killing pathogens, this peptide has multiple roles, such as immune
activation, proliferation of the inflammatory cells, chemotaxis, angiogenesis, wound
healing and antitumor activity (20,42,124).
Defensins
In humans, defensins are produced by epithelial cells of the skin, gastrointestinal,
urogenital, and respiratory systems, and immune cells. Defensins are small 3.5-6 kDa
peptides, of which cysteine residues form three to four disulfide bridges within the
molecule. Based on the arrangement of the bridges and their structure, human defensins are
divided into two classes, namely α-defensins and β-defensins. Both types of defensins can
protect the human body against Gram-positive and Gram-negative bacteria, viruses
including the immunodeficiency virus HIV-1 and pathogenic yeasts (19,103,428). In
addition to their antimicrobial activity, defensins participate in many other processes such
as chemoattraction and activation of the immune or inflammatory responses to infection
sites, wound healing, acceleration of angiogenesis, promotion of the production and release
of cytokines and chemokines and neutralization of bacterial lipopolysaccharides
(110,375,376,428).
Histatins
Histatins are peptides rich in histidine residues and are found in human saliva.
They contribute significantly to a healthy oral cavity because they have antibacterial and
strong antifungal activity, and they inhibit plaque formation. Importantly, histatins prevent
inflammation and inhibit host and bacterial enzymes involved in periodontal diseases. As a
consequence, histatins are under clinical investigation as treatment for oral fungal
infections (103,117,251).
The nonribosomal peptides (NRP) of Gram-positive bacteria, bacitracin
The NRPs are a class of secondary metabolites of microorganisms. Bacitracin, an
example of the NRP, is an antimicrobial substance produced by Bacillus licheniformis and
some strains of Bacillus subtilis. Bacitracin is synthesized as a mixture of closely related
cyclic dodecylpeptides, by a specialized nonribosomal peptide synthetase (NRPS) complex.
In general, the NRPS complex is organized in several modules, e.g. an initiation, an
elongation and a termination module. Each of these modules is responsible for the
introduction of one additional amino acid. Bacitracin is used as an antibiotic for treatment
Chapter 1
18
of skin and eye infections as well as for prevention of wound contagions caused by Gram-
positive cocci and bacilli. However, this AMP has a rather narrow antimicrobial spectrum,
for which it requires a divalent metal ion (271,314,315,464).
cAMPs of Gram positive bacteria
AMPs produced by bacteria are called bacteriocins. In general, they are short
(between 30 and 60 amino acids), hydrophobic and/or amphipathic peptides. Bacteriocins
are ribosomally synthesized as an inactive precursor peptide (prepeptide) that consists of an
N-terminal leader sequence and a C-terminal propeptide. The leader sequence targets
bacteriocins to a dedicated transporter and keeps bacteriocins in an inactive form until they
are secreted (121,131,374). As there were previously diverse classifications of bacteriocins,
N. C. K. Heng and J. R. Tagg proposed a universal classification consisting of four classes,
namely i) modified bacteriocin named lantibiotics, ii) unmodified peptides, iii) large
proteins and iv) cyclic peptides (203,258). However, the majority of bacteriocins belong to
the first two classes and thus these classes will be discussed in more detail below (Table_1).
Introduction
19
Table 1. Overview of some of the class I and class II bacteriocin peptides mentioned in this introduction a synthesized without the leader sequence; +, feature present/used; ND, not determined
Class I – Lantibiotics
Peptide Producer strain Mass (Da)
Leader type Modification enzyme Processing and
transport Ref.
PR-type GG-type LanB,
LanC LanM
LanP,
LanT LanT(P)
Class IA
Nisin Lactococcus lactis 3353 + + + (161) Subtilin Bacillus subtilis 3317 + + + (159,160)
Pep5 Staphylococcus
epidermidis 3488 + + + (244)
Mutacin I Streptococcus mutans 2364 + + + (423,424)
Epidermin S. epidermidis 2164 + + + (3)
Class IB
Mersacidin Bacillus ssp. 1825 + + + (68,69)
Lacticin 481 L. lactis 2901 + + + (413)
Salivaricin A Streptococcus salivarius 2315 + + + (449) Streptococcin A-FF22 Streptococcus pyogenes 2795 + + + (225)
Class IC
Cytolysin LL/LS Enterococcus faecalis 4164/2631 + + + (154) Lacticin 3147 A/B L. lactic 3322/2847 + + + (453)
Staphylococcin C55α/C55β Staphylococcus aureus 3339/2993 + + + (364)
Chapter 1
20
Class II – unmodified bacteriocins
Peptide Producer strain Mass (Da)
Leader type Processing and
transport Ref. No leader/other
leader GG-type
Class IIa
pediocin PA-1 Pediococcus acidilactici 4629 + + (198,332)
leucocin A Leuconostoc gelidum 3390 + + (187,397,530)
mesentericin Y105 Leuconostoc mesenteroides 3868 + + (9,194,195) sakacin A Lactobacillus sakei 4306 + + (13,14)
Class IIb
lactococcin A L. lactic 5778 + + (216,528) lactococcin 972 L. lactic 7500 + (QA-site) ND (327,329)
lacticin Q L. lactic 5926 +a ND (144)
Blp S. pneumoniae ND + + (97,307)
Class IIc
lactococcin G α/β L. lactic 4346/4110 + + (373)
Mutacin IV NlmA/NlmB S. mutans 4169/4826 + + (278,424) termophilin 13 ThmA/ThmB Streptococcus thermophilus 5776/3910 + ND (316)
CibAB CibA/CibB S. pneumoniae ND + ND (168,193)
Introduction
21
Class I - lantibiotics
Bacteriocins of the class I were named lantibiotics because they contain unusual
amino acids i.e. lanthionine (Lan) and/or methyllanthionine (MeLan). Additionally these
bacteriocins can contain unsaturated amino acids such as 2,3-didehydroalanine (Dha)
and/or (Z)-2,3-didehydrobutyrine (Dhb), as well as structures such as lysinoalanine, β-
hydroxy-aspartate, D-alanine, 2-oxobutyrate, 2-oxopropionate, 2-hydroxypropionate, S-
aminovinyl-D-cysteine, and/or S-aminovinyl D-methylcysteine (67,553).
The inactive form of the lantibiotic prepeptide is generally called LanA (―Lan‖ is a
general abbreviation for proteins involved in lantibiotics biosynthesis). The leader sequence
serves as recognition and a redirection site of the prepeptide to dedicated modification(s)
(LanBC or LanM and/or LanD) and transport (LanT) proteins. The LanBC or LanM
enzymes initiate the amino acid modifications, which results in the Lan and MeLan
residues. Modifications occur as follows: serine and threonine residues of the propeptide
are dehydrated to Dha and Dhb, respectively, by LanB or LanM. Subsequently, in the
propeptide, lanthionine or methyllanthionine might be formed by a cyclization reaction of
Dha or Dhb with a cysteine residue performed by either LanC or LanM (Fig. 2). Once the
dedicated modification enzymes have posttranslationaly transformed the C-terminus of the
peptide, the peptide is released from the cell by an ABC transporter (LanT) with or without
the N-terminus. Such a peptide becomes an active lantibiotic, once the leader sequence is
removed by a dedicated protease (LanP) or a LanT variant that contains a protease domain
(Fig. 2 and 3). The leader peptide is cleaved off behind characteristic GG, GA, GS, GI, or
PR or PA cleavage sites. However, some lantibiotics are processed in other, more
uncommon sites (67,163,553).
In addition to the modifications carried out by LanB and LanC or LanM, some
lantibiotics possess a LanD enzyme that performs the oxidative decarboxylation of LanA.
The LanD proteins oxidize and decarboxylate the C-terminal cysteine residues, before they
are coupled with Dha or Dhb, forming S-[(Z)-2-aminovinyl)]-ᴅ-cysteine (AviCys) or to S-
[(Z)-2-aminovinyl)]-(3S)-3-methyl-ᴅ-cysteine (AviMeCys) (291-296,311).
Based on their structure, lantibiotics are divided into type A (elongated peptides),
type B (globular peptides) and type C (multi-component peptides) (203). Lantibiotics can
be further classified according to the enzymes used for their modifications. Thus, there are
lantibiotics that use two distinct proteins, LanB and LanC, and others that use only one
protein, namely LanM, which combines the function of both LanB and LanC (67,163,553).
Class IA of lantibiotics
The IA class includes elongated lantibiotics modified by both the LanB and LanC
proteins or only by LanM. Nisin (161) (Fig.2), subtilin (159,160), epidermin (3),
gallidermin (252), staphylococcin T (146), mutacin 1140 (206), mutacin B-Ny266 (358),
mutacin I and III (422,422,423), Pep5 (244) and epicidin 280 (197) are modified by LanB
Chapter 1
22
and LanC. The LanM protein transforms bacteriocins such as lactocin S (480,481),
plantaricin C (521) and nukacin ISK-1 (460).
Figure 2. Posttranslational modification process of lantibiotics based on a representative bacteriocin i.e. nisin A.
The prepeptide NisA is synthesized; subsequently, NisB catalyzes dehydratation of underlined serine and
threonine residues, S and T, respectively, which is followed by a cyclization, in the propeptide part of the NisA,
carried by NisC. In the cyclization reaction the Lan and/or MeLan are made. After that, NisP proteolytically
removes the leader peptide and a mature nisin is formed.
Nisin, produced by Lactococcus lactis, is one of the best-characterized lantibiotics.
Nisin has been used successfully for over 45 years in the food industry as a preservative
(105). There are three known forms of nisin namely nisin A, nisin Z and nisin Q. The two
latter forms differ from nisin A by their amino acid sequence, namely nisin Z by one amino
acid and nisin Q by four (104,359,568). The nisin biosynthetic gene cluster, located on a
conjugative transposon, is composed of genes encoding proteins involved in synthesis of
the structural peptide (NisA; Nis, is the abbreviation for proteins engaged in nisin
production), nisin modification (NisB and NisC), transport (NisT), processing (NisP),
regulation (NisR and NisK) and immunity (NisI, NisF, E and G). The NisBTC proteins
Introduction
23
form the modification and transport membrane-associated complex (67,70,306). The
structural prepeptide of nisin (NisA; Fig. 2) is composed of 57 amino acid residues. The
mature and active form of nisin has 21 common amino acids, 1 lanthionine, 4
methyllanthionines, 1 dehydrobutyrine, and 2 dehydroalanines, making a total of five
lanthionine rings. A schematic representation of the posttranslational modifications and
processing of nisin is shown in Fig. 2.
Class IB of lantibiotics
The type IB includes globular lantibiotics such as mersacidin (68,69), cinnamycin
(143,243), duramycin, duramycin B and C (143), lacticin 481 (413), salivaricin A (449),
streptococcin A-FF22 (225), butyrivibriocin OR79A (246), variacin (420), mutacin II (556)
and actagardine (also known as gardimycin (254)). These lantibiotics have single LanM
protein to catalyze dehydratation and cyclization reactions. Mersacidin is produced by
Bacillus strains and contains three MeLan rings and one Dha, and AviMeCys. Interestingly,
mersacidin and cinnamycin do not have a typical processing site, instead these lantibiotics
are cleaved off after an EAA and AFA site, respectively (34,548).
Class IC of lantibiotics
The representatives of type IC are two-component lantibiotics, e.g. plantaricin W
(214), staphylococcin C55 (364), cytolysin (154), BHT-A (223), haloduracin (342) and
lacticin 3147 (453). The two-component lantibiotics require both peptides for their full
antimicrobial activity. This group of lantibiotics generally uses the LanM type of
modification enzyme and often each of the prepeptides has its own LanM protein. The
prepeptides are usually designed as LanA1 and LanA2, and the mature versions are named
Lanα and Lanβ. Generally, all α peptides described here have three rings and β peptides
have two, three or four (325). Of the multi-component lantibiotics, cytolysin is atypical
since it is also active against eukaryotic cells including erythrocytes and the prepeptides are
processed twice in order to establish their activity (91). It seems that plantaricin W and
haloduracin also may require double processing (214,342).
Modifications, processing and transport of lantibiotics
The LanBCT/LanMT proteins form the lantibiotic synthetase complex. The Lan
and/or MeLan are formed, in two steps, by either LanB and LanC or LanM (Fig. 2). First,
the hydroxyl of serine and threonine residues is dehydrated forming the α, β-unsaturated
amino acids, Dha and Dhb, respectively. Second, a thioether bridge might be formed by
joining a sulfhydryl group of a cysteine residue with a double bond of either Dha or Dhb
(Fig. 2). The LanB dehydratases do not show sequence similarity to other proteins. They
demonstrate rather low sequence identity, of approximately 30%, to each other, when the
prepeptides are not analogous. The LanC cyclases are zinc metalloproteins, of which the C-
terminus shows roughly 27% sequence identity to LanM. The LanB, LanC and LanM
Chapter 1
24
proteins have low substrate specificity. In other words, the Lan enzymes can modify the C-
terminus of any lantibiotic or nonlantibiotic, the only requirement is that propeptide is fused
to a leader sequence of a dedicated Lan enzyme (66,67,123,163,265,393,553).
Generally, lantibiotics are transported out of the cell by a dedicated LanT ABC
transporter (Fig. 3). Nevertheless, some lantibiotics such as Pep5 and epicidin 280 likely do
not require LanT for secretion (67,197,346). The LanT of nisin (NisT) has low substrate
specificity because it can transport modified and unmodified bacteriocins and non-
bacteriocin peptides (282). LanP is a subtilisin-like serine protease that cleaves the leader
sequence from the prepeptide and unlike LanT, LanP exhibits high substrate specificity.
The LanP enzyme of nisin (NisP) removes the leader peptide only from the prenisin with
already formed thioether rings (Fig. 2 and 3).
Figure 3. Schematic overview of the regulation and production of class I (on the left) and class II (on the right)
bacteriocins. HK, histidine kinase; RR, response regulator; ABC, ABC transporter; AP, accessory protein; IM,
immunity protein; LanB, dehydrogenase; LanC, cyclase; LanP, protease; LanT, transporter; LanFEG, immunity
proteins. The numbers next to the arrows indicate processes as follow: (1) regulation by TCS, i.e. RR and HK, as it
is marked in this figure or by a non-TCS (single) regulator; (2) expression of the bacteriocin locus genes; (3)
synthesis; (4) processing and export; (4a) modifications and export (by LanBCT as shown in this figure or by
LanMT), and processing; (5) immunity.
Introduction
25
An exceptional processing occurs for cytolysin, the two-peptide lantibiotic. Here,
each of the peptides is processed twice, namely the first time by a LanT protein (CylT) at
the transport stage and the second time by LanP (CylP) outside the cell. Lantibiotics such as
lacticin 481 and mutacin II do not have a dedicated LanP enzyme but instead they are
processed by a LanT protein, which combines function of an ABC transporter and protease
because it contains an N-terminal protease domain (67,123,163,393,553).
The LanD proteins introduce unusual amino acids, namely AviCys or AviMeCys,
to the C-terminus of some lantibiotics, e.g. epidermin, gallidermin, mersacidin and mutacin
1140. The LanD enzymes contain a noncovalently bound cofactor, either flavin
mononucleotide (FMN) or flavin adenine dinucleotide (FAD). The LanD of epidermin
(EpiD) has low substrate specificity in contrast to LanD of mersacidin (MrsD), which is
able to modify only mersacidin.
A number of other modifications have been shown to occur in lantibiotics, e.g. lacticin
3147 and lacticin S have ᴅ-Ala, which is introduced by the LanJ enzyme. Less common
modifications include erythro-3-hydroxy-ʟ-aspartic acid in cinnamycin and duramycin,
head-to-tail lysinoalanine bridge in cinnamycin, and in cypemycin bis-methyletion, and
allo-isoleucine (67,123,163,393,553).
Class II, unmodified peptides
Bacteriocins of the class II are non-lanthionine containing peptides and, unlike
lantibiotics, they do not require posttranslational modifications in order to be
antimicrobially active. Commonly, the gene cluster of the class II bacteriocins is composed
of a structural gene(s) encoding a precursor peptide, one or two dedicated ABC transporters
often containing a protease domain, optionally a protease, an immunity gene(s) and an
accessory protein. The unmodified bacteriocins become active through the following
process: the peptide is secreted and then the precursor peptide‘s N-terminus leader is
cleaved off behind the GG cleavage site by the protease, although not always in this order.
The accessory proteins are thought to be important in bacteriocin translocation and/or
processing (Fig. 3). However, their exact role is still being investigated. The class II of
bacteriocins is divided into three subclasses, i.e. pediocin-like peptides (IIa), miscellaneous
peptides (IIb) and multi-component peptides (IIc) (114,121,131,203,368).
Class IIa, the pediocin-like peptides
The class IIa consists of more than 20 bacteriocins and includes e.g. leucocin A
(187,397), sakacin A and P (14,211,222,511), curvacin A (510), mesentericin Y105
(194,195), pediocin PA-1 (198,332), enterocin A and P (15,59,75), divergicin (212),
carnobacteriocin B2 and BM1 (425), acidocin A (248), listriocin 743A (245), bacteriocin
31 (514), and enterocin SE-K4 (126). The characteristic feature of this subclass is a
―pediocin box‖, YGNGV/L(x)C(x)4C(x) (x, stands for any amino acid), in the N-terminal
part of the propeptide. Each of two cysteine residues of the pediocin box forms, with a
Chapter 1
26
dedicated residue of the C-terminus, a disulfide bridge. Moreover, all of the bacteriocins
from this class display strong antilisterial activity (121,131,368).
Most bacteriocins are secreted by a dedicated ABC transporter, though some of the
class IIa bacteriocins e.g. enterocin P (75), mesentericin Y105 (36), carnobacteriocin B2
(343) and divergicin (558) are transported by the Sec-dependent translocation system.
Accordingly, these four peptides have the N-terminal leader sequence of the Sec-system
and not the common leader peptide with a GG cleavage site (121,131,374).
Class IIb, miscellaneous peptides
This subclass combines all peptides other than the pediocin-like and the two-
peptide bacteriocins. There are more than 30 miscellaneous peptides e.g. lactococcin A
(216,528), lactococcin B (527), lactococcin 972 (327,328), enterocin B (59), enterocin EJ97
(147), lacticin Q (144), lacticin Z (231), BHT-B (424), aureocin A70 (369),
carnobacteriocin A (557) and bovicin 255 (545) and S. pneumoniae bacteriocins, BlpM and
BlpN (97,307). Some of the enterocins produced by Enterococcus species, i.e. L50A,
L50B, Q and EJ97, and aureocin A70, BHT-B, lacticin Q and Z are produced without an N-
terminal leader sequence. In addition, the three latter peptides have formylated N-terminal
methionine residues and they show rather high 46% sequence identity. Aureocin A70 is an
atypical bacteriocin because it is composed of four peptides encoded by four genes located
in the same operon. The aureocin peptides have high sequence similarity to each other. It
was shown that three of these peptides have antimicrobial activity. However, it is not
known whether the antimicrobial activity of aureocin 70 is due to a synergistic work of four
of them (369,374). Lactococcin 972 is a unique peptide in this subclass since the active
form of this peptide is a homodimer, it has another mode of action than most of the class II
bacteriocins and it is secreted by the Sec-dependent pathway (326-328).
Class IIc, multi-component peptides
The multi-component class IIc bacteriocins (two-peptide bacteriocins) are those
that consist of two very different peptides, designed as the α and β, and both peptides need
to act synergistically for full antimicrobial activity. More than 15 two-peptide bacteriocins
have been isolated and described. Examples of the class IIc include lactococcin M (355),
lactococcin Q and lactococcin G (373,569), mutacin IV (424), plantaricin E/F and
plantaricin J/K (188,353), plantaricin S (237), lactacin F (360,361), leucocin H (37),
enterocin 1071 (18), enterocin L50 (76), brochocin-C (479), acidocin J1132 (497,498),
termophilin 13 (316) and S. pneumoniae CibAB (80,168). In almost all cases the two
peptides of each bacteriocin have one or two GxxxG motifs in their C-terminus. These
motifs are commonly involved in helix-helix interactions in membrane proteins. It has been
shown that because of the GxxxG motif, peptide α interacts with peptide β forming a helix-
helix structure (149,368,374). Generally, the α and β peptides of the same bacteriocin do
not show amino acid sequence similarity to each other or to other two-peptide bacteriocins.
Introduction
27
Nevertheless, the α and β peptide of enterocin L50 share more than 70% identity to each
other; and the α and β peptide of enterocin 1071 and lactococcin G show significant
similarity of above 60%. Interestingly, the β peptides of mutacin IV, termophilin 13 and
lacticin F show sequence homology to each other and to the α peptides of termophilin 13
and mutacin IV. Notably, acidocin J1132 is an atypical bacteriocin because the α and β
peptide are transcribed from one gene and thus, it is unclear whether acidocin J1132 should
be qualified as a two-peptide or a one-peptide bacteriocin (149,368,374).
Mode of action of AMPs
All characteristic features of bacteriocins such as their small size, amino acid
sequence structure, cationic charge, hydrophobicity and amphipathicity, determine their
mode of action. In general, the antimicrobial activity of AMPs is due to their action towards
either the bacterial cell membrane (pore formation) or synthesis of peptidoglycan, or other
mode of actions.
Pore formation
The majority of cAMPs, e.g. LL-37, most of the lantibiotics and class II
bacteriocins, forms pores in the cytoplasmic membrane of sensitive cells. The attraction of
bacteriocins to bacterial membranes is enhanced by the fact that both have opposite charge,
i.e. bacteriocins are cationic and membranes are anionic. Through these pores, which can be
up to three nm wide, the efflux of ions and small molecules occurs. Additionally depletion
of ATP and dissipation of pH and/or membrane potential might take place. The mechanism
that causes membrane permeation can be divided into three models namely the barrel-stave,
carpet or toroidal-pore (47,196,352). Each of the models starts with the attraction of a
bacteriocin to the membrane, followed by attachment of a bacteriocin and interaction with
lipid bilayers. Once a threshold amount of the bacteriocin is reached, the peptides begin
with their insertion and membrane permeabilization. In the barrel-stave model, the attached
peptides aggregate and install themselves into the membrane bilayer in such a way that the
hydrophobic peptide regions are aligned with the lipid core region and the hydrophilic
peptide regions form the interior region of the pore. In the carpet model, bacteriocins
disrupt the membrane by orienting themselves parallel to the surface of the lipid bilayer
forming an extensive layer of a carpet. Hydrophilic regions of the peptides are on the side
of the pore and hydrophobic ones are directed in the lipid region. In the toroidal-pore
model, bacteriocins enter the membrane and induce the lipid monolayers to bend so that
both bacteriocins and the lipid head groups line the water core. Here also the hydrophilic
regions of bacteriocins face the pore (47,562). Lacticin Q, a class II bacteriocin produced
by L. lactis species (144), exhibits an unique pore formation model named ―huge toroidal-
pore‖, which occurs as follows. Briefly, lacticin Q binds to the negatively charged bacterial
membrane, after which it forms the largest pore described so far of 4.6 up to 6.6 nm. The
large pores cause protein leakage from the susceptible cell and a lipid transbilayer
Chapter 1
28
movement named flip-flop, during which lacticin Q migrates to the inner side of the
bacterial membrane. This bacteriocin does not require a target or docking molecule and it is
active in nanomolar amounts. Until now, lacticin Q is the only example of a bacteriocin
produced by Gram-positive bacteria, which in nanomolar concentration range is able to
form huge pores, protein leakage, lipid flip-flop and the bacteriocin translocation to inner
side of the membrane (563-565).
Interestingly, some cAMPs, e.g. nisin, may require a docking molecule for their
full antimicrobial activity. Nisin primarily uses lipid II as a docking molecule. This is the
most known and common target for bacteriocins with this mode of action (533). Lipid II is
a precursor for peptidoglycan, and thus the bacterial cell wall synthesis, because it carries
the subunit components of the cell wall across the bacterial membrane, i.e. N-
acetylglucosamine (GlcNAc)-N-acetylmuramic acid (MurNAc)-pentapeptide. The single
molecule of lipid II is composed of one GlcNAc-MurNAc-pentapeptide subunit linked to a
polyiosoprenoid anchor of 11 subunits long, via a three pyrophosphate moieties (45,46,49).
Nisin binds to the pyrophosphate molecules of lipid II forming the so-called pyrophosphate
cage (220,550). Briefly, one molecule of nisin first binds to the lipid II, which generates
docking sites for other nisin molecules. Nisin‘s first two lanthionine rings (A and B)
interact with the pyrophosphate of lipid II (220). Subsequently, the pore complex in the cell
membrane is formed and it constitutes of eight nisin and four lipid II molecules.
Additionally to membrane permeabilization, nisin inhibits cell wall synthesis by binding to
lipid II (45,46,49,185,549,552).
Some, if not all, two-component lantibiotics, e.g. lacticin 3147, use a mode of
action similar to nisin with lipid II as a docking molecule. Shortly, first the A1 peptide of
lacticin 3147 binds to lipid II, which is followed by binding of the A2 peptide and
subsequent pore formation. In this case, the pore complex is composed of four molecules of
each: A1, A2 and lipid II. This complex is analogous to that of nisin, in which there are also
eight bacteriocin molecules binding to four lipid II molecules (43,551). Interestingly, the
bactericidal activity of the two-peptide bacteriocins is higher, when both components are
involved in generating lipid II binding and permeabilization.
Class IIa and IIb pore forming bacteriocins, e.g. lactococcin A and lactococcin B,
enterocin P, mesentericin Y105 and sakacin A, use another docking molecule, namely the
mannose phosphotransferase system (man-PTS). It was shown that lactococcin A employs
the membrane-located proteins IIC and IID of man-PTS system to recognize a sensitive cell
and subsequently forms pores in the membrane (95,115,195,429,430,561).
Inhibition of cell wall synthesis
Lantibiotics such as mersacidin, actagardine, epidermin, gallidermin,
staphylococcin T and mutacin 1140 trigger bactericidal effects by inhibition of
peptidoglycan synthesis via binding to the lipid II but do not form pores. Epidermin,
gallidermin, staphylococcin T and mutacin 1140 have similar ring structures as nisin and
Introduction
29
they bind in a similar manner to lipid II. However, they are too short to be able to form
pores in the bacterial cell membrane. Consequently, by binding to lipid II these lantibiotics
remove lipid II from the site of peptidoglycan synthesis, i.e. the cell division site, and
segregate lipid II in a process named sequestration (186). Mersacidin, actagardine,
plantaricin C and lacticin 3147 are lantibiotics, which block the transglycosylation step of
peptidoglycan synthesis by binding to the lipid II. These peptides bind to all three subunits
of lipid II, i.e. GlcNAc, MurNAc and pyrophosphate (48). Thereby, the binding is different
from that of nisin, epidermin, gallidermin, staphylococcin T and mutacin 1140, which all
bind to the pyrophosphates of lipid II (220,550). Importantly, Ca2+
ions improve the
bactericidal activity of mersacidin, plantaricin C and lacticin 3147 by facilitating the
interaction of the peptides with the cell membrane and with lipid II (40,551).
Lactococcin 972 is a non-lantibiotic that has an unusual mode of action. It inhibits
cell division by blocking septum formation. Thus, lactococcin 972 is active only against
dividing cells and causes cell elongation and broadening. This bacteriocin binds to lipid II
and additionally inhibits the activity of two enzymes that use lipid II as a substrate namely
PBP2 and FemX thereby lactococcin 972 inhibits polymerization of the peptidoglycan. It
has been shown that lactococcin 972 most likely has a different lipid II binding site than
that of nisin and mersacidin meaning that a novel, third binding motif may be used by
bacteriocins (326-328).
Other mode of actions
It is worth mentioning that nisin, subtilin and sublancin have additional modes of
action. These lantibiotics are able to inhibit germination of spores from Bacillus and
Clostridium species (394,433). Other lantibiotics, i.e. cinnamycin and duramycin, induce
hemolysis of erythrocytes, inhibit phopholipase A2 or interfere with leucotriene and
prostaglandin synthesis in addition to their bactericidal activity. Furthermore, cinnamycin
can inhibit bacterial ATP-dependent protein translocation and calcium uptake, and
duramycin inhibits chloride transport and sodium and potassium ATPase
(363,365,490,560). All together, the mode of action of bacteriocins may be also other than
bactericidal, indicating an important role for these small peptides in the lifestyle of bacteria.
Self-immunity to produced bacteriocins
All AMPs producers are resistant to their own product. Although the structure,
production, modification and mode of action of bacteriocins are relatively well studied, the
self-protection mechanism to cognate bacteriocins is still not well understood.
For most of bacteriocins, the immunity gene(s) is located either in the same operon
as the structural bacteriocin gene or in close vicinity. Generally, self-immunity to
bacteriocins of class I consists of a single protein LanI, e.g. for cytolysin and Pep5
(82,346), and/or an ABC transporter composed of LanFEG, e.g. in case of mersacidin
(162). The LanI and LanFEG proteins can act together or separately. A fourth uncommon
Chapter 1
30
immunity protein namely LanH is an accessory molecule of the LanFEG ABC transporter
and it is found in the cluster of nukacin ISK-1, epidermin and gallidermin (6,7,136,384-
386,405). Usually, gene clusters of class II bacteriocins have one gene encoding an
immunity protein.
Nisin producers use two membrane bound, autonomous immunity systems, i.e.
LanFEG (NisFEG for nisin) and LanI (NisI associated with nisin) (173,426,475,488).
However, both systems are necessary for complete immunity towards nisin. It was shown
that NisI is important to interact with nisin and that 21 amino acids of the C-terminus of
NisI are involved in this process (500). Accordingly, it has been proposed that NisI
recognizes nisin and that NisFEG exports the peptide (120,272,489,500). The exact
protective action of lantibiotics‘ immunity proteins is not yet well understood. It was
suggested that LanI-type proteins either aggregate lantibiotics to prevent pore formation or,
for the bacteriocins that bind to lipid II, LanI might compete for lantibiotic-lipid II
interaction. Additionally the ABC transporter, LanFEG, of lantibiotics targeting lipid II
could possibly separate a peptide from its target and export it outside the cell (120).
Nukacin ISK-1 requires the LanFEG (NukFEG) and LanH (NukH) proteins for
full immunity. The LanH protein of nukacin ISK-1 (NukH) is membrane located and is able
to bind to nukacin ISK-1 and bacteriocins structurally similar to nukacin. It was shown that
NukH captures nukacin molecules and transfers them to the ABC transporter (NukFEG) in
an energy-dependent manner. NukH recognizes unusual amino acids in the C-terminus of
nukacin and binds to the bacteriocin by a disulfide bridge. Importantly, nukacin ISK-1
related lantibiotics of class IA can be recognized by NukH indicating that the immunity
protein recognizes the ring pattern on the lantibiotics (384-386).
The immunity protein of class IIa bacteriocins is located in the cytosolic part of
the bacterial cell and does not interact considerably with the membrane, which is in contrast
to the immunity protein of bacteriocins from class IIb and IIc, which is associated with the
bacterial membrane (139,239,372,536). The specificity of the immunity proteins of class IIa
bacteriocins is similar to that of LanI and is determined by the C-terminal part of these
proteins (239). It is not yet well understood how the process of self-protection for class IIa
bacteriocins is determined since the immunity protein does not interact specifically with the
bacteriocin (139). It is speculated that the immunity protein might either block pores in the
bacterial membrane formed by a bacteriocin, or interact with the putative receptor for a
bacteriocin, as is the case for other class IIa members namely lactococcin A (139,536).
Lactococcin A uses the IIC and IID proteins of man-PTS system as a target. The immunity
protein of lactococcin A (LciA) binds to the targets forming a strong complex and thereby
preventing bactericidal action of the bacteriocin. The complex is formed only in the
presence of lactococcin A or during the bacteriocin production. This mechanism of self-
immunity was proposed also for other class II bacteriocins including some of class IIa such
as enterocin P and sakacin A (95,115,195).
Introduction
31
The immunity protein of the two-component non-lantibiotic bacteriocins, e.g.
lactococcin G and enterocin 1071, requires an unknown cytosolic compound in order to
protect the bacterial cell. Importantly, it was shown that the immunity protein of
lactococcin G, namely LagC, is able to recognize each peptide, i.e. α and β, of the two-
component bacteriocin (387).
Some of the members of a CAAX amino-terminal protease family, also known as
the Abi (an abortive infection) family, confer a novel self-immunity mechanism to
bacteriocins of class II, e.g. plantaricin EF and JK, Blp-bacteriocins, streptolysin S and
sakacin 23K (96,112,257,307,377,379). Because the Abi family proteins are not yet well
studied in prokaryotes, in contrast to eukaryotes, little is known about their function and
mechanism of protection in these organisms. The Abi group consists of putative membrane-
bound metalloproteases that share three conserved motifs in their amino acid sequence. It is
thought that the motifs are the active site of the proteases (112,119,257,401). Notably, the
Abi immunity proteins of plantaricin EF and JK, and sakacin 23K conferred cross-
immunity against each other‘s bacteriocins. It is suggested that the CAAX proteases
recognise and protect, most likely by a proteolytic cleavage, a common receptor(s) or
pathway(s) in these bacteriocins producer strains (257).
In general, immunity proteins are very specific to their corresponding bacteriocin
and they do not show amino acid similarity to other immunity proteins even when the
bacteriocin peptides are alike, which makes it difficult to identify them. However, there are
exceptions: the amino acid sequence of the sakacin A and curvacin A peptide is different
but their immunity proteins are similar (211,510). Additionally the immunity proteins do
not confer cross-immunity to other bacteriocins from the same or other class of
bacteriocins. However, the immunity proteins of some lantibiotics, e.g. Pep5 or epidermin,
give cross-immunity to other related lantibiotics, namely epicidin 280 or gallidermin,
respectively (209,391). Moreover, there are a few class IIa bacteriocins, of which immunity
proteins may provide some protection against bacteriocins from the same class (368).
Regulation of bacteriocins production
Mostly, bacteriocins are produced either under specific environmental condition(s)
or in a defined bacterial growth stage, often from mid exponential to stationary growth
phase, or as response to an extracellular signal. Regulation of some bacteriocins, e.g.
lacticin 481 (207), nukacin ISK-1 (5,8,459,460), mutacin II (421), sakacin A and P (55,111)
etc., is strictly under control of an environmental signal(s), such as pH, osmotic stress,
temperature and nutrition composition. Given this, it might be difficult to find expression
condition(s) for putative bacteriocins. Expression of bacteriocins is under the control of
either a single specific regulator or a two-component response regulatory system.
Additionally transcription of bacteriocins is regulated co-ordinately with their dedicated
biosynthetic and/or immunity operon(s).
Chapter 1
32
Generally, the two-component response regulatory system is composed of an
intracellular response regulator (RR; for lantibiotics named LanR) and a membrane bound
histidine kinase (HK; for lantibiotics named LanK). In response to a specific extracellular
signal, the HK protein becomes autophosphorylated and subsequently activates, by
phosphorylation, its cognate RR, which after some conformational changes is able to
activate or repress transcription of a target gene cluster (Fig. 3). For nisin, subtilin and
salivaricin A the extracellular signal is the bacteriocin peptide itself, which acts as a peptide
pheromone (284,427,487,524). Thus, transcription of the bacteriocin cluster is
autoregulated by its own bacteriocin-peptide pheromone, a process which is known as a
quorum-sensing (284,487). Quorum-sensing in bacteria is a type of coordinated gene
expression in response to the local density of its own population. Uninduced bacteriocin
producer cells make a small amount of the pheromone peptide often in an early exponential
growth phase or earlier, which at a certain threshold concentration, mostly reached during
exponential stage, is able to induce the HK protein. Thereby, the quorum-sensing system
functions for bacteria as a cell density sensor (145,308,416). Generally, bacteriocin
pheromones activate their own gene cluster by inducing the HK (Fig. 3). Nisin A and nisin
Z induce the nisin cluster, boosting also the rr and hk genes (for nisin the genes are
designed as nisR and nisK, respectively) (100). Making use of the fact that nisin is required
for transcription of its own cluster and that very small amounts of the peptide are sufficient
to induce transcription, a heterologous-controlled protein expression system was developed:
the NICE system, which stands for nisin-controlled expression (284).
Production of the lantibiotic epidermin is likely under control of two different
regulatory systems, namely the Agr (accessory gene regulator) two-component system that
controls the stress response, production of many surface proteins and biofilm formation,
and a single regulator, EpiQ. The Agr system controls the extracellular processing of the N-
terminal leader peptide of epidermin by the LanP protease (EpiP) (255,404,405,408). The
EpiQ protein regulates transcription of genes involved in epidermin production,
modification and immunity. This unusual regulatory system of epidermin, involving one
dedicated regulator and one general two-component system, might be found for other
bacteriocins, for which regulation is not yet well studied and/or understood. Comparable to
epidermin, transcription of the lantibiotic mersacidin is controlled by two regulators, the
single MrsR1 regulator and the two-component system MrsR2 and MrsK2. Nevertheless, in
contrast to the Agr system the two regulators only control expression of the mersacidin
locus. MrsR2 activates transcription of the immunity genes and MrsR1 controls
biosynthesis of mersacidin (162). Regulation by only one orphan cognate regulator was
shown for e.g. lacticin 3147 (340) and mutacin II (421). Transcription activation of the
mutacin II operon is dependent on a dedicated regulator, MutR, from the Rgg family of
(regulator gene of glucosyltransferase). In addition, transcription of the mutacin II cluster is
affected by yet unknown component(s) of the medium (421,496).
Introduction
33
Interestingly, the lantibiotics salivaricin A, A1, A2, A3 and A4 produced by
Streptococcus salivarius and Streptococcus pyogenes strains, activate their own production
by interaction with a cognate two-component system. These structurally related lantibiotics
can act also as the pheromone peptides and thereby, they are able to induce each other‘s
expression. (524,543). Notably, for bacteria, the recognition of the peptide pheromone of
another or the same species enables inter- as well as intraspecies communication and
apparently, salivaricins are involved in the cross-talk between the producers of these
lantibiotics.
Only a few dedicated repression systems for bacteriocin production have been
described and among them are those for cytolysin and plantaricin A. Production of the
lantibiotic cytolysin depends on the presence of target cells, i.e. microbes or erythrocytes.
Briefly, the cytolysin specific regulators, namely CylR1 and CylR2, repress expression of
the bacteriocin‘s biosynthesis cluster in the absence of target cells. However, despite this
repression, the peptides of the two-component cytolysin, i.e. CylLs and CylLL, are produced
at a low-level and both peptides form an inactive complex. Once the target cells are present,
CylLL binds to phosphatidylcholine in the membrane of erythrocytes, which causes
accumulation of free CylLs in the medium. Subsequently, when CylLs reaches a threshold
concentration in the medium, derepression of cytolysin biosynthesis genes takes place
(83,171). Expression of plantaricin A is controlled by two response regulators, i.e. PlnC and
PlnD, and one histidine kinase, PlnB. However, contrary to cytolysin, PlnC acts as an
activator and PlnD as a repressor of plantaricin transcription, and both regulators are
phosporylated by PlnB (113,440).
Production of bacteriocins in coordination with competence development might
bring benefits to a bacterial competent cells, namely the uptake of DNA from non-
competent cells of the same or diverse species through lysis (278). Competence is a stage
for bacteria for natural genetic transformation, an ability to take up extracellular DNA from
the environment of the same strain or of foreign origin. Competence, an example of the
quorum-sensing system mentioned before, is a highly coordinated process. Shortly,
competence in the Streptococcus genus is mediated by the extracellular concentration of a
competence-stimulation peptide (CSP), which is sensed by a dedicated two-component
system (ComDE). In response to the CSP concentration, a certain amount of genes (in S.
pneumoniae more than 120 genes) is expressed. These genes are involved in processes such
as binding, uptake of DNA and recombination, and production of bacteriocins
(80,81,127,238,250,357). Consequently, production of AMPs from Streptococcus species,
i.e. SmbAB (409,566), mutacin IV (276,279,531), mutacin N (174), mutacin V (403),
termophilin 9 (141,142), Blp (97,307), CibAB (80,168), is coordinated by the competence
development since these bacteriocin clusters belong to the competence regulon.
Chapter 1
34
Figure 4. Schematic regulation of the S. pneumoniae BlpMN production. Three mechanisms might mediate
regulation of the blp locus, i.e. by BlpRH, HtrA and possibly by ComDE. Briefly, in the first mechanism, BlpC
stimulates BlpH (1), which after autophosporylation activates BlpR (2) and then BlpR activates expression of the
blp genes (2), and as a consequence the BlpMN bacteriocins are produced (3), and transported outside the cell via
the BlpAB transporter. In the second mechanism (6), ComDE might be able to activate transcription of some of
the blp locus, when the cell becomes competent. In the third mechanism (5), HtrA, which is regulated by CiaR,
influences blp regulation at the posttranscriptional level, probably by proteolytic cleavage of BlpC (4). As a
consequence, degraded BlpC is not able to activate BlpH (5), which leads to a loss of the bacteriocins production.
Regulation of the Blp bacteriocins in S. pneumoniae is rather complex (Fig. 4).
The BlpMN bacteriocins are a part of a blp locus, which consists of the two-component
regulatory system (BlpRH), pheromone peptide (BlpC), dedicated ABC transporter
(BlpAB), bacteriocin-like peptides and immunity proteins. Regulation of the blp regulon
might be mediated by three independent mechanisms. The first mechanism concerns a
cognate two-component regulator system, i.e. BlpRH. Briefly, addition of synthetic BlpC to
the growth medium stimulates BlpH, which after autophosporylation, phosporylates BlpR.
Subsequently, activated BlpR induces expression of the entire blp locus (102,436). The
second mechanism, namely the competence two-component regulatory system, ComDE, is
able to upregulate, likely indirectly, only some of the blp genes, i.e. blpZYA involved in a
production of a transport and immunity proteins for BlpMN bacteriocins (410). The third
mechanism consists of the global two-component regulatory system CiaRH that regulates
the blp locus via HtrA, which is directly controlled by CiaR, at the posttranscriptional level
(98,176,334). It is thought that HtrA influences BlpMN production at the signalling level,
i.e. by affecting BlpC peptide pheromone (98) (Fig. 4).
Bacteriocins of the genus Streptococcus
Species of the genus Streptococcus such as S. mutans, S. pyogenes, Streptococcus
rattus, S. salivarius, Streptococcus uberis, Streptococcus agalactiae, Streptococcus
Introduction
35
dysgalactiae, Streptococcus gordonii, S. thermophilus and Streptococcus mascedonicus,
produce a great number and diversity of bacteriocins. S. mutans produces a great variety of
class I bacteriocins, namely mutacin I (423,424), II (280,371,421), III (mutacin 1140)
(206,422), mutacin K8 (441), SmbA and SmbB (566), and B-Ny266 (358), and one class II
i.e. mutacin IV (424). Interestingly, production of some of these bacteriocins depends on
the environmental conditions since mutacin IV is biosynthesized only in planktonic cultures
and mutacin I only in a biofilm-resembling conditions (424). S. pyogenes produces a
lantibiotic such as streptococcin A-FF22 (225). S. rattus produces two lantibiotics, i.e.
streptin (542) and BHT-A (223), a variant of Smb from S. mutans (566), and bacteriocin of
class II, BHT-B (223). The streptin encoding gene was detected in 40 out of 58 strains,
however, only 10% were able to produce this bacteriocin (542). Salivaricin A, B and A2 are
the lantibiotics of S. salivarius (224,543). Salivaricin A (SalA) is active e.g. against most of
S. pyogenes strains, and although 90% of these strains carry a variant of the salA gene,
namely salA, still they are sensitive to SalA. Other derivatives of salivaricin A, i.e.
salivaricin A2 to A5, are produced by S. pyogenes, S. salivarius, S. agalactiae and S.
dysgalactiae (543).
The rumen dwelling Streptococcus bovis produces two types of class I bacteriocins
namely bovicin HJ50 and bovicin 255, and bovicin-like bacteriocins were found among
majority of rumen streptococci (86,545). Streptococcus uberis, another rumen bacterium, is
a producer of a nisin variant, nisin U, which shows 78% identity to nisin (555).
Food streptococci such as S. mascedonicus produces the class I macedocin (151)
and S. thermophilus produces the class II termophilins and Blp peptides (141,142,316).
In contrast to other strains of the Streptococcus genus, biologically active
bacteriocins of S. pneumoniae, namely the BlpMN and the CibAB peptides, were identified
only recently (97,307). However, purification of these AMPs was not successful and thus
little is known about their structure, antimicrobial mode of action and mechanism of
immunity. Interestingly, both bacteriocins i.e. the Cib and the Blp peptides show
intraspecies antimicrobial activity and BlpMN additionally demonstrate interspecies
activity (97,168,307). The blp locus demonstrates some genetical variations among
different S. pneumoniae strains (97,307), which would result in a production of various Blp
peptides. With agreement to the statement, this likely might aid the intraspecies competition
among S. pneumoniae strains. In contrast, the CibAB bacteriocins are produced only by
competent cells and they are active against those cells of the S. pneumoniae strain, which
are non-competent, the fratricide phenomenon (168). It is known that isogenic bacteria
growing under the same in vitro condition might demonstrate a different gene expression
pattern, which is named bistability (122). Therefore, some cells might become competent
and others not. However, the CibAB peptides could hypothetically be involved in
intraspecies competition also between two different S. pneumoniae strains. Briefly, when in
vivo two S. pneumoniae strains of another competence stimulating peptide type would meet
(54) the strain, which first develops competence would be able to kill a non-competent
Chapter 1
36
population of the other strain by means of CibAB. A similar process was described for S.
gordonii (202) and it was proposed that this phenomenon occurs generally among
streptococcal species (80).
The scope of this thesis
Bacteriocins are well described among the Gram-positive bacteria including a
variety of AMPs produced by the genus Streptococcus. Nevertheless, little is known about
bacteriocins produced by S. pneumoniae. Therefore, the main aim of the thesis was to find
and characterize bacteriocin(s) produced by S. pneumoniae. The thesis contributes to the
complex story of bacteriocins in S. pneumoniae. Moreover, it presents information about
three new clusters likely involved in nitrogen metabolism in this bacterium. It adds data on
the subject of S. pneumoniae resistance to selected AMPs. Additionally, it contributes to
development of novel lantibiotics that once might find use in food industry or in medicine.
Chapter 2 presents the analysis of a variety of bacteirocin-like gene clusters of
class I and II that occur in the genome of S. pneumoniae strains, namely R6, TIGR4, D39,
G54, CGSP14, Hungary 19A-6, Taiwan19F-14, P1031, JJA, ATCC 700669 and 70585. In
total, nine bacteriocin-like clusters were described, of which two were introduced before,
i.e. the Blp (Pnc) and CibAB cluster. Among the S. pneumoniae strains, some of the
clusters are genetically identical and some show deletion/insertion mutations. Two
bacteriocin-like gene clusters, i.e. a pneumococcal peptide of unknown function (ppu)
cluster and a pneumococcin cluster, were selected for further study. Chapter 3 describes
experiments aiming to show that the ppu cluster produces active bacteriocin, but no active
bacteriocin was found to be produced by the cluster. Further, chapter 3 describes that the
expression of the ppu cluster is reduced in a presence of a nitrogen compounds and that a
negative regulator, i.e. CodY - a branched-chain amino acid responsive regulator, controls
its expression. This suggests that the cluster is involved in nitrogen metabolism. In addition,
PpuR, a regulator encoded by the first gene in the ppu cluster, has been shown to be an
activator of the cluster. What is more, chapter 3 indicates that two other clusters, for which
the same function is suggested, are functionally linked to the ppu cluster and that they
might form a regulon.
Chapter 4 describes for the first time that it is possible to produce and modify,
otherwise difficult to obtain, antimicrobially active lantibiotics of S. pneumoniae. Here, the
class IA nisin production machinery, NisBTC, was used to generate, modify and secrete
biologically active, previously not yet isolated and characterized pneumococcin
bacteriocins of class IC, which have no sequence homology to nisin.
Chapter 5 focuses on the response of S. pneumoniae towards AMPs such as nisin,
LL-37, and bacitracin and elucidates some resistance mechanisms to these AMPs. By use of
genome-wide transcriptome analysis (a DNA microarray), the response of S. pneumoniae to
Introduction
37
non-bactericidal concentrations of three AMPs, we demonstrated that for a limited number
of genes, expression was changed in all conditions. Consequently, several novel ABC
transporters, i.e. namely SP0785-0787, SP0912-0913 and SP1715, were associated with the
resistance of S. pneumoniae to these three different AMPs. In addition, a GntR-like
regulator, SP1714, was shown to regulate two of these ABC transporters. Notably, the
chapter describes involvement of the blp locus in determining the resistance of S.
pneumoniae D39 to LL-37.
In chapter 6, a summary of the thesis is provided. In addition, the most important
findings and the possibility to use novel and genetically manipulated bacteriocins in
medicine is discussed.
Chapter 1
38
Chapter 2
Identification and comparative analysis of putative
bacteriocin-gene clusters in Streptococcus pneumoniae
Joanna A. Majchrzykiewicz, Jetta J.E. Bijlsma and Oscar P. Kuipers
Chapter 2
40
Bacteriocin-like gene clusters
41
Bacteriocins are antimicrobial peptides (AMPs) thought to contribute to the
survival of bacteria in niches inhabited by other species, by the elimination of
competitors. A large variability of known bacteriocins is relatively well studied among
Gram-positive bacteria. In addition, novel putative bacteriocin peptides are
continuously identified in newly sequenced genomes. Streptococcus pneumoniae is a
prominent human pathogen and is known to be highly genetically variable. Up to
date, only two active bacteriocin clusters have been described for this organism, i.e.
blp (pnc) and cib. To identify additional putative bacteriocin encoding gene clusters,
we have chosen to screen the genomes of 11 S. pneumoniae strains using our BAGEL
program, specifically designed for the identification of putative bacteriocins. Here, we
describe, in addition to the known Blp (Pnc) and Cib bacteriocins, seven novel
putative bacteriocin gene clusters within the sequenced S. pneumoniae strains. One of
the seven clusters displays features of a class I bacteriocin and the other six of class II
bacteriocins. Interestingly, one of the identified putative clusters belongs to class II
bacteriocins in all but one examined strain, i.e. CGSP14, where it resembles a class I
bacteriocin. Notably, some of the identified clusters demonstrate considerable genetic
variability within the examined strains. Given that we described nine different
bacteriocin-like clusters in 11 S. pneumoniae strains, it is tempting to speculate that
there are even more AMPs waiting to be discovered within the species S. pneumoniae.
Introduction
S. pneumoniae is a human pathogen that colonizes the nasopharynx and in certain
circumstances can cause otitis media, sinusitis, pneumonia or meningitis. The
nasopharyngeal niche can be inhabited and colonized by, apart from S. pneumoniae, many
other Gram-positive and Gram-negative bacteria of genera such as Staphylococcus,
Lactobacillus, Neisseria, Corynebacterium, non-hemolytic and alpha-hemolytic
Streptococcus and others (494). In order to colonize the human nasopharynx and/or to
cause subsequent disease, S. pneumoniae has to survive and grow in this competitive niche.
One of the mechanisms that enable bacteria to compete with other species is the production
of antimicrobial peptides (AMPs), also named bacteriocins. Their lethal spectrum ranges
from bacteria of the same and/or other species, to even fungi, yeast and eukaryotic cells
(350). They are thought to contribute to survival by eliminating competitors, which would
reduce the competition for nutrients, and allow for the adaptation to changes in an
environment through gaining genetic material of other residents. Thus, bacteriocins might
contribute to the evolution of species by facilitating the acquisition of foreign DNA. In
general, bacteriocins are divided into four classes according to their biochemical and
genetic characteristics (203). Bacteriocins of class I are posttranslationally modified, which
results in unusual amino acids such as lanthionine and/or methyllanthionine, and thus they
are named lantibiotics (307). Class II consists of small heat stable peptides that are non-
Chapter 2
42
lantibiotics and this class is divided into three subclasses: i) the pediocin-like bacteriocins
with strong anti-listerial activity, ii) miscellaneous peptides and iii) multi-component
bacteriocins (203). Class III contains large heat-labile proteins and class IV cyclic peptides
(203,267).
Bacteriocins are produced in the form of inactive prepeptides (for lantibiotics they
are denoted as LanA) that consist of an N-terminal leader peptide and a C-terminal
propeptide commonly joined by a PR/PQ/GG/GA/GS-cleavage site. Generally, bacteriocins
become active once the leader peptide is removed from the prepeptide by the protease
(LanP) or a transporter with a protease domain (LanT). Usually, the gene cluster of each
class of bacteriocins is composed of a structural gene(s) encoding a precursor peptide, one
or two dedicated transporters often carrying a domain for protease function, optionally
autonomous protease, and an immunity protein(s). The lantibiotics have additional
modification enzyme(s) (LanM or LanB and LanC), which catalyze the formation of
unusual amino acids. Specifically, serine and threonine residues, in the C-terminal
propeptide, might be dehydrated by LanM or LanB to didehydroalanine (Dha) and
didehydrobutyrine (Dhb), respectively, and subsequently in a cyclization reaction of a
cysteine residue together with a dehydrated amino acid, lanthionine and/or methyl-
lanthionine can be formed by LanM of LanC (67). Generally, once the dedicated
modification enzyme transforms posttranslationally the C-terminal part, the peptide, with or
without the N-terminus, is secreted from the cell by a specialized transporter (LanT). Such
a peptide becomes an active lantibiotic, when the leader sequence is removed by a
dedicated protease, i.e. LanP, or by LanT that contains a protease domain. Generally, self-
immunity to bacteriocins of class I and II consists of a single protein and/or, for lantibiotics,
of three-component ABC transporter (LanFEG). Commonly, immunity proteins are very
specific to their corresponding bacteriocin and they do not show amino acid similarity to
other immunity proteins, which makes it difficult to identify them. With a few exceptions,
all genes contributing to bacteriocin biosynthesis, modification (in case of lantibiotics),
transport, regulation and immunity are clustered together in the genome.
Despite the extensive knowledge about AMPs, little is known about putative
bacteriocins produced by S. pneumoniae. So far, two functional bacteriocin clusters have
been identified, namely blp (pnc) and cib (97,168,307). Furthermore, there is a large
genetic variation within S. pneumoniae serotypes, and some isolates differ in the capability
to colonize the nasopharynx and cause infectious diseases (22,253,458). Comparison of the
genomes of strain R6 and TIGR4 showed that they differ in about 10% of the genes (51),
even more strikingly, analysis of the genomic contents of 17 different isolates showed that
less than 50% of all S. pneumoniae gene clusters were conserved (205).
In the present study, we identified bacteriocin-like gene clusters within S.
pneumoniae R6 and TIGR4, using the BAGEL software (99), specifically designed to
identify putative bacteriocins. Subsequently, we performed in silico comparative analysis of
these clusters in ERGOTM
, a bioinformatics suite designed for comprehensive genome
Bacteriocin-like gene clusters
43
analysis (392), by use of all 11 available S. pneumoniae genome sequences, i.e. (serotype
indicated in superscript) D392, G54
19F, CGSP
14, Hungary
19A-6, Taiwan
19F-14, P1031
1,
JJA14
, ATCC 70066923F
and 705855.
All together, seven novel bacteriocin-like gene
clusters, within the analyzed S. pneumoniae genomes, were identified (Table 1 and Fig. 1).
Subsequently, an in silico characterization of these and the blp (pnc) and cib clusters is
described below. Furthermore, the presence of the clusters within the genus of
Streptococcus was investigated.
Materials and Methods
Data source
Comparisons of the putative bacteriocin-like clusters within the S. pneumoniae strains i.e. (genbank
entry in brackets) R6 (AE007317), TIGR4 (AE005672), D39 (CP000410), G54 (CP001015),
CGSP14 (CP001033), Hungary19A-6 (CP000936), Taiwan19F-14 (CP000921), P1031 (CP000920),
JJA (CP000919), ATCC 700669 (FM211187) and 70585 (CP001015), were performed with the
ERGOTM bioinformatics suite designed by Integrated Genomics, Inc.
(http://ergo.integratedgenomics.com/ERGO/) (392). The annotations of the genomes were derived
from the ERGOTM and/or NCBI database.
A web-based tool
Identification of the putative bacteriocin-like peptides was executed with the web-based genome
mining tool, Bagel (99). Sequence clustering and analysis was performed with ClustalW 2.0 (302).
Identification of functional domain(s) of proteins was performed using the Pfam database,
http://pfam.sanger.ac.uk/. Protein-protein BLAST searches were carried out by use of BLASTP,
http://blast.ncbi.nlm.nih.gov/Blast.cgi. Membrane-spanning regions and their orientation were
predicted with TMpred-Prediction of Transmembrane Regions and Orientation (210),
http://www.ch.embnet.org/software/TMPRED_form.html. The map of the S. pneumoniae genome
was obtained from a BacMap genome atlas (493), an interactive visual database containing bacterial
genomes, http://wishart.biology.ualberta.ca/BacMap/.
Results
Identification of nine putative bacteriocin-like clusters within the S.
pneumoniae genome
Bagel, a web-based genome mining tool, identified 96 and 82, putative bacteriocin
open reading frames (ORFs) within the genome sequence of S. pneumoniae R6 and TIGR4,
respectively (99). In order to determine which of the identified bacteriocin-like ORFs are
most likely to encode a functional bacteriocin-like peptide, an in silico analysis of each of
the ORFs, as well as their neighbouring genes, was performed. This analysis was focused
on the characteristic features of known bacteriocins i.e. relative abundance of positively
Chapter 2
44
charged amino acid residues and a high iso-electric point of the mature peptide, as well as
PR/PQ/GG/GA/GS-cleavage site occurrence (67,114,233). In addition, as a further
selection criterion, the neighbouring genes were also analyzed to establish whether they
encode putative modification, regulation, transport and/or immunity proteins. The analysis
revealed nine (putative) bacteriocin-like clusters, annotated here as clusters I - IX (Table 1
and Fig. 1) in the S. pneumoniae strains R6, TIGR4, D39, G54, CGSP14, Hungary 19A-6,
Taiwan19F-14, P1031, 70585, JJA and ATCC 700669.
Figure 1. Circular diagram (atlas) corresponding to a general S. pneumoniae chromosome with nine
putative bacteriocin-like clusters marked in their approximate position. The origin of replication is at
the top, as indicated by kbp numbers. The outer rings show the arrangement of coding sequences on
the two strands of the genome, colored according to the annotated function. The second outer (red)
ring indicates genes encoding proteins on the forward strand and the second inner (blue) ring points to
genes encoding proteins on the reverse strand. This figure was adapted from the BacMap genome
atlas map (493) for S. pneumoniae genome. Accession number: NC_003028 coresponds to the
complete genome of S. pneumoniae Tigr4.
Bacteriocin-like gene clusters
45
Table 1. General summary of nine bacteriocin-like gene clusters identified in S. pneumoniae strains. ‘+‘ indicates
gene(s) present in the cluster; a only present in S. pneumoniae CGSP14; ND, not detected/found
Cluster
No.
Bacteriocin
class
Distribution of a bacteriocin gene
among/11 strains
Transport/ processing
gene
Modification
gene Closest homolog, (ref)
I Class I 5/11 +/+ + Nisin, (477)
II Class II 2/11 + /+ ND ND
III Class II 6/11 +/ ND ND lactococcin 972, (327,328)
IV Class II 9/11 +/ ND ND lactococcin 972, (327,328)
V Class II, Class Ia 9/11, 1/11a +/+ +a Mutacin, (421-424)
VI Class II 10/11 +/+ ND Bacteriocin-like cluster of
S. thermophilus
VII Class II 2/11 +/+ + ND
VIII
(cib) Class II 11/11 +/+ ND ND
IX
(blp) Class II 4/11 +/+ ND
blp of S.thermophilus,
(141,142)
Cluster I, a putative two-peptide lantibiotic cluster
Cluster I, with features characteristic for a lantibiotic cluster, was found in the
genome of S. pneumoniae R6, D39, TIGR4, JJA and ATCC 700669 (Fig. 2) and appears to
be conserved in these strains. In silico analysis of the cluster indicated that it consists of 12
genes encoding structural lantibiotic peptides and proteins presumably involved in
regulation, modification, transport and immunity (Fig. 2). In R6, the genes SPR1765-1766
(both genes indicated in Fig. 2 as number 14) encode peptides containing a putative PR-
cleavage site and serine, threonine and cysteine residues in the C-terminal part of the
peptides (supplemental material, Fig. S1), all of which are well-known features of
lantibiotic-like peptides. Hence, we propose to name SPR1765 and SPR1766,
pneumococcin A1 and A2 (PneA1 and PneA2), respectively, (310). Gene SPR1763, gene
number 8 in Fig. 2, encodes a putative transcriptional regulator, annotated as PlcR (482)
and might thus control expression of the cluster. Gene SPR1767 (gene number 10 in Fig. 2)
encodes a protein with 23% amino acid sequence identity to previously described LanM-
type modification enzymes, i.e. MrsM, LctM, SalM, LcnDR2, ScnM, CylM, NukM,
McdM, MukM and SivM (4,8,224,225,396,441,467,468,523,543). Therefore, the function
of SPR1767 could be dehydratation of threonine and serine residues followed by formation
of the lanthionine and/or methyllanthionine residues. Gene SPR1768 (number 11 in Fig. 2)
encodes most likely a putative LanD-type enzyme because it contains a FAD-dependent
flavoprotein domain characteristic for these types of enzymes. The LanD proteins are able
to modify the structural peptide by the oxidative decarboxylation of a C-terminal cysteine
residue (67,163,553). SPR1769 is a transmembrane protein that contains a site-2 protease
(S2P) class of zinc metalloproteases (family M50) domain. The SPR1770 protein (gene
number 2 in Fig. 2) is predicted to be an ABC transporter containing the N-terminal double-
glycine peptidase C39F domain, which might cleave behind the double glycine motif and
remove the leader peptide from the bacteriocin propeptide (192). Interestingly, SPR1770
shows approximately 30% amino acid identity to known lantibiotics transport and/or
Chapter 2
46
immunity proteins i.e. MrsT, BhtT CylB and SmbG (4,223,467,566). It is possible that
SPR1769 and SPR1770 are involved in the propeptide processing and the subsequent
transport of the modified lantibiotic(s) across the bacterial membrane. The SPR1771
protein (gene number 3 in Fig. 2) shows amino acid sequence identity of over 35% to
known lantibiotic proteases of the PR-recognition site type, i.e. NisP, EpiP, MutP and
GdmP, and thus this protein is probably involved in the processing of the prepeptides
(150,422,477,526). Genes SPR1772 and SPR1773 (gene numbers 4 and 5, respectively in
Fig. 2) might encode putative immunity proteins (LanE and LanF-like proteins) since
SPR1772 has two transmembrane domains and SPR1773 is a putative ATP-binding protein
of an ABC transporter. The function of SPR1764, a short peptide, and of the SPR1774
protein is unknown and they do not share homology with proteins with known functions.
Homologs of SPR1764 were found in other S. pneumoniae strains and their amino acid
sequence alignment is shown in Fig. S2 of the supplemental material.
A.
B.
Gene IDa Gene
numberb Functionc
SPR1761 9 ATP/GTP hydrolase
SPR1762 12 Hypothetical protein
SPR1763 8 Transcriptional regulator, XRE-family like protein SPR1764 13 Hypothetical protein
SPR1765-1766 14 PneA1 and PneA2, two-peptide like bacteriocin (S. pneumoniae: TIGR4
SP1948-1949; ATCC700669 SPN23F_19700-19710; D39 SPD1747-1748; JJA SPJ_1942-1943)
SPR1767 10 LanM-like modification enzyme
SPR1768 11 LanD-like FAD-dependent flavoprotein SPR1769 1 S2P/M50 family protease
SPR1770 2 LanT-like, ABC transporter
SPR1771 3 LanP-like protein SPR1772 4 LanE-like protein
SPR1773 5 LanF-like protein, ABC transporter ATP-binding protein
SPR1774 15 Hypothetical protein SPR1775 6 Nucleoside diphosphate kinase
SPR1776 7 DNA-directed RNA polymerase beta chain
Figure 2. A. Graphical comparison of the cluster I in the S. pneumoniae strains. B. Table summarizing the cluster
I genes and their function. a Gene ID refers to S. pneumoniae R6 locus tags. b Gene number refers to gene numbers
shown in Figure 2 and described in order from left to right. c Putative/predicted function based on ERGOTM,
Bacteriocin-like gene clusters
47
R6/TIGR4 annotation and/or domain prediction, and/or homologous proteins. Genes with the same color are
predicted to have the same function, colors are arbitrarily designated.
Nucleotide sequence analysis of the 12-gene cluster indicated that it is organized
presumably in four transcriptional units as sequences resembling -35 and -10 boxes,
indicating promoter regions, were found in front of four genes. The first unit could be the
regulatory protein, SPR1763, the second one would consist only of SPR1764, a peptide
with unknown function, the third unit would include the two structural lantibiotic peptides
and the modification enzyme (SPR1765-1767), and the fourth unit would consist of
SPR1768-1774. In conclusion, analysis of the putative pneumococcin gene cluster indicates
that it likely belongs to the class I of bacteriocins.
Attempts to find antimicrobial activity mediated by the cluster I
In order to determine whether the cluster is functional and produces biologically
active peptides, various antimicrobial assays, such as patch and agar diffusion assay, were
performed using a variety of growth conditions (data not shown). In these assays the wild
type S. pneumoniae D39 was compared with a pneA1-pneA2 mutant. Nevertheless, no
antimicrobial activity specific to PneA1 and PneA2 was observed.
Transcriptional lacZ fusions were constructed to three of the four predicted
putative promoters for this region, i.e. Pspr1763, Pspr1764 and PpneA1-pneA2 (data not
shown). Their activity was studied in a variety of conditions, e.g. various media,
carbohydrate sources, temperatures and pH stress. None of the tested conditions induced
expression directed by the promoters (data not shown). The average activity of Pspr1763
and Pspr1765-1766 was 0.8 and 5.2 Miller Units, respectively (data not shown), which
indicates that the pneumococcin promoter might not be very active. In contrast, expression
of Pspr1764 was rather high, about 80 Miller Units in all tested conditions (data not
shown), which suggests that SPR1764 might have a repressing activity. In brief, no
(growth) condition, in which Pspr1765-1766 was induced, was identified. This might
indicate either that a further screen for expression condition is required or that there is
negative regulator or that the pneumococcin cluster is not functional in the D39 strain. To
identify a putative negative regulator of the pneumococcin cluster, random transposon
mutagenesis was performed using the Pspr1765-1766 lacZ fusion. Approximately 36.000
mutants were screened, on both GM17 and BHI medium, but none of them showed
increased expression of the putative pneumococcin promoter, i.e. PpneA1 -pneA2 (data not
shown).
Another approach was to put the expression of the pneumococcin A1 and A2
peptides under control of a chromosomally integrated fucose-inducible promoter (data not
shown) (63); upon addition of fucose, no expression of the PneA1 and PneA2 peptides was
detected in the examined supernatants (data not shown). However, the PneA1 and PneA2
peptides do have intrinsic antimicrobial activity as we showed in chapter 3 of this thesis. In
short, two chimeras were constructed by fusion of the leaderless pneA1 and pneA2 genes to
Chapter 2
48
the nisin leader peptide. Subsequently, the chimeras were produced in the Lactococcus
lactis NZ9000 strain containing the nisin biosynthetic enzymes, NisBTC (310,439).
Importantly, the chimeric peptides were modified and showed antimicrobial activity against
Micrococcus flavus (310), suggesting that in S. pneumoniae the pneumococcin peptides
might be produced and display antimicrobial activity under yet unidentified conditions.
Further investigation is needed to confirm this.
Cluster II, a non-lantibiotic cluster found in the genomic region of the pneumococcin
cluster in S. pneumoniae strains G54, CGSP14, 70585, P1031, Taiwan19F-14 and
Hungary 19A-6
Interestingly, in the same genomic region of the pneumococcin cluster, a second
non-lantibiotic bacteriocin-like cluster is located in the other analyzed S. pneumoniae
strains, namely G54, CGSP14, 70585, P1031, Taiwan19F-14 and Hungary19A-6 (Fig. 3).
This cluster II contains some homologs of the pneumococcin cluster, namely the genes
encoding putative regulatory and immunity proteins and those with unknown function
(SPR1763, SPR1764, SPR1772, SPR1773 and SPR1774, Fig. 3). In all these strains, the
bacteriocin-like cluster II contains a putative regulator (genes number 23 in Fig. 3), a LanE-
like protein (gene number 1 in Fig. 3), a LanF-like protein (gene number 4 in Fig. 3) and
several additional genes are present in some strains (Fig. 3). For instance, gene number 12
(strain 70585, P1031 and CGSP14) and gene number 15 (strain G54, 70585, P1031,
Hungary19A-6 and Taiwan19F-14) both encode a protein of unknown function, gene
number 25 (strain G54 and Hungary19A-6), which encodes a bacteirocin-like peptide, and
gene number 26 (strain G54) encoding a putative ABC transporter (Fig. 3). Gene number
25 of cluster II, annotated in Hungary19A-6 as SPH_2096 and in G54 as SPG_1856, has a
GG-cleavage site and a high pI of 8.8, and positive amino acids in the C-terminal end
suggesting that it encodes a putative bacteriocin-like peptide. Notably, only G54 and
Hungary19A-6 have this specific bacteriocin like-peptide, thus an antimicrobial activity
specific to this putative bacteriocin would likely be unique to these strains. As it is shown in
the amino acid sequence alignment in Fig. 4, SPH_2096 and SPG_1856 are identical and
they show similarity to other putative bacteriocin-like peptides from cluster III and IV (Fig.
4). To conclude, the lack of the pneumococcin cluster in the same genomic region in these
six S. pneumoniae strains and instead the presence of other genes, i.e. SPH_2096 or
SPG_1856, and homologs of SPR1772 and SPR1774, indicates that this region is
genetically variable among S. pneumoniae. Furthermore, two homologs of gene number 1
and 4, encoding LanE and LanF-like protein, respectively, were also found in L. lactis and
Streptococcus equi.
Bacteriocin-like gene clusters
49
A.
B.
Gene IDa Gene
numberb Functionc
SPR1764 12 peptide with unknown function
SPR1765-1766 13,24 PneA1 and PneA2;two-peptide like bacteriocin
SPR1767 10 LanM-like modification enzyme
SPR1768 11 LanD-like FAD-dependent flavoprotein SPR1769 14 S2P/M50 family protease
SPR1770 5 LanT-like, ABC transporter containing N-terminal double-glycine
peptidase C39 family SPR1771 6 LanP-like protein
SPR1772 1 LanE-like protein
SPR1773 4 LanF-like protein SPR1774 15 Unknown function
SP1959 7 Nucleoside diphosphate kinase
SP1960 8 DNA-directed RNA polymerase beta‘ chain SP1961 9 DNA-directed RNA polymerase beta chain
SPG_1848 16 Na+ driven multidrug efflux pump
SPG_1849 17 RecA protein SPG_1850 18 CinA; competence-damage protein
SPG_1851 19 Transcriptional regulator, LytR family
SPR1760 20 Acetyltransferase, GNAT SPR1761 21 ATP/GTP hydrolase
SPR1762 22 Hypothetical protein
SPR1763 23 Transcriptional regulator, XRE-family like protein SPG_1856
25 Bacteriocin-like peptide (S. pneumoniae: G54 SPG_1856; Hungary19A-6
SPH_2096)
SPG_1858 26 ABC transporter, ATP-binding protein yujA 3 Adenine-specific methyltransferase
ackA 2 Acetate kinase
Figure 3. A. Comparison of the cluster II genomic region in the S. pneumoniae strains. Dashed lines indicate
insertion elements and genes marked as grey indicate those that do not have have homology to other ones shown
in this figure. B. Table listing the genes and their function of the genomic region of the cluster II. a Gene ID refers
to S. pneumoniae R6 or TIGR4 or G54 locus tags. b Gene number refers to gene numbers shown in Figure 3 and
Chapter 2
50
described in order from left to right. c Putative/predicted function based on ERGOTM, R6/TIGR4 annotation and/or
domain prediction, and/or homologous proteins. Genes with the same color are predicted to have the same
function, colors are arbitrarily designated.
Figure 4. Amino acid sequence alignment of the bacteriocin-like peptide (gene number 25) of cluster II,
SPG_1856, with other putative bacteriocins of cluster III and IV of S. pneumoniae strains. Alignment was
performed using ClustalW (302). Asterisk, identical residues; colon, conserved residues; period, semi-conserved
residues.
Cluster III shares structural and amino acid similarity to the well-characterized
bacteriocin cluster, namely lactococcin 972
Cluster III was found in strains CGSP14, G54, Hungary19A-6, JJA, 70585 and
ATCC 700669, and consists of a putative bacteriocin structural gene (indicated as number 1
in Fig. 5), a putative ABC transporter, a hypothetical protein and a hypothetical protein
with eight transmembrane domains (gene numbers 3, 4 and 5, respectively, in Fig. 5). The
amino acid sequence of the bacteriocin-like peptide (SP70585_2060; ATCC 700669
SPN23F_20090; CGSP14 SPCG_1952; G54 SPG_1890; Hungary19A-6 SPH_2130; JJA
SPJ_1981) of cluster III in all strains is identical and contains a putative GG-proccesing
site, and positive residues in the C-terminal part of the peptide, all features characteristic for
known bacteriocins (Fig. 4 and supplemental material Fig. S3). Notably, the amino acid
sequence of these bacteriocin-like peptides is similar (approximately 41% identity) to the
known L. lactis plasmid-encoded bacteriocin, lactococcin 972 (327,328), (supplemental
material Fig. S3). Additionally the putative immunity protein of cluster III (number 5 in
Fig. 5) demonstrates significant similarity to the immunity protein of lactococcin 972.
Interestingly, cluster III is not present in the genomes of S. pneumoniae R6, D39 and
TIGR4, three strains often used for studying S. pneumoniae pathogenesis. Taken together,
the resemblance between the bacteriocin-like peptides of cluster III and the lactococcin 972
suggests that cluster III is likely to produce a functional antimicrobial peptide belonging to
Bacteriocin-like gene clusters
51
class II bacteriocins and that the cluster might be classified to the lactococcin 972 family of
bacteriocins.
A.
B.
Gene IDa Gene
numberb Functionc
SPG_1894 9 LPXTG-motif cell wall anchor domain
SPG_1893 8 Hydrolase, TatD family
SPG_1892 7 Topoisomerase-primase homolog SPG_1891 6 Transcriptional regulator, XRE-family like protein
SPG_1890 1 Bacteriocin-like peptide (S. pneumoniae: 70585 SP70585_2060; ATCC
700669 SPN23F_20090; CGSP14 SPCG_1952; G54 SPG_0625; Hungary19A-6 SPH_2130; JJA SPJ_1981)
SPG_1889 5 Immunity protein
SPG_1888 4 ABC transporter ATP binding protein SPG_1887 3 Hypothetical protein
SPG_1886 2 Dimethyladenosine transferase
Figure 5. A. Graphical representation of the cluster III in the S. pneumoniae strains. Genes marked as grey
indicate those that do not have have homology to other ones from this figure B. Genes of the genomic region of
the cluster III and their function. a Gene ID refers to S. pneumoniae G54 locus tags. b Gene number refers to gene
numbers shown in Figure 5 and described in order from left to right. c Putative/predicted function based on
ERGOTM, R6/TIGR4 annotation and/or domain prediction, and/or homologous proteins. Genes with the same
color are predicted to have the same function, colors are arbitrarily designated.
Cluster IV, located in another region of the genome, also shares homology with
lactococcin 972
The putative cluster IV is present in the majority of analysed strains: R6, G54,
D39, CGSP14, TIGR4, 70585, ATCC 700669, P1031 and Taiwan19F-14 (Fig. 6). The
bacteriocin-like cluster IV consists of three genes, i.e. a putative bacteriocin peptide, a
putative immunity protein and a putative ABC transporter (gene numbers 1, 18 and 5 in
Fig. 6, annotated in R6 strain as SPR0600, SPR0601 and SPR0602, respectively). The
putative propeptide amino acid sequence of the bacteriocin-like peptide IV (gene indicated
as number 1 in Fig. 6) demonstrates known bacteriocin features e.g. a GG-cleavage site,
positive amino acids in the C-terminus and high net pI of 9.2.
Chapter 2
52
A.
B.
Gene IDa Gene
numberb Functionc
SPR0590 7 Hypothetical protein SPR0591 8 Ribonuclease Z
SPR0592 9 Short chain dehydrogenase SPR0593 10 Transcriptional regulators, LysR family
SPR0594 13 Hypothetical cytosolic protein
SPR0595 14 Rhodanese-related sulfurtransferase SPR0596 15 Hypothetical protein
SPR0597 12 RsuA; ribosomal small subunit pseudouridine synthetase
SPR0598 11 TypA; GTP-binding protein SPR0599 16 Hypothetical membrane spanning protein
SPR0600 1 Bacteriocin-like peptide (S. pneumoniae: TIGR4 SP0685; 70585 SP70585_0742; ATCC 700669 SPN23F_06180; CGSP14 SPCG_0640; D39
SPD0595; G54 SPG_0625; P1031 SPP_0704; Taiwan19F-14 SPT_0707)
SPR0601 18 Immunity protein SPR0602 5 ABC transporter ATP binding protein
SPR0603 4 MurD; UDP-N-acetylmuramoylalanine—D-glutamate ligase
SPR0604 3 MurG; UDP-N-acetylglucosamine--N-acetylmuramyl-(pentapeptide) Pyrophosphoryl-undecaprenol N-acetylglucosamine transferase
SPR0605 2 DivIB; cell-division initiation protein
SPR0606 19 Hypothetical protein SPR0607 17 Hypothetical protein
SPG_0631 21 PyrF; Orotidine 5-phosphate decarboxylase
SPG_0632 22 Orotate phosphoribosyltransferase
Figure 6. A. Organization of the cluster IV in the S. pneumoniae strains. B. Table summarizing the list of genes
and their function of the genomic region of the cluster IV. a Gene ID refers to S. pneumoniae R6 or G54 locus tags. b Gene number refers to gene numbers shown in Figure 6 and described in order from left to right. c
Putative/predicted function based on ERGOTM, R6/TIGR4 annotation and/or domain prediction, and/or
homologous proteins. Genes with the same color are predicted to have the same function, colors are arbitrarily
designated.
Notably, peptide IV shows approximately 34% amino acid identity towards the known
bacteriocin lactococcin 972 (327,328), (Fig. 4 and supplemental material Fig. S3), and
about 58% towards bacteriocin-like peptides of cluster III, i.e. gene number 1 in Fig. 5. The
amino acid sequence of peptide IV is identical in all strains except for TIGR4, ATCC
Bacteriocin-like gene clusters
53
700669 and CGSP14 (Fig. 4 and supplemental material Fig. S3). In these three strains, the
peptide IV lacks approximately half of its N-terminus, which consequently could result in a
loss of activity of the cluster. The putative immunity protein of cluster IV (gene number 18
in Fig. 6) is homologous to the immunity protein of cluster III (gene number 5 in Fig. 5). In
addition, the putative ABC transporter of cluster IV (gene number 5, Fig. 6) might be
involved in transport of the bacteriocin-like peptide IV across the cell envelope. However,
the protein(s) responsible for processing of the bacteriocin-like peptide of cluster IV and as
well of the peptide of cluster III is unknown because in the close vicinity of these clusters
there are no putative proteases in the genome. Nevertheless, processing as well as transport
of the peptides of cluster III and IV might happen in a manner analogous to that of
lactococcin 972, where it is probably mediated by the Sec-secretion system (327). This
hypothesis is supported by the fact that the putative bacteriocins of cluster III and IV
contain signal peptide with characteristics that probably match requirements of the Sec-
export system (supplemental material Fig. S3), i.e. the N-terminus of the leader peptide is
positively charged, the C-terminus of the leader contains a consensus cleavage site (AXA)
and the sequence between the N- and C-terminus is hydrophobic (512). However, whether
the putative bacteriocins of cluster III and IV are secreted by the Sec-pathway or by the
putative ABC transporter of these clusters needs to be determined.
The bacteriocin-like cluster IV is highly conserved in all examined S. pneumoniae
strains, except for strain JJA and Hungary19A-6 (Fig. 6), indicating that it is probably also
present in the genomes of other not yet sequenced S. pneumoniae strains. In agreement with
that, a BLAST search showed that the cluster is present in some S. pneumoniae strains, for
which a partially sequenced genome is available in the public databases (data not shown).
In conclusion, this cluster likely produces a functional antimicrobial peptide of class II
bacteriocins, since it possesses the structural bacteriocin gene in addition to a transport and
an immunity protein. Additionally the cluster IV, as it is for the cluster III, can be classified
to the lactococcin 972 family.
Cluster V contains a pneumococcal peptide of unknown function (ppu)
In all sequenced S. pneumoniae strains except CGSP14, the putative bacteriocin-
like cluster V consists of six genes (Fig. 7). The cluster encodes a putative regulator, PpuR,
a bacteriocin-like peptide, PpuA, two CAAX amino terminal proteases, PpuBC, a
transporter belonging to the major facilitator family (MFS), PpuD, and a putative branched-
chain amino acid transporter, PpuE, (numbers 1, 8, 14, 22, 5 and 4, respectively in Fig. 7).
The PpuR regulator, of the Rgg/GadR/MutR protein family, shows 30% amino acid identity
to known positive regulators of bacteriocins i.e. MutR of Streptococcus mutans (277,421-
424) and BhtR of Streptococcus rattus (223). The amino acid sequence of PpuA (indicated
as number 8 in Fig. 7) displays characteristic features of bacteriocins, e.g. positive amino
acids in the C-terminal end and a putative GG-processing site (supplemental material Fig.
S4). Interestingly, in strain CGSP14 four genes encoding a putative bacteriocin
Chapter 2
54
(SPCG_0144), a putative serine (threonine) dehydratase (SPCG_0145) and a putative
lathionine synthetase (SPCG_0146), and lantibiotic efflux protein (SPCG_0147), have
replaced the ppuA gene. In addition, the amino acid sequences of SPCG_0145 and
SPCG_0146 show approximately 25% identity to known lantibiotics biosynthesis proteins,
e.g. EpiB and EpiC of Staphylococcus epidermidis (463), MutB and MutC of S. mutans
(421-423), GdmB and GdmC of Staphylococcus gallinarum (526), SrtB and SrtC of S.
pyogenes (249), SpaB and SpaC of Bacillus subtilis (74,169), NisB and NisC of L. lactis
(130,285), NsuB, and NsuC of Streptococcus uberis (555), and PepB, and PepC of S.
epidermidis (346). Moreover, the amino acid sequence of the putative bacteriocin
(SPCG_0144) shows high similarity to a putative lantibiotic precursor of S. thermophilus
strains LMD-9, LMG18311 and CNRZ1066 (supplemental material Fig. S5). Notably, all
examined S. thermophilus strains possess a putative lantibiotic locus homologous (identity
of 70-88%) to that of strain CGSP14 (116) except for strain LMD-9, where the similarity is
lower. Therefore, we hypothesize that a genetic exchange occurred between S.
thermophilus and the CGSP14 strain within this genomic region.
To examine whether the ppu cluster produces a functional bacteriocin-like peptide,
further extensive investigations were performed, which are described in chapter 3 of this
thesis. Shortly, we have shown that the ppu cluster is highly expressed in chemically
defined medium (CDM, (260)) and that CodY, a branched-chain amino acid regulator
(199), is a negative regulator of this cluster (chapter 3). Investigations of putative
antimicrobial activity of this cluster were performed using following S. pneumoniae strains,
namely R6, D39, TIGR4 and the derivatives D39ΔcodY, R6ΔppuA and D39ΔppuA. Several
antimicrobial activity assays such as patch and agar diffusion assay, and co-culture assays
were performed (data not shown) with various indicator strains such as L. lactis, M. flavus,
Moraxella catarrhalis and S. pneumoniae D39ΔcodY, R6ΔppuA and D39ΔppuA. However,
no antimicrobial activity specifically related to the PpuA peptide was observed (data not
shown).
Since extracellular amounts of PpuA might not have been sufficient either to reach
bactericidal concentration or to visualize the peptide on a gel, isolation of PpuA via diverse
concentration methods, e.g. TCA precipitation, stirred cells filtering and concentration
techniques, and FPLC by use of ion-exchange columns, were performed. Alternatively, the
peptide was cloned with a Strep-tag for expression, detection and purification purposes
(data not shown). Nevertheless, no PpuA-like peptide was found. Thus, we assumed that
the isolation/concentration methods were not suitable or sufficient to purify PpuA.
Therefore, PpuA was synthesized but it still did not show significant antimicrobial activity
(MIC>1 mg/ml) against the indicator strains mentioned above (data not shown). Thus, we
speculate that PpuA, and consequently the ppu cluster, performs another function(s) in S.
pneumoniae, most likely in nitrogen metabolism (chapter 3).
Bacteriocin-like gene clusters
55
A.
B.
Gene IDa Gene
numberb Functionc
SPR0134 9 Transposase
SPR0135 10 EpsG; Glycosyltransferase SPR0136 13 Glycosyltransferase
SPR0137 6 ABC transporter ATP-binding protein
SPR0138 12 Hypothetical protein
SPR0139 7 Ugd; UDP-glucose 6-dehydrogenase
SPR0140 1 PpuR; Transcriptional activator Rgg/GadR/MutR family
SPR0141 8 PpuA; a putative bacteriocin peptide (S. pneumoniae: TIGR4 SP0142; 70585 SP70585_0216; ATCC 700669 SPN23F_01520; D39 SPD0145; G54
SPG_0144; JJA SPJ_0175; P1031 SPP_ 0212; Taiwan19F-14 SPT_0189)
SPR0142-0143 14, 22 PpuBC; CAAX amino terminal protease family SPR0144 5 PpuD; Macrolide-efflux protein
SPR0145 4 PpuE; putative branched-chain amino acid transport protein AzlC
SPR0146 19 ABC transporter substrate-binding protein SPR0147 3 ABC transporter substrate-binding protein
SPR0148 2 DapE; Acetylornithine deacetylase/Succinyl-diaminopimelate desuccinylase
and related deacylases
SPR0149 21 ABC transporter ATP binding protein
SPG_0134 18 Transposase
SPG_0135 11 Glycosyltransferase SPCG_0139 15 Hypothetical protein
SPCG_0140 16 Hypothetical protein
SP70585_0219 17 Hypothetical protein SPG_0149 20 putative branched-chain amino acid transport protein AzlD
Figure 7. A. Comparison of the genomic region of the cluster V in S. pneumoniae strains. Dashed lines indicate
insertion elements and genes marked as grey indicate those that do not have have homology to other ones shown
in this figure. B. Table summarizing graphical overview of the cluster V. a Gene ID refers to S. pneumoniae R6 or
G54 or CGSP14 or 70585 locus tags. b Gene number refers to gene numbers shown in Figure 7 and described in
order from left to right. c Putative/predicted function based on ERGOTM, R6/TIGR4 annotation and/or domain
prediction, and/or homologous proteins. Genes with the same color are predicted to have the same function, colors
are arbitrarily designated.
Chapter 2
56
Cluster VI is rich in genes that encode putative bacteriocin peptides
The putative bacteriocin cluster VI is present in all analyzed S. pneumoniae
strains (Fig. 8). Cluster VI contains one or more putative bacteriocin peptides, depending
on the strain (gene numbers 16, 17, 20, 21 in Fig. 8, supplemental material Fig. S6), one
hypothetical membrane protein and two putative ABC transporters, each of which has a
multidrug transporter domain (gene numbers 6, 1 and 7, respectively, in Fig. 8).
The bacteriocin-like peptide number 17 in Fig. 8 is present in all analyzed S.
pneumoniae strains but Hungary19A-6. The bacteriocin-like peptide contains a GA-
processing site and, in the C-terminal part, positively charged amino acids. However, this
propeptide has an atypical low pI value of 4.7 (supplemental material Fig. S6 A). The
amino acid sequence of the bacteriocin-like peptide of gene number 16 in Fig. 8 has
lantibiotic-like features such as the PR-processing site and serine, and threonine residues
important for lantibiotic modifications (Fig. 8, supplemental material Fig. S7). However,
the lack of cysteine residues in the C-terminus, which are essential for the ring formation in
lantibiotics, as well as the absence in this genomic region of enzymes required for the
amino acid modifications specific for lantibiotics, excludes the possibility that the peptide
with gene number 16 is a lantibiotic. Nevertheless, the bacteriocin-like peptide can be still
functional.
The bacteriocin-like peptide with gene number 20 in Fig. 8 contains a GG-processing site
and is rich in both positive and negatively charged residues but again the net pI value is
low, 4.5, compared to known bacteriocins (supplemental material Fig. S6 B). The
bacteriocin-like peptide 21 has a high pI of 8.9 but lacks a known bacteriocin-processing
site (supplemental material Fig. S6 C). Interestingly, the C-terminal part of the peptide of
gene number 17 and the N-terminal part of the peptide of gene number 21 show amino acid
sequence similarities to peptides of S. mutans and S. gordonii with unknown function
(supplemental material Fig. S8 A and B). Homologs of two putative ABC transporters
(gene numbers 1 and 7 in Fig 8) were found in several S. thermophilus strains (Fig. 8).
Interestingly, these homologs in S. thermophilus are also adjacent to a putative bacteriocin
(gene marked as number 30 in Fig. 8). Therefore, we hypothesize that the two ABC
transporters are involved in transport of or immunity for the bacteriocin-like peptide VI. In
conclusion, the bacteriocin-like cluster VI might be involved in antimicrobial peptide
production, although the genes do not contain all the typical characteristics of bacteriocin
clusters. However, this cluster is probably functional and further research is necessary to
establish whether this cluster has a bacteriocin-like function and which of the putative
bacteriocin genes encodes an active antimicrobial substance.
Bacteriocin-like gene clusters
57
A.
B.
Gene IDa Gene
numberb Functionc
SPR1667 19 Galactose-1-phosphate uridylyltransferase
SPR1666 18 Thioesterase superfamily protein
SPR1665 14 DpnC; Type II restriction-modification system restriction subunit
SPR1664 2 DpnD
SPR1663 3 Xanthine permease
SPR1662 4 Xanthine phosphoribosyltransferase
SPR1661 22 Bleomycin resistance protein
SPR1660 5 ExoA; exodeoxyribonuclease III
SPR1659 17 Bacteriocin-like peptide (S. pneumoniae: TIGR4 SP1842; 70585 SP70585_1896; ATCC
700669 SPN23F_18580; CGSP14 SPCG_1817; D39 SPD1624; G54 SPG_0144; JJA
SPJ_1726; P1031 SPP_1841; Taiwan19F-14 SPT_1759)
SPR1658 6 Hypothetical membrane associated protein
SPR1656-1657 1, 7 ABC-type multidrug transport system, ATPase and permease components
SPR1655 8 Probable CPS biosynthesis glycosyltransferase
SPR1654 9 3-amino-5-hydroxybenzoic acid synthase family
SPR1653 16 Bacteriocin-like peptide, (S. pneumoniae: TIGR4 SP1836; D39 SPD1618; JJA SPJ_1741)
SP1835 15 Hypothetical peptide (S. pneumoniae: TIGR4 SP1835; Taiwan19F-14 SPT_1753)
SPR1652 11 Hypothetical protein
SPR1651 20 Bacteriocin-like peptide (S. pneumoniae 70585 SP70585_1889; ATCC 700669
SPN23F_18500; CGSP14 SPCG_1810; D39 SPD1616; JJA SPJ_1739)
SPR1650 12 Unknown protein
SPR1649 13 Phosphate transport system protein phoU
SPG_1735 26 dpnM
SPD1625 21 Bacteriocin-like peptide (S. pneumoniae: 70585 SP70585_1897; ATCC 700669
SPN23F_18590; D39 SPD1625; G54 SPG_1727; JJA SPJ_1748; P1031 SPP_1842;
Taiwan19F-14 SPT_1760)
STER_1653 32 Bacteriocin processing peptidase
STER_1652 31 Bacteriocin export accessory protein
STER_1651 30 Bacteriocin-like peptide
Figure 8. A. The cluster VI and its flanking region in the S. pneumoniae and the S. thermophilus strains. Dashed
lines indicate insertion elements and genes marked as grey indicate those that do not have have homology to other
ones from this figure. B. Genes of the genomic region of the cluster VI and their function. a Gene ID refers to S.
pneumoniae R6 or G54 or TIGR4 or S. thermophilus locus tags. b Gene number refers to gene numbers shown in
Figure 8 and described in order from left to right. c Putative/predicted function based on ERGOTM, R6/TIGR4
Chapter 2
58
annotation and/or domain prediction, and/or homologous proteins. Genes with the same color are predicted to have
the same function, colors are arbitrarily designated.
Cluster VII is likely not functional
The putative bacteriocin cluster VII, or some of its components, is present in
strains R6, D39, TIGR4, ATCC 700669, Hungary19A-6, 70585 and CGSP14 (Fig. 9). In
R6 the cluster VII is composed of genes (indicated as gene numbers 16, 8, 7, 2 and 1,
respectively, in Fig. 9) encoding proteins that are likely involved in the modification,
processing and transport of putative lantibiotic-like bacteriocin peptides (gene number 14
and/or 28 in Fig. 9) and the associated immunity.
A.
B.
Gene IDa Gene numberb Functionc
SPR1209 29 Hypothetical protein
SPR1207 20 Arsenate reductase
SP1346 13 CAAX amino terminal protease family
SPR1206 19 CAAX amino terminal protease family
SPR1205 16 Serine/threonine protein kinase
SPR1204 8 Protease II
SPR1203 7 ABC transporter ATP binding protein
SPR1202 2 ABC transporter ATP binding protein
SPR1201 1 Hypothetical protein
SPR1200 14 Bacteriocin-like peptide (S. pneumoniae D39 SPD1338)
SP1339 15 Unknown peptide
SP1333 9 Unknown peptide
SPR1199 28 Bacteriocin-like peptide (S. pneumoniae D39 SPD1174)
SPR1196 27 N-acetylmannosamine-6-phosphate 2-epimerase; NanE
SPR1195 26 Hypothetical protein
SPR1194 25 OppA; Oligopeptide-binding protein
SPR1193 24 OppB; Oligopeptide transport system permease protein
SPR1192 23 OppC; Oligopeptide transport system permease protein
SPR1191 22 OppF; Oligopeptide transport ATP-binding protein
SP1331 6 Transcriptional regulator RpiR family
SP1330 5 N-acetylmannosamine-6-phosphate 2-epimerase
SP1329 4 N-acetylneuraminate lyase
SP1328 3 Sodium-coupled N-acetylneuraminate transporter
Figure 9. A. The genomic region of the cluster VII in the S. pneumoniae strains. Dashed lines indicate insertion
elements and genes marked as grey indicate those that do not have have homology to other ones shown in this
figure. B. Table listing genes, and their function, of the genomic region of the cluster VII. a Gene ID refers to S.
Bacteriocin-like gene clusters
59
pneumoniae R6 or TIGR4 locus tags. b Gene number refers to gene numbers shown in Figure 9 and described in
order from left to right. c Putative/predicted function based on ERGOTM, R6/TIGR4 annotation and/or domain
prediction, and/or homologous proteins. Genes with the same color are predicted to have the same function, colors
are arbitrarily designated.
The putative bacteriocin peptides resemble lantibiotics as they contain a GG-processing
site, a high pI value and positively charged residues in the C-terminus, and serine and/or
threonine and cysteine residues, which might be involved in ring formation (supplemental
material Fig.S9 A and B).
The bacteriocin-like cluster VII is well conserved within R6 and D39 (Fig. 9) in
contrast to CGSP14 and Hungary 19A-6. In the CGSP14 strain, cluster VII lacks genes
putatively encoding modification, processing and transport proteins (gene number 16, 8, 7
and 2, respectively), and bacteriocin-like peptides (gene number 14 and 28 in Fig. 9).
However, strains CGSP14, P1031, G54 and ATCC 700669 contain a homolog of the
bacteriocin-like peptide of cluster VII (gene number 28), which is located in another
genomic region than cluster VII (supplemental material Fig. S10). Strain Hungary 19A-6
lacks the modification, processing and the bacteriocin-like peptides of cluster VII (gene
number 16, 8, 14 and 28, respectively, in Fig. 9). The genomic region of the bacteriocin-
like cluster VII contains many insertion elements in all strains, except D39 (Fig. 9), which
consequently, probably resulted in the destruction of the functionality of cluster VII in S.
pneumoniae.
Cluster VIII encodes CibAB, a two-peptide bacteriocin required for allolysis
Cluster VIII was found in all analyzed S. pneumoniae strains and is composed of
three genes, namely cibABC (Fig. 10). The name Cib stands for competence-induced
bacteriocins. When S. pneumoniae cells become competent, they produce a set of
molecules, which trigger the lysis of non-competent S. pneumoniae cells. This killing
mechanism was named fratricide and the type of cell-programmed lysis was termed
allolysis (79,80,168,193). It was shown that CibAB affect allolysis especially in solid phase
but infrequently in liquid culture (168,193). The cibAB genes encode a class II two-peptide
bacteriocin (gene numbers 7 and 8, respectively, in Fig. 10 and supplemental material Fig.
S11). According to S. Guiral et al., an open reading frame (ORF) of a protein conferring
resistance to CibAB, namely cibC, is located upstream of CibB (between genes number 8
and 1 in R6 genomic region in Fig. 10) (168). Nevertheless, this gene is not annotated in the
genomes of the analyzed S. pneumoniae strains and consequently it is not shown in Fig. 10,
which is automatically generated by ERGOTM
. The CibAB peptides contain a typical GG-
processing site, but how they are processed or transported outside the cell is not yet known.
It has been suggested that the proteolytic ABC transporter, ComAB, which processes and
exports competence stimulating peptide (CSP) also performs this function for CibAB
(78,79).
Chapter 2
60
A.
B.
Gene IDa Gene
numberb Functionc
SPR0135 14 Glycosyltransferase
15 EpsG; Glycosyltransferase
SPR0134 13 transposase
SPR0133 17 transposase
SPR0132 6 transposase
SPR0131 3 O-sialoglycoprotein endoprotease
SPR0130 4 RimI; ribosomal protein-S18-alanineacetyltransferase
SPR0129 5 non-proteolytic protein, peptidase family M22
SPR0128 7 CibA bacteriocin (S. pneumoniae: TIGR4 SP0125; CGSP14 CPSG_0129; D39
SPD0133; G54 SPG_0129; Hungary19A-6 SPH_0241; JJA SPJ_0158; P1031 SPP_0194;
Taiwan19F-14 SPT_0173; 70585 SP70585_0204; ATCC 700669 SPN23F_01380)
SPR0127 8 CibB bacteriocin (S. pneumoniae: TIGR4 SP0124; CGSP14 CPSG_0128; D39 SPD0132;
G54 SPG_0128; Hungary19A-6 SPH_0240; ; JJA SPJ_0157; P1031 SPP_0193;
Taiwan19F-14 SPT_0172; 70585 SP70585_0203; ATCC 700669 SPN23F_01379)
SPR0126 1 Putative regulatory protein
SPR0125 2 Zn -dependent hydrolase
SPR0124 9 GidA; Glucose inhibited division protein
SPR0123 10 Phosphohydrolase
SPR0122 12 tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase; TrmU
SPR0121 11 Pneumococcal surface protein A
RPN02123 16 Hypothetical protein
SPCG_0127 28 Hypothetical protein; bacteriocin-like peptide (Hungary19A-6 SPH_0239)
SPG_0122 29 Hypothetical protein
SPJ_0154 30 Hypothetical protein
Figure 10. A. Graphical representation of the cibABC (cluster VIII) locus in Streptococcace. Dashed lines indicate
insertion elements and genes marked as grey indicate those that do not have have homology to other ones shown
Bacteriocin-like gene clusters
61
in this figure. B. List of genes and their function of the genomic region of the cibABC locus in Streptococcace. a
Gene ID refers to S. pneumoniae R6 locus tags. b Gene number refers to gene numbers shown in Figure 10 and
described in order from left to right. c Putative/predicted function based on ERGOTM, R6/TIGR4 annotation and/or
domain prediction, and/or protein homologous. Genes with the same color are predicted to have the same function,
colors are arbitrarily designated.
The CibAB peptides have not been isolated neither could their contribution to
allolysis be detected in a cell-free supernatant or when the peptides were synthetically
produced (168). Hence, it was proposed that cell-to-cell interaction is required for CibAB
antimicrobial activity (168). Analysis of the cibABC region in five S. pneumoniae strains,
i.e. of serotype 2, 4, 19F, 23F and 6B, showed only two changes in protein sequences,
indicating that allolysis might be a conserved process within S. pneumoniae species (168),
which is in accordance with the fact that all strains are thought to be competent.
Interestingly, strains CGSP14, Hungary19A-6 and Taiwan19F-14 harbor, adjacent to
cibAB, a gene (with a gene number 28 in Fig. 10) encoding a peptide of unknown function.
The amino acid sequence of this peptide shows bacteriocin-like characteristics
(supplemental material Fig. S11 C). This raises the possibility that this peptide belongs to
the cibABC cluster in these three strains and it might perform a bacteriocin-like function,
which could change the fratricide mechanism in these strains. Strikingly, cluster VIII is
present in all analysed S. pneumoniae genomes, except for Hungary19A-6. A comparison
of the cibABC genomic region across some other species of the family Streptococcaceae
(Fig. 10) revealed that CibAB bacteriocins are specific for S. pneumoniae, although the
neighbouring genes of cibABC are conserved. Accordingly, we hypothesize that CibAB-
specific allolysis occurs only in S. pneumoniae or that other Streptococcus species use
different peptides. The latter is more likely since peptides similar to CibAB were found in
another genomic region of S. mitis and S. gordonii, but not in S. thermophilus, S. sanguinis
and S. mutans (80), suggesting that in other Streptococcus species, an allolysis-like
phenomenon might exists.
Cluster IX encodes the Blp (Pnc) bacteriocins belonging to the class IIb of bacteriocins
The blp (bacteriocin-like peptide, or pnc) cluster was first described by R. Lange
et al. (300) and antimicrobial activity has been shown for the Blp-bacteriocins (97,98,307)
(Fig. 11). Within the blp cluster, genes with number 9 and 10 in Fig. 11 encode bacteriocin
peptides, namely BlpM and BlpN. However, it is predicted that more bacteriocin-like
peptides of the Blp cluster, namely BlpI, BlpJ and BlpK (indicated by gene number 12 in
Fig. 11), and BlpU located in another genomic region than the Blp cluster, are involved in
Blp mediated antimicrobial activity (97,307). Interestingly, the Blp bacteriocins show both
inter- and intraspecies activity. For instance, the BlpM and BlpN bacteriocins of S.
pneumoniae type 6A and 19A were shown to inhibit the growth of the TIGR4 strain (97).
Similarly, the Blp bacteriocins of S. pneumoniae 632 were active against other S.
pneumoniae strains, namely R6 and 2306 but also against other species i.e. S. mitis,
Chapter 2
62
A.
B.
Gene IDa Gene numberb Functionc
SP0547 34 (PncP) CAAX amino terminal protease family SP0546 31 BlpZ (PncQ) Immunity protein
SP0545 23 BlpY (PncO) CAAX amino terminal protease family
SP0544 28 BlpX (PncN) Immunity protein SP0542 30 Hypothetical protein
SP0540 10 BlpN (PncJ) Bacteriocin-like peptide
SP0539 9 BlpM (PncI) Bacteriocin-like peptide
SP0536, SP0543 29 BlpL (PncM and PncH, respectively) immunity protein
SP0535 33 (PncG) Immunity protein
SP0531,SP0532, SP0533, SP0541
12 BlpI, BlpJ, BlpK and BlpO (PncA, PncD, PncE2 and PncV, respectively) bacteriocin-like peptides
SP0530 1 BlpC ABC transporter (SpiDCBA) SP0529 4 Bacteriocin export accessory protein
SP0528 32 BlpC (SpiP) Pheromone peptide
SP0527 6 BlpH (SpiH) Histidine kinase SP0526 5 BlpR (SpiR2) Response regulator
SP0525 26 BlpS (SpiR1) Response regulator
- 13,16,24,25, 41,57,58
Transposase
Figure 11. A. The blp (pnc; cluster IX) genomic region in Streptococcace. Dashed lines indicate insertion
elements and genes marked as grey indicate those that do not have have homology to other ones from this figure.
B. Genes of the blp cluster and their putative function. a Gene ID refers to S. pneumoniae TIGR4 locus tags. b Gene
number refers to gene numbers shown in Figure 11 and described in order from left to right. c Putative/predicted
Bacteriocin-like gene clusters
63
function based on ERGOTM, R6/TIGR4 annotation and/or domain prediction, and/or homologous proteins. Genes
with the same color are predicted to have the same function, colors are arbitrarily designated.
Streptococcus oralis, and S. pyogenes (307). Additionally the Blp peptides of S.
pneumoniae 632, 2306, TIGR4 and 628 inhibited growth of L. lactis and Micrococcus
luteus (307).
The BlpRH (TCS13), two-component system (marked as numbers 5, 6 and 26 in
Fig. 11), regulates production of the Blp-bacteriocins. It is thought that BlpH senses the
pheromone peptide, BlpC (number 32 in Fig. 11), and subsequently activates BlpR, which
induces expression of the blp cluster. This has been shown using synthetic BlpC, since the
natural conditions that induce blp expression remain unknown (102,436). In addition, the
blp cluster is negatively regulated on a posttranscriptional level by the serine protease HtrA
(98).
Extensive analysis of the genomic region of the blp cluster in various S.
pneumoniae strains and its isolates has been performed (97,307,436), which showed that
the size of the region can differ up to 5 kb, as it is in R6 and TIGR4 (307). Accordingly,
there are also variations in the number of bacteriocin encoding genes and their amino acid
sequence, as well as the number of immunity proteins and dedicated transporter proteins
(307). We determined that the blp-like cluster is also present in other streptococcal species
(Fig. 11). In some of these species, e.g. S. thermophilus, S. pyogenes, S. mutans and S. equi,
the cluster has been described and, in addition, for two latter species, it was shown to be
functional (141,142,217,273,531,532). Interestingly, the blp-like cluster in S. mutans and S.
thermophilus also contains significant variations in the number of bacteriocin-encoded
genes (142,531). All together, the data indicates that the blp-like cluster is ubiquitous
among streptococci and likely it contributes to their survival in the environment probably
by eliminating closely related species. In addition, the variation in the Blp-dependent intra-
and interspecies competition might be explained by the genetic variability of the IX cluster
in S. pneumoniae strains (97,307).
Discussion
Here, we present a comparative analysis of nine putative bacteriocin gene clusters
identified within 11 S. pneumoniae strains. Following a BAGEL analysis in strains R6 and
TIGR4, nine clusters were identified based on their amino acid similarities to known
bacteriocins (Fig. 1) and their genomic region was further analyzed. We hypothesize that
two clusters, I and VII, likely encode lantibiotics and that the other seven may encode
bacteriocins of the class II non-lantibiotics. Strikingly, for only two clusters, i.e. VIII (cib)
and IX (blp), antimicrobial activity has been shown (97,102,168,436) and for the remaining
seven clusters there is no experimental data indicating that they encode functional AMPs.
Therefore, we have chosen two clusters, namely I and V, for further study (chapter 3 and 4
of this thesis). Cluster I likely produces a lantibiotic type two-peptide bacteriocin that we
Chapter 2
64
named PneA1 and PneA2. Although we were unable to show PneA1 and PneA2 specific
antimicrobial activity in S. pneumoniae, chimeras of the leaderless pneA1 and pneA2 genes
and the nisin leader peptide, which were modified by the nisin modification and export
enzymes, NisBTC (439), were found to be active against M. flavus (chapter 4 of this thesis).
This strengthens our prediction that PneA1 and PneA2 are active lantibiotics. We were
unable to show that cluster V produces a bacteriocin-like peptide. Instead, we demonstrated
that cluster V is likely involved in nitrogen metabolism of S. pneumoniae D39 (chapter 3 of
the thesis).
Bacteriocins enable bacteria to survive in a competitive niche by eliminating other
microorganisms colonizing the same environment. Additionally bacteriocins could
indirectly facilitate bacterial evolution since the AMP-mediated destruction of the sensitive
bacteria causes release of DNA that can be taken up and integrated into the genome. This is
strongly suggested by the role of bacteriocins in fratricide, the predation of non-competent
cells by competent ones. The CibAB bacteriocin plays an important role in fratricide and
this mechanism has been suggested to be common among streptococci (168). Strikingly, a
similar process was described for S. sanguis (462) and analogous mechanisms driven by
e.g. nutrient limitation, were described for other bacteria (2,128,129,157,270). Notably, as
shown in Fig. 10, the genomic region of the cibABC cluster (cluster VIII) is similar in many
streptococci, such as S. pyogenes, S. thermophilus, S. suis, S. uberis, S. equi, S. agalactiae
and S. gordonii. However, all these species lack homologs of CibAB, which suggests that
they might use other peptides or mechanisms for fratricide. Altogether, it seems that in S.
pneumoniae strains the CibAB cluster is conserved and possibly all strains are able to
commit fratricide.
Comparison of the genomic region containing the blp (pnc) locus (cluster XI) in
many S. pneumoniae strains of different serotypes showed clear variations since the locus
can vary in size from 2.5 kb up to 8 kb. Furthermore, there are differences in the numbers
of bacteriocin encoding genes, as for instance the ATCC 700669 strain lacks blpM and
blpN, but surprisingly still showed Blp-like antimicrobial activity (92,97,307). In other
words, the spectrum of antimicrobial activity of the Blp bacteriocins differs in the S.
pneumoniae strains and is strain-dependent due to variations in the amino acid sequence of
the Blp bacteriocins and/or even of the whole blp bacteriocin encoding gene cluster. The
variation of the Blp-specific antimicrobial activity, might mediate inter- and intraspecies
competition (92,97,307). We showed (Fig. 11) that a genomic region similar to that of
cluster IX (blp) could be found in S. pyogenes, S. thermophilus, S. uberis and S. equi. This
indicates once more that genetic exchange occurs rather frequently between streptococci.
In general, Gram-positive bacteria commonly produce lantibiotics, for which the
encoding loci can be found either on the chromosome, (e.g. subtilin and salivaricin A) on
plasmids (e.g. epidermin and cytolysin), or on conjugative transposons (e.g. nisin). It has
been proposed that the bacteriocin biosynthetic genes might have spread out among Gram-
positive bacteria from a common ancestor, using these mobile genetic elements (56,72).
Bacteriocin-like gene clusters
65
Interestingly, cluster V in strain CGSP14 varies from the other analyzed S. pneumoniae
strains due to the presence of four genes that disrupted the cluster (Fig. 7). The four genes
encode proteins homologous to that of the putative lantibiotic production locus of S.
thermophilus LMG18311 and CNRZ1066, and a gene for a putative lantibiotic precursor
(supplemental material Fig. S5). Interestingly, analysis of sixteen other S. pneumoniae
strains, of the same serotype 14, showed that only one strain, namely SPnINV200, contains
the same four genes as the CGSP14 strain in cluster V (116). Notably, the locus in the S.
thermophilus LMG18311 and CNRZ1066 strains is flanked by genes encoding a phage
transcriptional repressor and a phage integrase family protein. Additionally downstream of
the locus in S. thermophilus LMG18311, CNRZ1066 and LMD-9, are two IS elements.
Consequently, we hypothesize that this putative lantibiotic locus could be easily
transferable among the genus Streptococcus.
Genome sequencing of S. pneumoniae strains/isolates regularly reveals novel
bacteriocin-like encoding loci. The comparative genomic analysis of eight S. pneumoniae
clinical isolates of different serotypes revealed that three of them, i.e. SP23-BS72, SP3-
BS71 and SP6-BS73, harbor genes encoding proteins potentially involved in lantibiotic
biosynthesis (472). Some of these are homologs of either MrsM or MrsT, which are
required for the production of the lantibiotic mersacidin in Bacillus licheniformis (4,472).
However, only SP23-BS72 seems to have a complete lantibiotic encoding locus (472) with
similarity to proteins and peptides of the haloduracin, Bht and lacticin biosynthesis
machinery (4,15,62). Notably, this locus is not present in the TIGR4 and R6 strains (472),
which is why the BAGEL screen did not identify them. Similarly, genome sequencing of S.
pneumoniae ATCC 700669 of serotype 23 identified a novel lantibiotic-like encoding
locus, of which the structural gene has features of mersacidin and lichenicidin (4,24,92).
Notably, the locus seems to be carried on a transposon (92) and again the locus is not
present in the TIGR4 and R6 strains, which once more, underscores the genetic variability
of S. pneumoniae.
For only two out of the nine bacteirocin-like clusters described here, namely Blp
and CibAB, antimicrobial activity has been demonstrated (97,168,193,307,526). In
addition, we have shown that chimeric peptides of cluster I have antimicrobial activity
against M. flavus (310). It is surprising that no bacteriocin-like activity has been found for
at least one of the other seven clusters. Our comparative analysis of potential bacteriocin
encoding clusters demonstrates that bacteriocins are likely a large part of the lifestyle of S.
pneumoniae. However, at the same time it appears difficult to find growth conditions in the
laboratory that stimulate production of these peptides, suggesting that they are perhaps
induced by signals specific for the host niche or competitive bacteria. This remains a great
challenge for future research. Furthermore, it seems that S. pneumoniae can potentially
produce a variety of bacteriocins and the nine putative bacteriocin clusters described here
are probably just a minor fraction of the number and diversity of the potential AMPs that
more than 90 serotypes of S. pneumoniae could produce.
Chapter 2
66
Supplemental Material
Figure S1. The amino acid sequences of the putative two-peptide lantibiotics of Figure 2, A. PneA1 and B.
PneA2. The putative cleavage site is underlined. Positively charged amino acids in propeptide part are bolded.
Residues possibly contributing to modification and ring formation in propeptide part, i.e. serine, threonine and
cysteine, are highlighted as italic letters.
Figure S2. Amino acid sequence alignment of SPR1764 from the cluster I in Figure 2 with other homologous
peptides of S. pneumoniae strains. Alignment was performed using ClustalW (302). Asterisk, identical residues;
colon, conserved residues.
Figure S3. Alignment of an amino acid sequence of the putative bacteriocin peptide of the cluster III of gene
number 1 in Figure 5, SPG_1890, with other putative bacteriocins of the same or other cluster from the S.
Bacteriocin-like gene clusters
67
pneumoniae strains and known bacteriocin of L .lactis, lactococcin 972. Alignment was performed using ClustalW
(302). Asterisk, identical residues; colon, conserved residues; period, semi-conserved residues.
Figure S4. Amino acid sequence alignment of the bacteriocin-like peptide of gene number 8 of the cluster V in
Figure 7, SPR0141, with bacteriocin-like peptides of the same cluster but in different S. pneumoniae strains.
Alignment was performed using ClustalW (302). Asterisk, identical residues; colon, conserved residues; period,
semi-conserved residues.
Figure S5. Amino acid alignment of the bacteriocin-like peptide, SPCG_0144, from the cluster V in Figure 7 of S.
pneumoniae CGSP14 with homologous peptides of S. thermophilus. Alignment was performed using ClustalW
(302). Asterisk, identical residues; colon, conserved residues; period, semi-conserved residues.
Figure S6. The amino acid sequence of the S. pneumoniae putative bacteriocin peptides of the cluster VI in Figure
8, A. peptide of a gene number 17 (SPR1659; pI 4.7), B. peptide of a gene number 20 (SPR1651; pI 4.5), C.
peptide of a gene number 21 (SPD1625; pI 8.9), and D. peptide of a gene number 17 in TIGR4 (SP1842; pI7.5).
The putative cleavage site is underlined and positively charged amino acids in the C-terminus are bolded.
Chapter 2
68
Figure S7. Amino acid sequence alignment of the bacteriocin-like peptide, SPR1653, of a gene number 16 from
the cluster VI in Figure 8 with other peptides of S. pneumoniae TIGR4 and D39 from the same cluster. Alignment
was performed using ClustalW (302). Asterisk indicates identical residues. The putative cleavage site is underlined
and positively charged amino acids in the C-terminus are bolded.
Figure S8. Amino acid sequence alignment of the bacteriocin-like peptide of a gene number A. 17 (SPR1659) and
B. 21 (SPD1625) of cluster VI in Figure 8 with other peptides of S. pneumoniae, S. gordonii Challis substr. CH1
and S. mutans UA159. Asterisk indicates identical residues; colon, conserved residues and period, semi-conserved
residues. Alignment was performed using ClustalW (302).
Figure S9. The amino acid sequence of the S. pneumoniae putative bacteriocin peptides of the cluster VII in
Figure 9, A. peptide of a gene number 14 (SPR1200; pI 7.2), B. peptide of a gene number 28 (SPR1199; pI 10)
and C. peptide of a gene number 15 (SP1339; pI 3.7). The putative cleavage site is underlined and positively
charged amino acids in the C-terminus are bolded.
Bacteriocin-like gene clusters
69
Figure S10. ClustalW sequence alignment of the putative bacteriocin-like peptide of a gene number 28 in Figure 9
(SPR1199) of the bacteriocin-like cluster VII with bacteriocin-like peptides of various cluster from S. pneumoniae
strains. Asterisk points to identical residues, colon to conserved residues and period to semi-conserved residues.
Figure S11. The amino acid sequence of the CibAB S. pneumoniae bacteriocins A. CibA of gene number 7 in
Figure 10 (SPR0128; pI 3.4), B. CibB of gene number 8 in Figure 10 (SPR0127; pI 10.3) of the cluster VIII. C. An
amino acid sequence of a putative bacteriocin-like peptide of gene number 28 of cluster VIII (SPH_0239; pI 12).
The putative cleavage site is underlined and positively charged amino acids in the C-terminus are bolded.
Chapter 2
70
Chapter 3
Exploring the function and regulation of a putative
pneumococcal peptide and its gene cluster in
Streptococcus pneumoniae
Joanna A. Majchrzykiewicz, Jetta J. E. Bijlsma and Oscar P. Kuipers
Chapter 3
72
Characterization of the ppu cluster
73
In silico analysis of the genome sequence of S. pneumoniae R6 indicated that
SPR0140-0146 genes might form a functional cluster containing a pneumococcal
peptide of unknown function with some resemblance to bacteriocins. Hence, we
named the peptide PpuA and the gene cluster ppuRABCDE. The cluster encodes
PpuR, a putative regulator, the PpuA peptide, PpuBC, two CAAX endopeptidases,
PpuD, a putative transporter, and PpuE a putative branched-chain amino acid
transporter. Here, we show that expression of the ppuRABCDE cluster is strictly
linked to the concentration of amino acids and peptides in the growth medium,
suggesting that the function of the cluster is related to the general nitrogen
metabolism of this bacterium. In line with this, we demonstrate that expression of
ppuRABCDE is under negative control of CodY, a branched-chain amino acid
responsive regulator. Moreover, transcriptional studies showed that PpuR is likely a
positive regulator of ppuABCDE. Transcriptome analysis of a ppuR and a ppuA
mutant revealed that expression of two other, not yet described putative clusters, are
influenced by the ppu cluster. Thus, they were designated as a peptide responsive
cluster, prcRABCD, and a transporter of amino acids, taaBC. Transcriptional analysis
of the prcRABCD and taaBC promoter regions confirmed that PpuR and the
ppuRABCDE cluster influenced their expression. Interestingly, expression of both the
prcA and taaBC promoter changed upon addition of nitrogen containing compounds
to the medium, which suggests that both clusters, i.e. prcRABCD and taaBC, might be
involved in the nitrogen metabolism of S. pneumoniae, as is the ppuRABCDE cluster.
In conclusion, we revealed that three gene clusters, i.e. ppu, prc and taa, are most
likely involved in nitrogen metabolism in S. pneumoniae. In addition, two regulators
of these clusters were identified: namely PpuR regulates ppuRABCDE expression
together with CodY, and influences that of the prc and the taa cluster, which suggests
that they form a novel regulon in this bacterium.
Introduction
S. pneumoniae is a common inhabitant of the human upper respiratory tract and
can cause serious diseases i.e. sinusitis, acute otitis media, pneumonia and meningitis. The
successful spread from the nasopharynx to a variety of different tissues in the human body
requires efficient adaptation to changes in the quality and availability of nutrients, such as
amino acids and to environmental stresses generated by the antimicrobial defenses of other
bacterial species, e.g. bacteriocins, and those of the host. Bacteriocins are small cationic
antimicrobial peptides (AMPs) produced by Gram-positive bacteria. In general, they can be
divided into four classes according to their biochemical and genetic characteristics. Class I,
the lantibiotics, comprises peptides that require several posttranslational modifications to
acquire biological activity. Class II, the non-lantibiotics do not require modification for
Chapter 3
74
their antimicrobial activity. Class III consists of large proteins and class IV of cyclic
peptides (163,203,529).
Signature-tagged mutagenesis (STM) screens and in vivo transcriptome analysis of
S. pneumoniae identified SPR0140-0146 as required for invasive diseases in mice
(38,191,390). Additionally this cluster was identified, by use of differential fluorescence
induction (DFI), during in vitro conditions resembling a mouse infection model (319).
Because these genes were found to be important for S. pneumoniae virulence but their
function and regulation are unknown, we decided to further study the ppuRABCDE
cluster‘s function and regulation.
The SPR0140-0146 cluster, named here the pneumococcal peptide of unknown
function (ppuRABCDE) cluster, seems to consist in S. pneumoniae R6 of six genes, namely
ppuR, -A, -B, -C, -D, -E. The cluster is likely organized in two transcriptional units, one
consisting of the ppuR gene encoding a putative transcriptional regulator of the
Rgg/GadR/MutR family and a presumed operon containing the other genes of the cluster
starting with ppuA (Fig. 1A). Since PpuR shows more than 30% amino acid sequence
identity to the positive transcriptional regulators of known bacteriocins, namely BhtR of
Streptococcus ratti (223) and MutR of Streptococcus mutans (421-424), and because the
amino acid sequence of PpuA (Fig. 1B) possesses several characteristic features of
bacteriocin-like peptides such as a GG-processing site, positively charged amino acids and
a high (~11.3) pI value of the putatively processed peptide, we hypothesized that this
cluster might be involved in production of a bacteriocin-like peptide, namely PpuA.
The ppuBC genes encode proteins belonging to the CAAX endopeptidase family,
also known as the Abi family (401) that consists of various, prokaryotic and eukaryotic,
mostly hypothetical proteins, of which the function is unknown. Members of this family are
putative metal-dependent proteases that are likely linked to a protein/peptide modification
and/or secretion processes (77,401,478). Examples are the PlnIL proteins of Lactobacillus
plantarum that are probably involved in maturation, transport and immunity of plantaricin
A, a bacteriocin (112) and the SkkI protein, which gives immunity to sakacin 23K (257).
The ppuD gene encodes a putative transporter of the major facilitator superfamily (MFS)
and ppuE encodes a putative branched-chain amino acid transport protein with homology to
AzlC (25). The MFS transporters are single-polypeptide secondary carriers that occur
ubiquitously in prokaryotes and eukaryotes. The MFS proteins transport small molecules in
response to chemiosmotic-ion gradients. The MFS consists of at least 34 families involved
in transport of, amongst others, sugars, drugs, nitrate, nucleosides, peptides or amino acids
(395,456). The ppuRABCDE cluster shows reasonable biosynthetic locus similarity to
clusters involved in bacteriocin production, namely mutacin II and Bht-B (223,421). The
lantibiotic mutacin II is one of the bacteriocins produced by S. mutans and Bht-B, a non-
lantibiotic, is produced by S. rattus (60,73,223,556). Like the ppuRABCDE cluster, the
biosynthetic gene locus of mutacin II and Bht-B consists of genes divided into two
transcriptional units. The mutR gene, which encodes a homolog of the transcriptional
Characterization of the ppu cluster
75
regulator of glucosyltransferase G (rgg) of Streptococcus gordonii (495), is followed by
genes that contain the structural bacteriocin gene and genes that encode proteins involved in
bacteriocin modification/processing, transport and immunity. In this study, we investigated
the regulation of the ppu gene cluster in more detail in order to shed some light on its
function.
A.
B.
Figure 1. Organization of putative ppuRABCDE cluster in S. pneumoniae R6 and D39 and amino acid sequence of
PpuA. (A) Genetic map of the ppuRABCDE cluster in S. pneumoniae R6 and D39; thick white arrows indicate the
ppuRABCDE genes in their transcriptional direction, and two black thin arrows indicate putative promoters of the
ppuRABCDE cluster, namely PppuR and PppuA. (B) Amino acid sequence of the PpuA peptide; positive amino
acids are indicated in bold and the GG-putative processing sites, i.e. 1, 2 and 3, are underlined.
Materials and Methods
Bacterial strains and growth conditions
Strains and plasmids used in this study are listed in Table 1. Strains were stored in 10% glycerol (v/v)
at -80 °C. Streptococcus pneumoniae strains were grown without agitation at 37°C either in liquid
media: i.e. in M17 (504) (Difco) broth supplemented with 0.5% (w/v) glucose (GM17) and/or Todd-
Hewitt (Oxoid) broth supplemented with 0.5% yeast extract (THY) and/or chemically defined
medium (CDM) (260), or in solid media: GM17 or THY agar containing 3% defibrinated sheep blood
(Johnny Rottier, Kloosterzande, The Netherlands). Lactococcus lactis and Escherichia coli were
grown as described previously (260). When appropriate media were supplemented with antibiotics:
chloramphenicol (2 μg/ml for S. pneumoniae, 5 μg/ml for L. lactis), erythromycin and spectinomycin
(for S. pneumoniae 0.25 μg/ml and 150 μg/ml, respectively), trimethoprim (18 μg/ml for S.
pneumoniae), and tetracycline (2.5 μg/ml for S. pneumoniae), and ampicillin (100 μg/ml for E. coli).
DNA isolation and manipulation
All techniques concerning DNA manipulations were performed as described previously (260,261).
Plasmids and primers used in this study are listed in Table 1 and 2, respectively. The chromosomal
DNA of S. pneumoniae D39 was used as a template for primer design and PCR amplifications. All
the constructs were confirmed by sequencing.
Construction of transcriptional lacZ fusions
The PP2 plasmid was used to generate a transcriptional fusion of the putative promoter region of
ppuR to lacZ. The putative promoter region was amplified with primer pair PppuR-fv/PppuR-rev.
Subsequently, the amplified fragment was digested with XbaI and EcoRI and cloned into these sites in
pPP2 yielding pPP2PppuR. E. coli EC1000 was used as a cloning host. Similarly, transcriptional lacZ
Chapter 3
76
fusions of the following putative promoter regions of ppuA (yielding pPP2PppuA), prcR (yielding
pPP2PprcR), prcA (yielding pPP2PprcA), taaBC (yielding pPP2taaBC) and SPR1352 (yielding
pPP2Pspr1352) were constructed with use of primer pairs PppuA-fv/PpuA-rev, PprcR-fv/Pprc-rev,
PprcA-fv/PprcA-rev, PtaaBC-fv/PtaaBC-rev and Pspr1352-fv/Pspr1352-rev, respectively. Next the
constructs were introduced into S. pneumoniae D39 strains by natural transformation.
Table 1. Strains and plasmids used in this study
EryR, erythromycin resistance; TetR, tetracycline resistance; SpecR, spectinomycin resistance; trmpR,
trimethoprim resistance
Strain Description Reference or source
S. pneumoniae
D39 Serotype 2 strain, cps2 (12,301) R6 D39(Δcps2 2538-9862) with increased transformation
efficiency
(219)
TIGR4 (505,541)
ppuR D39ppuR;SpecR T. G. Kloosterman
ppuA D39ppuA;EryR This work
WH101 D39codY;TrmpR (199)
ΔcodYppuR WH101 ppuR This work
PppuR D39bgaA::PppuR-lacZ;TetR This work
PppuR_1 D39bgaA::PppuR_1-lacZ:TetR This work
PppuR_2 D39bgaA::PppuR_2-lacZ:TetR This work
PppuA D39bgaA::PppuA-lacZ;TetR This work
PppuA_1 D39bgaA::PppuA_1-lacZ;TetR This work
PppuA_2 D39bgaA::PppuA_2-lacZ;TetR This work
PprcA D39bgaA::PprcA-lacZ;TetR This work
PprcR D39bgaA::PprcR-lacZ;TetR This work
PtaaBC D39bgaA::PtaaBC-lacZ;TetR This work
PSPR1352 D39bgaA::PSPR1352-lacZ;TetR This work
ppuR/PppuR ppuR bgaA::PppuR-lacZ;TetR This work
ppuR/PppuR_1 ppuR bgaA::PppuR_1-lacZ;TetR This work
ppuR/PppuR_2 ppuR bgaA::PppuR_2-lacZ;TetR This work
ppuR/PppuA ppuR bgaA::PppuA-lacZ;TetR This work
ppuR/PppuA_1 ppuR bgaA::PppuA_1-lacZ;TetR This work
ppuR/PprcR ppuR bgaA::PprcR-lacZ;TetR This work
ppuR/PprcA ppuR bgaA::PprcA-lacZ;TetR This work
ppuR/PtaaBC ppuR bgaA::PtaaBC-lacZ;TetR This work
ppuR/PSPR1352 ppuR bgaA::PSPR1352-lacZ;TetR This work
ppuR/PppuA_2 ppuR bgaA::PppuA_2-lacZ;TetR This work
ppuA/PppuR ppuA bgaA::PppuR-lacZ;TetR This work
ppuA/PppuR_1 ppuA bgaA::PppuR_1-lacZ;TetR This work
ppuA/PppuR_2 ppuA bgaA::PppuR_2-lacZ;TetR This work
ppuA/PppuA ppuA bgaA::PppuR-lacZ;TetR This work
ppuA/PppuA_1 ppuA bgaA::PppuA_1-lacZ;TetR This work
ppuA/PppuA_1 ppuA bgaA::PppuA_2-lacZ;TetR This work
ppuA/PprcR ppuA bgaA::PprcR-lacZ;TetR This work
ppuA/PprcA ppuA bgaA::PprcA-lacZ;TetR This work
ppuA/PtaaBC ppuA bgaA::PtaaBC-lacZ;TetR This work
ppuA/PSPR1352 ppuA bgaA::PSPR1352-lacZ;TetR This work
codY/ PppuR WH101 bgaA::PppuR-lacZ;TetR This work
codY/ PppuR_1 WH101 bgaA::PppuR_1-lacZ;TetR This work
codY/ PppuR_2 WH101 bgaA::PppuR_2-lacZ;TetR This work
codY/ PppuA WH101 bgaA::PppuA-lacZ;TetR This work
codY/ PppuA_1 WH101 bgaA::PppuA_1-lacZ;TetR This work
Characterization of the ppu cluster
77
codY/ PppuA_2 WH101 bgaA::PppuA_2-lacZ;TetR This work
codYppuR/ PppuA ΔcodYppuR bgaA::PppuA-lacZ;TetR This work
L. lactis NZ9000 MG1363 pepN::nisRK (290)
E. coli
EC1000 KmR; MC1000 derivative carrying a single copy of the pWV01 repA gene in glgB
(303)
Plasmid
pPP2 AmpR TetR; promoter-less lacZ. For replacement of bgaA
(SPR0565) with promoter-lacZ fusions. Derivative of
pPP1.
(175)
pPP2PppuR pPP2 PppuR-lacZ This work
pPP2PppuA pPP2 PppuA-lacZ This work
pPP2PppuR_1 pPP2 PppuR_1-lacZ This work
pPP2PppuR_2 pPP2 PppuR_2-lacZ This work
pPP2PppuA_1 pPP2 PppuA_1-lacZ This work
pPP2PppuA_2 pPP2 PppuA_2-lacZ This work pPP2PprcR pPP2 PprcR-lacZ This work
pPP2PprcA pPP2 PprcA-lacZ This work
pPP2PtaaBC pPP2 PtaaBC-lacZ This work
pPP2Pspr1352 pPP2 PSPR1352-lacZ This work
Table 2. Oligonucleotide primers used in this study
Name Nucleotide sequence (5’ to 3’);
restriction enzyme sites underlined
Restriction site
KN-ppuR-fv-1 TGCTCTAGACCTTCTTTTGGATTTGGA -
KN-ppuR-rev-2 CGGGATCCCATCCTACCACCTCCTAGC -
KN-ppuR-fv-3 GGGGTACCCATCCCTTTTTGAATTGCG -
KN-ppuR-rev-4 GAAGATCTAACTGGAAACGACCACAC -
KN-ppuA-fv-1 CCCACTAGCAGAGGAGGATAGCG -
KN-ppuA-rev-2 GAGATCTAATCGATGCATGCCCACTTCTGCGACCTAGGAT -
KN-ppuA-fv-3 AGTTATCGGCATAATCGTGGCTCTTATAGGAGATAATAGG -
KN-ppuA-rev-4 ACACTGAACTTCTGGTCAGC -
PppuR-fv CCGGAATTCCCTTCTTTTGGATTTGGAGGA EcoRI
PppuR-rev GCTCTAGACATCCTACCACCTCCTAGC XbaI
PppuA-fv CGGAATTCGCCGAGTTGGAGAGGATGTTACG EcoRI PppuA-rev GCTCTAGAGGTTGCCTCCTCTAACATCTTGC XbaI
PprcA-fv CGGAATTCTGTCCATAATCCCATCTCATAT EcoRI
PprcA-rev GCTCTAGATAGTTCCAACAGCACTTATCATT XbaI
PprcR-fv CGGAATTCAGTAGTTCCAACAGCACTTATC EcoRI
PprcR-rev GCTCTAGATGTCCATAATCCCATCTCATAT XbaI
PtaaBC-fv CGGAATTCGTAGAAAATGGAACCGTTAAGCA EcoRI PtaaBC-rev GCTCTAGATCTGATGCTAAAATCGTTGTAAC XbaI
Pspr1352-fv CGGAATTCCAACTCCTCCAAGTGATGTGTTGA EcoRI
Pspr1352-rev GCTCTAGATTGTTCCATGAGATTACCTCGC XbaI PppuA_S1-fv ATTTTTTAAAATAAGCCAATTTTCGTGTTATACTG -
PppuA_S1-2-rev CGGCGAATTCGCAGGTACCGATGCAT -
PppuA_S2-fv CTTCATCTATTATATTCCTCCTTGTTAGT - PppuR_S1-fv CCAAAGTGTCAGAATGTTTTGACA -
PppuR_S1-2-rev GGGAAGACAATATCCTCCAAATCC -
PppuR_S2-fv GTAGGTTCTTTGTAACCGCTCC -
In order to construct subclones, shorter pieces of the ppuR and ppuA promoter regions and the round
PCR method with 5‘ phosphorylated primers was used as described earlier (439). Amplified
fragments with primer pair PppuA_S1-fv/PppuA_S1-2-rev yielded construct pPP2PppuA_1,
PppuA_S2-fv/PppuA_S1-2-rev yielded construct pPP2PppuA_2, PppuR_S1-fv/PppuR_S1-2-rev
Chapter 3
78
resulted in construct pPP2PppuR_1 and PppuR_S2-fv/PppuR_S1-2-rev resulted in construct
pPP2PppuR_2. Subsequently, the constructs were introduced into either S. pneumoniae D39, ΔppuR
or ΔppuA strains by natural transformation as described before (260,418).
Construction of ppuA and ppuR mutants
To construct the ppuA (ΔppuA) mutant, allelic-replacement mutagenesis was used. Shortly, primers
KN-ppuA-fv-1/KN-ppuA-rev-2 and KN-ppuA-fv-3/KN-ppuA-rev-4 were used to generate PCR
fragments of approximately 600 bps of the left and right flanking regions of ppuA. Next the flanking
regions were fused to an erythromycin resistance cassette, generated with primer pair Ery-rev/Ery-for
from pORI28, by means of overlap extension PCR (485) and the resulting PCR product was
transformed to S. pneumoniae D39 yielding ΔppuA.
Construction of the ppuR mutant (ΔppuR) was performed as follows. The left and right flanking
regions of ppuR were PCR amplified with primer pairs KN-ppuR-fv-1/KN-ppuR-rev-2 and KN-
ppuR-fv-3/KN-ppuR-rev-4, respectively and cloned as XbaI/BamHI and KpnI/BglII fragments in
pORI28spec1 (261) using E. coli EC1000 as the cloning host. The resulting construct was used as a
template for PCR and amplifies a product with primer pair KN-ppuR-fv-1/ KN-ppuR-rev-4, yielding
a linear cassette, which was transformed to S. pneumoniae D39. Transformants, having replaced the
ppuR gene with the speR gene, were selected with PCR and verified with Southern blotting.
β-galactosidase assay
β-galactosidase assays were performed as described previously (229,261).
Growth studies
Growth of S. pneumoniae D39 was performed in 96-well microtiterplates in CDM. The assay was
prepared as follows. A culture of S. pneumoniae D39 of approximately OD600 0.2 was stored in
aliquots at -80°C. For the growth assay, aliquots were thawed, spun down and resuspended in a fresh
medium to OD600~0.1, and were applied into microtiterplates to a total volume of 200 μl/well. The
microtiterplate was incubated in a GENios (TECAN Benelux) at 37°C and the OD600 was measured
every 30 min. All the growth studies were performed in triplicate at least.
DNA microarray analyses and transcriptional profiling
By DNA microarray analysis the transcriptome of ΔppuR and ΔppuA was independently compared to
the transcriptome of the D39 wild-type. For DNA microarray analysis each of the strains was grown
in 3 biological replicates in CDM and cells were harvested at an OD600 of ~0.3. DNA microarrays
were produced, prepared and analyzed as described before (261,534,535). Differential gene
expression with the Bayesian p-value < 0.0001 and with a differential expression greater than 2-fold
was considered as significantly differentially expressed.
Synthesis of the PpuA peptide
Two putative versions of predicted mature PpuA peptide, namely PpuA1:
(GGGGRSGISGWGVPGIYPGWGNQGMSPNRGAFDWTIDLADGLFGRRRR) and
PpuA2: (GGRSGISGWGVPGIYPGWGNQGMSPNRGAFDWTIDLADGLFGRRRR), were
synthesized by Pepscan Presto via service of ServiceXC B.V., Pepscan's official distributor in the
Benelux. The peptides, delivered as crude, were dissolved in DMSO and desalted with 50 mM Tris-
HCl of pH 5.5 on Microcon columns (Millipore), and stored in aliquots at -20°C at concentration of 2
mg/ml.
Putative promoters sequence analysis
Motif identification in the putative promoter sequence of ppuR and ppuA was carried with the Gibbs
Motif Sampler, http://bayesweb.wadsworth.org/gibbs/gibbs.html (508) and Motif Sampler
Characterization of the ppu cluster
79
http://homes.esat.kuleuven.be/~thijs/Work/MotifSampler.html (506,507), and Clone Manager from
Scientific & Educational Software (Sci-Ed Software).
Results
The ppuRABCDE cluster does not seem to produce a bacteriocin-like peptide
We have indicated that a possible function of the ppuRABCDE cluster in S.
pneumoniae is a bacteriocin-like peptide production. In order to determine whether the
PpuA peptide possesses bacteriocin activity, various antimicrobial assays both in solid
phase, namely patch and agar diffusion assays, and in a liquid phase, i.e. dilution and co-
culture assay, were performed under a variety of growth conditions including CDM (data
not shown). As putative PpuA producer strains, S. pneumoniae R6, D39, TIGR4 and S.
pneumoniae D39 ΔcodY were examined. As a negative control, S. pneumoniae D39
deficient in ppuA, ΔppuA, was analyzed. In the assays various bacterial strains, e.g.
Lactococcus lactis, Micrococcus flavus, Moraxella catarrhalis and S. pneumoniae
strains/mutants etc., were examined as indicator strains for their sensitivity to the possible
PpuA activity. In addition, a potentially sensitive indicator strain was constructed, Δppu, by
deleting ppuABCDE and two downstream genes, SPR0147-0148, in S. pneumoniae D39
(SPD0144-0149). This strain lacks the putative immunity genes and thus should be
sensitive to any PpuA bacteriocin-like activity. Antimicrobial activity specifically related to
the PpuA peptide was not observed against any tested indicator strain or under any tested
growth condition (data not shown). In order to isolate PpuA diverse concentration
methods/tools, such as TCA, Amicon Stirred Ultrafiltration Cells, FPLC by use of ion-
exchange column, and by generation and induction of a Strep-tagged PpuA, were
undertaken (data not shown). Nevertheless, no PpuA-like peptide was identified. Thus, we
assumed that the isolation/concentration methods were not suitable or sufficient enough to
purify PpuA. Hence, to learn whether PpuA has antimicrobial activity, based on the
presence of two putative processing sites in the peptide, two versions of PpuA were
chemically synthesized, PpuA_1 and PpuA_2 (Fig. 1B). Subsequently, the PpuA_1 and
PpuA_2 peptides were tested for possible bacteriocin-like activity in the spot assay and in
the dilution assay (data not shown). Both peptides did not show significant antimicrobial
activity (MIC>1 mg/ml) against the various indicator strains mentioned above (data not
shown). Thus, we propose that PpuA most likely performs another function in S.
pneumoniae.
The ppuRABCDE cluster is highly induced in chemically defined medium (CDM)
In order to study the ppuRABCDE cluster, it was necessary to determine under
which conditions the genes were expressed. Therefore, chromosomal transcriptional lacZ
fusions were constructed to the predicted ppu promoter regions, namely PppuR and PppuA
(Fig 1A). Subsequently the activity of these promoters was studied in various conditions
Chapter 3
80
(Table 3). Of all the conditions tested, e.g. BHI, THY, TSB (data not shown), GM17 and
CDM, only the latter induced the expression of both promoters (Table 3). Expression of
PppuR increased at most 2-fold, whereas activity of PppuA was induced about 20-fold in
CDM (Table 3). This suggested that certain compounds in the complex/undefined media
such as peptides might repress both PppuR and PppuA. To confirm this hypothesis, the
activity of the promoters was tested in CDM supplemented with casitone, and compared to
the activity in CDM itself (Table 3).
Table 3. β-galactosidase activity of transcriptionally fused to lacZ promoter of ppuA and/or ppuR, and their
derivatives ppuA_1, ppuA_2 and ppuR_1, ppuR_2 in the wild type S. pneumoniae D39 and its isogenic mutants of
ppuA, ppuR and codY grown in grown in GM17, CDM and in CDM supplemented with 3% casitone. The
PppuA_1, PppuA_2, PppuR_1 and PppuR_2, are truncated versions of PppuA and PppuR, and schematic overview
of the truncations is shown in Figure 3. Miller Units are the averages of at least three independent experiments and
the standard deviations are shown in brackets
Strain Promoter
β-galactosidase activity (Miller Units)
GM17 CDM CDM without a.a. with casitone
D39
PppuA
46 (11)
987 (75)
220 (16)
PppuA_1 26 (2) 41 (3) 31 (1)
PppuA_2 62 (9) 1160 (23) 52 (5)
PppuR 15 (0.6) 32 (0.5) 17 (1)
PppuR_1 11 (0.9) 10 (0.4) 11 (0.5)
PppuR_2 8 (0.8) 9 (0.5) 9 (0.9)
ΔcodY
PppuA 861 (24) 907 (17) 950 (56)
PppuA_1 ND ND ND
PppuA_2 753 (44) 1083 (49) 716 (2)
PppuR 112 (10) 94 (8) 118 (20)
PppuR_1 163 (5) 146 (11) ND
PppuR_2 130 (8) 12.7 (3) ND
ΔppuR
PppuA 24 (0.5) 20 (4). 18 (3)
PppuA_1 22 (0.9) 33 (1) 26 (3)
PppuA_2 72 (12) 44 (7) 36 (2)
PppuR 20 (2) 33 (1) 17 (2)
PppuR_1 ND ND ND
PppuR_2 ND ND ND
ΔppuA
PppuA 44 (3) 817 (60) 200 (19)
PppuA_1 ND ND ND
PppuA_2 ND ND ND
PppuR 13 (0.2) 27 (4) 16 (0.5)
PppuR_1 ND ND ND
PppuR_2 ND ND ND
In a medium supplemented with casitone, the PppuR activity decreased 2-fold and the
PppuA expression was reduced about 5-fold. This indicated that the components of
Characterization of the ppu cluster
81
casitone, amino acids and peptides, caused repression of ppuR and ppuA transcription in
CDM (Table 3).
CodY is a negative regulator of the ppuRABCDE cluster
Expression of PppuR and PppuA decreased upon addition of extra amino acids and
peptides (i.e. casitone) to CDM (Table 3). CodY is a major bacterial regulator responding to
the level of branched-chain amino acids in the environment (108,242,473,484). In addition,
transcriptome analysis of a S. pneumoniae codY mutant suggested that the ppuRABCDE
cluster might be a part of its regulon (199). To investigate whether CodY is indeed involved
in regulation of the ppuRABCDE cluster, expression of PppuR and PppuA was examined in
a codY deficient strain (Table 3). Transcription of both promoters increased significantly,
about 8-fold for PppuR and 4-fold for PppuA, in the ΔcodY strain independently of the type
of medium used and the presence of casitone (Table 3). Therefore, we concluded that CodY
is a negative regulator of the ppuRABCDE cluster.
Figure 2. Growth comparison of S. pneumoniae D39 and its
isogenic mutants in CDM. Comparison of the wild type S.
pneumoniae D39 (black rhomboids) and ΔppuR (white
squares) and ΔppuA (white triangles). The arrow indicates
the time point when cells were collected for transcriptome
analysis. The bacterial growth curves are representatives of
three independent experiments.
Expression profile of the ΔppuR and the ΔppuA strain grown in CDM
Transcriptome analysis of S. pneumoniae D39 wild-type and an isogenic ppuR
mutant was performed, on bacteria grown to an OD600~0.28 in CDM (Fig. 2), to obtain
more information about the putative function(s) of ppuR and/or the ppu cluster in S.
pneumoniae, and to ascertain whether PpuR is indeed a regulator of the ppu cluster. All
significant differentially expressed genes can be found in Table 4. The transcriptome
analysis showed that in the ΔppuR strain the expression of many genes, particularly
hypothetical genes or genes with unknown function had changed significantly (Table 4).
The ppuABCDE genes were about 10-fold downregulated in the ΔppuR strain, indicating
that PpuR most likely acts as a positive regulator of the ppuRABCDE cluster.
In addition, we hypothesized that PpuA might act as a pheromone. Therefore, the
effect of deletion of ppuA on global gene expression was also studied by transcriptome
analysis.
Chapter 3
82
Table 4. Summary of transcriptome comparison of S. pneumoniae D39 strains: ΔppuR and ΔppuA with D39 wild-
type. Only genes, for which the Bayes.P was ≤0.0001 (value between brackets) and transcript levels changed two-
fold or more in one or both strains were considered as significantly differentially expressed and are shown below. a
gene identifiers refer to TIGR4 or R6 locus tags, or to both TIGR4/R6; b ratios (D39 ΔppuR compared to D39) in
bold, Bayes.P values in parenthesis; c ratios (D39 ΔppuA compared to D39) in bold, Bayes.P values in parenthesis; d annotation according to NCBI database; NP, not present in TIGR4 genome; NDE, not significantly differentially
expressed
Gene IDa Gene
name ΔppuRb ΔppuAc Annotationd
TIGR4/R6
SP0034 0.2 (4.2e-12) 0.2 (5.3e-11) hypothetical membrane spanning protein
SP0044 purC 0.3 (1.4e-5) NDE phosphoribosylaminoimidazole-succinocarboxamide synthase
SP0047 purM 0.4 (2.4e-10) NDE phosphoribosylaminoimidazole synthetase
SP0048 purN 0.5 (6.9e-7) NDE phosphoribosylglycinamide formyltransferase
SP0051 0.3 (5.0e-9) NDE phosphoribosylamine--glycine ligase
SP0053 0.4 (4.7e-6) NDE phosphoribosylaminoimidazole carboxylase catalytic subunit
SP0054 0.5 (2.8e-7) NDE phosphoribosylaminoimidazole carboxylase ATPase subunit
SP0088 3.2 (7.9e-4) NDE hypothetical protein
SP0090 0.4 (7.0e-7) NDE ABC transporter, permease protein
SP0098 0.5 (1.7e-6) 0.5 (1.0e-6) hypothetical protein
SP0099 0.5 (7.2e-6) 0.5 (7.2e-6) hypothetical protein
SP0100 0.5 (3.5e-6) 0.5 (1.9e-4) hypothetical protein
SP0133 5.2 (8.0e-4) NDE hypothetical protein
SP0138 NDE 3.2 (6.2e-6) hypothetical protein
SP0139 NDE 4.5 (1.3e-8) UDP-glucose dehydrogenase
SP0141 ppuR 0.1 (2.6e-7) NDE transcriptional regulator
SP0142 ppuA 0.0 (9.0e-14) 0.0 (5.7e-13) bacteriocin-like peptide
SP0143 ppuB 0.0 (0.0e+0) 0.0 (0.0e+0) CAAX amino terminal protease family
SP0144 ppuC 0.0 (1.1e-16) 0.0 (0.0e+0) CAAX amino terminal protease family
SP0145 ppuD 0.0 (0.0e+0) 0.0 (0.0e+0) transporter, major facilitator family protein
SP0146 ppuE 0.3 (6.9e-06) 0.2 (9.9e-10) putative branched-chain amino acid transport protein azlC
SP0159 0.5 (2.5e-5) 0.7 (6.4e-10) homolog of a transporter for Mn(II), Mn(III), and Fe(II)
SP0177 3.1 (9.7e-5) NDE riboflavin synthase subunit alpha
SP0179 ruvA 2.7 (2.1e-5) NDE holliday junction DNA helicase motor protein
SP0200 NDE 2.0 (3.1e-4) competence-induced protein Ccs4
SP0202 nrdD 2.0 (8.5e-9) NDE anaerobic ribonucleoside triphosphate reductase
SP0204 3.6 (1.1e-6) 1.9 (6.1e-4) predicted acetyltransferase, GNAT family
SP0205 nrdG 2.2 (5.8e-5) NDE anaerobic ribonucleoside-triphosphate reductase activating
protein
SP0206 7.1 (4.5e-5) NDE hypothetical protein; uridine kinase
SP0207 5.4 (6.3e-4) NDE hypothetical protein; uridine kinase
SP0237 rplQ NDE 2.1 (1.2e-7) 50S ribosomal protein L17
SP0246 3.1 (6.3e-4) NDE DeoR family transcriptional regulator
SP0261 0.5 (7.7e-9) 0.4 (7.8e-9) undecaprenyl diphosphate synthase
SP0281 pepC 0.4 (4.3e-10) 0.4 (3.2e-7) aminopeptidase C
SP0303 celA 0.1 (2.5e-12) 0.1 (2.3e-8) 6-phospho-beta-glucosidase
SP0306 0.2 (1.1e-10) 0.1 (2.8e-8) transcriptional regulator
SP0307 0.3 (3.5e-4) 0.2 (1.1e-6) PTS system, IIA component
SP0309 0.2 (1.6e-7) 0.1 (3.9e-7) hypothetical protein
SP0335 ftsL 0.7 (2.6e-5) 0.5 (6.5e-7) Cell division protein FtsL, putative
SP0336 aliA 0.5 (7.1e-8) 0.5 (9.2e-8) oligopeptide ABC transporter
SP0338 2.5 (2.4e-6) NDE ATP-dependent Clp protease
SP0355 0.2 (2.4e-4) NDE hypothetical protein
SP0423 accB 0.4 (6e-6) NDE acetyl-CoA carboxylase biotin carboxyl carrier protein subunit
SP0437 NDE 0.5 (2.3e-5) glutamyl-tRNA (Gln) aminotransferase subunit A
SP0449 NDE 2.4 (8.0e-7) hypothetical protein
SP0506 vanD NDE 2.0 (8.4e-5) phage integrase family integrase/recombinase
SP0525 blpS 3.1 (9.9e-5) NDE regulatory protein
SP0547 8.1 (8.3e-5) NDE CAAX amino terminal protease family
SP0550 nrrD 2.4 (2.1e-4) NDE anaerobic ribonucleoside triphosphate reductase
SP0557 rbfA 4.1 (2.9e-5) NDE ribosome-binding factor A
SP0595 4.8 (4.6e-5) NDE hypothetical protein
SP0604 vnc NDE 0.4 (9.7e-7) sensor histidine kinase VncS
SP0607 NDE 0.4 (1.3e-7) amino acids ABC transporter,permease protein
SP0610 0.2 (1.6e-6) 0.3 (9.2e-6) amino acids ABC transporter,ATP
SP0621 0.3 (1.7e-9) 0.2 (1.0e-7) hypothetical protein
SP0634 3.1 (4.9e-5) NDE hypothetical protein
Characterization of the ppu cluster
83
SP0669 thyA 0.4 (9.0e-9) NDE thymidylate synthase
SP0670 0.4 (1.4e-6) NDE hypothetical protein
SP0718 2.2 (5.6e-7) 2.1 (3.8e-7) thiamine-phosphate pyrophosphorylase
SP0727 copY 0.3 (3.3e-5) NDE CopY rergulator
SP0750 livH 0.4 (2.6e-9) 0.5 (3.4e-5) branched-chain amino acid ABC transporter, permease protein
SP0751 livM 0.6 (4.1e-7) 0.7 (1.9e-4) branched-chain amino acid ABC transporter, permease protein
SP0766 NDE 2.0 (2.4e-8) superoxide dismutase, manganese-dependent
SP0835 NDE 2.0 (2.2e-5) purine nucleoside phosphorylase
SP0887 2.0 (7.4e-4) NDE type I restriction-modification system, S subunit, putative
SP0907 7.5 (4.1e-4) NDE hypothetical protein
SP0912 0.2 (9.2e-10) 0.3 (2.0e-7) ABC transporter, permease protein
SP0913 0.2 (3.7e-7) 0.3 (2.6e-7) ABC transporter, ATP protein
SP0921 0.5 (1.3e-8) 0.4 (9.2e-10) agmatine deiminase
SP0943 NDE 0.5 (7.0e-8) tRNA (uracil-5)-methyltransferase Gid
SP0968 NDE 0.5 (2.9e-7) diacylglycerol kinase
SP1004 2.4 (6.6e-5) NDE conserved hypothetical protein
SP1012 3.6 (1.3e-11) 3.7 (3.2e-9) hypothetical protein
SP1027 2.0 (5.3e-10) 1.4 (3.9e-5) hypothetical protein
SP1041 2.2 (6.5e-4) NDE hypothetical protein
SP1045 NDE 2.2 (5.6e-4) hypothetical protein
SP1059 0.3 (2.8e-4) NDE hypothetical protein
SP1137 0.4 (3.1e-4) NDE GTP-binding protein, putative
SP1229 0.3 (3.5e-12) NDE hypothetical protein
SP1320 0.3 (8.1e-7) NDE v-type sodium ATP synthetase, subunit E
SP1325 0.3 (1.2e-5) 0.2 (7.4e-5) Gfo/Idh/MocA family oxidoreductase
SP1342 2.4 (7.3e-5) NDE drug efflux ABC transporter, ATP-binding/permease protein
SP1343 2.0 (3.6e-5) NDE prolyl oligopeptidase family protein
SP1416 queA 0.3 (5.1e-11) 0.3 (2.5e-12) S-adenosylmethionine:tRNA ribosyltransferase-isomerase
SP1442 8.9 (4.6e-4) NDE IS66 family Orf2
SP1453 0.4 (2.2e-6) NDE hypothetical protein
SP1460/
SPR1314 taaB 2.1 (1.3e-8) 2.3 (2.2e-8) amino acids ABC transporter,ATP
SP1461/
SPR1315 taaC 1.8 (7.4e-9) 2.0 (9.4e-8) amino acids ABC transporter,permease
SP1462 3.0 (3.4e-4) NDE hypothetical protein
SP1463 ogt 2.1 (2.7e-6) 2.1 (8.6e-5) methylated-DNA--protein-cysteine S-methyltransferase
SP1476 2.0 (8.0e-4) NDE hypothetical protein
SP1499/
SPR1352 bta 1.9 (1.1e-6) 2.1 (1.0e-6) bacteriocin transport accessory protein
SP1556 0.3 (1.1e-5) NDE hypothetical protein
SP1588 NDE 2.0 (3.1e-5) pyridine nucleotide-disulfide oxidoreductase
SP1589 murE NDE 0.5 (2.5e-6) UDP-N-acetylmuramyl tripeptide synthase
SP1704/
SPR1546 prcD 0.1 (4.9e-13) 0.1 (7.3e-14) ABC transporter, ATP-binding protein;
SP1705/
SPR1547 prcC 0.0 (1.0e-13) 0.0 (1.1e-14) hypothetical protein
SP1706/
SPR1548 prcB 0.0 (1.6e-14) 0.0 (7.7e-16) hypothetical protein
NP/SPR1549 prcA 0.1 (2.0e-5) 0.1 (3.2e-5) hypothetical protein
NP/SPR1550 prcR 3.2 (2.7e-3) NDE transcriptional activator, Rgg/GadR/MutR family protein
SP1714 NDE 0.4 (1.5e-8) transcriptional regulator, GntR family
SP1715 NDE 0.5 (2.0e-7) ABC transporter, ATP-binding protein
SP1754 0.4 (5.4e-7) 0.4 (8.8e-7) hypothetical protein
SP1758 0.2 (7.2e-5) NDE glycosyl transferase, group 1
SP1764 wcaA 0.3 (1.3e-5) NDE glycosyl transferase family protein
SP1786 2.1 (1.5e-8) NDE hypothetical protein
SP1856 3.9 (1.1e-4) NDE MerR family transcriptional regulator
SP1857 4.2 (6.2e-4) NDE cation efflux system protein
SP1870 1.6 (4.1e-4) 2.5 (1.9e-6) iron-compound;ABC transporter
SP1871 1.9 (6.0e-5) 2.9 (4.7e-7) iron-compound;ABC transporter
SP1872 2.1 (8.0e-8) 2.5 (4.1e-8) iron-compound;ABC transporter
SP1919 0.3 (6.8e-7) 0.3 (1.5e-4) ABC transporter, permease protein
SP1924 0.5 (1.4e-8) 0.6 (8.5e-8) hypothetical protein
SP1925 0.4 (5.5e-10) 0.5 (6.6e-9) hypothetical protein
SP1926 0.4 (1.2e-10) 0.5 (1.5e-9) hypothetical protein
SP2032 0.2 (8.3e-5) NDE BglG family transcriptional regulator
SP2087 ulaA 0.2 (5.6e-7) NDE ascorbate-specific PTS system enzyme II
SP2115 0.4 (2.3e-6) NDE hypothetical protein
SP2132 0.4 (3.7e-9) NDE hypothetical protein
Chapter 3
84
SP2160 0.4 (2.3e-5) NDE hypothetical protein
SP2171 adcC 0.5 (1.7e-5) NDE Zinc ABC transporter, ATP-binding protein
SP2217 mreD 2.4 (1.4e-6) NDE putative rod shape-determining protein
This showed that in the ΔppuA strain the entire putative ppu cluster, except for ppuR, was
about 10-fold downregulated possibly due to a polar effect of the ppuA mutation (Table 4).
The data suggests that PpuA does not have an influence on ppuR expression, which was
confirmed by the finding that expression of the PppuR in the ΔppuA strain did not change
significantly when compared to the wild type (Table 3).
Comparison of the transcriptome profile of the ΔppuR to that of the ΔppuA strain
(Table 4) showed that in both mutants, genes encoding proteins involved in ribonucleotide
biosynthesis, cellobiose metabolism, amino acids transporters, iron ABC transporters and
many encoding hypothetical proteins had changed expression. Interestingly, in the ΔppuA
strain two genes (SP0138 and SP0139) upstream of ppuR were induced but not in the
ΔppuR mutant, suggesting that PpuA might influence their expression.
Genes SPR1546-1549 were downregulated nearly 10-fold in both mutants, i.e.
ΔppuA and ΔppuR. The SPR1547-1549 genes encode hypothetical proteins. Gene SPR1546
encodes an ATP-binding protein of an ABC transporter that belongs to a family of ATP-
binding proteins of multisubunit transporters involved in drug resistance (BcrA and DrrA),
nodulation, lipid transport, and lantibiotic immunity. In silico analysis of the genomic
region of SPR1546-1549 showed that these genes might form one transcriptional unit.
Interestingly, spr1550, which is adjacent to the SPR1546-1549 genes, was induced 3-fold
only in the ΔppuR mutant. The SPR1550 gene encodes a putative positive regulator of
Rgg/GadR/MutR family proteins and in silico analysis of the genomic region adjacent to
SPR1546-1550 showed that these genes might form a putative operon. Therefore, the
SPR1546-1550 cluster was selected for further study and we propose the name prc for the
cluster, which stands for peptide responsive cluster (prc) and the proteins encoded
prcRABCD (prcR for SPR1550 and prcABCD for SPR1546-1549). Noteworthy, analysis of
the genomic region of prcRABCD in R6, D39 and TIGR4 showed that TIGR4 lacks prcR
and prcA (SPR1550 and SPR1549, respectively; data not shown).
The SPR1314-1315 genes, induced approximately 2-fold in both ΔppuA and
ΔppuR, encode putative amino acids ABC transporter, thus the name of transporter of
amino acids, taa, was proposed (taaBC for SPR1314-1315). To investigate whether taaBC
responds to ΔppuA and ΔppuR mutation and/or amino acids in the medium we chose it for
further study. In addition, SPR1352 encoding a putative bacteriocin transport accessory
protein was approximately 2-fold upregulated in both mutants, namely ΔppuA and ΔppuR,
and was chosen for further analysis.
Characterization of the ppu cluster
85
Validation of the microarray results confirmed that PpuR is most likely a positive
regulator of ppuA
To confirm the observed differential expression patterns of some ΔppuR and/or
ΔppuA targets, namely taaBC, SPR1352, prcR and prcABCD, and ppuA, chromosomal
transcriptional fusions of lacZ with their putative promoters were generated. Expression of
PtaaBC and PSPR1352 did not change in either the ΔppuR or the ΔppuA mutant (Table 5),
in contrast to the transcriptome profiling where 2-fold induction was observed in both
mutants. The activity of PprcR increased about 4-fold in both mutants, which corresponds
to the transcriptome data of ΔppuR, whereas this ORF in the transcriptome analysis of the
ΔppuA strain was not significantly differentially expressed (Table 5). Despite the
transcriptome results, which showed a 9-fold reduction of prcA expression, the
transcriptional lacZ fusion data showed that PprcA decreased roughly 2-fold in ΔppuR.
Table 5. β-galactosidase activity of, transcriptionally fused to lacZ, promoter of taaBC, SPR1352, prcA or prcR
in the wild-type D39 (wt), ΔppuR and ΔppuA strain grown in CDM. Miller Units are the averages of at least three
independent experiments and the standard deviations are shown in brackets
Promoter of β-galactosidase activity (Miller Units)
wt ΔppuR ΔppuA
taaBC 228 (24) 182 (12) 191 (13)
SPR1352 199 (15) 243 (21) 256 (34)
prcA 11700 (2470) 5608 (1440) 192 (17)
prcR 29 (2) 173 (21) 124 (8)
However, in the ΔppuA strain the activity of PprcA was reduced nearly 50-fold, which is in
agreement with the transcriptome data (Table 5) and might suggest a role of PpuA in gene
regulation. In accordance with the transcriptome analysis, PppuA transcription was reduced
approximately 45-fold in the ppuR mutant (Table 3). However, expression of PppuR was
not affected in the ΔppuR strain (Table 3). Thus, PpuR is likely an essential positive
regulator of ppuA but not of its own expression. What is more, ppuABCDE or PpuR likely
has a regulatory influence on the prcRABCD cluster. These results demonstrate that the
activity of PppuA and PprcA in general corresponds well with the transcriptome analysis in
contrast to the PtaaBC and PSPR1352 expression.
Expression of the ppuRABCDE putative regulon, i.e. prc and taa, depends on the
presence of nitrogen compounds and/or possibly on the ppu gene products
The transcriptome data showed that the mutation of both ppuR and ppuA
influenced the expression of the prcA and prcR genes (Table 4). However, because in the
transcriptional data the effect of the ppuA mutation on PprcA was stronger than that of
ppuR (Table 5), we decided to investigate whether this regulation is mediated by the PpuA
peptide. Thus, the effect of addition of the synthesized PpuA peptides, namely PpuA_1 and
PpuA_2, on the PprcA and PprcR activity was measured in the wild-type D39, ΔppuR and
Chapter 3
86
ΔppuA strains grown in CDM (Table 6). Because the effect of both peptides was highly
similar, only the results of PpuA_1 are shown (Table 6). To determine whether the effects
were specific for PpuA_1, this experiment was also performed in CDM with the addition of
casitone (Table 6). Expression of PprcA and PprcR changed significantly in each tested
strain upon addition of both PpuA_1 and casitone. In all three strains, expression of PprcA
was notably higher in CDM than in CDM supplemented with either PpuA_1 or casitone
and the effect was most pronounced in the wild type (Table 6). The data suggest firstly that
peptides (or di-, tri-peptides, or free amino acids) influence expression of PprcA and
secondly that either PpuA or product(s) of the ppu locus might stimulate prcA expression,
and thirdly that prcABCD might belong to the ppu regulon. Expression of PprcR in the
wild-type strain did not change after addition of PpuA_1 or casitone but increased in both
mutants, i.e. ΔppuR and ΔppuA with or without PpuA_1 or casitone. This suggests that
activity of this promoter is not dependent on a peptide source but rather on the proteins
encoded by the ppu gene(s).
Table 6. β-galactosidase activity of, transcriptionally fused to lacZ, promoter of prcA, prcR, taaBC or SPR1352 in
wild-type D39 (wt), ΔppuR and ΔppuA strain grown in CDM and in CDM supplemented with 10 µg/ml of either
PpuA_1 (PpuA_1) or casitone (casitone). Miller Units are the averages of at least three independent experiments
and the standard deviations are shown in brackets
Promoter of Medium β-galactosidase activity (Miller Units)
wt ΔppuR ΔppuA
prcA
CDM
PpuA_1 casitone
11700 (2470)
4008 (750) 764 (11)
5608 (1440)
2553 (150) 455 (30)
192 (20)
303 (34) 46 (3)
prcR
CDM
PpuA_1 casitone
29 (2)
43 (5) 31 (2)
173 (11)
110 (15) 123 (9)
124 (8)
104 (17) 65 (2)
taaBC
CDM PpuA_1
casitone
228 (14) 530 (24)
508 (30)
182 (12) 170 (18)
73 (5)
191 (11) 108 (5)
55 (4)
SPR1352
CDM
PpuA_1
casitone
199 (17)
198 (13)
134 (5)
243 (33)
229 (12)
105 (8)
256 (24)
184 (9)
143 (10)
Given the observed effect of peptides (or di-, tri-peptides, or free amino acids) on
the PprcA promoter, we decided to examine their effect on PtaaBC and PSPR1352
expression in the same growth conditions. In the wild-type, the expression of PtaaBC
increased when either PpuA_1 or casitone was added (Table 6). Interestingly, although
PpuR and PpuA do not seem to be involved in PtaaBC expression in CDM, induction of
expression in response to either PpuA_1 or casitone did depend on these two proteins
(Table 6). The activity of PSPR1352 did not change significantly in either tested conditions
(Table 6). This demonstrates that the taaBC genes might be involved in amino acid
Characterization of the ppu cluster
87
transport and that there is a functional and or a regulatory link between the taaBC and the
ppuRABCDE genes.
Prediction of putative PpuR and CodY operators in S. pneumoniae
Since CodY is a negative regulator of ppuR and possibly of ppuA, and PpuR is an
essential positive regulator of ppuA, this suggests that CodY binding box(es) may be
present in the promoter region of at least one of these two genes. In conjunction, there is
likely a putative operator site for PpuR, which has not yet been identified, in the promoter
of ppuA. A previous study on CodY in S. pneumoniae showed that except for ppuR and
ppuA the members of the CodY regulon contain a sequence resembling the consensus
binding motif of CodY in L. lactis (AATTTTGWCAAAATT, CodY binding consensus
motif) (108,199). Analysis of the ppuR and ppuA promoter region, with the Sampler Motif,
Gibbs Motif, and Clone Manager and by eye, indicated putative CodY-boxes in both
regions (marked as a dashed line in Fig. 3) but no putative operator site for PpuR (Fig. 3).
Figure 3. Nucleotide sequences of putative promoter regions of (A) ppuR and (B) ppuA in S. pneumoniae D39.
Numbers indicate the base positions relative to the translational start. Predicted -35 and -10 boxes are shown as
bolded. Predicted CodY binding motifs are underlined with dashed line. In the ppuR and the ppuA promoter
sequence, underlined bases indicate inverted repeat/palindromic sequence. Designed truncated promoter fragments
of ppuR and ppuA, which were constructed in order to find putative operator sites for PpuR and CodY, are
indicated for PppuR_1 and PppuA_1 in grey shade and for PppuR_2 and PppuA_2 indicated in italic.
Chapter 3
88
In order to establish the location of the putative CodY/PpuR operator(s) in the
promoter regions of ppuA and ppuR, each of them was truncated from the 5‘ end into two
shorter fragments, PppuA_1 and PppuA_2 for PppuA and for PppuR, PppuR_1 and
PppuR_2, and fused to lacZ reporter gene using the pPP2 vector (Fig. 3 A and B).
Subsequently, expression of the truncated promoter fragments was measured in various
media and genetic backgrounds (Table 3). Activity of PppuA_1 was abolished in all
conditions and strains tested, indicating that the PpuR putative operator is not present in this
promoter fragment as expression of ppuA is strictly dependent on this regulator (Table 3).
In contrast PppuA_2 expression was similar to that of the wild type PppuA, in all conditions
and strains tested, strongly suggesting that the PpuR putative operator is located in this
fragment (Table 3). As all expression of PppuA_1 is abolished it was hard to specify
whether a putative CodY operator is present in this promoter. The promoter expression
studies of PppuR_1 and PppuR_2 showed that the CodY putative box is most likely located
in the PppuR_1 promoter fragment since the CodY repression effect was visible in both
truncated promoter fragments and as well in the wild type one (Table 3).
Discussion
The aim of this study was to determine the function(s) and regulation of the S.
pneumoniae putative pneumococcal peptide of unknown function cluster (ppuRABCDE)
that has been suggested to be important in invasive disease (38,191,319,390). Interestingly,
the ppuRABCDE genes were found to be highly induced in blood, indicating their
contribution to survival in this environment (390). However, growth in blood of the ppuA
mutant tested in vitro was similar to that of the wild type (390). Genes ppuR, ppuD and
ppuE were found in an STM study as important for lung infection (191). Thus, although the
functional role of the operon is still unclear it is likely that it plays an important role during
pathogenesis.
Since we have shown that CodY, a branched-chain amino acid responsive
regulator, is a negative regulator of the ppuRABCDE cluster (Table 3), we hypothesize that
ppuRABCDE is likely to be involved in nitrogen metabolism in S. pneumoniae. CodY is a
global regulator that adjusts bacterial cell metabolism to the environmental changes in
nutrient supply and additionally influences expression of genes involved in virulence
(199,417,484). Generally, CodY is activated by branched-chain amino acids (BCAAs) and
in Bacillus subtilis also by binding GTP (107,125,411,432,469). CodY regulates expression
of a broad range of genes, which in B. subtilis are involved in transport and metabolism of
nutrients, sporulation, motility and competence development (354). In L. lactis, CodY
represses peptidases, peptides and amino acids uptake systems, and aminotransferases,
during growth in complex media (62,164,165). In Streptococcus pyogenes and
Staphylococcus aureus CodY influences, besides genes involved in amino acid transport
and metabolism, the expression of genes encoding proteins involved in virulence and
Characterization of the ppu cluster
89
virulence regulation (312,417). In S. pneumoniae CodY contributes to colonization of the
nasopharynx (199). The putative regulon of CodY in this bacterium includes genes
encoding proteins involved in amino acid uptake, metabolism and biosynthesis, and the
ppuRABCDE cluster (199). Interestingly, the regulon of CodY in L. lactis includes genes
probably involved in a production of a putative bacteriocin or cell communication peptide
(166).
Based on our study we propose the following putative regulation mechanism of
the ppu cluster (Fig. 4). Expression of the ppuABCDE operon is dependent on positive
regulation by PpuR, which is repressed by CodY; this regulator might also repress
ppuABCDE. Interestingly, ppuR was constitutively upregulated in a multidrug resistant S.
pneumoniae M22 strain (321), indicating possible involvement of PpuR in resistance
mechanisms in this organism. Notably, the ppuRABCDE cluster was up-regulated in D39
ΔglnAP grown in GM17 (201). Induction of the cluster is surprising since: i) the mutant
grew in a rich medium, i.e. GM17, ii) CodY was likely expressed in this condition and iii)
this cluster is repressed in the wild type (Table 3). However, it might indicate that
glutamine/glutamate deficiency influences regulation of the CodY regulon or of the cluster.
Therefore, it would be interesting to verify whether these amino acids affect ppuRABCDE
expression. Similarly, downregulation of the ppuABCDE genes in the psaR mutant in D39
strain grown in CDM was unexpected (200), since we showed that the ppuRABCDE cluster
is expressed in CDM. However, additional regulators such as PsaR might be involved in
maintaining ppuRABCDE expression.
Figure 4. Schematic prediction of regulation of the ppuRABCDE, taaBC and prcRABCD cluster expression. Thick
white arrows indicate genes of the clusters. Black thin arrows indicate putative promoters of the clusters. The
regulators of the ppuRABCDE cluster, i.e. PpuR and CodY, are marked in two white circles. PpuR activates ppuA
expression (open arrow). CodY represses expression of the ppuR and possibly (indicated with a question mark) of
the ppuA gene (perpendicular). A functional or a regulatory link (indicated with a question mark) between the
ppuRABCDE, taaBC and prcRABCD clusters are marked with either open arrows or perpendicular.
Chapter 3
90
In silico analysis of the ppuA and ppuR putative promoter region identified
putative CodY motifs in the PppuR_1 fragment (Fig. 3). Whether the box is functional
needs to be confirmed and is the subject of ongoing experiments. As with other confirmed
CodY boxes, the putative binding sites are present in close vicinity of the -35 box of both
promoters (166). However, a previous study in B. subtilis demonstrated that even up to five
mismatches within the CodY consensus box could result in a functional element indicating
that we might have overlooked other authentic CodY boxes (26). It is uncertain whether
PppuA harbors a CodY box and whether CodY influences ppuA expression directly or only
through regulation of PpuR since expression of PppuA_1 was abolished in all mutants
probably because this promoter fragment lacks the PpuR operator site (Fig. 3, Table 3).
Transcription of PppuA_2 in ΔppuR was approximately 2-fold higher, in all tested media,
than that of the intact promoter, which is confusing since the positive regulator, i.e. PpuR,
was absent in this condition. Thus, more experiments are needed to determine whether
CodY directly influences expression of PppuA and this is a subject of ongoing research. A
putative operator region(s) of PpuR has not yet been found in PppuA. Hence, to prove a
direct regulatory effect of PpuR on the ppuA promoter region, direct binding of PpuR
protein to this promoter needs to be performed and it is also the subject of ongoing
experiments.
Transcriptome analysis of the ΔppuA and ΔppuR strains was performed in order to
determine the potential function(s) of PpuA, as well as that of the ppu cluster. The response
of the ΔppuR and ΔppuA mutant was comparable suggesting that they are in the same
pathway (Table 4). In both mutants there were many differentially expressed genes of
diverse or unknown function, which made it impossible to pinpoint a putative function for
ppuRABCDE. The expression of two putative, not yet studied clusters, namely prcABCD
(SPR1546-1549) and taaBC (SPR1314-1315), decreased and increased, respectively, in
both, ppuA and ppuR, mutants. Notably, gene prcB was found in an STM screen as
important for lung infection (191) and prcB and prcC belong to one of the accessory
regions (AR) in S. pneumoniae (38). The ARs are regions of diversity between S.
pneumoniae strains and they can have an effect on the ability to colonize and to cause
invasive diseases by this bacterium (38). Interestingly, prcABCD were among the few
genes induced in a spxR mutant (431). SpxR is a positive regulator of spxB and strH,
encoding a pyruvate oxidase and a glycoprotein exoglycosidasae, respectively. It is not
known, what stimulates the regulatory function of SpxR, but it was hypothesized that
perhaps SpxR senses the metabolic state of the cell (431). Notably, SpxR is required for
virulence in a murine model of infection diseases (431). Transcriptional studies with the
two prc promoter regions, i.e. PprcR and PprcA, in ΔppuR and ΔppuA demonstrated an
influence of the ppuRABCDE cluster on their expression (Table 5). Consequently,
transcription of PprcA and likely that of PprcR might be mediated by Ppu product(s), thus
ppuRABCDE and prcRABCD might belong to the same regulon. Notably, activity of PprcA
decreased upon supplementation of the growth medium with amino acids and peptides
Characterization of the ppu cluster
91
(Table 6), which might indicate involvement of the prcRABCD cluster in controlling
nitrogen metabolism in S. pneumoniae, as is suggested for the ppu cluster. Similarly,
because the PtaaBC activity changed after addition of casitone and/or the PpuA_1 peptide,
we propose that the taaBC genes encoding putative amino acid transporters are also
involved in nitrogen metabolism. What is more, expression of PtaaBC indicated a
functional or a regulatory link between the taaBC and ppu genes, since the activity of this
promoter decreased in both the ppuA and the ppuR mutant. Expression of the prc and the
taa cluster have not been changed in the transcriptome either of the codY mutant (199) or of
the ΔglnAP mutant, in which expression of the whole CodY regulon was altered (201),
indicating that probably they are not directly regulated by CodY and thus they do not
belong to the CodY regulon.
All together, we showed that PpuR is most likely a positive, essential regulator of
ppuABCDE and that CodY is a negative regulator of the ppuRABCDE cluster (Fig. 4).
Additionally we demonstrated that ppuRABCDE, prcRABCD and taaBC are all possibly
involved in controlling nitrogen metabolism in S. pneumoniae, which has to be confirmed
by further research. Most importantly, these three novel clusters might be linked to each
other on a regulatory and/or functionally level and eventually form a regulatory unit.
Acknowledgements
We thank Tomas G. Kloosterman for providing the S. pneumoniae D39 ppuR
mutant and for assistance with the data analyses. We thank Rachel Hamer for her technical
help in construction of the PppuA mutant.
Chapter 3
92
Chapter 4
Production of a class IC two-component lantibiotic of
Streptococcus pneumoniae using the class IA nisin
synthetic machinery and leader sequence
Joanna A. Majchrzykiewicz, Jacek Lubelski, Gert N. Moll, Anneke Kuipers,
Jetta J. E. Bijlsma, Rick Rink and Oscar P. Kuipers
Based on: Antimicrob. Agents Chemother. (2010) 54 (4): 1498–1505
Chapter 4
94
Chimeric lantibiotics
95
Recent studies showed that the nisin modification machinery can successfully
dehydrate serines and threonines and introduce lanthionine rings in small peptides
that are fused to the nisin leader sequence. This opens up exciting possibilities to
produce and engineer larger antimicrobial peptides in vivo. Here we demonstrate the
exploitation of the class IA nisin production machinery to generate, modify, and
secrete biologically active, previously not-yet-isolated and -characterized class IC two-
component lantibiotics that have no sequence homology to nisin. The nisin synthesis
machinery, composed of the modification enzymes NisB and NisC and the transporter
NisT, was used to modify and secrete a putative two-component lantibiotic of
Streptococcus pneumoniae. This was achieved by genetically fusing the propeptide-
encoding sequences of the SPR1765 (pneA1) and SPR1766 (pneA2) genes to the nisin
leader-encoding sequence. The chimeric prepeptides were secreted out of Lactococcus
lactis, purified by cation exchange fast protein liquid chromatography, and further
characterized. Mass spectrometry analyses demonstrated the presence and partial
localization of multiple dehydrated serines and/or threonines and
(methyl)lanthionines in both peptides. Moreover, after cleavage of the leader peptide
from the prepeptides, both modified propeptides displayed antimicrobial activity
against Micrococcus flavus. These results demonstrate that the nisin synthetase
machinery can be successfully used to modify and produce otherwise difficult to
obtain antimicrobially active lantibiotics.
Introduction
Small antimicrobial peptides produced by Gram-positive bacteria are named
bacteriocins. One group of bacteriocins, the non-lantibiotics, comprises peptides that do not
require modification for their antimicrobial activity (529). Members of another group, the
lantibiotics, require post-translational modifications to acquire biological activity (163,553).
Lantibiotics are produced as inactive prepeptides, consisting of an N-terminal leader
peptide and a C-terminal propeptide part. Most of the serine and threonine residues of the
propeptide are dehydrated to dehydroalanine (Dha) and dehydrobutyrine (Dhb),
respectively, by LanB- or LanM-type enzymes (―Lan‖ is a general abbreviation for proteins
involved in lantibiotics biosynthesis). LanC or LanM enzymes can subsequently couple
these dehydroresidues to cysteines, thus forming a (methyl)-lanthionine ring. After the
leader peptide is removed from the prepeptide by the extracellular LanP or transmembrane
LanT proteins, the active lantibiotic is released. The immunity against the produced
lantibiotics is provided by the LanI and/or LanFEG proteins (67,285,286). Based on the
structure, lantibiotics are divided into three types, i.e. type A (elongated peptides), type B
(globular peptides) and type C (multi-component peptides) (203,553). Type A lantibiotics
are modified by two enzymes, LanB and LanC. In type B lantibiotics, dehydratation and
cyclization are performed by a single enzyme called LanM. The C-terminal sequences of
Chapter 4
96
LanM type enzymes share homology with the LanC proteins. LanM enzymes share no
homology with the LanB proteins (339,476). Type C includes two-component lantibiotics,
of which antimicrobial activity mainly depends on synergistic action of both peptides
(341,342). Each of the peptides of the two-component lantibiotics, except cytolysin,
possesses its own dedicated modification LanM enzyme (553,554).
Due to an increasing resistance of bacteria to available antibiotics, there is an
urgent need to search for substances active against multidrug resistant pathogens. Since
some lantibiotics exhibit a stable activity at nanomolar concentrations against antibiotic
resistant pathogens, it is currently of great interest to apply these lantibiotics (105,338,450).
It has already been shown in a mouse model that mersacidin is active against methicillin-
resistant Staphylococcus aureus strains (MRSA) (281). Another lantibiotic, lacticin 3147, is
a successful antimicrobial agent against MRSA, vancomycin-resistant Enterococcus
faecalis, penicillin-resistant Streptococcus pneumoniae, Propionibacterium acnes and
Streptococcus mutans (148).
One of the most studied lantibiotics is nisin (161,163,287,306), a type A lantibiotic
produced by certain Lactococcus lactis strains. It has a long record of safe industrial usage
as a food preservative (105). Due to the broad activity spectrum against Gram-positive
pathogens, including S. pneumoniae, nisin has good potential for a number of other
applications (156). Recently, it was shown that designed hexapeptides and non-lantibiotic
peptides fused to the leader peptide of nisin could be successfully modified by NisB and
NisC and exported out of L. lactis via NisT (265,283,438). The discovery that the
lantibiotic modification enzymes LanB, LanC and LanM possess rather low substrate
specificities brings a new opportunity to use them as a tool to improve stability and activity
of peptides potentially valuable for medical applications (66,265,439).
Here, we present successful application of the nisin expression/modification
system to produce, modify and secrete entirely unrelated putative lantibiotics that, based on
bioinformatic predictions, belong to the class IC lantibiotics. The produced peptides were
dehydrated multiple times, as shown by matrix-assisted laser desorption ionization-time of
flight (MALDI-TOF). Importantly, the modified peptides showed antimicrobial activity
against Micrococcus flavus. Our study demonstrates that the nisin production/modification
machinery can be used to produce and posttranslationally modify silent lantibiotics, i.e.
those for which production conditions are not known, and which otherwise would be
difficult to obtain from their natural sources.
Materials and Methods
Bacterial strains and growth conditions
Strains and plasmids used in this study are listed in Table 1. Strains were stored in 10% (vol/vol)
glycerol at -80 °C. S. pneumoniae, E. faecalis, S. aureus and Streptococcus mitis strains were grown
at 37°C in standing M17 (Difco) broth supplemented with 0.5% (wt/vol) glucose (GM17) and, when
Chimeric lantibiotics
97
appropriate, 2 μg/ml chloramphenicol. L. lactis and Micrococcus flavus were grown at 30°C in GM17
or minimal medium (439) supplemented with 5 μg/ml chloramphenicol and/or 5 μg/ml erythromycin
when appropriate.
Construction of chimeric peptides
Standard genetic manipulations were essentially performed as described by Sambrook et al. (457).
Plasmid pIL3BTC encoding the nisin modification machinery (439) and plasmid pNZnisA-E3 (282)
were used to produce and modify chimeric peptides. Briefly, two open reading frames, SPR1765 and
SPR1766 (pneA1 and pneA2, respectively), were amplified by PCR from genomic DNA of S.
pneumoniae R6 and cloned into pNG8048E, resulting in a plasmid, named pNGspr1765-1766, for
which L. lactis NZ9000 (439) was used as a host. All the subsequent genetic cloning procedures were
performed in this organism. This plasmid, pNGspr1765-1766, served as a template to amplify
separately the genes SPR1765 and spr1766. Subsequently, each of the amplified products of genes
SPR1765 and SPR1766 was subcloned to pNZnisA-E3 expression plasmid. This resulted into two
new plasmids, pNZE3-nis-spr1765 and pNZE3-nis-spr1766, which carried the nisin structural gene
and a lantibiotic gene next to one another.
Table 1. Strains and plasmids used in this study
Strain/plasmid Description Reference
or source
S. pneumoniae
R6 D39 (Δcps2 2538-9862) with increased transformation
efficiency
(219)
L. lactis
NZ9000 MG1363 pepN::nisRK (259)
S. aureus RN6390B Lab collection
S. mitis
NTCC10712 Lab collection
E. faecalis
V583
(455)
M. flavus
NIZO B423
NIZO a Food
Research
Plasmid
pIL3BTC nisBTC, encoding for nisin modification machinery; EryR (439)
pNZ8048 nisin-inducible PnisA; CmR (101)
pNG8048E nisin-inducible PnisA, pNZ8048 derivative containg EryR gene
to facilitate cloning; CmR EryR
Lab collection
pNZnisA-E3 nisA, encoding for nisin (282)
pNZE3-nis-spr1765 contains nisA gene and SPR1765 gene This work pNZE3-nis- spr1766 contains nisA gene and SPR1766 gene This work
pNZE3-spr1765 contains a part of nisA gene which encodes for leader peptide of
nisin and leaderless part of SPR1765 gene fused in frame
This work
pNZE3-spr1766 contains a part of nisA gene which encodes for leader peptide of
nisin and leaderless part of SPR1766 gene fused in frame
This work
pNGspr1765-1766 pNG8048E contains SPR1765 and SPR1766 gene under own
promoter
This work
To construct genetic fusions of the nisin leader sequence and the structural leaderless sequence of the
SPR1765 and SPR1766 (pneA1 and pneA2, respectively) genes in frame, the round PCR method with
Chapter 4
98
5' phosphorylated primers was used as described earlier (439) using Phusion DNA polymerase
(Finnzymes). The final chimeric peptide expression plasmids pNZE3-spr1765 and pNZE3-spr1766
were thus constructed. These plasmids were used separately in combination with a plasmid pIL3BTC,
to produce and secrete modified chimeric peptides. Plasmid isolation was performed by means of the
Plasmid DNA Isolation Kit (Roche Applied Science). Restriction analysis was performed with
restriction enzymes from Fermentas. DNA ligation was performed with T4 DNA ligase (Fermentas).
Peptides
Peptides encoding the sequence of leaderless PneA1 and PneA2 were purchased from Pepscan
Lelystad NL. Peptides were purified to homogeneity by high-pressure liquid chromatography (HPLC)
on a Jupiter Proteo C12 (4 μm, 90- Å, 250- by4.6-mm) column with an acetonitrile gradient.
Expression and purification of microbially produced peptides were performed as follows. Overnight
cultures of L. lactis NZ9000 containing pIL3BTC and a chimeric peptide expression plasmid, namely
pNZnisA-E3 or pNZE3-spr1765, or pNZE3-spr1766, in GM17 were diluted 1:50 in minimal medium
containing appropriate antibiotics and for induction 0.5 ng/ml nisin (Sigma). Cultures were grown for
24h at 30°C. Subsequently, supernatants were separated from cells by centrifugation. Next,
supernatants were filtered through a 0.2 μm filter (Millipore). Prior to purification on a 5-ml HiTrap
SP cation exchange column (GE Healthcare) using fast protein liquid chromatography (FPLC; on an
Akta purifier; Amersham Bioscience), supernatants were diluted 1:1 with a 100 mM lactic acid
solution and filtered through 0.2 μm filters. After passage of supernatant through a column, unbound
compounds were washed away with 100 mM lactic acid. The fractions containing prepeptides were
concentrated and desalted with 50 mM Tris-HCl of pH 5.5 on Microcon columns (Milipore). Intact
prepeptides and peptides without leader sequence were analyzed with MALDI-TOF mass
spectrometry and used for screening of antimicrobial activity.
N-terminal sequence removal
The N-terminal sequence from the FPLC-purified prepeptides prePneA1 and prePneA2 was removed
by trypsin. Prepeptides were incubated for 2h at 37°C with 20 μg/ml trypsin in 100 mM Tris-HCl
buffer (pH 8) containing 10 mM CaCl2. Alternatively to remove the leader from prePneA2, 180 µl of
the prepeptide was incubated for 30 min at 37°C with 20 µl of 0.5 M phosphate buffer (pH 7.4) and
with 10 µl of leucine aminopeptidase (Sigma; suspension in 3.5 M ammonium sulfate).
Mass spectrometry analysis
To investigate whether chimeric peptides possess free cysteine residues, reactions with 1-cyano-4-
dimethylaminopyridinium tetrafluoroborate (CDAP) were performed. To obtain higher mass spectra
resolutions with MALDI-TOF, both prepeptides and propeptides, prior to CDAP treatment, were
purified on a Hewlett Packard 1050 HPLC apparatus using a Jupiter Proteo C12 (4-µm, 90-Å, 250-by
4.6-mm) column. Reverse-phase purification was used with a gradient of 10% - 40% acetonitril in
purified water. All buffers contained 0.1% TFA (trifluoroacetic acid). The reactions with CDAP were
performed as described before (265,347). Briefly, the pH of vacuum-dried trypsinated, non-
trypsinated peptides and a control peptide, termed NisB2 (H-CRYTDPKPHIRLRIK-OH)
resuspended in 16 μl MilliQ was adjusted to 2 or 3 with 0.1% TFA. Prior to treatment with CDAP, 1
µl of 100 mg/ml of the reducing agent, triscarboxyethyl phosphine (TCEP), was added to each
mixture and a reaction was carried out for 5 min at room temperature. Subsequently, 2 µl of 100
mg/ml CDAP was added to the mixtures, followed by incubation for 15 min at room temperature.
Analysis was essentially performed as described before (282). Briefly, ZipTips (C18 ZipTip;
Millipore) were wetted with 100% acetonitrile and washed with 0.1% TFA. Subsequently, the
supernatant containing the peptides was mixed with 0.1% TFA and applied to a ZipTip. Peptides that
Chimeric lantibiotics
99
were bound to the column were washed with 0.2% TFA and eluted with 50% acetonitrile and 0.1%
TFA. The eluent was mixed in a ratio of 1:1 with 10 mg/ml α-cyano-4-hydroxycinnamic acid
(matrix). A total of 1.5 μl of such prepared mixture was spotted on the target and allowed to dry.
Mass spectra were recorded with a Voyager-DE Pro (Applied Biosystems) MALDI-TOF. In order to
increase the sensitivity external calibration was applied with six different peptides (Protein MALDI-
MS calibration kit; Sigma).
Amino acid sequence alignment
Amino acid sequence alignment of nisin with pneumococcin A1 and A2 (SPR1765 and SPR1766,
respectively) was performed with Clustal W (302), a program for multiple sequence alignments. The
peptide sequences were derived from the NCBI database.
Gel Electrophoresis
Prepeptides and mature peptides were analyzed on Tris-tricine gels (461) and stained with Coomassie
(Fermentas).
Peptide concentration determination
Peptide concentrations were determined using the DC protein assay of Bio-Rad. HPLC-purified nisin
was used as standard.
MIC determination
MIC assays for M. flavus, S. pneumoniae, E. faecalis, S. aureus and S. mitis were performed in 96-
well microtiter plates in GM17. The assay was performed as follows. Overnight cultures of the above-
mentioned strains were diluted 1:50, and growth was continued to an optical density at 600 nm
(OD600) of 0.2. Subsequently, 150 μl cultures were mixed with 50 μl of appropriate medium and
various concentrations of peptides. The microtiter plates were incubated in a GENios (TECAN
Benelux) at a suitable temperature for overnight growth of the strain and the OD600 was measured
every 30 min. The MIC values were determined at the time when the cells without antimicrobial
substance reached half of the maximal optical density. MICs were calculated from the lowest
concentration of the antimicrobial substance that was able to inhibit the growth of the tested strain.
All the susceptibility assays were performed in triplicate at least.
Results
In silico analysis of the putative two-peptide lantibiotic-like cluster from S.
pneumoniae
After in silico analysis of 11 putative bacteriocin genes, identified by BAGEL
(99), of S. pneumoniae R6 and their adjacent ORFs, we selected two of them, i.e. SPR1765
and SPR1766. In silico analysis of these two genes, as well as their nine adjacent ORFs,
which most likely constitute a single cluster (Fig. 1), indicated that SPR1765 and SPR1766
belong to the class IC of two-component like lantibiotics. We here propose to name
SPR1765 and SPR1766, pneumococcin A1 and A2 (PneA1 and PneA2), respectively. So
far there are no experimental data indicating that these bacteriocin-like peptides are
expressed under laboratory growth conditions or under any other growth media or
conditions we have tried (data not shown). In silico analysis showed that the nine adjacent
genes (Fig.1), are likely involved in pneumococcin A1 and A2 modification, transport,
processing and immunity. Gene SPR1767 encodes a protein with amino acid sequence
Chapter 4
100
similarity to a classic bifunctional LanM-like modification enzyme. The SPR1768 gene
encodes a putative flavin adenine dinucleotide (FAD)-dependent flavoprotein that could
catalyze the oxidative decarboxylation of C-terminal residues. The functions for SPR1764
and SPR1769, and SPR1774 are unknown. SPR1770 protein is a predicted ABC transporter
containing putative N-terminal double-glycine peptidase activity (peptidase of C39 family)
and is most likely responsible for transport of modified bacteriocins and for prepeptide
processing. The SPR1771 protein shares 48% identity with NisP, the nisin leader peptidase.
The last two genes of the regulon, SPR1772 and SPR1773, encode a putative immunity
protein and a putative ABC transporter, respectively. This analysis clearly shows that
PneA1 and PneA2 very likely belong to the class IC two-component lantibiotics.
Figure 1. Putative pneumococcin A1 and A2 gene cluster. Organization of the chromosomally located
biosynthetic gene cluster of pneumococcin A1 and A2 genes (SPR1765 and SPR1766, respectively) in S.
pneumoniae. ORFs are represented by thick gray arrows, and their SPR gene identification numbers are shown in
the gray arrows. The putative promoters are represented by thin and bent black arrows.
Additionally, the gene cluster of pneumococcins contains the gene encoding a putative
flavoprotein (the SPR1768 gene product). The gene encoding this type of enzyme is also
found in the gene clusters of epidermin and mersacidin. The modifications made by the
flavoproteins are required for full activity of these peptides (296,311). Whereas NisT
displays rather broad transport specificity of peptides fused to the leader peptide of nisin,
NisP has been shown to only process fully modified prenisin (282). Figure 2 shows the
amino acid sequence alignment of pneumococcin A1 and A2 with the nisin structural
peptide. There is a low similarity between the peptides both in the part of the leader
sequence and in the part of the propeptide. On the basis of lantibiotic leader sequences (Fig.
2), the predicted pneumococcin A1 and A2 peptides possess three candidate cleavage sites,
one behind GlyGly/GlyAla (after which SPR1770 might cleave), one behind a shared
GlyAla sequence, and one behind ProArg. Strikingly, prenisin, prePneA1 and prePneA2
share the sequence GAxPRxT (the x being a variable residue), which comprises in the
middle the site behind ProArg. These data, together with the shared 48% identity of the
putative leader peptidase, SPR1771, with NisP, indicate that the Pne leader peptides end
with ProArg.
Chimeric lantibiotics
101
Figure 2. Amino acid sequence alignment of nisin with pneumococcins A1 and A2. The cleavage site in the
peptide sequence is highlighted in gray. Identical amino acids residues for all three sequences are indicated with an
asterisk and conserved residues are shown by a colon and semi-conserved amino acids by a point.
Production, secretion and purification of the chimeric peptides
To investigate the production and secretion of chimeric peptides, pNZE3-spr1765
and pNZE3-spr1766 were introduced into L. lactis NZ9000 containing pIL3BTC. Cultures
of L. lactis NZ9000 plasmid-containing derivatives induced with nisin were grown for 24 h
in minimal medium. Subsequently, the supernatants were collected and the prepeptides
were purified on a HiTrap SP cation exchange column. The same procedure was applied for
prenisin, a positive control. All prepeptides were produced and secreted as visualized by
Tris-tricine electrophoresis (Fig. 3). Prepeptide concentrations were determined with
HPLC-purified nisin of known concentration, as reference. Without nisin induction, no
secreted peptides were observed (data not shown). The production levels of both prePneA1
and prePneA2 were approximately 50% lower, than that of prenisin.
Figure 3. Tris-Tricine gel illustrating trypsin-treated and non trypsin-treated purified chimeric peptides and nisin.
Lane M, marker.; lane 1, prePneA1;
lane 1A, trypsin-treated PneA1; lane
2, prePneA2; lane 2A, trypsin-treated
PneA2; lane 3, prenisin; lane 3A,
trypsin-treated prenisin.
Streptococcal chimeric
peptides are modified by
nisin synthetase enzymes
To test whether the purified chimeras prePneA1 and prePneA2 were modified,
they were analyzed by MALDI-TOF spectrometry. Table 2 presents a summary of the
obtained masses. Interestingly, analysis of prePneA1 and prePneA2 showed that both
prepeptides were modified multi-fold. PrePneA1 showed 4- and 3-fold dehydration and
prePneA2 4-, 3- and 2-fold. Chimeric prepeptides were processed by trypsin or leucine
aminopeptidase and further characterized. Since the leader peptide keeps the prepeptide
inactive, its removal allows the assessment of the antimicrobial activity of the mature
peptides. Trypsin cleaves a peptide bond behind lysine or arginine, with arginine being
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102
preferred over lysine allowing arginine-specific cleavage under controlled conditions
(Table 2).
The prePneA1 was processed by trypsin, and only the nisin leader sequence was
clipped off, leaving the mature peptide with multiple dehydrations (Table 2). However,
prolonged digestion at higher concentration of trypsin resulted in two additional mass peaks
corresponding to two fragments (Table 2). The N-terminal part of PneA1 (Table 2) was
cleaved off and showed no dehydrations, likely due to protection against dehydratation of
Ser/Thr by their directly flanking residues (439).
Table 2. NisB-mediated dehydration of chimeric prepeptides and their fragments analyzed by MALDI-TOF mass
spectrometry. The average masses are shown
Peptide (fragment)
No. of
observed
dehydration
Mass (M + H+) without
Met1 (Da)
Observed Calculated
PrePneA1
(nisin leader
WTPTPIILKSAAASSKVCISAAVSGIGGLVSYNNDCLG)
4 3
6,009.6 6,026.5
6,009.9 6,027.9
PneA1
(WTPTPIILKSAAASSKVCISAAVSGIGGLVSYNNDCLG) 6 5
4
3
3,655.9 3,674.6
3,692.8
3,711.2
3,658.3 3,676.3
3,694.3
3,712.3
PneA1
(fragment 1WTPTPIILK)
0 1,068.9 1,069.3
PneA1
(fragment 2 SAAASSKVCISAAVSGIGGLVSYNNDCLG) 4 3
2
2,643.6 2,662.0
2,680.5
2,644.0 2,662.0
2,680.0
PrePneA2
(nisin leader STIICSATLSFIASYLGSAQTRCGKDNKKK) 4 3
2
5,437.6 5,454.0
5,472.2
5,437.2 5,455.3
5,473.3
PneA2
(STIICSATLSFIASYLGSAQTRCGKDNKKK) 6 5
4
3
3,086.7 3,103.9
3,121.5
3,139.6
3,085.6 3,103.7
3,121.7
3,139.7
prePneA2
(fragment 1 SKKDSGASPRSTIICSATLSF)
4
3
2,085.1
2,103.1
2,084.7
2,102.7
PneA2
(fragment 2 STIICSATLSFIASYLGSAQTR)
6 5
4
3 2
1
2,184.1 2,200.8
2218.9
2236.7 2254.9
2273.0
2,183.6 2,201.6
2219.6
2237.6 2255.6
2273.6
Chimeric lantibiotics
103
The identity of this N-terminal fragment was confirmed by sequence data obtained
by post source decay (data not shown). The other lysine residue in the mature peptide
(SSK) appears protected against proteolysis, likely due to posttranslational modifications in
its vicinity (305). The five dehydrations that were observed in the mature PneA1 are
therefore located in the C-terminal part of the peptide, in which six serines are present.
Since one of those serines is located next to the lysine that is cleaved by trypsin, it is likely
that the observed dehydrations are all within the last 25 amino acids of PneA1, except for
the first serine at SSK.
PneA2 contains a number of residues that are substrates for trypsin and some of
them are not protected by modified residues (Fig. 2). Therefore, we initially obtained a
smaller fragment (Table 2, PneA2 fragment 2), which lacked the C-terminal extension but
contained multiple dehydrated residues. Additionally in HPLC-purified chimeric prePneA2,
there was also a clear fraction containing a peptide fragment consisting of part of the nisin
leader sequence and the N-terminal part of the PneA2 peptide (Table 2, prePneA2 fragment
1). Interestingly, this peptide fragment contained up to four dehydrations, which clearly
shows that four out of five dehydrated residues are located in the first 11 amino acids of the
mature peptide.
Figure 4. Modified PneA1 contains thioether bridges. The SAAASSKVCISAAVSGIGGLVSYNNDCLG
fragment dehydrated fourfold, threefold and 2-fold (solid line) is treated with CDAP (dotted line) to detect the
presence of modifiable cysteines (yielding peptide plus 25 Da) and non-modifiable thioether-linkage-forming
cysteines. The 2,643.6-Da peak, which is dehydrated 4-fold shows a mild single CDAP addition. The 2,661.9-Da
(3-fold dehydrated) peak shows single and double CDAP additions, as indicated by black arrows. The 2,680.5-Da
(2-fold dehydrated) peak shows clear single and double CDAP additions; 1 CDAP, 2,705.3 Da (plus 25 Da) and 2
CDAP, 2,729.8 Da (plus 50 Da). The 5-fold dehydration peak is becoming visible after CDAP addition, indicating
that this peptide has two thioether rings.
Subsequently, removal of the N-terminal leader peptide from chimeric peptides by
NisP overexpressed in L. lactis was also investigated. However, neither the release of the
leader peptide nor antimicrobial activity of PneA1 or PneA2 was detected (data not shown).
Intact PneA2 was obtained using leucine aminopeptidase and appeared to be 3- to 6-fold
dehydrated (Table 2). Importantly, the activity of the leucine aminopeptidase apparently
Chapter 4
104
had stopped after the last Arg. This indicates the presence of a thioether bridge starting at
either Ser1 or Thr2. Studies by Rink et al. indicated that flanking hydrophilic residues on
both sides of a Ser or Thr would not favor dehydration. Furthermore, serines are less readily
dehydrated than threonines (439). Therefore, a large extent of dehydration of Ser1 flanked
by Arg and Thr is not likely. Taken together, this indicates that Thr2 is fully dehydrated and
thioether cross linked to Cys5.
To investigate whether thioether rings were formed, CDAP, which reacts only with
free cysteines, was used. The control NisB2 peptide that contains one free cysteine was
used as a positive control (data not shown) for CDAP modification. CDAP modification
converts a free thiol group of cysteine into an isothiocyanate, yielding a mass increase of 25
Da. The 5-fold dehydrated PneA1 showed hardly any CDAP modification, indicating that
there were no free cysteine residues and thus the formation of two thioether rings. The 3-
fold dehydrated PneA1 peptide showed single and double CDAP modifications, indicating
the formation of either one or no thioether rings (Fig. 4). Similar studies on CDAP
modification of prePneA2 indicated the presence of two thioether rings in the extensively
dehydrated peptides (data not shown). These data demonstrate that the putative lantibiotics,
which are entirely unrelated to nisin, can be successfully produced, modified, and secreted
by the nisin synthetase machinery.
The produced and modified peptides have significant antimicrobial activity
To investigate the antimicrobial activity of the modified peptides, the chimeric
prepeptides were incubated with trypsin or leucine aminopeptidase to remove the N-
terminal leader sequence. Various dilutions of trypsin-treated peptides, namely, prenisin
(positive control) and pneumococcin A1 and A2, were tested for antimicrobial activity in
the MIC assay (Table 3). Of all microorganisms tested, i.e. M. flavus, S. pneumoniae, E.
faecalis, S. aureus and S. mitis, only M. flavus was susceptible to the tested peptides, i.e.
PneA1 and PneA2 (Table 3). In control experiments, no significant inhibition was found
with either buffer or bovine serum albumin (BSA) treated with trypsin or with empty
samples, i.e. fractions from prepeptide purifications that did not contain peptides (Table 3).
Additionally undigested chimeric prepeptides did not show significant antimicrobial
activity against the indicator strain (Table 3). Unmodified PneA1 (MIC of ˃ 50 µM) and
PneA2 (MIC of 1.5 mM) propeptides obtained by chemical synthesis were at least 30-fold
and 170-fold less active, respectively, than the HPLC-purified active fraction of the
corresponding NisBC-modified peptides, without leader peptide. This proves that NisBC-
induced modifications are required for lantibiotic activity.
PneA1 inhibited growth of M. flavus at a peptide concentration of 0.6 µM. PneA2,
from which the leader sequence was removed either by trypsin or leucine aminopeptidease,
inhibited growth at approximately 10 or 8.5 µM, respectively (Table 3). The combination of
both modified chimeric peptides, PneA1 and PneA2, did not act synergistically (Table 3).
Chimeric lantibiotics
105
Thus, the data demonstrate that it is possible to utilize the nisin synthetase machinery for
the production of antimicrobially active peptides unrelated to nisin.
Table 3. Susceptibility of M. flavus to NisBC-modified Pne-A1 and Pne-A2 peptides. a NI, no inhibition; MICs are
calculated using the molecular weights of the mature peptides
Sample
MIC (µM) for indicated digestion type:
None Trypsin Leucine
aminopeptidase
Prenisin
Nisin leader peptide PneA1
Nisin leader peptide Pne-A2 Nisin leader peptide PneA1 + nisin leader peptide
PneA2
BSA Buffer
Empty sample
20
80
85 80
NIa
NIa
NIa
0.004
0.6
10 0.6
NIa
NIa
NIa
8.5
Discussion
To the best of our knowledge, we present here for the first time the successful
expression, modification, secretion and biological activity of novel class IC lantibiotics by
the nisin synthetases, which normally produce nisin, a class IA lantibiotic. To present a
significant challenge as substrate peptides, pneumococcin A1 and A2 from S. pneumoniae
R6, which presumably belong to type C two-component lantibiotics, were chosen as
substrates for the nisin enzymes. The class IC two-component lantibiotics require a LanM-
type enzyme that performs both dehydratation and cyclization, whereas class IA lantibiotics
require LanB dehydratases and LanC-cyclases. It has been already shown that LanBC-type
enzymes can modify peptides other than nisin which are fused to nisin leader sequence.
Kluskens et al. and Kuipers et al. demonstrated that both medically relevant nonlantibiotic
peptides and a truncated lantibiotic, lacticin 3147, fused with the nisin leader sequence,
modified by NisB and NisC, were exported via NisT and contained dehydrated amino acids
and lanthionine rings (265,283). The same was proven by Rink et al. for various
hexapeptides (438,439). Thus, based on the discovery that the nisin synthetase machinery
can accept various peptides as templates for modification, the propeptide part, which is the
predicted maturating part of either the PneA1 or the PneA2 peptide, was fused to the nisin
leader sequence and introduced in L. lactis that overexpresses NisBTC. The produced
peptides were multifold dehydrated and contained thioether rings. Some dehydrated
residues and one thioether ring could be localized by studying peptide fragments and by
applying leucine aminopeptidase.
To be biologically active, lantibiotics require prepeptide processing, i.e. removal
of the leader sequence. The prepeptide sequences and the homology of the peptidase with
Chapter 4
106
NisP indicated that the site behind PR might be the processing site from which the
propeptide starts. To liberate the mature peptides, we used trypsin or leucine
aminopeptidase. Exported, purified and processed peptides were tested for antimicrobial
activity and M. flavus was found to be highly susceptible to both the PneA1 and PneA2
peptides. Despite the fact that PneA1 and PneA2 are predicted two-component lantibiotics,
we did not observe any significant synergistic effect when these peptides were combined
(data not shown). The PneA1 and PneA2 peptides are a mixture of extensively modified
and hardly modified peptides. These mixtures probably contain active, less active and
inactive peptides. Thus, the determined antimicrobial activity is the mean of those of all
these peptides, indicating that the specific activity for a single active peptide might be
higher. With respect to this, a preliminary experiment was performed with processed and
NisBC-modified PneA1 and PneA2 peptides, which were dehydrated four- and fivefold,
separated and purified from the total mixture. However, the MICs of both peptides were not
significantly different from the MICs of the unpurified peptides (data not shown). A
challenge for future work might be to sort out the active peptide fraction from the inactive
fraction in order to get a better picture of which modifications yield antimicrobially active
peptides.
The putative cluster of pneumococcins consists of 11 ORFs. In the cluster two
genes might be required for proper modification of PneA1 and PneA2. These genes encode
a single putative LanM-type modification enzyme and a putative LanD-type flavoprotein.
Flavoproteins catalyze the oxidative decarboxylation of a C-terminal cysteine residue
involved in ring formation. A FAD-dependent flavoprotein catalyzes this reaction for
mersacidin, a lantibiotic produced by Bacillus sp. (311). Another flavoprotein, which is
flavin mononucleotide (FMN) dependent, catalyzes the same reaction for epidermin, a
lantibiotic of S. epidermidis, and this enzyme is essential for formation of a biologically
activity peptide (296). It is not known whether the putative LanD-type flavoprotein of
PneA1 and PneA2 performs a similar function in this cluster. Because the original cluster of
pneumococcins contains LanM and LanD-type modification enzymes, peptides modified by
NisB and NisC might not be fully active by lack of the oxidative decarboxylation.
Furthermore, we do not know whether the native dehydration and ring pattern is exactly
similar to the one installed heterologously by NisB and NisC. These factors might explain
the presumably suboptimal antimicrobial activities and lack of synergism within this
putative two-component lantibiotic system. However, both peptides, PneA1 and PneA2 still
showed significant antimicrobial activity.
The production of non-lantibiotic or lantibiotic chimeras with a heterologous
system has been reported using either closely related or non-lantibiotic peptides. For
example, production of chimeric nonlantibiotic bacteriocins pediocin PA-1, which is fused
to the leader of lactococcin A and/or to enterocin P, or enterocin A, which is fused to the
leader of enterococcin P, resulted in the secretion of active peptides (218,323,324). These
cases of successful production of biologically active bacteriocins concerns nonlantibiotic
Chimeric lantibiotics
107
bacteriocins, which in contrast to lantibiotics do not require posttranslational modifications
for antimicrobial activity. Production of class IA lantibiotic chimeras, such as nisin/subtilin
or subtilin/nisin, with either subtilin or nisin expression machineries, was performed
successfully (61,289). Of the amino acids residues of the leaders and mature peptides of
subtilin and nisin, 57% is identical (289). Studies using lacticin 481 synthetase
demonstrated its ability to prepare other lantibiotics in the class IB of lacticin 481 family,
including nukacin ISK-1, mutacin II, and ruminococcin A (400).
In contrast, we show here for the first time that it is possible to use the nisin
synthetase system to produce, modify and secrete lantibiotics, from a very different source
and class, which exhibit considerable antimicrobial activity.
Acknowledgments
We thank Patrick J. Bakkes, Hadi Eskandari and Agnieszka Moskal for their
technical help in conducting some experiments presented in this study. Jacek Lubelski was
supported by the Dutch Technology Foundation, STW project 06927.
Chapter 4
108
Chapter 5
Generic and specific adaptative response of
Streptococcus pneumoniae to challenge with three
distinct antimicrobial peptides: bacitracin, LL-37 and
nisin
Joanna A. Majchrzykiewicz, Oscar P.Kuipers and Jetta J.E. Bijlsma
Based on: Antimicrob. Agents Chemother. (2010) 54 (1): 440–451
Chapter 5
110
Responses of S. pneumoniae to AMPs
111
To investigate the response of Streptococcus pneumoniae to three distinct
antimicrobial peptides (AMPs), bacitracin, nisin and LL-37, transcriptome analysis of
challenged bacteria was performed. Only a limited number of genes were found to be
up- or down-regulated in all cases. Several of these common highly induced genes
were chosen for further analysis, i.e. SP0385-0387, SP0912-0913, SP0785-0787,
SP1714-1715 and the blp cluster. Deletion of these genes in combination with MIC
determinations showed that several putative transporters, i.e. SP0785-0787 and
SP0912-0913, were indeed involved in resistance to lincomycin, LL-37 and to
bacitracin, nisin, lincomycin, respectively. Mutation of the blp immunity genes
resulted in increased sensitivity to LL-37. Interestingly, a putative ABC transporter
(SP1715) protected against bacitracin and Hoechst 33342, but conferred sensitivity to
LL-37. A GntR-like regulator, SP1714, was identified as a negative regulator of itself
and two of the putative transporters. In conclusion, we show that resistance to three
different AMPs in S. pneumoniae is mediated by several putative ABC transporters,
some of which have not been associated with antimicrobial resistance in this organism
before. In addition, a GntR-like regulator was identified, which regulates two of these
transporters. Our findings extend the understanding of defense mechanisms of this
important human pathogen against antimicrobial compounds and points toward novel
proteins, i.e. putative ABC transporters, which can be used as targets for the
development of new antimicrobials.
Introduction
Increased resistance of bacteria to commonly used antibiotics creates severe
problems in treating infectious diseases. The resistance of one of the most important human
pathogens, Streptococcus pneumoniae, to commonly used antibiotics has increased
significantly in recent decades (118). This bacterium colonizes the nasopharynx and the
upper respiratory tract asymptomatically. Nevertheless, under certain circumstances S.
pneumoniae can cause otitis media, meningitis, pneumonia and sepsis (350). To cause
diseases, S. pneumoniae has to colonize successfully the mucosal surface of the
nasopharynx, followed by dissemination to other parts of the human body. Mucosal
surfaces of the human body form the first barrier that protects against pathogens. In this
layer, mainly neutrophils and epithelial cells produce antimicrobial peptides (AMPs).
Generally, AMPs display a cationic and an amphipathic nature, but they are variable in
sequence, secondary structure, size and mode of action (407). Antimicrobial peptides play
an essential role in the host‘s innate immune response (269).
One human AMP, the 18 kDa hCAP-18 (124), is produced as an inactive
preproprotein that consists of a precursor protein, cathelin, and a carboxyterminal peptide,
LL-37 (486). LL-37 is a linear 37 amino acid long cationic peptide with activity against
Gram-positive and Gram-negative bacteria (522). It has been shown that the bactericidical
Chapter 5
112
action of LL-37 is due to immobilization of the peptide within the membrane lipid bilayer,
where, as a consequence, it causes destabilization of the bacterial membrane (388).
In addition to coping with the human immune system, S. pneumoniae has to
compete with other bacterial inhabitants, which also produce AMPs as a defense against
competitors, to achieve successful colonization of the nasopharynx. AMPs generated by
Gram-positive bacteria are named bacteriocins, and nisin produced by Lactococcus lactis
and commonly used as a food preservative, is one of the best characterized ones (433). The
antimicrobial activity of nisin is rather broad among Gram-positive bacteria (161,337).
Nisin is able to inhibit peptidoglycan biosynthesis by interaction with lipid II and forms
pores in bacterial membranes, which leads to the cell death (44,46,186). Another attack and
defense system used by bacteria is the production of antibiotics such as bacitracin. This
toxic compound is a mixture of cyclic polypeptides produced by Bacillus licheniformis .
Bacitracin is a nonribosomally synthesized antibiotic, which in Gram-positive cocci and
bacilli blocks biosynthesis of the bacterial cell wall by interaction with C55-isoprenyl
pyrophosphate (16,228,491,492).
To establish whether S. pneumoniae contains general defense mechanisms against
heterologous AMPs, transcriptome analysis of S. pneumoniae D39 was performed upon
challenge with three different antimicrobial peptides, i.e. LL-37, nisin and bacitracin. The
transcript levels of genes involved in various processes such as gene regulation, transport,
virulence, fatty acids synthesis, and phosphotransferase systems had changed significantly.
Several highly induced genes were chosen for further analysis. We show, for the first time
to our knowledge that some of these genes, encoding putative ABC transporters, are
involved in the defense of S. pneumoniae against multiple antimicrobial compounds, e.g.
bacitracin, nisin, LL-37, lincomycin or Hoechst 33342. Furthermore, we demonstrate that
the putative regulatory protein, SP1714, is a repressor of its own expression and that of two
putative ABC transporter genes, one of which belongs to another operon. In summary,
these results give new insight into the transcriptional stress response of S. pneumoniae to
structurally different AMPs and enable the identification of common features of the
molecular defense mechanisms against various antimicrobial substances in this organism.
This will eventually lead to selection and/or design of more suitable antimicrobial agents
and development of more effective preventive measures.
Materials and Methods
Bacteria and growth conditions
The strains used in this study are listed in Table 1 and were stored in 10% glycerol at -80°C.
Streptococcus pneumoniae strains were grown at 37°C in standing Todd-Hewitt (Oxoid) broth
supplemented with 0.5% yeast extract (THY) and/or on M17 agar (504) containing 0.25% glucose
(GM17) and 3% defibrinated sheep blood (Johnny Rottier, Kloosterzande, The Netherlands).
Lactococcus lactis was grown in GM17 without agitation at 30°C. Escherichia coli was grown
shaking in TY (tryptone/yeast extract) at 37°C. Where appropriate, media were supplemented with
Responses of S. pneumoniae to AMPs
113
antibiotics at the following final concentrations: erythromycin and spectinomycin for S. pneumoniae
(0.25 μg/ml and 150 μg/ml, respectively), chloramphenicol (2 μg/ml for S. pneumoniae, 4 μg/ml for
L. lactis), tetracycline (2.5 μg/ml for S. pneumoniae), trimethoprim (18 μg/ml for S. pneumoniae), and
ampicillin (100 μg/ml for E. coli). Nisin (Sigma) was used for induction of gene expression at a
concentration of 5 ng/ml.
Strain construction
Strains, plasmids and oligonucleotide primers used in this study are listed in Table 1 and 2. The
genome sequence of S. pneumoniae D39 was used to design all primers (301). All the indicated PCR
fragments and plasmids were introduced into S. pneumoniae D39 as described previously (260,418).
Table 1. Strains and plasmids used in this study
EryR, erythromycin resistance; CmR, chloramphenicol resistance; TetR, tetracycline resistance; SptR, spectinomycin
resistance; trmpR, trimpethoprim resistance
Strain Description Reference or source
S. pneumoniae D39 Serotype 2 strain, cps2 (12,301) source: group
of P.W. Hermans
D39nisRK D39 bgaA::nisRK; TrmpR (260)
385-387 D39SP0385-0387; SpecR This work
785-787 D39SP0785-0787; EryR This work
912-913 D39SP0912-0913; EryR This work
1714-1715 D39SP1714-1715; EryR This work
1715 D39SP1715; EryR This work
blp D39SPD0473-0476; Ery This work
OV912 D39nisRK /pNZ912; CmR This work
OV1715 D39nisRK /pNZ1715; CmR This work
CO912 OV912 912-913 This work
CO1715 OV1715 1715 This work
CO1716 OV1715 1714-1715 This work
DM39 385-387 912-913 This work
DM19 912-913 1714-1715 This work
PR385 D39bgaA::PSP0385-lacZ;TetR This work
PR785 D39bgaA::PSP0785-lacZ:TetR This work
PR912 D39bgaA::PSP0912-lacZ;TetR This work
PR1714 D39bgaA::PSP1714-lacZ;TetR This work
PR7851714 PR785/1714-1715 This work
PR9121714 PR912/1714-1715 This work
PR17141714 PR1714/1714-1715 This work
E. coli
EC1000 KmR; MC1000 derivative carrying a single
copy of the pWV01 repA gene in glgB
(303)
L. lactis
NZ9000 MG1363 pepN::nisRK (290)
Plasmid
pPP2 AmpR TetR; promoter-less lacZ. For replacement of bgaA
(SPR0565) with promoter-lacZ fusions. Derivative of pPP1.
(175)
pNZ8048 CmR; Nisin-inducible PnisA (101) pPA1 pPP2 PSP0385-lacZ This work
pPA2 pPP2 PSP0785-lacZ This work
pPA3 pPP2 PSP0912-lacZ This work
pPA4 pPP2 PSP1714-lacZ This work
pNZ912 pNZ8048 carrying SP0912-0913 downstream of PnisA This work
pNZ1715 pNZ8048 carrying SP1715 downstream of PnisA This work
Chapter 5
114
S. pneumoniae clones were selected on GM17 agar with the appropriate antibiotic(s). L. lactis and E.
coli were transformed by electroporation as described before (215). All constructs and deletions were
verified by DNA sequencing.
Table 2. Oligonucleotide primers used in this study
Name Nucleotide sequence (5’ to 3’);
restriction enzyme sites underlined
Restriction
site
KN-sp912-913-for-1 GGAAGCCAGCCACAGGCTGTA -
KN-sp912-913-rev-2 GAGATCTAATCGATGCATGCGTGTCATGAGAATCTCCTTTC -
KN-sp912-913-for-3 AGTTATCGGCATAATCGTTACTTCCTCATCGCCTATGTGCTG -
KN-sp912-913-rev-4 CGTAGATGGTTACCTAAGGGAACC -
KN-sp785-787-for-1 TGACAGGGACTTTGTGAGTGTG -
KN-sp785-787-rev-2 GAGATCTAATCGATGCATGCCCCTCCAGCAAACAATACA -
KN-sp785-787-for-3 AGTTATCGGCATAATCGTCAACAAGATGGACACTCGTCT -
KN-sp785-787-rev-4 GGAAGACTGTTCCATTCCAGAA -
KN-sp385-387-for-1 GTGCCACCATAGCAGATCTACAA -
KN-sp385-387-rev-2 CCTCCTCACTATTTTGATTAGTATGAGAGCAATAATGACATAGGC -
KN-sp385-387-for-3 TGGGAAATATTCATTCTAATTGGCCATTTGGTGGGGCAAGAGGAG -
KN-sp385-387-rev-4 TCACGCTAGAGGTACTTGCTTGC -
KN-sp1714-1715-for-1 TCAGTGCCTCCTGACCGATAATCGGG -
KN-sp1714-1715-rev-2 GAGATCTAATCGATGCATGCTTGGTCTCCTTTCTCTTACCC -
KN-sp1714-1715-for-3 AGTTATCGGCATAATCGTTACTCGGAACCTACTACATCTTGA -
KN-sp1714-1715-rev-4 GTGACAGCTCTAGGTGCAGCT -
KN-sp1715-for-1 CTTGACACAGGACGTTTCTGGGCT -
KN-sp1715-rev-2 GAGATCTAATCGATGCATGCCATTTTCAAATGCTAGTAATGACAT -
KN-sp1715-for-3 AGTTATCGGCATAATCGTTACTCGGAACCTACTACATCTTGA -
KN-sp1715-rev-4 GTGACAGCTCTAGGTGCAGCT -
KN-blp-for-1 CTCATCCAAGATTCCTTGGAGAT -
KN-blp-rev-2 GAGATCTAATCGATGCATGCAGCCACCTCTATTTCAAGCCACC -
KN-blp-for-3 AGTTATCGGCATAATCGTCGAGACAAGTATGGAAAGAG -
KN-blp-rev-4 CAAAGCGTTCTACTGTACCAGACAT -
Oversp912-913;fv CATGCCATGGCACTTTTAGATGTAAAACACG NcoI
Oversp912-913;rev GCTCTAGAATACCTCGATTTTGAAGTCGAGG XbaI
Oversp1715;fv CATGCCATGGCATTACTAGCATTTGAAAATG NcoI
Oversp1715;rev GCTCTAGATGAGTATGTTACATATCTAGG XbaI
Psp385-387-fv CGGAATTCGTGCCACCATAGCAGATCTACA EcoRI
Psp385-387-rev GCTCTAGACTCATAGGTTCATCCTCTCCCT XbaI
Psp785-787-fv CGGAATTCTCCGCTACCTCCACCGATAGCAAT EcoRI
Psp785-787-rev GCTCTAGACTTCATAATGAAACTCCTTTTC XbaI
Psp912-913-fv CGGAATTCTGGATGCTGATAACAACTGATAAC EcoRI
Psp912-913-rev GCTCTAGAGTGTCATGAGAATCTCCTTTCT XbaI
Psp1714-1715-fv CGGAATTCCTACGAATGGTGTTCCCTTCT EcoRI
Psp1714-1715-rev GCTCTAGATGTCAAATGTCCAGGACATC XbaI
Construction of deletion strains. The knockout of the SP0385-0387 genes was made with primer pairs
KN-sp385-387-for-1/KN-sp385-387-rev-2 and KN-sp385-387-for-3/KN-sp385-387-rev-4 by overlap
extension PCR, as described by Song H.L. et al. (2005), and allelic replacement with a spectinomycin
resistance cassette, yielding strain 385-387 (485). The deletion strains with an erythromycin
resistance cassette of SP0785-0787 (yielding strain 785-787) and SP0912-0913 (yielding strain
912-913), and SP1714-1715 (equivalent genes in S. pneumoniae D39: SPD1524-1526; yielding
strain 1714-1715), and SP1715 (equivalent genes in S. pneumoniae D39: SPD1525-1526; yielding
strain 1715), and blp genes (equivalent genes in S. pneumoniae D39: SPD0473-0476; yielding strain
Δblp) were made in a similar way as the SP0385-0387 mutant, using primer pairs KN-sp785-787-for-
1/KN-sp785-787-rev-2 and KN-sp785-787-for-3/KN-sp785-787-rev-4, KN-sp912-913-for-1/KN-
sp912-913-rev-2 and KN-sp912-913-for-3/KN-sp912-913-rev-4, KN-sp1714-1715-for-1/KN-sp1714-
1715-rev-2 and KN-sp1714-1715-for-3/KN-sp1714-1715-rev-4, KN-sp1715-for-1/KN-sp1715-rev-2
and KN-sp1715-for-3/KN-sp1715-rev-4, and KN-blp-for-1/KN-blp-rev-2 and KN-blp-for-3/KN-blp-
rev-4, respectively.
Responses of S. pneumoniae to AMPs
115
Construction of lacZ fusions. To construct the chromosomal transcriptional fusions of lacZ to the
putative promoters of the presumed operons of SP0385-0387, SP0785-0787, SP0912-0913 and
SP1714-1715, the putative promoter fragments were amplified from the chromosomal DNA of S.
pneumoniae D39 with the primer pairs listed in Table 2. Putative promoter of the SP0385-0387 genes
(promoter length 167 nt) was amplified with the primer pair Psp385-387-fv/ Psp385-387-rev; putative
promoter of the SP0785-0787 genes (promoter length 311 nt) was amplified with the primer pair
Psp785-787-fv/Psp785-787-rev; putative promoter of the SP0912-0913 genes (promoter length 585
nt) was amplified with the primer pair Psp912-913-fv/Psp912-913-rev; putative promoter of the
SP1714-1715 genes (promoter length 237 nt) was amplified with the primer pair Psp1714-1715-
fv/Psp1714-1715-rev. The obtained fragments were cloned into the EcoRI/XbaI sites of pPP2 giving
rise to pPA1, pPA2, pPA3, pPA4. These plasmids were transformed into S. pneumoniae D39 to
generate PR385 and PR785 and PR912 and PR1714 strains. In addition, introduction of plasmids
PA2, PA3 and PA4 into a 1714-1715 mutant resulted in the PR7851714, PR9121714 and
PR17141714 strains, respectively.
Construction of overexpression plasmids. For overexpression of SP0912-913 and SP1715 with the
nisin inducible system (101,290) these gene fragments were amplified with the primer pairs
Oversp912-913;fv/Oversp912-913;rev and Oversp1715;fv/Oversp1715;rev, respectively, and were
fused to NcoI/XbaI sites in pNZ8048, yielding pNZ912 and pNZ1715. These plasmids were
transformed into S. pneumoniae D39 generating the OV912 and the OV1715 strains. For the
complementation assay pNZ912 was transformed to the 912 strain yielding strain CO912 and
pNZ1715 was transformed into 1714-1715 and to 1715 yielding CO1716, CO1715, respectively.
Antimicrobial agents
Stock solutions of antimicrobial peptides/agents were stored in aliquots at -20°C. The solutions of
bacitracin (Sigma), Hoechst 33342 (2‘-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5‘Bi-1H-
bezimidazole; Molecular Probes, Inc.), gramicidin (Sigma), lincomycin (Sigma), vancomycin
(Sigma), daunomycin (Sigma) and ethidium bromide (Sigma) were prepared in MilliQ water. The
stock solution of nisin (Sigma) was prepared in 0.05% acetic acid and LL-37 (Innovagen) in 0.01%
acetic acid with 0.01% BSA (NEB). Dilutions of each antimicrobial compound were always freshly
prepared from these stocks.
DNA microarrays and transcriptional profiling
DNA microarrays were produced and analyzed as described before (288,534).
Experimental design. One millilitre aliquots of S. pneumoniae D39 (OD600~0.25) were used to
inoculate 100ml of THY medium and were grown at 37°C until early logarithmic phase
(OD600~0.25). Subsequently, cultures were split in two and exposed to 0.7 μg/ml bacitracin, 0.1 μg/ml
nisin, or 4.5 μg/ml LL-37 (end concentrations) for 15 (early response) and 30 (late response) min.
These concentrations of AMPs were chosen, based on the results of growth experiments performed
with all three AMPs and gave a 10% reduction of the maximal OD compared to that with no AMP. In
this manner, the bacteria were stressed with the AMPs but not killed to a great extent, because this
would negatively influence the quality of the RNA for the transcriptome experiments. For each AMP,
three replicates were performed, and as a control, bacteria without any AMP were used.
RNA isolation, cDNA preparation and hybridization. RNA was isolated from 50 ml of three
independent cultures exposed to either no AMPs or to each AMP. After centrifugation, pellets were
frozen in liquid nitrogen and stored at -80°C. Subsequently, pellets were suspended in 500 μl of 10
mM Tris-HCl, 1 mM EDTA pH 8.0, after which 50 μl of 10% SDS, 500 μl of
Chapter 5
116
phenol:chloroform:isoamyloalcohol (24:24:1), 500 mg of glass beads (Sigma,75-150 μm), 175 μl of
Macaloid solution (Bentone) were added. RNA was isolated with a High Pure RNA Isolation Kit
(Roche). Subsequently, cDNA was obtained from 15-20 μg of total RNA and the labelling Cy3/Cy5-
dCTPs of cDNA was performed with the CyScribe Post labelling kit (Amersham Biosciences).
Hybridization was carried out at 45°C for 16 h in Ambion Slide hybridization buffer (Ambion
Europe) on superamine glass slides (Array-It; SMMBC). Slides contained replicates of amplicons of
2.087 open reading frames (ORFs) of S. pneumoniae TIGR4 and 184 unique ORFs of S. pneumoniae
R6. Amplicon sequences are available on the World Wide Web at molgen.biol.rug.nl. Slides were
scanned using a GeneTac LSIV confocal laser scanner (Genomics Solutions).
Data analysis. ArrayPro 4.5 (Media Cybernetics Inc., Silver Spring, MD) was used to analyze the
data. For the processing and normalization of the data the MicroPrep software was used as described
previously (534,535). Genes with p < 0.0001 and with a differential expression greater than 1.2 or
lower than 0.8 were considered significantly differentially expressed. The DNA microarray data are
available on the DNA microarray data are submitted to the GEO database, preliminary accession
number GSE16491.
Microarray data accession number.
The DNA microarray data were submitted to the GEO database and are available under accession
number GSE16491.
β-galactosidase assays
S. pneumoniae isolates were incubated at 37°C in THY and grown to an early logarithmic phase
(OD600~0.25). Subsequently, D39 derivatives were incubated for 15 (data not shown), 30 and 90
minutes with or without 0.7 μg/ml bacitracin, 0.1 μg/ml nisin or 4.5 μg/ml LL-37 (the same end
concentration of these AMPs were used for transcriptome analyses). Next the pellets were collected
and β-galactosidase assays were performed as described previously by Israelsen et al. (229) with the
following modifications. Two millilitres of the cell cultures were centrifuged; pellets were suspended
in 250 μl Z buffer (60 mM Na2HPO4*2 H2O, 40 mM NaH2PO4* H2O, 10 mM KCl, MgSO4*7 H2O)
and 15 μl (final concentration 0.06 mg/ml) cetyltrimethyl ammonium bromide and incubated for 5
min. at 30°C. The assay was started by the addition of 50 μl of 4 mg/ml ONPG (O-Nitrophenyl β-D-
Galactopyranoside, Sigma) and stopped by addition of 250 μl of Na2CO3 (1M).
Determination of MICs
Determination of the MICs of the various compounds for S. pneumoniae D39 and the mutants were
performed in 96-well microtiter plates. Incubations took place in a microplate reader (GENios,
TECAN). Aliquots of strains OV912, OV1715, CO912, CO1715, and CO1716 were made using THY
broth with an induction concentration of nisin (5 ng/ml) for the nisin-inducible expression of the
genes of interest. For the MIC assays, the aliquots were thawed, spun down and resuspended in a
fresh THY broth. The medium of strains OV912, OV1715, CO912, CO1715 and CO1716 was again
supplemented with the induction concentration of nisin. Exponentially growing strains (at an OD600
~0.2) were applied into the wells of microtiter plates at a total volume of 200 μl/well with increasing
concentrations of the antimicrobial substance being tested. The microtiter plates were incubated at
37°C for overnight growth, and the O.D600 was measured every 30 min. The MICs were determined
when the reference strains (cells without antimicrobial substance) reached half of the maximal optical
density. MICs were calculated from the lowest concentration of the antimicrobial substance that was
able to inhibit the growth of the tested strain. Strains were grown in the absence of the antibiotics
used to select for the genetic modifications of S. pneumoniae to prevent any influence on the MIC.
Pneumococcal strains with pNZ8048, a negative control for the overexpression, showed no change in
Responses of S. pneumoniae to AMPs
117
susceptibility to the tested drugs (data not shown). To control for a positive influence of the nisin
induction on susceptibility of the strains carrying overexpression vectors, these strains were also
examined in the MIC assay without nisin induction, but no change in the susceptibility was observed
compared to that with nisin (data not shown). All the susceptibility assays were performed at least in
triplicate.
Results
Genome - wide identification of S. pneumoniae genes responding to bacitracin, nisin or
LL-37 challenge
Nisin, bacitracin and LL-37 differ in structure and mode of action, but their
targets, subunits of the bacterial cell envelope, are thought to be similar. To investigate
whether there is a general stress response of S. pneumoniae to different AMPs,
transcriptome analyses of strain D39 exposed for 15 and 30 min to sublethal amounts of
bacitracin, nisin or LL-37, were performed.
Exposure to all three AMPs resulted in significantly changed (Bayes P value of
≤0.0001 and fold change of ≤0.8 or ≥1.2) transcript levels of genes involved in various
processes such as regulation, transport, fatty acid biosynthesis, virulence, bacteriocin
production, metabolic processes, protein fate, phosphotransferase systems and many genes
encoding hypothetical proteins. LL-37 seemed to have the most profound influence on the
transcriptome of S. pneumoniae D39, as expression of ~ 10% of the genome changed upon
exposure. A complete overview of significantly up- and downregulated genes is shown in
Table S1 in the supplemental material. The response to each individual AMP had a number
of genes in common at both time points (see Table S2, section A, in the supplemental
material), and several genes were differentially regulated upon change with more than one
AMP at both 15 and 30 min (see Table S2, section B). Subsequently, we investigated how
many significantly down- and upregulated genes were identified as common in each stress
response to bacitracin, nisin and LL-37 after two time points (Fig. 1; also see Table S3).
The data revealed that treatment with nisin and LL-37 for 15 min. resulted only in a few
(11) downregulated genes in common (Fig. 1; also see Table S1, sections C and E, and
Table S3 section A). Prolongation of the time of exposure to these two AMPs to 30 min did
not yield any genes in common (Fig. 1A; also see Table S1 sections D and F). Interestingly,
there were no down-regulated genes identified when D39 was exposed for 15 min to
bacitracin. After 30 min treatment with this AMP, 66 genes were downregulated (see Table
S1, section B), 32 of which were also downregulated by exposure to LL-37 for 30 min (Fig.
1A; also see Table S1, sections B and F and Table S3, section A). Treatment with all three
AMPs induced the expression of several common genes, the number of which increased
with longer exposure (Fig. 1.B; also see Table S3). Although bacitracin, nisin and LL-37
are distinct antimicrobial compounds, the S. pneumoniae transcriptome response to them
revealed certain analogous features. Since we were interested in genes that might be
involved in the resistance mechanisms of D39 to two or all three AMPs, which are expected
Chapter 5
118
to be upregulated, we focused on the most interesting and prominently induced genes,
which are described below.
Figure 1. Venn diagrams indicating the number of genes downregulated (A) and upregulated (B) in the 15- and
30-min stress response of D39 to bacitracin, nisin and LL-37. Numbers quantify the genes with significantly
altered expression (Bayes P value of ≤0.0001; expression ratio greater than 1.2 or lower than 0.8) that were either
shared or exclusive to each D39 response. List of genes in common indicated in this figure can be found in Table
S3, sections A, B and C, in the supplemental material.
Genes induced in the response to all three AMPs
Comparison of the transcriptome profiles of S. pneumoniae D39 in response to
bacitracin, nisin and LL-37 revealed that genes SP0641, encoding the pneumococcal
surface serine protease PrtA (33); gene SP2062, a member of VicRK regulon (351,370),
encoding a putative transcriptional regulator of the MarR (multiple antibiotic resistance
regulators) family; and genes SP0419 and SP0422 involved in fatty acids biosynthesis
(304), were all moderately (SP0641, 1.3- to ~3 fold; SP2062, 1.5- to ~2 fold; SP0419, ~1.8-
fold to 2-fold; and SP0422, 1.4 to ~2.2-fold) upregulated upon exposure to each AMP at
either 15 or 30 min (Fig. 2; also see Table S3). Gene SP0913, encoding a permease protein,
was induced moderately (2-fold) upon LL-37 treatment and up to 13-fold upon treatment
with nisin and bacitracin. SP0912, an ATP-binding protein, was upregulated 9-fold upon
nisin and bacitracin exposure, and it probably forms an ABC transporter with SP0913 (23)
(Fig. 2 and Table 3). SP0912-0913 share amino acid identity with the known ABC
Responses of S. pneumoniae to AMPs
119
transporters BceAB from Bacillus subtilis and MbrAB from Streptococcus mutans, which
are known to be involved in resistance to bacitracin, and the YsaBC transporter from L.
lactis that mediates a protective effect against nisin (275,333,518). SP0912 shares
considerable identity with BceA (52 %), MbrA (58 %) and YsaC (61 %), whereas SP0913
has a moderate identity with BceB (25%), MbrB (30%) and YsaB (31%). Thus, SP0912-
0913 were chosen for further study.
Figure 2. General comparison of differentially and antagonistically expressed genes of D39, involved in
regulation, virulence, and resistance mechanisms, upon bacitracin, nisin and LL-37 stress for 15 and/or 30 min.
Thickness of the arrow indicates the strength of differential expression.
Genes induced in the response to both bacitracin and LL-37
Bacitracin and LL-37 both induced expression of the SP0385 gene, encoding a
putative membrane protein and the adjacent, SP0386-0387, genes encoding the two-
component system number three (TCS03) (Fig. 2 and Table 3) (300). In addition, several
transporters (SP0785-0787 and SP1715) were induced upon exposure to both AMPs, as was
a putative transcriptional regulator, SP1714 (Fig. 2 and Table 3; also see Tables S1,
sections A, B, E and F, and Table S3 in the supplemental material). TCS03, one of the 13
two-component systems in S. pneumoniae was upregulated more than 2-fold upon
bacitracin and moderately (1.5-fold) upon LL-37 treatment. The TCS03 shares amino acid
sequence similarity with TCS11 from S. mutans, CesSR from L. lactis, VraRS from
Staphylococcus aureus, and LiaRS (YvqEC) from B. subtilis, which have been proposed to
Chapter 5
120
be sensors of cell envelope-mediated stresses (241). The transcript level of the adjacent
SP0385 gene changed similarly to that of TCS03. The SP0385 membrane protein with
unknown function shares 27% sequence identity to LiaF (YvqF), a membrane protein of the
liaRS gene cluster. Analysis of the genomic sequence of D39 revealed that SP0385 is
probably transcribed from the same promoter as TCS03. To investigate whether the
SP0385-0387 genes play a role in S. pneumoniae resistance to AMPs we chose them for
further study. The expression of the SP0785-0787 genes increased more than 2-fold upon
bacitracin stress and more than 4-fold upon LL-37 stress (Fig. 2 and Table 3). Analysis of
the D39 genomic sequence indicated that the SP0785-0787 genes might be transcribed from
the same promoter, which is in accordance with the transcriptome data. SP0785 encodes a
protein annotated in the NCBI database as a membrane fusion protein (MFP) subunit of an
efflux transporter. The SP0786-0787 genes are annotated as encoding an ABC transporter,
SP0786 as an ATP-binding and SP0787 as a permease protein with three transmembrane
domains. Interestingly, the SP0787 protein showed 34% amino acid sequence identity to
BacI, involved in secretion of bacteriocin 21, and 32% amino acid sequence identity to
MacB, involved in the resistance to macrolides, (268,515). Therefore, we decided to
investigate the function of SP0785-0787 further.
The SP1714-1715 genes, presumably in an operon, were upregulated more than 2-
fold in response to bacitracin and even 13-fold in response to LL-37 (Table 3), and were
chosen for further study. The SP1714 gene encodes a putative transcriptional regulator of
most likely, the GntR (gluconate regulator) family of regulators, while SP1715 encodes a
putative ABC transporter, and were chosen for further study.
Genes induced upon challenge with bacitracin and nisin
Among the genes that were upregulated upon both bacitracin and nisin exposure
were the SP0912 gene, described above, and the SP2063 gene (Fig. 2; also see Table S1,
sections B and D, and Table S3 in the supplemental material). SP2063, a member of the
VicRK regulon (351,370), was upregulated 7-fold upon bacitracin stress and almost 2-fold
upon nisin stress. This gene encodes a protein with a LysM (lysin motif) domain, so it is
probably cell wall attached, but otherwise the function is unknown (Fig. 2) (57).
Up-regulated genes in common for the nisin and LL-37 response
Treatment with nisin or LL-37 positively stimulated expression of several identical
genes. Among them was the SP2173 gene, encoding DltD (Fig. 2; also see Table S1,
sections C and E, and Table S4, section A in the supplemental material). Interestingly, all
four genes of the dlt operon, dltABCD (SP2173-2176), showed induction upon LL-37
exposure (see Table S1, section E), but only one gene of this operon, dltD, was upregulated
upon nisin exposure (Fig. 2 also see Table S1, sections C and E). The dlt operon encodes
proteins mediating D-alanylation of the teichoic acids, which improves resistance to
neutrophil traps in TIGR4 (539). Furthermore, the dlt operon confers resistance to nisin and
Responses of S. pneumoniae to AMPs
121
gallidermin in strains Rx and D39, and in S. aureus to defensins, protegrins and other
cationic AMPs (274,406). Thus, the upregulation of dlt genes upon LL-37 and dltD upon
nisin is in accordance with the previous data and indicates that this operon also plays a role
in the resistance of S. pneumoniae D39 to LL-37.
Table 3. Differential expression of genes selected for further analysis upon S. pneumoniae treatment for different
times with bacitracin, nisin and LL-37a. a Genes were selected for further analysis based on a Bayes P value of ≤0.0001 and ≤0.8- and ≥1.2-fold change
after 15 min and 30 min with bacitracin, nisin and LL-37. NDE, not significantly expressed; b blpU encodes a
homolog of SP0533. D39 does not posses blpK, but part of the amplicon sequence of SP0533 is identical to that of
SPD0046; c The SP1715 amplicon on the array is homologous only to SPD1525; there is no information on the
transcript levels of SPD1526
TIGR4
locus tag
D39
locus tag Putative/predicted function Gene
Fold induction upon exposure for indicated time (min) to:
Bacitracin Nisin LL-37
15 30 15 30 15 30
SP0385 SPD0350 Membrane protein 2.1 NDE NDE NDE 1.5 1.6
SP0386 SPD0351 Sensor histidine kinase hk03 2.4 NDE NDE NDE 1.5 1.7
SP0387 SPD0352 DNA-binding response
regulator rr03 2.1 NDE NDE NDE 1.5 1.5
SP0525 SPD0467 Regulatory protein blpS NDE NDE NDE NDE 1.6 1.8
SP0526 SPD0468 Response regulator blpR NDE NDE NDE NDE 1.5 1.6
SP0527 SPD0469 Histidine kinase blpH NDE NDE NDE NDE 1.5 1.8
SP0528 SPD0470 Peptide pheromone blpC NDE NDE NDE NDE NDE 1.8
SP0529 SPD0471 ABC transporter, permease
protein blpB NDE NDE NDE NDE 1.7 2.2
SP0530 SPD0472 ABC transporter, ATP-
binding protein blpA NDE NDE NDE NDE 3.4 3.6
SP0533 SPD0046b Bacteriocin blpK NDE NDE NDE NDE 1.5 1.5
SP0545 SPD0473 CAAX protease blpY NDE NDE NDE NDE 5.0 5.9
SP0546 SPD0474 Immunity protein blpZ NDE NDE NDE NDE 3.5 3.8
SP0547 SPD0475 CAAX protease NDE NDE NDE NDE 3.7 4
SP0785 SPD0686 RND efflux-like protein 1.8 2.1 NDE NDE 4.1 5.5
SP0786 SPD0687 ABC transporter, ATP-
binding protein 1.9 2.3 NDE NDE 5.1 5.0
SP0787 SPD0688 ABC transporter, permease
protein 1.9 2.4 NDE NDE 4.6 6.1
SP0912 SPD0804 ABC transporter, ATP-
binding protein 8.2 8.7 9.1 6 NDE NDE
SP0913 SPD0805 ABC transporter, permease
protein 12.4 9.6 13.3 11.8 1.9 1.8
SP1714 SPD1524 GntR transcriptional
regulator 2.9 NDE NDE NDE 7.2 9.1
SP1715 SPD1525-
1526c ABC transporter, ATP-
binding protein 2.3 NDE NDE NDE 11.4 13
Differences in the D39 transcriptome response to bacitracin, nisin and LL-37
The glnRA (SP0501-0502), htrA (SP2063), SP2240 and blp (SP0525-0529,
SP0533, SP0545-0547) genes had an opposite expression levels upon challenge with
different AMPs. Surprisingly, the glnRA genes were upregulated upon LL-37 stress,
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122
whereas they were downregulated upon challenge with nisin (Fig. 2; also see Table S1,
sections C, D, E and F). The glnR gene encodes the repressor of the genes encoding the
glutamine synthesis and uptake complex, glnA and glnPQ (261). Although the genes
involved in glutamine metabolism are well studied within pathogens
(184,261,501,502,520), it is not clear why glnRA are oppositely expressed upon nisin and
LL-37 exposure.
Similarly, expression of htrA and its adjacent gene SP2240 was antagonistic in the
D39 stress response to bacitracin and LL-37 (Fig. 2; also see Table S1, sections B, E and
F). These genes were 2-fold downregulated upon bacitracin treatment and more than 3-fold
upregulated upon LL-37 exposure. HtrA (high-temperature requirement A), a major
virulence factor of S. pneumoniae, is a serine protease that plays a significant role in
resistance to high temperatures, oxidative stress, and it is involved in the transformation
efficiency (98,227). One of the pneumococcal TCS, CiaRH (SP0798-0799), positively
controls the expression of htrA and SP2240 (226,334,466). Since ciaRH was upregulated
upon challenge with LL-37 and not with bacitracin, the induction of htrA and SP2240
expression in response to LL-37 was most likely mediated by CiaRH. The expression of the
SP0107 gene, also a member of the VicRK regulon (351,370), which encodes a protein
with a LysM (lysin motif) (57) domain and unknown function, increased more than 2-fold
upon bacitracin exposure and was reduced, approximately 2-fold upon LL-37 exposure
(Fig. 2).
One feature completely distinguished the response to LL-37 from that to bacitracin
and to nisin; genes of the blp (bacteriocin-like peptide; pnc) locus were induced only upon
LL-37 stress (Fig. 2, and Table 3; also see Table S1, sections E and F, in the supplemental
material). The blp genes encode proteins for Blp bacteriocin(s) production, regulation,
transport and immunity (97,98,102,307). Since the putative blp immunity genes, SP0545-
0547, were strongly induced only upon LL-37 (Table 3), we speculated that, in strain D39,
they might be involved in a resistance mechanism against this AMP, and therefore, the blp
genes involved in bacteriocin production and immunity were selected for further study.
Changes mediated by bacitracin, nisin and LL-37 on the expression of SP0385-0387,
SP0785-0787, SP0912-0913 and SP1714-1715
In order to confirm the differential patterns of expression upon bacitracin, nisin
and LL-37 challenge, lacZ-promoter fusions of the promoters of the genes selected for
further study were made. The same experimental procedure as applied for the transcriptome
analysis was used for AMPs exposure, with one modification. Exposure of the D39
derivatives to the AMPs for 30 and 90 min resulted in higher β–galactosidase activities than
a 15-min exposure. Similar observations were made by R. Bernard et al. for the bceAB
promoter with bacitracin (28). The reason for this might be that the bacteria need more than
15 min to fully produce the β-galactosidase enzyme. The expression of one unresponsive
promoter under these conditions did not show the same, time dependent, increase,
Responses of S. pneumoniae to AMPs
123
indicating that this is not a general effect of the AMPs on the β-galactosidase assay (data
not shown). Therefore, we decided to measure the promoter‘s responses after 30 and 90
min incubation with each AMP.
Table 4. β-galactosidase activities of the promoter of the SP0385-0387, SP0785-0787, SP0912-0913, and SP1714-
1715 genes in the wild-type D39 strain transcriptionally fused to lacZa. a The activities of the promoter of the SP0785-0787 and SP1714-1715 genes were also studied in a ΔSP1714-1715
strain. In all cases, the bacteria were grown in THY without AMPs or with either 0.7 μg/ml bacitracin, 0.1 μg/ml
nisin or 4.5 μg/ml LL-37. b Values are the averages of the results of five independent experiments, and the
standard deviations are indicated in parentheses. ND, not determined
Activity (Miller Units)b of promoter with indicated treatment for indicated time (min)
Strain Promoter
of genes
Without AMP Bacitracin Nisin LL-37
30 90 30 90 30 90 30 90
D39
SP0385-
0387 32(6) 45(16) 194(31) 259(46) 96(3) 82(14) 99(36) 85(11)
SP0785-
0787 24(3) 26(3) 45(6) 59(8) 46(8) 38(1) 37(9) 37(7)
SP0912-
0913 4(1) 4(2) 23(2) 52(12) 59(10) 64(20) 4(0.5) 3(0.1)
SP1714-
1715 25(4) 59(5) 90(16) 114(24) 68(27) 60(24) 146(40) 267(21)
ΔSP1714
-1715
SP0785-
0787 126(26) ND 144(15) ND 145(19) ND 180(15) ND
SP1714-
1715 539(137) ND 734(45) ND 715(34) ND 656(8) ND
The expression of the SP0385-0387 promoter increased upon exposure to all
AMPs tested (more than 3-fold upon bacitracin exposure and approximately 2-fold with the
other two AMPs), which is in contrast to the transcriptome profiling, where these ORFs
were induced only upon bacitracin and LL-37 exposure (Table 4). The activity of the
SP0785-0787 promoter increased slightly, approximately 2-fold, upon bacitracin and nisin
stimulation, but there was no effect of LL-37 exposure (Table 4), which differs from the
results observed in the transcriptome analysis. Induction of PSP0912-0913 activity upon
LL-37 stress was not observed, but in response to bacitracin and nisin, its activity was
greater than 12- and 15-fold higher (Table 4), respectively, which corresponds to the
transcriptome data. After 30 min of induction, the expression of SP1714-1715 was
enhanced 3-fold upon bacitracin exposure and 6-fold upon LL-37 exposure (Table 4),
which is in agreement with the transcriptome data. However, the expression of this
promoter also increased, approximately 2-fold after 30 min of treatment with nisin, which
was not observed for this AMP in transcriptome analysis. These results demonstrate that the
activity of the tested promoters is induced upon exposure to AMPs and corresponds with
the transcriptome analysis.
Determination of MICs for S. pneumoniae mutant derivatives
In order to determine whether the genes mentioned before play a direct role in the
resistance to any of the AMPs used, mutant and/or complementation constructs of these
genes were made and the strains obtained were tested for their susceptibility to bacitracin,
Chapter 5
124
nisin and LL-37 (Table 5). To establish whether these genes are also involved in resistance
to other antimicrobial agents and could potentially encode multidrug (MDR) transporters,
we also exposed the strains to Hoechst 33342, daunomycin, lincomycin, gramicidin and
vancomycin, and ethidium bromide (Table 5). None of the mutant strains was more
susceptible than the wild type to vancomycin, daunomycin or ethidium bromide (data not
shown). The SP0385-0387 mutant (385-387) was 2-fold more susceptible to bacitracin.
The SP0785-0787-deficient strain (785-787) exhibited considerable sensitivity to LL-37
(more than 4-fold) and to lincomycin (~10-fold). The SP0912-0913 mutant (912-913)
showed enhanced sensitivity to nisin, bacitracin, gramicidin and lincomycin, which could
be complemented by expression of the genes in the mutant (CO912). As expected, the blp-
deficient strain (blp strain) was more sensitive only to LL-37. Mutation of SP1714-1715
(1714-1715) caused decreased resistance of D39 to bacitracin and Hoechst 33342 but,
interestingly, increased resistance to LL-37. To exclude a role of the GntR-like regulator
(SP1714) in the observed increased susceptibility of the mutant to bacitracin, Hoechst
33342, or LL-37, a mutant of only the putative ABC transporter, SP1715 (1715), was
generated.
Table 5. MICs for S. pneumoniae D39 and derivatives treated with various antimicrobial substances. a Values are averages of the results of at least three independent experiments. MICs are given in micrograms per
milliliter unless stated otherwise. Bold font indicates a difference of more than approximately 2-fold compared to
the MIC of the wild type. ND, not determined. b strain overexpresses SP1715 in the 1715 mutant. c strain
overexpresses SP1715 in the 1714-1715 mutant. d strain overexpresses SP0912-0913 in the 912-913 mutant. e
double mutant of 385-387 with 912-913. f double mutant of 912-913 with 1714-1715. g strain overexpresses
SP0912 or SP1715 in the wild-type S. pneumoniae D39
Strain
MICs (μg/ml)a for:
Bacitracin Nisin LL-37 Hoechst
33342 (μM) Gramicidin Lincomycin
D39
4
0.8
14
1
2.2
0.5
385-387 1.5 0.8 14 1 2.2 0.5
785-787 4 0.8 3 1 1.5 0.03
1715 1.7 0.8 30 0.5 2.2 0.5
1714-1715 1.7 0.8 30 0.5 2.2 0.5
CO1715b 4 NDa 1 1 ND ND
CO1716c 5 ND 2 1 ND ND
OV1715g 5 ND 2 2 ND ND
blp strain 4 0.8 3 1 2.2 0.5
912-913 0.7 0.2 14 1 1 0.03
CO912d 4 0.6 ND ND 2 4
OV912g 15 1 ND ND 2.5 0.5
DM39e 0.7 0.18 9 ND
1 0.5
DM19f 0.7 0.35 26 0.5 2 ND
This SP1715 mutant had the same phenotype as the SP1714-1715 deficient strain (Table 5);
strongly suggesting that SP1715 encodes a putative ABC transporter that determines
Responses of S. pneumoniae to AMPs
125
resistance to bacitracin and Hoechst 33342 and sensitivity to LL-37. Introduction of either
the SP0385-0387 or the SP1714-1715 mutation into the ΔSP0912-0913 background (DM39
and DM19, respectively) did not result in increased sensitivity to bacitracin, nisin, Hoechst
33342, or LL-37 compared to that of the single mutants, indicating that these proteins are
functioning in the same pathway. Additionally, overexpression of the SP0912-0913 genes
(OV912) increased the resistance of D39 to bacitracin more than 3-fold, whereas it had only
a moderate effect on resistance to nisin and gramicidin and no effect on resistance to
lincomycin. Overexpression of SP1715 in both mutant and the wild-type backgrounds
(CO1715, CO1716, and OV1715) increased the sensitivity to LL-37 7-fold compared with
that of the wild type, the resistance to Hoechst 33342 increased 2-fold, and minor effects
were observed for bacitracin. Thus, multiple genes identified in the transcriptome analysis
indeed play a role in the resistance of D39 to the AMPs tested. Furthermore, some of these
genes also confer resistance also to other antimicrobial compounds.
The GntR-like regulator, SP1714, is a repressor of its own expression and that of
SP0785-0787
The SP1714-1715 and SP0785-0787 genes were upregulated upon treatment with
bacitracin and LL-37, and mutation of these genes changed the resistance of D39 to these
two AMPs. This indicated that the SP1714-1715 and SP0785-0787 genes might belong to
the same regulatory pathway. Therefore, we decided to study the influence of the SP1714
regulator on the expression of selected gene promoters (SP0785-0787, SP0912-0913 and
SP1714-1715). The activity of PSP1714-1715 in the ΔSP1714-1715 background increased
about 6-fold, and this induction was independent from the stress caused by the AMPs
(Table 4). Likewise, the activity of PSP0785-0787 in the ΔSP1714-1715 background
increased ~4-fold, which demonstrated that the GntR-like regulator repressed PSP0785-
0787 expression, which was again independent of AMP addition (Table 4). Unfortunately,
the open reading frame of SP1714 overlaps with that of SP1715, making it difficult to
delete only SP1714 without influencing SP1715 expression. Therefore, in order to avoid
mutant construction difficulties and to exclude the possibility that SP1715 played a part in
the observed regulatory effects, we examined the expression from these promoters in a
ΔSP1715 mutant. As expected, there was no effect of SP1715 deletion on the expression of
the promoter of SP0785-0787 and SP1714-1715. Likewise, there was no effect of either
SP1714-1715 or SP1715 deletion on the expression of the SP0912-0913 promoter (data not
shown). These data suggest that SP1714, encoding a GntR-like regulator, is a repressor of
its own expression, as well as that of SP1715 and SP0785-0787.
Discussion
The objective of this study was to investigate whether the stress response of S.
pneumoniae D39 to bacitracin, nisin and LL-37 would reveal common features. A second
Chapter 5
126
objective was to determine whether any genes identified have a direct role in conferring
resistance to these and various other antimicrobial compounds. Bacitracin, nisin and LL-37
differ in structure and mode of action but their targets, subunits of the bacterial cell
envelope, are similar. Comparison of the transcriptome response to each compound
revealed that they had a low number of significantly differentially expressed genes in
common (Fig. 1 and Fig. 2). The response of strain D39 to LL-37 was rather broad
compared to that of bacitracin or nisin. This extensive reaction to LL-37 suggests a more
general response of D39 to human peptides than to bacterial compounds, i.e. bacitracin and
nisin (Fig. 1). Analysis of the differentially expressed genes after either 15 min or 30 min
exposure to the tested AMPs, showed little overlap in the downregulated genes in
comparison to the induced genes (Fig. 1). Comparison of the early (15 min) response to the
late one (30min) for each AMP showed that there was little overlap of commonly up- or
downregulated genes (see Fig. S1 and Table S2, section A, in the supplemental material).
However, among these commonly induced genes, we identified several, SP0912-0913 and
SP0785-0787 or SP1714-1715, that were involved in the resistance of D39 to the AMPs
tested, as shown by susceptibility assays. Thus, the transcriptome response of D39 to the
AMPs changes with time but the genes determining resistance are induced in both, the early
(15 min) and the late (30 min), responses. Interestingly, the reaction of D39 to LL-37 and
bacitracin had more genes in common than the reaction to LL-37 and nisin or to bacitracin
and nisin (Fig. 1), which might suggest a more similar general stress response to bacitracin
and LL-37.
The genes SP0385-0387, SP0912-0913, SP0785-0787, SP1714-1715 and blp had
large changes in expression upon challenge with one or more AMPs; therefore, they were
characterized in more detail since they could be alternative candidates for resistance
inhibition by specific drugs. Notably, transcription of homologous of some of the genes
identified in this study, e.g. SP0386-0387, SP0912-0913, SP0785-0787 and SP1714-1715,
have also been found to be affected in response to various antimicrobial compounds,
including bacitracin, nisin, or LL-37, in several other Gram-positive bacteria, i.e. L. lactis,
B. Subtilis, and B. licheniformis (275,333,414).
We showed that the SP0912-0913 genes, encoding a putative ABC transporter,
were induced upon exposure to all three AMPs tested (Fig. 2 and Table 3) and that the
mutant was more sensitive to bacitracin and nisin and, additionally, to lincomycin and
gramicidin (Table 5). The finding that SP0912-0913 is involved in resistance to lincomycin,
nisin and bacitracin is in accordance with previous data for the SP0912-0913 homolog from
B. subtilis, BceAB (formerly YtsCD), which was induced upon bacitracin and LL-37
challenge, and which conferred resistance to bacitracin in this bacterium (29,414). The
other homologs of SP0912-0913, MbrAB from S. mutans and YsaBC from L. lactis,
modulated bacitracin and nisin resistance, respectively (275,518). Although it was shown
that SP0912-0913 genes were induced in S. pneumoniae TIGR4 and Tupelo strains upon
vancomycin challenge (170), we have not seen increased sensitivity of the SP0912-0913
Responses of S. pneumoniae to AMPs
127
mutant to this antibiotic (data not shown). Thus, the SP0912-0913 transporter does not
appear to be directly involved in resistance to vancomycin. The finding that SP0912-0913
is involved in resistance to antimicrobial compounds acting on cell envelope, i.e. nisin and
bacitracin, and antimicrobial compound involved in protein synthesis inhibition, i.e.
lincomycin (64), strongly suggests that the ABC transporter might be of the MDR type.
Recently, Becker et al. showed that this ABC transporter is indeed involved in resistance of
S. pneumoniae R6 to bacitracin (23).
Both the TCS03 and the upstream gene SP0385, which probably form an operon
with TCS03, were induced upon bacitracin and LL-37 challenge (Fig. 2 and Table 3). The
exact function of TCS03 in S. pneumoniae is not yet known, but it has been shown that the
expression of the SP0385-0387 genes was positively affected upon vancomycin stress but
repressed during invasive disease in the cerebrospinal fluid (CSF) (170,390). TCS03 shares
significant amino acid sequence similarity to TCS11 from S. mutans and to CesSR from L.
lactis, to VraRS from S. aureus, and to LiaRS (YvqEC) from B. subtilis. It has been shown
that these homologous TCSs are induced upon challenge with various AMPs, although they
did not confer significant resistance to the antimicrobial agents tested (330,333,414,525).
This study also showed that SP0385-0387 did not significantly confer resistance to the
compounds tested, except for bacitracin (Table 5), which corresponds to the phenotype of
TCS03 homologs in L. lactis CesSR, S. aureus VraRS and B. subtilis LiaRS
(297,330,333,335). Therefore, it has been proposed that these TCSs are the sensors of cell
envelope-mediated stresses, but their exact role in the response to AMPs remains unclear
(241). Interestingly, three genes that belong to the VicRK regulon (SP0107, SP2062, and
SP0203) were induced by AMPs in our study. The VicRK TCS and its homologs in other
Gram-positive bacteria regulate, among others, genes involved in murein biosynthesis and
are essential; in S. pneumoniae this is due to its regulation of PscB. In S. mutans, it was
shown that the VicRK homolog is under the positive control of the LiaRS system. Thus, it
might well be that to withstand exposure to AMPs and the subsequent stress on the cell
wall, the VicRK regulon is also necessary.
The SP0785-0787 genes, encoding a putative ABC transporter, were induced in
response to both bacitracin and LL-37 (Fig. 2 and Table 3), and the SP0785-0787-deficient
strain was significantly more sensitive to LL-37 and lincomycin, and moderately sensitive
to gramicidin (Table 4). The SP0785-787 genes were upregulated upon vancomycin stress,
but the susceptibility assay did not show increased sensitivity of the SP0785-0787 mutant
to this antibiotic (170). Interestingly, Marrer et al. demonstrated that the SP0785-0787
genes were induced upon bacitracin, chloramphenicol and fusidic acid exposure but
repressed by actinomycin and ciprofloxacin challenges (322). These data indicate that
SP0785-0787 might be involved in S. pneumoniae resistance to even more antimicrobial
compounds than were tested, which could imply that the SP0785-0787 proteins display
some characteristics of MDR and are of direct importance for the global defense
mechanism against antimicrobial compounds in S. pneumoniae.
Chapter 5
128
The SP1714 and SP1715 genes, encoding a GntR-like regulator and a putative
ABC transporter, respectively, were considerably upregulated upon challenge with LL-37
and bacitracin (Fig. 2 and Table 3). Strains deficient in SP1715 and both SP1714-1715
were more sensitive to Hoechst 33342 and bacitracin. Surprisingly, the SP1715 and
SP1714-1715 mutants were more resistant to LL-37 than the wild type, whereas
complementation and overexpression of SP1715 increased the sensitivity of strain D39 to
LL-37. These data indicate that, on one hand, SP1715 is involved in D39 sensitivity to LL-
37 and, on the other, in D39 resistance to bacitracin and Hoechst 33342. Furthermore, we
show that SP1714 is a negative regulator of its own gene and, most likely, also of SP1715
that determines sensitivity to LL-37, and of SP0785-0787, which protects against LL-37.
Since SP1714 was upregulated upon challenge with LL-37 and bacitracin, we speculate that
the stress caused by these antimicrobial compounds induces an unknown factor, which
subsequently interacts with SP1714. This interaction might cause release of SP1714 from a
dedicated promoter site and consequently derepression of genes regulated negatively by
SP1714, i.e. SP1714-1715 and SP0785-0787.
Most of the described GntR-like regulators are repressors of various bacterial
metabolic pathways, such as gluconate, histidine and arabinose biosynthesis (454).
Recently, Truong-Bolduc et al. identified a new GntR-like regulator, NorG that regulates
expression of quinolones and β-lactams multidrug efflux pumps (516). In previous studies,
the expression profile of SP1714-1715 increased after induction with vancomycin (170),
but treatment with penicillin had an opposite effect (445). In addition, these genes were
downregulated in the CSF fraction during a transcriptome study of S. pneumoniae during
invasive disease (390). These data could imply that the expression of SP1714-1715 depends
on external stimuli and that the GntR-like protein, SP1714, might regulate the response to a
wide variety of toxic components, most likely via an additional regulatory mechanism. The
exact function of the GntR-like regulator, SP1714, remains to be determined and is the
subject of ongoing studies.
Interestingly, the blp genes were only induced upon stimulation with LL-37 (Fig.
2, Table 3). Notably, from the eight TCS mutants tested for growth efficiency in a
respiratory tract infection (RTI) model, only a BlpR mutant was attenuated indicating that it
is an essential TCS under these conditions (509). The reason why BlpRH was essential for
pneumococcal survival within the RTI remained unclear. Our transcriptome data showed
that the presence of LL-37 induced the entire blp locus, especially the putative blp
immunity genes. Previously, it has been demonstrated that the chemically synthesized
peptide pheromone, BlpC, first induces the two-component system, BlpRH, which,
subsequently leads to upregulation of the complete blp gene cluster (102). Since LL-37 and
BlpC are short linear cationic peptides, we hypothesize that like BlpC, LL-37 could interact
with BlpH and consequently through BlpR activates the entire blp locus. We also speculate
that the blp immunity proteins could confer D39 resistance to LL-37, which is strongly
supported by the finding that the blp deficient strain was sensitive to LL-37. This could
Responses of S. pneumoniae to AMPs
129
explain why BlpRH is essential in the RTI, where many AMPs such as LL-37 are present.
In order to confirm our hypothesis we will continue to evaluate whether LL-37 can induce
the expression of the BlpRH and consequently if the expression of the rest of blp locus will
be enhanced. In addition, we will examine whether the blp mutant is more sensitive to other
human AMPs.
To conclude, the transcriptional response of S. pneumoniae D39 to three distinct
AMPs bacitracin, nisin and LL-37, was diverse and complex and revealed that only a few
genes were differentially expressed in response to all three. Most importantly, mutants of
some of these genes, i.e. SP0912-0913, SP0785-0787 and SP1714-1715, exhibited cross-
sensitivity/resistance to several antimicrobial substances, including some that were not used
in the initial challenge experiments, which, to our knowledge, has not been shown before.
Additionally we showed that the blp locus is involved in determining the resistance of D39
to human AMPs, LL-37. Therefore, some of these genes might be interesting candidates for
inhibition by specific blocking reagents, which would result in novel medicines for the
prevention and treatment of pneumococcal diseases.
Acknowledgements
We thank Rachel Hamer for her technical help in conducting some of experiments
presented in this study. We thank Rutger Brouwer and Anne de Jong for their help with the
submission of the array data to the GEO database.
Supplemental material
Supplemental material may be found at: http://aac.asm.org/.
Chapter 5
130
Chapter 6
General Discussion
Chapter 6
132
To treat infections, ancient Egyptians, Chinese and Greeks were using molds and
plants that contained antimicrobial substances, although they were probably unaware of the
working mechanism. However, as early as in 1877 the history of antimicrobials did begin
with an observation made by Louis Pasteur and Robert Koch of an air-borne bacillus that
inhibited the growth of Bacillus anthracis (299). Subsequently, in 1928, Alexander Fleming
discovered penicillin produced by fungi of Penicillium spp. Nevertheless, it took more than
ten years before penicillin and another antibiotic, namely gramicidin, were isolated by Ernst
Chain and Howard Florey, and used commercially to treat infections (140). Since then, the
search for antibiotic compounds with similar capabilities and produced by microorganisms
has led to the discovery of various antibiotics and antimicrobial peptides (AMPs).
Antimicrobial substances e.g. antibiotics and AMPs play a major role in the lives of almost
all living organisms. AMPs are small proteins produced by many living organisms in order
to inhibit the growth or kill microorganisms in their vicinity, while producers stay immune
themselves. They contribute to the survival of the organism, protection of their ecological
niche and safeguarding essential nutrients by elimination of competitors. AMPs are mostly
cationic peptides and those produced by bacteria are named bacteriocins. According to their
structural features, bacteriocins are divided into four classes, namely i) posttranslationaly
modified bacteriocins named lantibiotics, ii) unmodified peptides, iii) large proteins and iv)
cyclic peptides (203). In 1925, the first antimicrobial activity due to bacteriocin was
described for an antibiotic-like substance ―prinicipe V‖ produced by a bacterium and active
against bacteria (134,135). Later the substance was named ―colicin‖. Subsequently, in
1928, a bacteriocin that is now widely used as a food preservative was discovered and was
named nisin in 1947 (336,444,544). From then on, a variety of bacteriocins have been
reported to be produced by a wide range of bacterial genera.
Bacteriocins have been the subject of intense research for the last two decades
because of their potential applications in food preservation and medical treatments.
Bacteriocins used in food industry should meet several criteria, i.e. the bacteriocin
producing strain preferably should be recognized as a safe one, the bacteriocin should not
cause any health problems, the bacteriocin should have a broad spectrum of inhibition or
have specific activity, the bacteriocin should be stable during the manufacture process and
soluble, and the bacteriocin should not change the flavor of food. Food lactic acid bacteria
(LAB), i.e. natural bacteria of fermented food products, produce a great number of
bacteriocins. Currently, only two products of class I and class II bacteriocins, namely nisin
and pediocin PA-1, respectively, have been used safely as a preservative for e.g. meat, dairy
products, canned food, alcoholic drinks, salads and bakery products (90).
Since some lantibiotics are antimicrobially active at low-nanomolar concentrations
against antibiotic resistant pathogens, they are considered to have an excellent potential in
medical applications, see Table 1. Besides that, the unusual features of lantibiotics, i.e.
lanthionine rings, protect them from protease activity and render them stable in a broad
range of pH and heat. Nisin may have a therapeutic potential in e.g. treatment of
General Discussion
133
Helicobacter pylori, a pathogen of the human gastric mucosa, causing gastric diseases
(106) and in curing Staphylococcus aureus, Streptococcus pneumoniae or Clostridium
difficile infections, see Table 1 (106,156,470). Another lantibiotic, mersacidin, is active
against S. aureus (MRSA) strains and vancomycin-resistant enterococci, and is already in
the preclinical stage of development, (Table 1) (180). For two other lantibiotics, i.e.
epidermin and gallidermin, preliminary clinical tests have demonstrated their potential in
topical treatment of acne, a skin infection caused by Proppionibacterium acnes, (Table 1)
(252). For lacticin 3147 various clinical applications have been considered, including use in
veterinary medicine and as a food preservative, (Table 1) (450-453). Cinnamycin,
ancovenin and duramycin may have medical applications in blood pressure regulation,
treatment of inflammations and viral infections, see Table 1, (143). Currently, to our
knowledge, only three lantibiotics have been licensed for use in clinical applications,
namely nisin, lacticin 3147 and salivaricin (90). Nisin and lacticin 3147 are allowed to be
used in curing animal diseases (90). Producer strains of two related lantibiotics, i.e.
salivaricin A2 and B, are used in New Zealand as a probiotic treatment of throat infections
and chronic bad breath (123). Although there are many studies concerning the successful
biomedical use of lantibiotics (Table 1), government drug industrial regulators are not yet
convinced of the suitability of bacteriocins as antimicrobial agents in medicine (41,89).
For several years, production of bacteriocins by a human pathogen, namely S. pneumoniae,
has been investigated and in recent times, bacteriocin-like activities of S. pneumoniae
proteinaceous substances have finally been elucidated (97,168,307). The activity belongs to
two individual AMPs, namely Blp (also known as Pnc) and CibAB (97,168,307). The
ability to produce a variety of AMPs and the activity spectrum of these AMPs may vary
considerably among different S. pneumoniae strains (97,307). This can be explained by the
genetic variability of the blp (pnc) cluster and of other bacteriocin-like clusters among S.
pneumoniae strains described in chapter 2 of the thesis (172). Chapter 2 describes data
obtained by a bioinformatic study of the putative bacteriocin-like genomic regions in S.
pneumoniae and their comparisons in streptococci. This chapter reflects the genetic
variation in those genomic regions, which is observed for at least six out of nine described
bacteriocin-like clusters. It is remarkable that except Blp and CibAB no bacteriocin-like
activity has been found for at least one of the described clusters. The comparative analysis
of the variety and significant numbers of potentially encoding bacteriocin clusters
demonstrates that bacteriocins play an important role in the lifestyle of S. pneumoniae.
Additionally it seems that the species S. pneumoniae could potentially produce a wide
variety of bacteriocins and the nine bacteriocin-like clusters described in chapter 2 are
probably just the start of the description of the amount and diversity of the AMPs that S.
pneumoniae strains could produce. We speculate that in order to find antimicrobial activity
that is mediated by novel bacteriocins of S. pneumoniae, broad screening of many of the S.
pneumoniae strains grown under a wide variety of conditions is required.
Chapter 6
134
Table 1. Example for potential medical applications for some lantibiotics (adapted from (89)). ND, not determined
Lantibiotic Producing strain Inhibitory activity of
commercial interest Potential Biomedical Applications
Clinical
development
Nisin A Lactococcus lactis Gram-positive
Gram-negative
Bacterial mastitis, oral hygiene, cosmetic deodorants and topical formulations; treatment of methicillin-resistant
S. aureus (MRSA) and enterococcal infections, peptic ulcer,enterocolitis, and lung mucus clearing
(89,156,470)
(Pre)Clinical
trials
Lacticin 3147 L. lactis Gram-positive Bacterial mastitis, oral hygiene treatment of MRSA and
enterococcal infections, and acne (89,148,415) ND
Gallidermin/Epidermin
Staphylococcus gallinarum/
Staphylococcus
epidermidis
P. acnes, staphylococci,
streptococci Acne, eczema, follicultis, impetigo (39,89) ND
Mutacin 1140 Streptococcus mutans S. mutans Prevention of dental caries, treatment of streptococcal throat
infection (206,483)
Preclinical
trials
Mersacidin/Actagardine Bacillus subsp.
/Actinoplanes subsp.
Staphylococci including methicillin-resistant
strains, streptococci
Treatment of MRSA and streptococcal infections (39) Preclinical
trials
Duramycin
Streptomyces subsp. and
Streptoverticillium
subsp.
inhibitor of phospholipase A2
Treatment of MRSA and streptococcal infections, and dry eyes syndrome and reduced mucociliary clearance (39,89)
Phase II clinical trials
Cinnamycin Streptomyces
cinnamoneus
Inhibitor of herpes simplex
virus, phospholipase A2,
angiotension converting
enzyme (ACE)
Inflammation, blood pressure regulation, treatment of viral
infection (89) ND
Ancovenin S. cinnamoneus Inhibitor of ACE Blood pressure regulation (89) ND
NVB302 (modified type-B lantibiotic)
ND C. difficile Treatment of C. difficile Associated Diarrhoea (CDAD) (39) Preclinical
trials
General Discussion
135
Most of the time, the initiation and duration of bacteriocin production may be
associated with growth conditions that resemble the natural niche of the microorganism.
There are many growth condition factors that can influence bacteriocins production e.g.
nitrogen, buffer, sugar, temperature, pH and/or other factors (167,398). Therefore, it might
be difficult to induce biosynthesis of some AMPs, e.g. those of S. pneumoniae. In
agreement with this, the influence of environmental factors, such as temperature, on the Blp
bacteriocins production has been shown (307). Interestingly, some S. pneumoniae strains
produce the Blp bacteriocins at 37°C, whereas others produce them at 35°C (97,307), which
is the temperature of the upper nasopharynx. This niche can be colonized by S. pneumoniae
and the temperature regulation of bacteriocins production might aid in intra- and
interspecies competition. The growth conditions affecting bacteriocin production have also
been described for other streptococci. For instance production of streptococcin AFF-22 by
Streptococcus pyogenes has been shown to be affected by temperature, pH and medium
composition (232). Similarly, production of some S. mutans and Streptococcus
thermophilus bacteriocins depends on the type of medium (230,443). Therefore, production
of novel bacteriocins by S. pneumoniae could be strictly influenced by environmental
conditions and more research is needed to find the AMPs production conditions.
Accordingly, many growth conditions were screened to find the one, which stimulated
expression of one of the bacteriocin-like gene clusters, namely ppu, described in chapter 3.
However, the ppu cluster seemed not to be involved in bacteriocin-like peptide production
(chapter 3). Nevertheless, we showed that the function of the ppuRABCDE cluster is
related in some way to general nitrogen metabolism in S. pneumoniae and that the cluster is
under negative control of CodY, a branched-chain amino acid responsive regulator
(199,484). CodY is one of the bacterial regulators, which is able to adjust globally bacterial
cell metabolism to environmental changes, and additionally influences the expression of
genes involved in virulence. In S. pneumoniae, CodY contributes to colonization of the
nasopharynx and it regulates the expression of a broad range of genes encoding proteins
involved in amino acid uptake, metabolism and biosynthesis, as well as the ppu cluster
(199). We have shown that PpuR is a positive regulator of ppuABCDE and that CodY,
likely by repression of the ppuR transcription, inhibits the expression of the whole ppu
cluster. However, a putative operator region(s) of PpuR has not yet been identified in
PppuA. Moreover, we do not know whether CodY additionally represses expression of
ppuABCDE. Hence, to prove a direct regulatory effect of PpuR and CodY on the PppuA,
direct binding of these proteins to the promoters needs to be performed.
Additionally in chapter 3, we identified two novel clusters, i.e. prcRABCD and taaBC,
which might as well be involved in nitrogen metabolism and we hypothesize that together
with ppu they form a novel regulon in S. pneumoniae. Nevertheless, we do not know how
the ppu cluster influences the expression of prcRABCD and taaBC, and whether the link
between the three clusters is functional, regulatory or both. In a study of Hendriksen et al.,
expression of the whole CodY regulon, including the ppu cluster, but not prc and taa, was
Chapter 6
136
changed in a S. pneumoniae D39 ΔglnAP mutant (glnAP), genes encoding glutamine
synthetase and glutamine ABC transporter (201). The data suggests that CodY does not
regulate the prc and taa cluster directly, but only the ppu cluster. The adjustment of ppu,
prc and taa expression in response to nitrogen sources in a specific medium is probably
important for S. pneumoniae in order to survive in three different niches, namely the
nasopharynx, lungs and/or blood stream. Nevertheless, the regulation of the ppu, prc and
taa cluster expression and their exact roles in nitrogen metabolism is not yet well
understood and will require more study.
Despite characteristic features of lantibiotics e.g. i) a wide variety of structures, ii)
thioether rings, which stabilize the structure of lantibiotics and make them less sensitive to
heat, proteases and reducing agents, iii) activity in nanomolar quantity, and iv) activity
against multi-resistant microorganisms; lantibiotics are not generally approved for medical
applications. There are many possible reasons for this, for instance poor solubility, a
relatively narrow activity spectrum, and the possibility of resistance development and lack
of suitable and cheap technology to produce lantibiotics for commercial use. However, by
use of peptide engineering, many of the drawbacks can be overcome. What is more, the
engineering of lantibiotics via genetic and/or chemical modifications, gives the opportunity
to study structure-function relationship of lantibiotics and to develop novel, and improved
peptides with e.g. medical potential. Diverse strategies have been described for these
purposes for instance: peptide sequence modification by amino acids
substitutions/deletion/insertion, the chemical modifications, the backbone cyclization and
engineering of the modification enzymes. An example of peptide sequence modification is,
in nisin Z, substitution of a residue in position 27 or 31 to lysine, by which the bacteriocin
solubility was improved without diminishing the activity, which is of importance if the
peptide is going to be commercially used (446). The effects of amino acids substitutions in
a peptide sequence are difficult to predict. Although, it has been shown that mutation of
amino acids involved in thioether formation usually results in a substantial decrease of
activity (35,66,71,287).
Since it has been established that the specificity of the lantibiotics‘ modification
enzymes, i.e. LanBC and LanM, is relaxed and that they can modify peptides, which are
fused to a dedicated leader sequence, various studies have been conducted with use of this
information (66,265,439). Production of novel AMPs, or those already known, with the
heterologous expression systems could be beneficial for commercial use and especially in
medicine, where the engineered peptides could be used as e.g. a substitute of, or next to,
antibiotics. However, until recently, many peptides produced with the lantibiotics‘
heterologous expression systems were modified, but only closely related peptides still had
an antimicrobial activity. To our knowledge we presented for the first time (chapter 4) the
successful expression, modification, secretion and biological activity of novel unknown, not
closely related to nisin, class IC lantibiotics of S. pneumoniae (pneumococcins; PneA1 and
PneA2) by the nisin synthetases, i.e. LanBTC, which normally produce nisin, a member of
General Discussion
137
the class IA lantibiotics (Fig. 1). The PneA1 and PneA2 peptides were antimicrobially
active but only against Micrococcus flavus (Fig. 1), which is sensitive to most known
AMPs. The approach used in chapter 4 to ‗awaken‘ otherwise difficult to obtain novel
lantibiotic could be successfully used for other unknown AMPs. Moreover, in chapter 2,
screens through the S. pneumoniae genomes in search for putative, novel, bacteriocin
encoding genes showed that there is a great variety of them. Consequently, these many
unknown AMPs could reveal new modes of action, a broader spectrum of activity and/or
unusual structures. What is more, production of those AMPs with the use of the
lantibiotics‘ heterologous expression systems, could enable selection of ―improved‖
peptides that could putatively be used in medicine as therapeutic agents e.g. used as
substitutes of, or next to, antibiotics (87,348).
Figure 1. Antimicrobial activity of trypsinated chimeric peptides (pneumococcins, i.e. PneA1 and PneA2) and the
controls (BSA, buffer and nisin) in the agar diffusion assay against M. flavus. A culture of M. flavus was mixed
with medium and poured into plates to solidify. Subsequently, in the solidified medium holes were made and filled
with fourfold dilution of various substances. Continuing fourfold dilution for each substance is shown in six holes
divided over two rows. The directions of the dilutions, for each row, are marked below the name of the tested
substance.
The resistance of some bacteria to commonly used antibiotics is on the rise.
Therefore, it is not only important to find alternatives for antibiotics but it is also of great
interest to better understand the resistance mechanisms of bacteria, which would possibly
help to develop new or modify existing surrogates for antimicrobials e.g. AMPs. Following
this idea, in chapter 5 we investigated the response of S. pneumoniae to challenges by three
distinct AMPs, i.e. bacitracin, nisin and LL-37. A few transporters, namely SP0912-0913,
SP0785-0787 and SP1715, and some putative immunity proteins of the Blp bacteriocin
cluster were identified as those involved in resistance of S. pneumoniae to the examined
antimicrobial substances such as bacitracin, nisin, LL-37, Hoechst 33342, gramicidin
Chapter 6
138
and/or lincomycin. Surprisingly, we found out that in S. pneumoniae D39, SP1715 was
involved in sensitivity to LL-37 on one hand and on the other hand in resistance to
bacitracin and Hoechst 33342. The reason for that is not known but we speculate that the
ABC transporter, SP1715, might serve as a kind of receptor for LL-37, as it is done by
some bacteriocins, which use membrane-bound proteins as a docking molecule. It has been
shown that LL-37 is a pore-forming molecule, but whether it binds to a receptor has still to
be established. Interestingly, a novel regulator, i.e. SP1714, was identified and associated
with negative regulation of its own promoter and two ABC transporters, namely SP0785-
0787 and SP1715. The exact function of SP1714 remains to be determined. However, the
transcriptome data indicated that the expression of the regulator depends on external stimuli
and that SP1714 might regulate the response to a wide variety of toxic components, most
likely via an additional regulatory mechanism. The findings in chapter 5 extend the
understanding of defense mechanisms of this important human pathogen against
antimicrobial compounds and points toward novel ABC transporters, which can be used as
targets for the development of new antimicrobials.
In conclusion, the ability of S. pneumoniae to produce a whole set of bacteriocins
could improve the colonization by killing competing bacteria and could increase the
availability of foreign DNA for genetic exchange. Thus, bacteriocins might be considered
as one of the triggers for the evolution of S. pneumoniae pathogenesis. A great challenge
however, is still to find out under which conditions they are being produced. What is more,
bacteriocins, being used as additives important for food production, have also a great
potential to be applied in medicine.
References
139
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Samenvatting voor de leek
Introduction
Biologie is een natuurwetenschap die zich bezighoudt met leven en levende
organismen, inclusief hun structuur, functie , groei, oorsprong, ontwikkeling, voortplanting
en taxonomie Omdat Biologie zo‘n buitengewoon breed onderwerp is, wordt het
tegenwoordig opgedeeld in verschillende disciplines. Deze onderverdeling is gebaseerd op
twee criteria: i) op de biologische organisaties, bijvoorbeeld moleculen, cellen, individuen,
populaties en ii) op het specifieke onderwerp dat wordt onderzocht bijvoorbeeld, structuur
en functie, groei en ontwikkeling. Op basis van deze criteria kan men een onderverdeling
maken in: a) botanie, de plantenstudie, b) zoologie, de dierenstudie, c) microbiologie, de
studie naar microscopische organismen, zoals bacteria, d) virologie, de studie naar virussen,
e) moleculaire biologie, de studie naar biologische functie op moleculair niveau, f)
biochemie, de studie naar chemische reacties die nodig zijn voor de levensvatbaarheid en
het functioneren van organismen, g) genetica, de studie naar genen en erfelijkheid en h)
moleculaire genetica, de studie naar de structuur en functie van genen op een moleculair
niveau. Een molecuul kan worden beschreven als het kleinste deeltje van een stof dat
dezelfde chemische en fysieke eigenschappen als de stof zelf.
In de 17e eeuw kwam de biologie in een stroomversnelling toen een Nederlander,
Antony van Leeuwenhoek, de microscoop verbeterde. Hij wordt beschouwd als de
grondlegger van de microbiologie omdat hij de eerste was die ééncellige organismen,
zogenaamde microorganismen, onderzocht en beschreef, en is nu bekend als ―de vader
van de microbiologie‖. Microorganismen of microben zijn zo klein dat ze niet met het blote
oog kunnen worden waargenomen. Ze vormen een diverse groep en bestaan uit bacteria,
virussen, schimmels, algen en dieren zoals plankton. Virussen zijn echter niet-levende
organismen, omdat ze niet de structuur van een levende cel bezitten en worden daarom
gerefereerd als kleine infectieoverdragers.
De fundametele bouwsteen van leven is de cel en alle levende organismen bestaan
uit één of meer van deze bouwstenen (Fig. 1). Figuur 1 illustreert de verschillen tussen de
structuur en componenten van dierencellen, plantencellen en bacteriecellen. De
componenten van elke cel bestaan uit moleculen. Over het algemeen kan men een aantal
moleculen onderscheiden: 1) organische moleculen zoals proteïnen, carbohydrate, vetzuren,
nucleinezuren en 2) niet-organische moleculen zoals water, stikstof, metalen en niet-
metalen.
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166
Figuur 1. Bacteriologische cellen verschillen
van dieren en plantencellen in meerdere
opzichten. Een fundamenteel verschil is dat
bacteriologische cellen geen intracellulair
organellen hebben, zoals mitochondriën,
chloroplasten, en een kern, die wel aanwezig
zijn in dieren en plantencellen. De afbeelding
is verkregen uit Encyclopædia Britannica
Online (2007; http://www.britannica.com/).
De meeste moleculen zijn veel te klein om met het blote oog te worden
waargenomen, maar een uitzondering hierop is DNA (deoxyribonucleic acid). Binnen de
cel vindt men DNA in langwerpige structuren, chromosomen genoemd. De hoofdrol van
DNA is het vastleggen van genetische informatie die wordt gebruikt bij de ontwikkeling en
het functioneren van alle levende organismen en sommige virussen. De genetische
informatie is tweeledig: 1) de informatie die nodig zijn bij het construeren van andere
celcomponenten en 2) de informatie die de start en het eind van de onder punt 1 genoemde
constructie, bepalen. Deze informatie is vastgelegd in genen. Dus de informatie die nodig is
bij het bouwen van een basismolecuul, proteïne en RNA (ribonucleinezuur), is beschreven
in een gen en een gen codeert een proteïne of een RNA molecuul. Elke gen bestaat uit een
specifieke volgorde van nucleotide-moleculen: adenine (A), tyrosine (T), cytosine (C), en
guanine (G). De informatie in elke gen wordt beschreven op basis van een specifieke code,
de zogenaamde genetische code (Fig. 2). De genetische code is een verzameling van drie
nucleotide-moleculen, codons genoemd.
Figuur 2. De genetische code. De afbeelding is afkomstig
uit the Science at a Distance 2005, Professor John Blamire
(http://www.brooklyn.cuny.edu/bc/ahp/SDV2.html).
Samenvatting voor de leek
167
Proteïnen bestaan uit aminozuur-moleculen. Deze worden gevormd als langgerekte
draden die als een bolletje bijelkaar worden gehouden. Proteïnen bevatten tussen de 50 en
zelfs meer dan 2000 aminozuren. Proteïnen die minder dan 50 aminozuren bevatten worden
peptiden genoemd. Proteïnen zijn essentieel voor alle organismen omdat alle functies in
een cel afhankelijk zijn van proteïnen en elke proteïne zijn eigen specifieke functie vervult
bij: de verplaatsing van cellen en organismen, de celdeling, de katalyse van alle
biochemische reacties, het transporteren van materialen in vloeistoffen, het activeren van
genen (deze proteïnen worden regulators genoemd) en de synthese/vorming van andere
proteïnen.
Proteïnen worden gevormd, zoals reeds beschreven, aan de hand van de informatie
die is vastgelegd in genen. Elke proteïne heeft zijn eigen unieke aminozuursamenstelling
die is vastgelegd in de genetische code. Omdat de genetische code uit een set van drie
nucleotide-moleculen, zogenaamde codons, bestaat, en DNA uit vier nucleotiden bestaat,
kunnen er 64 verschillende codons ontstaan. Omdat elk codon gerelateerd is aan een
aminozuur is er een overlap zodat sommige aminozuren worden gecodeert door meerdere
codons.
Wanneer er een signaal wordt afgegeven om een bepaald gen te activeren, expressie
genoemd, wordt de proteïne synthese gestart. De proteïne wordt gevormd in twee stappen:
1) transcriptie and 2) vertaling. Tijdens de transcriptie worden de codons gekopieerd naar
RNA (messenger RNA; mRNA). Vervolgens wordt deze mRNA kopie vertaald naar welke
aminozuren worden gevormd, in het vertalingsproces. Kortom, de mRNA draagt de
informatie over naar ribosomen, en deze vormen een ―molecuulmachine‖. In deze
―molecuulmachine‖ begint het vertaalproces; de informatie van de genen die naar mRNA is
gecopieerd wordt uitgelezen en resulteert uiteindelijk in de vorming van proteïnen.
Vervolgens kunnen deze gevormde proteïnen hun functie uitoefenen in de cel, waar ze voor
bedoeld zijn.
Bacteriocines
Als het gaat om microben, wordt vaak verwezen naar ―bacillen‖ en deze worden
als ―slecht‖ en als ziekteveroorzakers ervaren, maar er zijn ook microben die een nuttige
functie vervullen voor mensen en hun omgeving. Bijvoorbeeld melkzuurbacteria (LAB)
worden aangetroffen in melkprodukten. Deze LAB worden veel gebruikt bij de bereiding
van zuivelprodukten zoals kaas, melk en yoghurt, omdat deze de smaak, geur en structuur
geven aan het product. Bovendien produceren LAB en andere bacteria substanties,
bacteriocines genoemd, die voorkomen dat er andere ―slechte‖ bacteria groeien in
bijvoorbeeld zuivelproducten, waardoor ze een langere houdbaarheid hebben. Bacteriocines
zijn peptiden, die door bacteria worden geproduceerd en gelijke of sterk gerelateerde
bacteria vernietigen. Bacteriocines worden beschouwd als een kleine verzameling
antibiotica. Ze werden ontdekt door A Gratia in 1925. Vanaf dat moment is er een grote
verscheidenheid aan bacteriocines beschreven. Op dit moment hebben bacteriocines grote
Samenvatting voor de leek
168
waarde voor de medische en voedselindustrie omdat ze ―slechte‖ microorganismen
vernietigen. Daarom zouden ze als substituut voor antibiotica of andere
antimicrobiologische verbindingen kunnen dienen.
Bacteriocines worden in een cel geproduceerd als inactieve peptides die uit een
signaal component (leader peptide), dat wordt afgesplitst wanneer de peptide de cel verlaat
waarin het geproduceerd is, en een andere component, wat de actieve peptide vormt. Om
actieve peptiden te produceren moeten bacteriocines achtereenvolgens worden
geproduceerd, gemodificeerd, verwerkt (de signaalcomponent moet worden verwijderd) en
getranporteerd (de bacteriocine moet van binnen naar buiten de cel worden gebracht).
Daarnaast moet de producerende cel immuun zijn voor zijn eigen bacteriocine. Voor dit
hele process zijn een aantal genen verantwoordelijk. Om efficient te produceren moeten
deze genen dicht bij elkaar worden verzameld in de DNA. Een set genen die betrokken zijn
bij een specifiek proces wordt een cluster genoemd. Daarom wordt de set genen die
verantwoordelijk zijn voor de productie van actieve bacteriocines een bacteriocine-cluster
genoemd.
Streptococcus pneumoniae
Veel individuen dragen Streptococcus pneumoniae bacteria (zogenaamde
pneumococcus) in hun neus en keel en vaak veroorzaakt deze bacteria geen ziekte. De
bacteria kunnen worden overgedragen aan andere personen door druppels speeksel,
wanneer de drager hoest of niest. Vaak leidt dit niet tot een ziekte, maar minder weerzame
individuen kunnen een pneumokokkenziekte ontwikkelen. S. pneumoniae kan, afhankelijk
van welk lichaamsdeel geinfecteerd is, een reeks ziekten veroorzaken. De ziekten zijn
onder andere: sinusitis (infectie van de sinusses, otitis media (middenoorinfectie)),
bacteraemia (bacteria-invasie in het bloed), pneumonia (longontsteking), meningitis
(hersenvliesontsteking). Sommige bevolkingsgroepen lopen een verhoogd risico op
infectie, met name kinderen onder de 5 jaar, ouderen boven de 65 jaar, mensen met een
verzwakt immuunsysteem en chronisch zieken. Er zijn momenteel 90 verschillende typen
pneumokokken en er is geen vaccin dat tegen alle deze typen bescherming biedt. Er zijn
echter twee effectieve vaccins die bescherming bieden tegen de meest voorkomende typen
pneumokokken.
In dit proefschrift
Bacteriocines worden veelvuldig beschreven bij bacteriolische onderzoeken. Toch
is er wienig bekend over bacteriocines die geproduceerd worden door S. pneumoniae. Het
hoofddoel van dit proefschrift is het vinden en bestuderen van bacteriocines die
geproduceerd worden door pneumokokken en het identificeren van genen van de
pneumokokken die verantwoordelijk zijn voor de immuniteit tegen antibiotica.
Hoofdstuk 1 begint met een gedetailleerde beschrijving van de pneumokokken en
bacteriocines.
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169
In Hoofdstuk 2 wordt getracht bacteriocine-soortige clusters in DNA reeksen van
bepaalde S. pneumoniae typen te vinden. Negen bacteriocine-soortige clusters waren
geïdentificeerd, waarvan er twee reeds in eerdere onderzoeken waren beschreven; de
zogenaamde Blp (Pnc) en CibAB. Van de overige zeven nieuwe bacteriocine-soortige
clusters werden er twee geselecteerd voor verdere analyse; de zogenaamde pneumococcal
peptide of unknown function (ppu) cluster en pneumococcin cluster.
In Hoofdstuk 3 worden experimenten beschreven die bedoeld zijn om aan te
tonen dat de ppu actieve bacteriocines produceert. Er werden echter geen bacteriocines
gevonden die door dit cluster gecodeerd waren. Vervolgens worden experimenten
beschreven die de functie van ppu proberen aan te tonen. Het onderzoek toonde aan dat de
expressie van het ppu cluster wordt bepaald door twee regulators; een negatieve regulator,
CodY genaamd, en een positieve regulator, PpuR genaamd. Bovendien vonden we twee
nieuwe clusters die functioneel verbonden waren aan het ppu cluster. We ontdekten dat
deze drie clusters betrokken zijn bij stikstofmetabolisme in de pneumekokken.
Hoofdstuk 4 beschrijft voor het eerst dat het mogelijk is om, moeilijk te
verkrijgen, antimicrobiologische actieve bacteriocines van S. pneumoniae te produceren en
te modificeren.
Omdat S. pneumoniae geen bacteriocines produceerde die gecodeerd waren door
pneumococcin clusters, namelijk pneumococcin cluster A1 en A2, moesten we een andere
manier vinden om deze bacteriocines te produceren. We hebben de methode ―klonen‖
hiervoor gebruikt. Bij klonen worden een reeks genen gemodificeerd, dat wil zeggen
nucleotiden worden toegevoegd of verwijderd en/of de gekloonde reeks wordt in een
andere bacterie uitgezet dan waar het oorspronkelijk was geproduceerd. Om de productie
van pneumococcins A1 en A2 te simuleren, hebben we deze genen gemodificeerd in het
kloonproces. Dit resulteerde in pneumococcins die bestaan uit een signaal component
(leader sequence), van een al reeds bekend bacteriocine, namelijk nisin, en een propeptide
sequence. Deze gemodificeerde reeks bacteriocine genen, A1_1 en A2_2, werden in een de
Lactococcus lactis bacterie, uitgezet. Deze bacteria wordt veelvuldig gebruikt voor de
kaasproductie. Zoals reeds vermeld moeten bacteriocines worden geproduceerd,
gemodificeerd en getransporteerd, naar het medium buiten de cel, waarin de bacteria groeit,
om antimicrobiologisch actief te worden. Omdat pneumococcins A1 en A2 zodanig waren
gemodificeerd, dat ze de nisin signaalcomponent bezitten en dit component bepaalt welke
specifieke proteïnen voor de modificatie en transport worden, hebben we deze uitgezet in
Lactococcus lactis. Lactococcus lactis is een bacterie dat proteïnen kan aanmaken die nisin
kunnen modificeren en transporteren. Daarna hebben we de bacterie verwijderd uit het
medium en hebben we de antimicrobiologische activitiet onderzocht van de substantie,
pneumococcins A1_1 en A2_1, tegen andere bacteria. We hebben aangetoond dat deze
bacteriocines in staat zijn om één bacterie aan te vallen en te vernietigen. Dit is een groot
succes en het is nooit eerder aangetoond dat het mogelijk is om met behulp van een ―nisin
specifieke productie machine‖, actieve bacteriociones te produceren en modificeren. Dit
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170
idee zou gebruikt kunnen worden in de farmaceutische industrie om antibiotica te
produceren.
Hoofdstuk 5 beschrijft nieuwe weerstandsmechanismen van S. pneumoniae ten
aanzien van drie verschillende substanties, zoals nisin, LL-37 en bacitracin die in staat zijn
om bacteria te vernietigen. Het is van groot belang om inzicht in deze
weerstandsmechanismen van bacteria te krijgen, teneinde deze te vernietigen of te
beheersen, bij het gebruik van antibiotica. We hebben hiervoor de pneumokokken met de
drie verschillende substanties laten ontwikkelen. Vervolgens hebben we de aangetaste
pneumokokken geïsoleerd en onderzocht op DNA, door middel van een bepaalde
methode, DNA microarray genoemd. We hebben naar de reactie van alle genen van S.
pneumoniae (~2200 genen) gekeken, in het bijzonder of de genen actief (expressief) of juist
uit gezet werden. Op basis van dit onderzoek zijn een aantal genen gekozen voor verder
onderzoek. Deze genen zijn betrokken bij de productie van proteïnen, die een rol zouden
kunnen spelen bij de weerstandsmechanismen van de bacteria tegen de drie geteste
substanties. Vervolgens hebben we de producerende genen gekloond om te bepalen welke
proteïnen deze rol zouden kunnen vervullen. Hieruit voortvloeiend hebben we een aantal
nieuwe proteïnen geïdentificeerd die transporters coderen, die worden geassocieerd met de
weerstand van S. pneumonia tegen de geteste substanties. Deze transporters zijn SP0785-
0787, SP0912-0913 en SP1715. Daarnaast hebben we aangetoond dat een nieuwe regulator,
SP1714, twee van deze transporters reguleerde. Alles bij elkaar geven deze resultaten
belangrijke inzichten in de weestandsmechanismen van S. pneumoniae.
Dankwoord/Acknowledgements
171
Acknowledgements/Dankwoord
I think that the love for science and the drive to do PhD brought me across this PhD
position at MolGen department in Groningen. I took a chance in the opportunity given to
me to become a scientist and to express my passion for science, which soon became my
whole live.
In what we do, we are never alone and there are always people who, consciously or not,
have a certain impact on our live. Like the Red Queen Hypothesis of evolution, mentioned
in the statements (stellingen), indicates that ―the natural selection will arise from co-
evolutionary interactions with other species, not from interactions with the environment‖.
Consequently, I think my change came through the interactions with people who I met
during my ‗dutchy times‘. As I believe that each change is supposed to be good for us, I
would like to take an opportunity and thank people who participated in ―my evolution‖.
I would like to thank all people, who made it possible for me to do my PhD and without
whom this book would never see a day light.
Prof. Jacek Bardowski, thank you for informing me about the PhD position at MolGen.
Prof. Oscar Kuipers, my promotor, thank you for giving me the opportunity to join your
group, to trust me that I could handle the project, which focused on the unpredictable and
naughty bacteriocins of pneumococcus, supporting and having a confidence in my research
idea to produce bacteriocins of pneumococcus via nisin production machinery, giving me
your intellectual and scientific support, encouragement, guidance and for understanding.
Dr. Jetta Bijlsma, my daily supervisor and co-promotor, thank you for your invaluable
scientific input, support, advises and discussions, for leading me into the right directions in
this PhD project, and for your patience and time spent in particular on the beginning when I
needed an extra support, and for invaluable editing assistance of this thesis.
As it is said, beginnings are never easy, so was my beginning in the new country and new
position. But it is people who make a place and at this beginning, there were those, who
gave me a helpful hand and made my time happy and wonderful. Naomi (I ―inherited‖ your
desk place when you left, it‘s a pity that it happened so quickly), I enjoyed a lot of our
conversations; thank you for your time, support and for many more. Rasmus and Anton, my
first office-mates, thank you for the introduction to the lab life and your scientific help.
Anton thanks for the introduction to Dutch language and for the first, second and third
and…many more, Dutch grammar explanations.
Dankwoord / Acknowledgements
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Tomas, my strep-office-lab-mate; I am particularly happy that our paths of live have
crossed also because you believed in me when I couldn‘t and with your unconventional
sense of humor you always made me laugh. Many thanks for the scientific and nonscientific
talks and tips! Sulman, my strep-office-lab-mate, thank you for being a great colleague, for
introducing me to the Pakistani cuisine and for helping me to make an important decisions
whether to go to Australia. Rutger, thank you for being a friend and for many small and
long talks! Jacek and Rick, I was lucky to have you as my collaborators. Thanks for your
invaluable input on making chapter 4 of this thesis!
A very special ―thank you word‖ goes to my paranimfen: Bogusia and Tomas. Thank you
for being there for me on this special day and for your crucial help in the preparations for
this defence! Bogusia, good luck with your dissertation, I am sure you will do a great job.
I would like to acknowledge Emma and Mirelle for all the instances, in which your
assistance helped me along the way. Furthermore, Harma, Anne H., Mozes, Peter, Anne de
J. and Siger, thank you for all of your computer and technical assistance. Very special
thanks go out to my students, Rachel, Corina, Celia and Amaya, thank you for the help in
the lab.
I am grateful to my colleagues, who made Groningen a very special place over all those
years, for providing a stimulating and fun environment to work and for any other help.
Many thanks go to Akos, Aleksandra, Anja, Aldert, Araz, Ana, Bogusia, Chris, Evert Jan,
Ganesh, Gierbe, Hein, Helga, Imke, João, Jolanda, Jan, Jan Willem, Kim, Maria, Patricia,
Robyn, Rustem, Robèr, Reindert, Sandra, Sacha, Sierd, Tom, Tariq, Wiep Klaas and Wout.
I would also like to thank my friends, Iwona, Kasia and Asia K. (dziewczyny, dzięki za
waszą przyjaźń i wsparcie!), Sacha G. (thank you for being my friend), Andrzej (długo
myślałam co mam Ci napisać, ale poprostu brak mi słów, dziękuje!), Marta (dzięki za
wprowadzenie mnie w tajniki holenderskiego), Asia Kapłon and Magda (powodzenia z
waszymi dr), and Liana, Verena, Madelon and Roel, who shared my time spent outside the
lab, without you guys it would never be the same.
I would like to show my gratitude to the entire Family Koehorst, Annemarie, Hennie,
Maureen, Brigitte, Charlotte, Jeroen, Bas, Hans, Rachel, Celine, Boet, Jasper, Mats and last
but not least Naut. Thank you for being there for me, when I needed it and for being my
surrogate family for all those years and for years to come.
I owe my deepest gratitude to my parents, Ania and Waldek. Dziękuje za wasze bezcenne,
emocjonalne i moralne, wsparcie, którym obdarzaliście mnie nieprzerwanie przez całe moje
życie, a w szczególnosci podczas pracy nad tym doktoratem. Ten doktorat z pewnością
Dankwoord/Acknowledgements
173
nigdy nieujżałby światła dziennego bez waszej pomocy. Dziękuje że stworzyliście dla mnie
środowisko, w którym podążanie tą ścieżką życiową wydawało się takie naturalne.
Moim braciszkom, Adam i Mariusz, dzięki za to, że zawsze mogę na was liczyć.
Above all, I would like to acknowledge the tremendous sacrifices that my husband Bert
made to ensure that I will complete this dissertation. I am thankful for your endless
encouragements throughout this entire journey. Without you, I would have struggled to find
the inspiration and motivation needed to complete this thesis. Thank you for being there for
me and for ...... translating my nederlandse samenvatting voor de leek.
Finally, I am grateful that I can close this chapter of my live,
Joanna
Notes
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