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

Reference List

1. Adamou, J. E., J. H. Heinrichs, A. L. Erwin, W. Walsh, T. Gayle, M. Dormitzer, R. Dagan, Y. A.

Brewah, P. Barren, R. Lathigra, S. Langermann, S. Koenig, and S. Johnson. 2001. Identification and characterization of a novel family of pneumococcal proteins that are protective against sepsis. Infect.

Immun. 69:949-958.

2. Aizenman, E., H. Engelberg-Kulka, and G. Glaser. 1996. An Escherichia coli chromosomal "addiction module" regulated by guanosine 3',5'-bispyrophosphate: a model for programmed

bacterial cell death. Proc. Natl. Acad. Sci. U. S. A 93:6059-6063. 3. Allgaier, H., G. Jung, R. G. Werner, U. Schneider, and H. Zahner. 1986. Epidermin: sequencing of a

heterodetic tetracyclic 21-peptide amide antibiotic. Eur. J. Biochem. 160:9-22.

4. Altena, K., A. Guder, C. Cramer, and G. Bierbaum. 2000. Biosynthesis of the lantibiotic mersacidin: organization of a type B lantibiotic gene cluster. Appl. Environ. Microbiol. 66:2565-2571.

5. Aso, Y., H. Koga, T. Sashihara, J. Nagao, Y. Kanemasa, J. Nakayama, and K. Sonomoto. 2005.

Description of complete DNA sequence of two plasmids from the nukacin ISK-1 producer, Staphylococcus warneri ISK-1. Plasmid 53:164-178.

6. Aso, Y., K. Okuda, J. Nagao, Y. Kanemasa, P. N. Thi Bich, H. Koga, K. Shioya, T. Sashihara, J.

Nakayama, and K. Sonomoto. 2005. A novel type of immunity protein, NukH, for the lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1. Biosci. Biotechnol. Biochem. 69:1403-

1410.

7. Aso, Y., K. Okuda, J. Nagao, Y. Kanemasa, P. N. Thi Bich, H. Koga, K. Shioya, T. Sashihara, J. Nakayama, and K. Sonomoto. 2005. A novel type of immunity protein, NukH, for the lantibiotic

nukacin ISK-1 produced by Staphylococcus warneri ISK-1. Biosci. Biotechnol. Biochem. 69:1403-

1410. 8. Aso, Y., T. Sashihara, J. Nagao, Y. Kanemasa, H. Koga, T. Hashimoto, T. Higuchi, A. Adachi, H.

Nomiyama, A. Ishizaki, J. Nakayama, and K. Sonomoto. 2004. Characterization of a gene cluster of

Staphylococcus warneri ISK-1 encoding the biosynthesis of and immunity to the lantibiotic, nukacin

ISK-1. Biosci. Biotechnol. Biochem. 68:1663-1671.

9. Aucher, W., V. Simonet, C. Fremaux, K. Dalet, L. Simon, Y. Cenatiempo, J. Frere, and J. M.

Berjeaud. 2004. Differences in mesentericin secretion systems from two Leuconostoc strains. FEMS Microbiol Lett. 232:15-22.

10. Audouy, S. A., S. van Selm, M. L. van Roosmalen, E. Post, R. Kanninga, J. Neef, S. Estevao, E. E.

Nieuwenhuis, P. V. Adrian, K. Leenhouts, and P. W. Hermans. 2007. Development of lactococcal GEM-based pneumococcal vaccines. Vaccine 25:2497-2506.

11. Auranen, K., J. Mehtala, A. Tanskanen, and S. Kaltoft. 2010. Between-strain competition in

acquisition and clearance of pneumococcal carriage--epidemiologic evidence from a longitudinal study of day-care children. Am. J. Epidemiol. 171:169-176.

12. Avery, O. T., C. M. Macleod, and M. McCarty. 1944. Studies on the chemical nature of the

substance inducing transformation of pneumococcal types. Induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus type III. Mol. Med 1:344-365.

13. Axelsson, L. and A. Holck. 1995. The genes involved in production of and immunity to sakacin A, a

bacteriocin from Lactobacillus sake Lb706. J. Bacteriol. 177:2125-2137. 14. Axelsson, L., A. Holck, S. E. Birkeland, T. Aukrust, and H. Blom. 1993. Cloning and nucleotide

sequence of a gene from Lactobacillus sake Lb706 necessary for sakacin A production and

immunity. Appl. Environ. Microbiol 59:2868-2875. 15. Aymerich, T., H. Holo, L. S. Havarstein, M. Hugas, M. Garriga, and I. F. Nes. 1996. Biochemical

and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial

bacteriocin in the pediocin family of bacteriocins. Appl. Environ. Microbiol 62:1676-1682. 16. Azevedo, E. C., E. M. Rios, K. Fukushima, and G. M. Campos-Takaki. 1993. Bacitracin production

by a new strain of Bacillus subtilis. Extraction, purification, and characterization. Appl Biochem.

Biotechnol 42:1-7. 17. Bagnoli, F., M. Moschioni, C. Donati, V. Dimitrovska, I. Ferlenghi, C. Facciotti, A. Muzzi, F.

Giusti, C. Emolo, A. Sinisi, M. Hilleringmann, W. Pansegrau, S. Censini, R. Rappuoli, A. Covacci,

V. Masignani, and M. A. Barocchi. 2008. A second pilus type in Streptococcus pneumoniae is

prevalent in emerging serotypes and mediates adhesion to host cells. J Bacteriol. 190:5480-5492.

18. Balla, E., L. M. Dicks, T. M. Du, M. J. Van Der Merwe, and W. H. Holzapfel. 2000.

Characterization and cloning of the genes encoding enterocin 1071A and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071. Appl. Environ. Microbiol

66:1298-1304.

References

140

19. Bals, R. 2000. Epithelial antimicrobial peptides in host defense against infection. Respir. Res. 1:141-

150.

20. Bals, R. and J. M. Wilson. 2003. Cathelicidins--a family of multifunctional antimicrobial peptides. Cell Mol. Life Sci. 60:711-720.

21. Barocchi, M. A., J. Ries, X. Zogaj, C. Hemsley, B. Albiger, A. Kanth, S. Dahlberg, J. Fernebro, M.

Moschioni, V. Masignani, K. Hultenby, A. R. Taddei, K. Beiter, F. Wartha, A. von Euler, A. Covacci, D. W. Holden, S. Normark, R. Rappuoli, and B. Henriques-Normark. 2006. A

pneumococcal pilus influences virulence and host inflammatory responses. Proc. Natl. Acad. Sci. U.

S. A 103:2857-2862. 22. Battig, P., L. J. Hathaway, S. Hofer, and K. Muhlemann. 2006. Serotype-specific invasiveness and

colonization prevalence in Streptococcus pneumoniae correlate with the lag phase during in vitro

growth. Microbes. Infect. 8:2612-2617. 23. Becker, P., R. Hakenbeck, and B. Henrich. 2009. An ABC transporter of Streptococcus pneumoniae

involved in susceptibility to vancoresmycin and bacitracin. Antimicrob. Agents Chemother.

53:2034-2041. 24. Begley, M., P. D. Cotter, C. Hill, and R. P. Ross. 2009. Identification of a novel two-peptide

lantibiotic, lichenicidin, following rational genome mining for LanM proteins. Appl. Environ.

Microbiol. 75:5451-5460. 25. Belitsky, B. R., M. C. Gustafsson, A. L. Sonenshein, and W. C. Von. 1997. An lrp-like gene of

Bacillus subtilis involved in branched-chain amino acid transport. J Bacteriol. 179:5448-5457.

26. Belitsky, B. R. and A. L. Sonenshein. 2008. Genetic and biochemical analysis of CodY-binding sites in Bacillus subtilis. J. Bacteriol. 190:1224-1236.

27. Bergmann, S., M. Rohde, G. S. Chhatwal, and S. Hammerschmidt. 2001. Alpha-Enolase of

Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface. Mol. Microbiol. 40:1273-1287.

28. Bernard, R., A. Guiseppi, M. Chippaux, M. Foglino, and F. Denizot. 2007. Resistance to bacitracin

in Bacillus subtilis: unexpected requirement of the BceAB ABC transporter in the control of

expression of its own structural genes. J. Bacteriol. 189:8636-8642.

29. Bernard, R., P. Joseph, A. Guiseppi, M. Chippaux, and F. Denizot. 2003. YtsCD and YwoA, two

independent systems that confer bacitracin resistance to Bacillus subtilis. FEMS Microbiol Lett. 228:93-97.

30. Berry, A. M., A. D. Ogunniyi, D. C. Miller, and J. C. Paton. 1999. Comparative virulence of

Streptococcus pneumoniae strains with insertion-duplication, point, and deletion mutations in the pneumolysin gene. Infect. Immun. 67:981-985.

31. Berry, A. M. and J. C. Paton. 1996. Sequence heterogeneity of PsaA, a 37-kilodalton putative

adhesin essential for virulence of Streptococcus pneumoniae. Infect. Immun. 64:5255-5262. 32. Berry, A. M., J. Yother, D. E. Briles, D. Hansman, and J. C. Paton. 1989. Reduced virulence of a

defined pneumolysin-negative mutant of Streptococcus pneumoniae. Infect. Immun. 57:2037-2042.

33. Bethe, G., R. Nau, A. Wellmer, R. Hakenbeck, R. R. Reinert, H. P. Heinz, and G. Zysk. 2001. The cell wall-associated serine protease PrtA: a highly conserved virulence factor of Streptococcus

pneumoniae. FEMS Microbiol Lett. 205:99-104.

34. Bierbaum, G., H. Brotz, K. P. Koller, and H. G. Sahl. 1995. Cloning, sequencing and production of the lantibiotic mersacidin. FEMS Microbiol Lett. 127:121-126.

35. Bierbaum, G., C. Szekat, M. Josten, C. Heidrich, C. Kempter, G. Jung, and H. G. Sahl. 1996.

Engineering of a novel thioether bridge and role of modified residues in the lantibiotic Pep5. Appl. Environ. Microbiol 62:385-392.

36. Biet, F., J. M. Berjeaud, R. W. Worobo, Y. Cenatiempo, and C. Fremaux. 1998. Heterologous

expression of the bacteriocin mesentericin Y105 using the dedicated transport system and the general secretion pathway. Microbiology 144 ( Pt 10):2845-2854.

37. Blom, H., T. Katla, A. Holck, K. Sletten, L. Axelsson, and H. Holo. 1999. Characterization,

production, and purification of leucocin H, a two-peptide bacteriocin from Leuconostoc MF215B. Curr. Microbiol 39:43-48.

38. Blomberg, C., J. Dagerhamn, S. Dahlberg, S. Browall, J. Fernebro, B. Albiger, E. Morfeldt, S. Normark, and B. Henriques-Normark. 2009. Pattern of accessory regions and invasive disease

potential in Streptococcus pneumoniae. J. Infect. Dis. 199:1032-1042.

39. Boakes, S. and S. Wadman. 2008. The Therapeutic Potential of Lantibiotics. Innovations in Pharmaceutical Technology 27:22-25.

40. Bottiger, T., T. Schneider, B. Martinez, H. G. Sahl, and I. Wiedemann. 2009. Influence of Ca(2+)

ions on the activity of lantibiotics containing a mersacidin-like lipid II binding motif. Appl. Environ. Microbiol 75:4427-4434.

References

141

41. Bower, C. K., M. K. Bothwell, and J. McGuire. 2001. Lantibiotics as surface active agents for

biomedical applications. Colloids and Surfaces B: Biointerfaces 22:259-265.

42. Brandenburg, L. O., D. Varoga, N. Nicolaeva, S. L. Leib, R. Podschun, C. J. Wruck, H. Wilms, R. Lucius, and T. Pufe. 2009. Expression and regulation of antimicrobial peptide rCRAMP after

bacterial infection in primary rat meningeal cells. J. Neuroimmunol. 217:55-64.

43. Breukink, E. 2006. A lesson in efficient killing from two-component lantibiotics. Mol. Microbiol 61:271-273.

44. Breukink, E. and B. de Kruijff. 1999. The lantibiotic nisin, a special case or not? Biochim. Biophys.

Acta 1462:223-234. 45. Breukink, E., H. E. van Heusden, P. J. Vollmerhaus, E. Swiezewska, L. Brunner, S. Walker, A. J.

Heck, and B. de Kruijff. 2003. Lipid II is an intrinsic component of the pore induced by nisin in

bacterial membranes. J. Biol. Chem. 278:19898-19903. 46. Breukink, E., I. Wiedemann, C. van Kraaij, O. P. Kuipers, H. Sahl, and B. de Kruijff. 1999. Use of

the cell wall precursor lipid II by a pore-forming peptide antibiotic. Science 286:2361-2364.

47. Brogden, K. A. 2005. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3:238-250.

48. Brotz, H., G. Bierbaum, K. Leopold, P. E. Reynolds, and H. G. Sahl. 1998. The lantibiotic

mersacidin inhibits peptidoglycan synthesis by targeting lipid II. Antimicrob. Agents Chemother. 42:154-160.

49. Brotz, H., M. Josten, I. Wiedemann, U. Schneider, F. Götz, G. Bierbaum, and H. G. Sahl. 1998. Role

of lipid-bound peptidoglycan precursors in the formation of pores by nisin, epidermin and other lantibiotics. Mol. Microbiol 30:317-327.

50. Brown, J. S., S. M. Gilliland, J. Ruiz-Albert, and D. W. Holden. 2002. Characterization of pit, a

Streptococcus pneumoniae iron uptake ABC transporter. Infect. Immun. 70:4389-4398. 51. Bruckner, R., M. Nuhn, P. Reichmann, B. Weber, and R. Hakenbeck. 2004. Mosaic genes and

mosaic chromosomes-genomic variation in Streptococcus pneumoniae. Int. J. Med. Microbiol.

294:157-168.

52. Brueggemann, A. B., D. T. Griffiths, E. Meats, T. Peto, D. W. Crook, and B. G. Spratt. 2003. Clonal

relationships between invasive and carriage Streptococcus pneumoniae and serotype- and clone-

specific differences in invasive disease potential. J. Infect. Dis. 187:1424-1432. 53. Brueggemann, A. B., T. E. Peto, D. W. Crook, J. C. Butler, K. G. Kristinsson, and B. G. Spratt.

2004. Temporal and geographic stability of the serogroup-specific invasive disease potential of

Streptococcus pneumoniae in children. J. Infect. Dis. 190:1203-1211. 54. Brugger, S. D., L. J. Hathaway, and K. Muhlemann. 2009. Detection of Streptococcus pneumoniae

strain cocolonization in the nasopharynx. J. Clin. Microbiol. 47:1750-1756.

55. Brurberg, M. B., I. F. Nes, and V. G. Eijsink. 1997. Pheromone-induced production of antimicrobial peptides in Lactobacillus. Mol. Microbiol 26:347-360.

56. Buchman, G. W., S. Banerjee, and J. N. Hansen. 1988. Structure, expression, and evolution of a

gene encoding the precursor of nisin, a small protein antibiotic. J. Biol. Chem. 263:16260-16266. 57. Buist, G., A. Steen, J. Kok, and O. P. Kuipers. 2008. LysM, a widely distributed protein motif for

binding to (peptido)glycans. Mol. Microbiol 68:838-847.

58. Canvin, J. R., A. P. Marvin, M. Sivakumaran, J. C. Paton, G. J. Boulnois, P. W. Andrew, and T. J. Mitchell. 1995. The role of pneumolysin and autolysin in the pathology of pneumonia and

septicemia in mice infected with a type 2 pneumococcus. J. Infect. Dis. 172:119-123.

59. Casaus, P., T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernandez, and H. Holo. 1997. Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A.

Microbiology 143 (Pt 7):2287-2294.

60. Caufield, P. W., N. K. Childers, D. N. Allen, and J. B. Hansen. 1985. Distinct bacteriocin groups correlate with different groups of Streptococcus mutans plasmids. Infect. Immun. 48:51-56.

61. Chakicherla, A. and J. N. Hansen. 1995. Role of the leader and structural regions of prelantibiotic

peptides as assessed by expressing nisin-subtilin chimeras in Bacillus subtilis 168, and characterization of their physical, chemical, and antimicrobial properties. J. Biol. Chem. 270:23533-

23539. 62. Chambellon, E. and M. Yvon. 2003. CodY-regulated aminotransferases AraT and BcaT play a major

role in the growth of Lactococcus lactis in milk by regulating the intracellular pool of amino acids.

Appl. Environ. Microbiol. 69:3061-3068. 63. Chan, P. F., K. M. O'Dwyer, L. M. Palmer, J. D. Ambrad, K. A. Ingraham, C. So, M. A. Lonetto, S.

Biswas, M. Rosenberg, D. J. Holmes, and M. Zalacain. 2003. Characterization of a novel fucose-

regulated promoter (PfcsK) suitable for gene essentiality and antibacterial mode-of-action studies in Streptococcus pneumoniae. J. Bacteriol. 185:2051-2058.

References

142

64. Chang, F. N., C. J. Sih, and B. Weisblum. 1966. Lincomycin, an inhibitor of aminoacyl sRNA

binding to ribosomes. Proc. Natl. Acad. Sci. U. S. A 55:431-438.

65. Charpentier, E. and E. Tuomanen. 2000. Mechanisms of antibiotic resistance and tolerance in Streptococcus pneumoniae. Microbes. Infect. 2:1855-1864.

66. Chatterjee, C., G. C. Patton, L. Cooper, M. Paul, and W. A. van der Donk. 2006. Engineering

dehydro amino acids and thioethers into peptides using lacticin 481 synthetase. Chem. Biol. 13:1109-1117.

67. Chatterjee, C., M. Paul, L. Xie, and W. A. van der Donk. 2005. Biosynthesis and mode of action of

lantibiotics. Chem. Rev. 105:633-684. 68. Chatterjee, S., D. K. Chatterjee, R. H. Jani, J. Blumbach, B. N. Ganguli, N. Klesel, M. Limbert, and

G. Seibert. 1992. Mersacidin, a new antibiotic from Bacillus. In vitro and in vivo antibacterial

activity. J. Antibiot. (Tokyo) 45:839-845. 69. Chatterjee, S., S. Chatterjee, S. J. Lad, M. S. Phansalkar, R. H. Rupp, B. N. Ganguli, H. W.

Fehlhaber, and H. Kogler. 1992. Mersacidin, a new antibiotic from Bacillus. Fermentation, isolation,

purification and chemical characterization. J. Antibiot. (Tokyo) 45:832-838. 70. Cheigh, C. I. and Y. R. Pyun. 2005. Nisin biosynthesis and its properties. Biotechnol. Lett. 27:1641-

1648.

71. Chen, P., J. Novak, M. Kirk, S. Barnes, F. Qi, and P. W. Caufield. 1998. Structure-activity study of the lantibiotic mutacin II from Streptococcus mutans T8 by a gene replacement strategy. Appl.

Environ. Microbiol. 64:2335-2340.

72. Chen, P., F. Qi, J. Novak, and P. W. Caufield. 1999. The specific genes for lantibiotic mutacin II biosynthesis in Streptococcus mutans T8 are clustered and can be transferred en bloc. Appl. Environ.

Microbiol. 65:1356-1360.

73. Chikindas, M. L., J. Novak, A. J. Driessen, W. N. Konings, K. M. Schilling, and P. W. Caufield. 1995. Mutacin II, a bactericidal antibiotic from Streptococcus mutans. Antimicrob. Agents

Chemother. 39:2656-2660.

74. Chung, Y. J. and J. N. Hansen. 1992. Determination of the sequence of spaE and identification of a

promoter in the subtilin (spa) operon in Bacillus subtilis. J. Bacteriol. 174:6699-6702.

75. Cintas, L. M., P. Casaus, L. S. Havarstein, P. E. Hernandez, and I. F. Nes. 1997. Biochemical and

genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum. Appl. Environ. Microbiol 63:4321-4330.

76. Cintas, L. M., P. Casaus, H. Holo, P. E. Hernandez, I. F. Nes, and L. S. Havarstein. 1998. Enterocins

L50A and L50B, two novel bacteriocins from Enterococcus faecium L50, are related to staphylococcal hemolysins. J. Bacteriol. 180:1988-1994.

77. Clarke, S. 1992. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues.

Annu. Rev. Biochem. 61:355-386. 78. Claverys, J. P. and L. S. Havarstein. 2002. Extracellular-peptide control of competence for genetic

transformation in Streptococcus pneumoniae. Front Biosci. 7:d1798-d1814.

79. Claverys, J. P. and L. S. Havarstein. 2007. Cannibalism and fratricide: mechanisms and raisons d'etre. Nat Rev Microbiol 5:219-229.

80. Claverys, J. P., B. Martin, and L. S. Havarstein. 2007. Competence-induced fratricide in

streptococci. Mol. Microbiol 64:1423-1433. 81. Claverys, J. P., B. Martin, and P. Polard. 2009. The genetic transformation machinery: composition,

localization, and mechanism. FEMS Microbiol Rev 33:643-656.

82. Coburn, P. S., L. E. Hancock, M. C. Booth, and M. S. Gilmore. 1999. A novel means of self-protection, unrelated to toxin activation, confers immunity to the bactericidal effects of the

Enterococcus faecalis cytolysin. Infect. Immun. 67:3339-3347.

83. Coburn, P. S., C. M. Pillar, B. D. Jett, W. Haas, and M. S. Gilmore. 2004. Enterococcus faecalis senses target cells and in response expresses cytolysin. Science 306:2270-2272.

84. Coffey, T. J., M. C. Enright, M. Daniels, J. K. Morona, R. Morona, W. Hryniewicz, J. C. Paton, and

B. G. Spratt. 1998. Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae. Mol.

Microbiol. 27:73-83. 85. Coffey, T. J., M. C. Enright, M. Daniels, P. Wilkinson, S. Berron, A. Fenoll, and B. G. Spratt. 1998.

Serotype 19A variants of the Spanish serotype 23F multiresistant clone of Streptococcus

pneumoniae. Microb. Drug Resist. 4:51-55. 86. Cookson, A. L., S. J. Noel, W. J. Kelly, and G. T. Attwood. 2004. The use of PCR for the

identification and characterisation of bacteriocin genes from bacterial strains isolated from rumen or

caecal contents of cattle and sheep. FEMS Microbiol Ecol. 48:199-207.

References

143

87. Cornut, G., C. Fortin, and D. Soulieres. 2008. Antineoplastic properties of bacteriocins: revisiting

potential active agents. Am. J Clin. Oncol. 31:399-404.

88. Corso, A., E. P. Severina, V. F. Petruk, Y. R. Mauriz, and A. Tomasz. 1998. Molecular characterization of penicillin-resistant Streptococcus pneumoniae isolates causing respiratory disease

in the United States. Microb. Drug Resist. 4:325-337.

89. Cotter, P. D., C. Hill, and R. P. Ross. 2005. Bacterial lantibiotics: strategies to improve therapeutic potential. Curr. Protein Pept. Sci. 6:61-75.

90. Cotter, P. D., C. Hill, and R. P. Ross. 2005. Bacteriocins: developing innate immunity for food. Nat

Rev Microbiol 3:777-788. 91. Cox, C. R., P. S. Coburn, and M. S. Gilmore. 2005. Enterococcal cytolysin: a novel two component

peptide system that serves as a bacterial defense against eukaryotic and prokaryotic cells. Curr.

Protein Pept. Sci. 6:77-84. 92. Croucher, N. J., D. Walker, P. Romero, N. Lennard, G. K. Paterson, N. C. Bason, A. M. Mitchell,

M. A. Quail, P. W. Andrew, J. Parkhill, S. D. Bentley, and T. J. Mitchell. 2009. Role of conjugative

elements in the evolution of the multidrug-resistant pandemic clone Streptococcus pneumoniae Spain23F ST81. J. Bacteriol. 191:1480-1489.

93. Dagan, R. and K. P. Klugman. 2008. Impact of conjugate pneumococcal vaccines on antibiotic

resistance. Lancet Infect. Dis. 8:785-795. 94. Dagerhamn, J., C. Blomberg, S. Browall, K. Sjostrom, E. Morfeldt, and B. Henriques-Normark.

2008. Determination of accessory gene patterns predicts the same relatedness among strains of

Streptococcus pneumoniae as sequencing of housekeeping genes does and represents a novel approach in molecular epidemiology. J. Clin. Microbiol. 46:863-868.

95. Dalet, K., Y. Cenatiempo, P. Cossart, and Y. Hechard. 2001. A sigma(54)-dependent PTS permease

of the mannose family is responsible for sensitivity of Listeria monocytogenes to mesentericin Y105. Microbiology 147:3263-3269.

96. Datta, V., S. M. Myskowski, L. A. Kwinn, D. N. Chiem, N. Varki, R. G. Kansal, M. Kotb, and V.

Nizet. 2005. Mutational analysis of the group A streptococcal operon encoding streptolysin S and its

virulence role in invasive infection. Mol. Microbiol 56:681-695.

97. Dawid, S., A. M. Roche, and J. N. Weiser. 2007. The blp bacteriocins of Streptococcus pneumoniae

mediate intraspecies competition both in vitro and in vivo. Infect. Immun. 75:443-451. 98. Dawid, S., M. E. Sebert, and J. N. Weiser. 2009. Bacteriocin activity of Streptococcus pneumoniae

is controlled by the serine protease HtrA via posttranscriptional regulation. J. Bacteriol. 191:1509-

1518. 99. de Jong, A., S. A. van Hijum, J. J. Bijlsma, J. Kok, and O. P. Kuipers. 2006. BAGEL: a web-based

bacteriocin genome mining tool. Nucleic Acids Res. 34:W273-W279.

100. de Ruyter, P. G., O. P. Kuipers, M. M. Beerthuyzen, I. van Alen-Boerrigter, and W. M. de Vos. 1996. Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis. J. Bacteriol.

178:3434-3439.

101. de Ruyter, P. G., O. P. Kuipers, and W. M. de Vos. 1996. Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl. Environ. Microbiol. 62:3662-3667.

102. de Saizieu, A., C. Gardes, N. Flint, C. Wagner, M. Kamber, T. J. Mitchell, W. Keck, K. E. Amrein,

and R. Lange. 2000. Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide. J. Bacteriol. 182:4696-4703.

103. de Smet, K. and R. Contreras. 2005. Human antimicrobial peptides: defensins, cathelicidins and

histatins. Biotechnol. Lett. 27:1337-1347. 104. de Vos, W. M., J. W. Mulders, R. J. Siezen, J. Hugenholtz, and O. P. Kuipers. 1993. Properties of

nisin Z and distribution of its gene, nisZ, in Lactococcus lactis. Appl. Environ. Microbiol 59:213-

218. 105. Delves-Broughton J. 2008. Nisin and its uses as a food preservative. Food technology 44:100-117.

106. Delves-Broughton, J., P. Blackburn, R. J. Evans, and J. Hugenholtz. 1996. Applications of the

bacteriocin, nisin. Antonie Van Leeuwenhoek 69:193-202. 107. den Hengst, C. D., P. Curley, R. Larsen, G. Buist, A. Nauta, D. vanSinderen, O. P. Kuipers, and J.

Kok. 2005. Probing direct interactions between CodY and the oppD promoter of Lactococcus lactis. J. Bacteriol. 187:512-521.

108. den Hengst, C. D., S. A. van Hijum, J. M. Geurts, A. Nauta, J. Kok, and O. P. Kuipers. 2005. The

Lactococcus lactis CodY regulon: identification of a conserved cis-regulatory element. J. Biol. Chem. 280:34332-34342.

109. Diacovich, L. and J. P. Gorvel. 2010. Bacterial manipulation of innate immunity to promote

infection. Nat. Rev. Microbiol. 8:117-128.

References

144

110. Diamond, G., N. Beckloff, A. Weinberg, and K. O. Kisich. 2009. The roles of antimicrobial peptides

in innate host defense. Curr. Pharm. Des 15:2377-2392.

111. Diep, D. B., L. Axelsson, C. Grefsli, and I. F. Nes. 2000. The synthesis of the bacteriocin sakacin A is a temperature-sensitive process regulated by a pheromone peptide through a three-component

regulatory system. Microbiology 146 ( Pt 9):2155-2160.

112. Diep, D. B., L. S. Havarstein, and I. F. Nes. 1996. Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11. J. Bacteriol. 178:4472-4483.

113. Diep, D. B., O. Johnsborg, P. A. Risoen, and I. F. Nes. 2001. Evidence for dual functionality of the

operon plnABCD in the regulation of bacteriocin production in Lactobacillus plantarum. Mol. Microbiol 41:633-644.

114. Diep, D. B. and I. F. Nes. 2002. Ribosomally synthesized antibacterial peptides in Gram positive

bacteria. Curr. Drug Targets. 3:107-122. 115. Diep, D. B., M. Skaugen, Z. Salehian, H. Holo, and I. F. Nes. 2007. Common mechanisms of target

cell recognition and immunity for class II bacteriocins. Proc. Natl. Acad. Sci. U. S. A 104:2384-

2389. 116. Ding, F., P. Tang, M. H. Hsu, P. Cui, S. Hu, J. Yu, and C. H. Chiu. 2009. Genome evolution driven

by host adaptations results in a more virulent and antimicrobial-resistant Streptococcus pneumoniae

serotype 14. BMC. Genomics 10:158. 117. Dodds, M. W., D. A. Johnson, and C. K. Yeh. 2005. Health benefits of saliva: a review. J. Dent.

33:223-233.

118. Doern, G. V., S. S. Richter, A. Miller, N. Miller, C. Rice, K. Heilmann, and S. Beekmann. 2005. Antimicrobial resistance among Streptococcus pneumoniae in the United States: have we begun to

turn the corner on resistance to certain antimicrobial classes? Clin. Infect Dis. 41:139-148.

119. Dolence, J. M., L. E. Steward, E. K. Dolence, D. H. Wong, and C. D. Poulter. 2000. Studies with recombinant Saccharomyces cerevisiae CaaX prenyl protease Rce1p. Biochemistry 39:4096-4104.

120. Draper, L. A., R. P. Ross, C. Hill, and P. D. Cotter. 2008. Lantibiotic immunity. Curr. Protein Pept.

Sci. 9:39-49.

121. Drider, D., G. Fimland, Y. Hechard, L. M. McMullen, and H. Prevost. 2006. The continuing story of

class IIa bacteriocins. Microbiol Mol. Biol. Rev 70:564-582.

122. Dubnau, D. and R. Losick. 2006. Bistability in bacteria. Mol. Microbiol 61:564-572. 123. Dufour, A., T. Hindre, D. Haras, and J. P. Le Pennec. 2007. The biology of lantibiotics from the

lacticin 481 group is coming of age. FEMS Microbiol Rev 31:134-167.

124. Durr, U. H., U. S. Sudheendra, and A. Ramamoorthy. 2006. LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochim. Biophys. Acta 1758:1408-1425.

125. Dworkin, J. and R. Losick. 2001. Linking nutritional status to gene activation and development.

Genes Dev. 15:1051-1054. 126. Eguchi, T., K. Kaminaka, J. Shima, S. Kawamoto, K. Mori, S. H. Choi, K. Doi, S. Ohmomo, and S.

Ogata. 2001. Isolation and characterization of enterocin SE-K4 produced by thermophilic

enterococci, Enterococcus faecalis K-4. Biosci. Biotechnol. Biochem. 65:247-253. 127. Eldholm, V., O. Johnsborg, K. Haugen, H. S. Ohnstad, and L. S. Havarstein. 2009. Fratricide in

Streptococcus pneumoniae: contributions and role of the cell wall hydrolases CbpD, LytA and LytC.

Microbiology 155:2223-2234. 128. Ellermeier, C. D., E. C. Hobbs, J. E. Gonzalez-Pastor, and R. Losick. 2006. A three-protein

signaling pathway governing immunity to a bacterial cannibalism toxin. Cell 124:549-559.

129. Engelberg-Kulka, H., S. Amitai, I. Kolodkin-Gal, and R. Hazan. 2006. Bacterial programmed cell death and multicellular behavior in bacteria. PLoS. Genet. 2:e135.

130. Engelke, G., Z. Gutowski-Eckel, M. Hammelmann, and K. D. Entian. 1992. Biosynthesis of the

lantibiotic nisin: genomic organization and membrane localization of the NisB protein. Appl. Environ. Microbiol. 58:3730-3743.

131. Ennahar, S., T. Sashihara, K. Sonomoto, and A. Ishizaki. 2000. Class IIa bacteriocins: biosynthesis,

structure and activity. FEMS Microbiol Rev 24:85-106. 132. Ephrussi-Taylor, H. 1949. Additive effects of certain transforming agents from some variants of

pneumococcus. J. Exp. Med. 89:399-424. 133. European Antimicrobial Resistance Surveillance System (EARSS). 2008. EARSS Annual Report

2006 and EARSS Annual Report2008, In .

134. Farkas-Himsley, H. 1980. Bacteriocins--are they broad-spectrum antibiotics? J Antimicrob. Chemother. 6:424-426.

135. Farkas-Himsley, H. 1980. Bacteriocins--are they broad-spectrum antibiotics? J Antimicrob.

Chemother. 6:424-426. 136. Fath, M. J. and R. Kolter. 1993. ABC transporters: bacterial exporters. Microbiol Rev 57:995-1017.

References

145

137. Fernebro, J., I. Andersson, J. Sublett, E. Morfeldt, R. Novak, E. Tuomanen, S. Normark, and B. H.

Normark. 2004. Capsular expression in Streptococcus pneumoniae negatively affects spontaneous

and antibiotic-induced lysis and contributes to antibiotic tolerance. J. Infect. Dis. 189:328-338. 138. Figueiredo, A. M., R. Austrian, P. Urbaskova, L. A. Teixeira, and A. Tomasz. 1995. Novel

penicillin-resistant clones of Streptococcus pneumoniae in the Czech Republic and in Slovakia.

Microb. Drug Resist. 1:71-78. 139. Fimland, G., L. Johnsen, B. Dalhus, and J. Nissen-Meyer. 2005. Pediocin-like antimicrobial peptides

(class IIa bacteriocins) and their immunity proteins: biosynthesis, structure, and mode of action. J.

Pept. Sci. 11:688-696. 140. Florey, H. W. 1945. Use of Micro-organisms for therapeutic purposes. Br Med J 2:635-642.

141. Fontaine, L., C. Boutry, E. Guedon, A. Guillot, M. Ibrahim, B. Grossiord, and P. Hols. 2007.

Quorum-sensing regulation of the production of Blp bacteriocins in Streptococcus thermophilus. J. Bacteriol. 189:7195-7205.

142. Fontaine, L. and P. Hols. 2008. The inhibitory spectrum of thermophilin 9 from Streptococcus

thermophilus LMD-9 depends on the production of multiple peptides and the activity of BlpG(St), a thiol-disulfide oxidase. Appl. Environ. Microbiol. 74:1102-1110.

143. Fredenhagen, A., G. Fendrich, F. Marki, W. Marki, J. Gruner, F. Raschdorf, and H. H. Peter. 1990.

Duramycins B and C, two new lanthionine containing antibiotics as inhibitors of phospholipase A2. Structural revision of duramycin and cinnamycin. J. Antibiot. (Tokyo) 43:1403-1412.

144. Fujita, K., S. Ichimasa, T. Zendo, S. Koga, F. Yoneyama, J. Nakayama, and K. Sonomoto. 2007.

Structural analysis and characterization of lacticin Q, a novel bacteriocin belonging to a new family of unmodified bacteriocins of gram-positive bacteria. Appl. Environ. Microbiol 73:2871-2877.

145. Fuqua, C., S. C. Winans, and E. P. Greenberg. 1996. Census and consensus in bacterial ecosystems:

the LuxR-LuxI family of quorum-sensing transcriptional regulators. Annu. Rev Microbiol 50:727-751.

146. Furmanek, B., T. Kaczorowski, R. Bugalski, K. Bielawski, J. Bohdanowicz, and A. J. Podhajska.

1999. Identification, characterization and purification of the lantibiotic staphylococcin T, a natural

gallidermin variant. J Appl. Microbiol 87:856-866.

147. Galvez, A., E. Valdivia, H. Abriouel, E. Camafeita, E. Mendez, M. Martinez-Bueno, and M.

Maqueda. 1998. Isolation and characterization of enterocin EJ97, a bacteriocin produced by Enterococcus faecalis EJ97. Arch. Microbiol 171:59-65.

148. Galvin, M., C. Hill, and R. P. Ross. 1999. Lacticin 3147 displays activity in buffer against gram-

positive bacterial pathogens which appear insensitive in standard plate assays. Lett. Appl. Microbiol. 28:355-358.

149. Garneau, S., N. I. Martin, and J. C. Vederas. 2002. Two-peptide bacteriocins produced by lactic acid

bacteria. Biochimie 84:577-592. 150. Geissler, S., F. Götz, and T. Kupke. 1996. Serine protease EpiP from Staphylococcus epidermidis

catalyzes the processing of the epidermin precursor peptide. J. Bacteriol. 178:284-288.

151. Georgalaki, M. D., E. van den Berghe, D. Kritikos, B. Devreese, J. van Beeumen, G. Kalantzopoulos, L. de Vuyst, and E. Tsakalidou. 2002. Macedocin, a food-grade lantibiotic

produced by Streptococcus macedonicus ACA-DC 198. Appl. Environ. Microbiol 68:5891-5903.

152. Gherardi, G., C. G. Whitney, R. R. Facklam, and B. Beall. 2000. Major related sets of antibiotic-resistant Pneumococci in the United States as determined by pulsed-field gel electrophoresis and

pbp1a-pbp2b-pbp2x-dhf restriction profiles. J. Infect. Dis. 181:216-229.

153. Gianfaldoni, C., S. Censini, M. Hilleringmann, M. Moschioni, C. Facciotti, W. Pansegrau, V. Masignani, A. Covacci, R. Rappuoli, M. A. Barocchi, and P. Ruggiero. 2007. Streptococcus

pneumoniae pilus subunits protect mice against lethal challenge. Infect. Immun. 75:1059-1062.

154. Gilmore, M. S., R. A. Segarra, M. C. Booth, C. P. Bogie, L. R. Hall, and D. B. Clewell. 1994. Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its

relationship to lantibiotic determinants. J. Bacteriol. 176:7335-7344.

155. GlaxoSmithKline. 2009. Synflorix™, GlaxoSmithKline's pneumococcal vaccine, receives European authorization. GSK release, London UK, in press.

156. Goldstein, B. P., J. Wei, K. Greenberg, and R. Novick. 1998. Activity of nisin against Streptococcus pneumoniae, in vitro, and in a mouse infection model. J. Antimicrob. Chemother. 42:277-278.

157. Gonzalez-Pastor, J. E., E. C. Hobbs, and R. Losick. 2003. Cannibalism by sporulating bacteria.

Science 301:510-513. 158. Gray, B. M., G. M. Converse, III, and H. C. Dillon, Jr. 1980. Epidemiologic studies of Streptococcus

pneumoniae in infants: acquisition, carriage, and infection during the first 24 months of life. J.

Infect. Dis. 142:923-933.

References

146

159. Gross, E., H. H. Kiltz, and L. C. Craig. 1973. Subtilin, II: the amino acid composition of subtilin

(author's transl). Hoppe Seylers. Z. Physiol Chem. 354:799-801.

160. Gross, E., H. H. Kiltz, and E. Nebelin. 1973. Subtilin, VI: the structure of subtilin (author's transl). Hoppe Seylers. Z. Physiol Chem. 354:810-812.

161. Gross, E. and J. L. Morell. 1971. The structure of nisin. J. Am. Chem. Soc. 93:4634-4635.

162. Guder, A., T. Schmitter, I. Wiedemann, H. G. Sahl, and G. Bierbaum. 2002. Role of the single regulator MrsR1 and the two-component system MrsR2/K2 in the regulation of mersacidin

production and immunity. Appl. Environ. Microbiol 68:106-113.

163. Guder, A., I. Wiedemann, and H. G. Sahl. 2000. Posttranslationally modified bacteriocins--the lantibiotics. Biopolymers 55:62-73.

164. Guedon, E., P. Renault, S. D. Ehrlich, and C. Delorme. 2001. Transcriptional pattern of genes

coding for the proteolytic system of Lactococcus lactis and evidence for coordinated regulation of key enzymes by peptide supply. J. Bacteriol. 183:3614-3622.

165. Guedon, E., P. Serror, S. D. Ehrlich, P. Renault, and C. Delorme. 2001. Pleiotropic transcriptional

repressor CodY senses the intracellular pool of branched-chain amino acids in Lactococcus lactis. Mol. Microbiol. 40:1227-1239.

166. Guedon, E., B. Sperandio, N. Pons, S. D. Ehrlich, and P. Renault. 2005. Overall control of nitrogen

metabolism in Lactococcus lactis by CodY, and possible models for CodY regulation in Firmicutes. Microbiology 151:3895-3909.

167. Guerra, N. P., M. L. Rua, and L. Pastrana. 2001. Nutritional factors affecting the production of two

bacteriocins from lactic acid bacteria on whey. Int. J. Food Microbiol 70:267-281. 168. Guiral, S., T. J. Mitchell, B. Martin, and J. P. Claverys. 2005. Competence-programmed predation of

noncompetent cells in the human pathogen Streptococcus pneumoniae: genetic requirements. Proc.

Natl. Acad. Sci. U. S. A 102:8710-8715. 169. Gutowski-Eckel, Z., C. Klein, K. Siegers, K. Bohm, M. Hammelmann, and K. D. Entian. 1994.

Growth phase-dependent regulation and membrane localization of SpaB, a protein involved in

biosynthesis of the lantibiotic subtilin. Appl. Environ. Microbiol. 60:1-11.

170. Haas, W., D. Kaushal, J. Sublett, C. Obert, and E. I. Tuomanen. 2005. Vancomycin stress response

in a sensitive and a tolerant strain of Streptococcus pneumoniae. J. Bacteriol. 187:8205-8210.

171. Haas, W., B. D. Shepard, and M. S. Gilmore. 2002. Two-component regulator of Enterococcus faecalis cytolysin responds to quorum-sensing autoinduction. Nature 415:84-87.

172. Hakenbeck, R., N. Balmelle, B. Weber, C. Gardes, W. Keck, and S. A. de. 2001. Mosaic genes and

mosaic chromosomes: intra- and interspecies genomic variation of Streptococcus pneumoniae. Infect. Immun 69:2477-2486.

173. Halami, P. M., T. Stein, A. Chandrashekar, and K. D. Entian. 2009. Maturation and processing of

SpaI, the lipoprotein involved in subtilin immunity in Bacillus subtilis ATCC 6633. Microbiol Res. 174. Hale, J. D., B. Balakrishnan, and J. R. Tagg. 2004. Genetic basis for mutacin N and of its

relationship to mutacin I. Indian J. Med. Res. 119 Suppl:247-251.

175. Halfmann, A., R. Hakenbeck, and R. Bruckner. 2007. A new integrative reporter plasmid for Streptococcus pneumoniae. FEMS Microbiol Lett. 268:217-224.

176. Halfmann, A., M. Kovacs, R. Hakenbeck, and R. Bruckner. 2007. Identification of the genes directly

controlled by the response regulator CiaR in Streptococcus pneumoniae: five out of 15 promoters drive expression of small non-coding RNAs. Mol. Microbiol. 66:110-126.

177. Hamel, J., N. Charland, I. Pineau, C. Ouellet, S. Rioux, D. Martin, and B. R. Brodeur. 2004.

Prevention of pneumococcal disease in mice immunized with conserved surface-accessible proteins. Infect. Immun. 72:2659-2670.

178. Hammerschmidt, S., S. R. Talay, P. Brandtzaeg, and G. S. Chhatwal. 1997. SpsA, a novel

pneumococcal surface protein with specific binding to secretory immunoglobulin A and secretory component. Mol. Microbiol. 25:1113-1124.

179. Hanage, W. P., C. Fraser, and B. G. Spratt. 2006. The impact of homologous recombination on the

generation of diversity in bacteria. J. Theor. Biol. 239:210-219. 180. Hancock, R. E. and H. G. Sahl. 2006. Antimicrobial and host-defense peptides as new anti-infective

therapeutic strategies. Nat Biotechnol. 24:1551-1557. 181. Hansman, D., H. Glasgow, J. Sturt, L. Devitt, and R. Douglas. 1971. Increased resistance to

penicillin of pneumococci isolated from man. N. Engl. J. Med. 284:175-177.

182. Harboe, Z. B., R. W. Thomsen, A. Riis, P. Valentiner-Branth, J. J. Christensen, L. Lambertsen, K. A. Krogfelt, H. B. Konradsen, and T. L. Benfield. 2009. Pneumococcal serotypes and mortality

following invasive pneumococcal disease: a population-based cohort study. PLoS. Med.

6:e1000081.

References

147

183. Hare, K. M., P. Morris, H. Smith-Vaughan, and A. J. Leach. 2008. Random colony selection versus

colony morphology for detection of multiple pneumococcal serotypes in nasopharyngeal swabs.

Pediatr. Infect. Dis. J. 27:178-180. 184. Harth, G., S. Maslesa-Galic, M. V. Tullius, and M. A. Horwitz. 2005. All four Mycobacterium

tuberculosis glnA genes encode glutamine synthetase activities but only GlnA1 is abundantly

expressed and essential for bacterial homeostasis. Mol. Microbiol 58:1157-1172. 185. Hasper, H. E., K. B. de, and E. Breukink. 2004. Assembly and stability of nisin-lipid II pores.

Biochemistry 43:11567-11575.

186. Hasper, H. E., N. E. Kramer, J. L. Smith, J. D. Hillman, C. Zachariah, O. P. Kuipers, B. de Kruijff, and E. Breukink. 2006. An alternative bactericidal mechanism of action for lantibiotic peptides that

target lipid II. Science 313:1636-1637.

187. Hastings, J. W., M. Sailer, K. Johnson, K. L. Roy, J. C. Vederas, and M. E. Stiles. 1991. Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc

gelidum. J. Bacteriol. 173:7491-7500.

188. Hauge, H. H., D. Mantzilas, V. G. Eijsink, and J. Nissen-Meyer. 1999. Membrane-mimicking entities induce structuring of the two-peptide bacteriocins plantaricin E/F and plantaricin J/K. J.

Bacteriol. 181:740-747.

189. Hausdorff, W. P., J. Bryant, C. Kloek, P. R. Paradiso, and G. R. Siber. 2000. The contribution of specific pneumococcal serogroups to different disease manifestations: implications for conjugate

vaccine formulation and use, part II. Clin. Infect. Dis. 30:122-140.

190. Hausdorff, W. P., J. Bryant, P. R. Paradiso, and G. R. Siber. 2000. Which pneumococcal serogroups cause the most invasive disease: implications for conjugate vaccine formulation and use, part I. Clin.

Infect. Dis. 30:100-121.

191. Hava, D. L. and A. Camilli. 2002. Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors. Mol. Microbiol. 45:1389-1406.

192. Havarstein, L. S., D. B. Diep, and I. F. Nes. 1995. A family of bacteriocin ABC transporters carry

out proteolytic processing of their substrates concomitant with export. Mol. Microbiol. 16:229-240.

193. Havarstein, L. S., B. Martin, O. Johnsborg, C. Granadel, and J. P. Claverys. 2006. New insights into

the pneumococcal fratricide: relationship to clumping and identification of a novel immunity factor.

Mol. Microbiol 59:1297-1307. 194. Hechard, Y., B. Derijard, F. Letellier, and Y. Cenatiempo. 1992. Characterization and purification of

mesentericin Y105, an anti-Listeria bacteriocin from Leuconostoc mesenteroides. J. Gen. Microbiol

138:2725-2731. 195. Hechard, Y., C. Pelletier, Y. Cenatiempo, and J. Frere. 2001. Analysis of sigma(54)-dependent

genes in Enterococcus faecalis: a mannose PTS permease (EII(Man)) is involved in sensitivity to a

bacteriocin, mesentericin Y105. Microbiology 147:1575-1580. 196. Hechard, Y. and H. G. Sahl. 2002. Mode of action of modified and unmodified bacteriocins from

Gram-positive bacteria. Biochimie 84:545-557.

197. Heidrich, C., U. Pag, M. Josten, J. Metzger, R. W. Jack, G. Bierbaum, G. Jung, and H. G. Sahl. 1998. Isolation, characterization, and heterologous expression of the novel lantibiotic epicidin 280

and analysis of its biosynthetic gene cluster. Appl. Environ. Microbiol 64:3140-3146.

198. Henderson, J. T., A. L. Chopko, and P. D. van Wassenaar. 1992. Purification and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC-1.0. Arch. Biochem. Biophys. 295:5-

12.

199. Hendriksen, W. T., H. J. Bootsma, S. Estevao, T. Hoogenboezem, A. de Jong, R. de Groot, O. P. Kuipers, and P. W. Hermans. 2008. CodY of Streptococcus pneumoniae: link between nutritional

gene regulation and colonization. J. Bacteriol. 190:590-601.

200. Hendriksen, W. T., H. J. Bootsma, A. van Diepen, S. Estevao, O. P. Kuipers, R. de Groot, and P. W. Hermans. 2009. Strain-specific impact of PsaR of Streptococcus pneumoniae on global gene

expression and virulence. Microbiology 155:1569-1579.

201. Hendriksen, W. T., T. G. Kloosterman, H. J. Bootsma, S. Estevao, R. de Groot, O. P. Kuipers, and P. W. Hermans. 2008. Site-specific contributions of glutamine-dependent regulator GlnR and GlnR-

regulated genes to virulence of Streptococcus pneumoniae. Infect Immun 76:1230-1238. 202. Heng, N. C., J. R. Tagg, and G. R. Tompkins. 2007. Competence-dependent bacteriocin production

by Streptococcus gordonii DL1 (Challis). J. Bacteriol. 189:1468-1472.

203. Heng, N. C. K. and John R.Tagg. 2006. What's in a name? Class distinction for bacteriocins. Nat Rev Microbiol 4:doi:10.1038/nrmicro1273-c1.

204. Henriques-Normark, B., C. Blomberg, J. Dagerhamn, P. Battig, and S. Normark. 2008. The rise and

fall of bacterial clones: Streptococcus pneumoniae. Nat. Rev. Microbiol. 6:827-837.

References

148

205. Hiller, N. L., B. Janto, J. S. Hogg, R. Boissy, S. Yu, E. Powell, R. Keefe, N. E. Ehrlich, K. Shen, J.

Hayes, K. Barbadora, W. Klimke, D. Dernovoy, T. Tatusova, J. Parkhill, S. D. Bentley, J. C. Post,

G. D. Ehrlich, and F. Z. Hu. 2007. Comparative genomic analyses of seventeen Streptococcus pneumoniae strains: insights into the pneumococcal supragenome. J. Bacteriol. 189:8186-8195.

206. Hillman, J. D., J. Novak, E. Sagura, J. A. Gutierrez, T. A. Brooks, P. J. Crowley, M. Hess, A. Azizi,

K. Leung, D. Cvitkovitch, and A. S. Bleiweis. 1998. Genetic and biochemical analysis of mutacin 1140, a lantibiotic from Streptococcus mutans. Infect. Immun. 66:2743-2749.

207. Hindre, T., J. P. Le Pennec, D. Haras, and A. Dufour. 2004. Regulation of lantibiotic lacticin 481

production at the transcriptional level by acid pH. FEMS Microbiol Lett. 231:291-298. 208. Hirst, R. A., A. Kadioglu, C. O'Callaghan, and P. W. Andrew. 2004. The role of pneumolysin in

pneumococcal pneumonia and meningitis. Clin. Exp. Immunol. 138:195-201.

209. Hoffmann, A., T. Schneider, U. Pag, and H. G. Sahl. 2004. Localization and functional analysis of PepI, the immunity peptide of Pep5-producing Staphylococcus epidermidis strain 5. Appl. Environ.

Microbiol 70:3263-3271.

210. Hofmann, K. and W. Stoffel. 1993. TMbase - A database of membrane spanning proteins segments, p. 164. In Biol.Chem.Hoppe-Seyler.

211. Holck, A., L. Axelsson, S. E. Birkeland, T. Aukrust, and H. Blom. 1992. Purification and amino acid

sequence of sakacin A, a bacteriocin from Lactobacillus sake Lb706. J. Gen. Microbiol 138:2715-2720.

212. Holck, A., L. Axelsson, and U. Schillinger. 1996. Divergicin 750, a novel bacteriocin produced by

Carnobacterium divergens 750. FEMS Microbiol Lett. 136:163-168. 213. Holmes, A. R., R. McNab, K. W. Millsap, M. Rohde, S. Hammerschmidt, J. L. Mawdsley, and H. F.

Jenkinson. 2001. The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein

that is essential for virulence. Mol. Microbiol. 41:1395-1408. 214. Holo, H., Z. Jeknic, M. Daeschel, S. Stevanovic, and I. F. Nes. 2001. Plantaricin W from

Lactobacillus plantarum belongs to a new family of two-peptide lantibiotics. Microbiology 147:643-

651.

215. Holo, H. and I. F. Nes. 1995. Transformation of Lactococcus by electroporation. Methods Mol. Biol.

47:195-199.

216. Holo, H., O. Nilssen, and I. F. Nes. 1991. Lactococcin A, a new bacteriocin from Lactococcus lactis subsp. cremoris: isolation and characterization of the protein and its gene. J. Bacteriol. 173:3879-

3887.

217. Hols, P., F. Hancy, L. Fontaine, B. Grossiord, D. Prozzi, N. Leblond-Bourget, B. Decaris, A. Bolotin, C. Delorme, E. S. Dusko, E. Guedon, V. Monnet, P. Renault, and M. Kleerebezem. 2005.

New insights in the molecular biology and physiology of Streptococcus thermophilus revealed by

comparative genomics. FEMS Microbiol. Rev. 29:435-463. 218. Horn, N., M. I. Martinez, J. M. Martinez, Hernández P.E., M. J. Gasson, J. M. Rodriguez, and H. M.

Dodd. 1998. Production of pediocin PA-1 by Lactococcus lactis using the lactococcin A secretory

apparatus. Appl Environ. Microbiol 64:818-823. 219. Hoskins, J., W. E. Alborn, J. Arnold, L. C. Blaszczak, S. Burgett, B. S. Dehoff, S. T. Estrem, L.

Fritz, D. J. Fu, W. Fuller, C. Geringer, R. Gilmour, J. S. Glass, H. Khoja, A. R. Kraft, R. E. Lagace,

D. J. LeBlanc, L. N. Lee, E. J. Lefkowitz, J. Lu, P. Matsushima, S. M. McAhren, M. McHenney, K. McLeaster, C. W. Mundy, T. I. Nicas, F. H. Norris, M. O'Gara, R. B. Peery, G. T. Robertson, P.

Rockey, P. M. Sun, M. E. Winkler, Y. Yang, M. Young-Bellido, G. S. Zhao, C. A. Zook, R. H.

Baltz, S. R. Jaskunas, P. R. Rosteck, P. L. Skatrud, and J. I. Glass. 2001. Genome of the bacterium Streptococcus pneumoniae strain R6. Journal of Bacteriology 183:5709-5717.

220. Hsu, S. T., E. Breukink, E. Tischenko, M. A. Lutters, B. de Kruijff, R. Kaptein, A. M. Bonvin, and

N. A. van Nuland. 2004. The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics. Nat Struct. Mol. Biol. 11:963-967.

221. Huebner, R. E., R. Dagan, N. Porath, A. D. Wasas, and K. P. Klugman. 2000. Lack of utility of

serotyping multiple colonies for detection of simultaneous nasopharyngeal carriage of different pneumococcal serotypes. Pediatr. Infect. Dis. J. 19:1017-1020.

222. Huhne, K., L. Axelsson, A. Holck, and L. Krockel. 1996. Analysis of the sakacin P gene cluster from Lactobacillus sake Lb674 and its expression in sakacin-negative Lb. sake strains. Microbiology

142 ( Pt 6):1437-1448.

223. Hyink, O., M. Balakrishnan, and J. R. Tagg. 2005. Streptococcus rattus strain BHT produces both a class I two-component lantibiotic and a class II bacteriocin. FEMS Microbiol. Lett. 252:235-241.

224. Hyink, O., P. A. Wescombe, M. Upton, N. Ragland, J. P. Burton, and J. R. Tagg. 2007. Salivaricin

A2 and the novel lantibiotic salivaricin B are encoded at adjacent loci on a 190-kilobase

References

149

transmissible megaplasmid in the oral probiotic strain Streptococcus salivarius K12. Appl. Environ.

Microbiol. 73:1107-1113.

225. Hynes, W. L., J. J. Ferretti, and J. R. Tagg. 1993. Cloning of the gene encoding streptococcin A-FF22, a novel lantibiotic produced by Streptococcus pyogenes, and determination of its nucleotide

sequence. Appl. Environ. Microbiol. 59:1969-1971.

226. Ibrahim, Y. M., A. R. Kerr, J. McCluskey, and T. J. Mitchell. 2004. Control of virulence by the two-component system CiaR/H is mediated via HtrA, a major virulence factor of Streptococcus

pneumoniae. J. Bacteriol. 186:5258-5266.

227. Ibrahim, Y. M., A. R. Kerr, J. McCluskey, and T. J. Mitchell. 2004. Role of HtrA in the virulence and competence of Streptococcus pneumoniae. Infect Immun 72:3584-3591.

228. Ishihara, H., M. Takoh, R. Nishibayashi, and A. Sato. 2002. Distribution and variation of bacitracin

synthetase gene sequences in laboratory stock strains of Bacillus licheniformis. Curr. Microbiol 45:18-23.

229. Israelsen, H., S. M. Madsen, A. Vrang, E. B. Hansen, and E. Johansen. 1995. Cloning and partial

characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80. Appl Environ. Microbiol 61:2540-2547.

230. Ivanova, I., V. Miteva, T. Stefanova, A. Pantev, I. Budakov, S. Danova, P. Moncheva, I. Nikolova,

X. Dousset, and P. Boyaval. 1998. Characterization of a bacteriocin produced by Streptococcus thermophilus 81. Int. J Food Microbiol 42:147-158.

231. Iwatani, S., T. Zendo, F. Yoneyama, J. Nakayama, and K. Sonomoto. 2007. Characterization and

structure analysis of a novel bacteriocin, lacticin Z, produced by Lactococcus lactis QU 14. Biosci. Biotechnol. Biochem. 71:1984-1992.

232. Jack, R. W. and J. R. Tagg. 1992. Factors affecting production of the group A streptococcus

bacteriocin SA-FF22. J Med Microbiol 36:132-138. 233. Jack, R. W., J. R. Tagg, and B. Ray. 1995. Bacteriocins of gram-positive bacteria. Microbiol. Rev.

59:171-200.

234. Jansen, A. G., G. D. Rodenburg, A. van der Ende, L. van Alphen, R. H. Veenhoven, L. Spanjaard, E.

A. Sanders, and E. Hak. 2009. Invasive pneumococcal disease among adults: associations among

serotypes, disease characteristics, and outcome. Clin. Infect. Dis. 49:e23-e29.

235. Jarva, H., R. Janulczyk, J. Hellwage, P. F. Zipfel, L. Bjorck, and S. Meri. 2002. Streptococcus pneumoniae evades complement attack and opsonophagocytosis by expressing the pspC locus-

encoded Hic protein that binds to short consensus repeats 8-11 of factor H. J. Immunol. 168:1886-

1894. 236. Jedrzejas, M. J. 2001. Pneumococcal virulence factors: structure and function. Microbiol. Mol. Biol.

Rev. 65:187-207.

237. Jimenez-Diaz, R., R. M. Rios-Sanchez, M. Desmazeaud, J. L. Ruiz-Barba, and J. C. Piard. 1993. Plantaricins S and T, Two New Bacteriocins Produced by Lactobacillus plantarum LPCO10 Isolated

from a Green Olive Fermentation. Appl. Environ. Microbiol 59:1416-1424.

238. Johnsborg, O. and L. S. Havarstein. 2009. Regulation of natural genetic transformation and acquisition of transforming DNA in Streptococcus pneumoniae. FEMS Microbiol. Rev. 33:627-642.

239. Johnsen, L., G. Fimland, D. Mantzilas, and J. Nissen-Meyer. 2004. Structure-function analysis of

immunity proteins of pediocin-like bacteriocins: C-terminal parts of immunity proteins are involved in specific recognition of cognate bacteriocins. Appl. Environ. Microbiol 70:2647-2652.

240. Jonsson, S., D. M. Musher, A. Chapman, A. Goree, and E. C. Lawrence. 1985. Phagocytosis and

killing of common bacterial pathogens of the lung by human alveolar macrophages. J. Infect. Dis. 152:4-13.

241. Jordan, S., M. I. Hutchings, and T. Mascher. 2008. Cell envelope stress response in Gram-positive

bacteria. FEMS Microbiol Rev. 32:107-146. 242. Joseph, P., M. Ratnayake-Lecamwasam, and A. L. Sonenshein. 2005. A region of Bacillus subtilis

CodY protein required for interaction with DNA. J. Bacteriol. 187:4127-4139.

243. Kaletta, C., K. D. Entian, and G. Jung. 1991. Prepeptide sequence of cinnamycin (Ro 09-0198): the first structural gene of a duramycin-type lantibiotic. Eur. J. Biochem. 199:411-415.

244. Kaletta, C., K. D. Entian, R. Kellner, G. Jung, M. Reis, and H. G. Sahl. 1989. Pep5, a new lantibiotic: structural gene isolation and prepeptide sequence. Arch. Microbiol 152:16-19.

245. Kalmokoff, M. L., S. K. Banerjee, T. Cyr, M. A. Hefford, and T. Gleeson. 2001. Identification of a

new plasmid-encoded sec-dependent bacteriocin produced by Listeria innocua 743. Appl. Environ. Microbiol 67:4041-4047.

246. Kalmokoff, M. L., D. Lu, M. F. Whitford, and R. M. Teather. 1999. Evidence for production of a

new lantibiotic (butyrivibriocin OR79A) by the ruminal anaerobe Butyrivibrio fibrisolvens OR79:

References

150

characterization of the structural gene encoding butyrivibriocin OR79A. Appl. Environ. Microbiol

65:2128-2135.

247. Kaltoft, M. S., U. B. Skov Sorensen, H. C. Slotved, and H. B. Konradsen. 2008. An easy method for detection of nasopharyngeal carriage of multiple Streptococcus pneumoniae serotypes. J. Microbiol.

Methods 75:540-544.

248. Kanatani, K., M. Oshimura, and K. Sano. 1995. Isolation and characterization of acidocin A and cloning of the bacteriocin gene from Lactobacillus acidophilus. Appl. Environ. Microbiol 61:1061-

1067.

249. Karaya, K., T. Shimizu, and A. Taketo. 2001. New gene cluster for lantibiotic streptin possibly involved in streptolysin S formation. J. Biochem. 129:769-775.

250. Karlsson, D., S. Karlsson, E. Gustafsson, B. H. Normark, and P. Nilsson. 2007. Modeling the

regulation of the competence-evoking quorum sensing network in Streptococcus pneumoniae. Biosystems 90:211-223.

251. Kavanagh, K. and S. Dowd. 2004. Histatins: antimicrobial peptides with therapeutic potential. J.

Pharm. Pharmacol. 56:285-289. 252. Kellner, R., G. Jung, T. Horner, H. Zahner, N. Schnell, K. D. Entian, and F. Götz. 1988.

Gallidermin: a new lanthionine-containing polypeptide antibiotic. Eur. J. Biochem. 177:53-59.

253. Kelly, T., J. P. Dillard, and J. Yother. 1994. Effect of genetic switching of capsular type on virulence of Streptococcus pneumoniae. Infect. Immun. 62:1813-1819.

254. Kettenring, J. K., A. Malabarba, K. Vekey, and B. Cavalleri. 1990. Sequence determination of

actagardine, a novel lantibiotic, by homonuclear 2D NMR spectroscopy. J. Antibiot. (Tokyo) 43:1082-1088.

255. Kies, S., C. Vuong, M. Hille, A. Peschel, C. Meyer, F. Götz, and M. Otto. 2003. Control of

antimicrobial peptide synthesis by the agr quorum sensing system in Staphylococcus epidermidis: activity of the lantibiotic epidermin is regulated at the level of precursor peptide processing. Peptides

24:329-338.

256. King, S. J., K. R. Hippe, and J. N. Weiser. 2006. Deglycosylation of human glycoconjugates by the

sequential activities of exoglycosidases expressed by Streptococcus pneumoniae. Mol. Microbiol.

59:961-974.

257. Kjos, M., L. Snipen, Z. Salehian, I. F. Nes, and D. B. Diep. 2010. The Abi proteins and their involvement in bacteriocin self-immunity. J. Bacteriol. 192:2068-2076.

258. Klaenhammer, T. R. 1993. Genetics of bacteriocins produced by lactic acid bacteria. FEMS

Microbiol. Rev. 12:39-85. 259. Kleerebezem, M., M. M. Beerthuyzen, E. E. Vaughan, W. M. de Vos, and O. P. Kuipers. 1997.

Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible expression

cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp. Appl Environ. Microbiol 63:4581-4584.

260. Kloosterman, T. G., J. J. Bijlsma, J. Kok, and O. P. Kuipers. 2006. To have neighbour's fare:

extending the molecular toolbox for Streptococcus pneumoniae. Microbiology 152:351-359. 261. Kloosterman, T. G., W. T. Hendriksen, J. J. Bijlsma, H. J. Bootsma, S. A. van Hijum, J. Kok, P. W.

Hermans, and O. P. Kuipers. 2006. Regulation of glutamine and glutamate metabolism by GlnR and

GlnA in Streptococcus pneumoniae. J. Biol. Chem. 281:25097-25109. 262. Klugman, K. P. 1990. Pneumococcal resistance to antibiotics. Clin. Microbiol. Rev. 3:171-196.

263. Klugman, K. P. 2002. The successful clone: the vector of dissemination of resistance in

Streptococcus pneumoniae. J. Antimicrob. Chemother. 50 Suppl S2:1-5. 264. Klugman, K. P. and H. J. Koornhof. 1988. Drug resistance patterns and serogroups or serotypes of

pneumococcal isolates from cerebrospinal fluid or blood, 1979-1986. J. Infect. Dis. 158:956-964.

265. Kluskens, L. D., A. Kuipers, R. Rink, E. de Boef, S. Fekken, A. J. Driessen, O. P. Kuipers, and G. N. Moll. 2005. Post-translational modification of therapeutic peptides by NisB, the dehydratase of

the lantibiotic nisin. Biochemistry 44:12827-12834.

266. Knaust, A. and M. Frosch. 2004. Genome-based vaccines. Int. J. Med. Microbiol. 294:295-301. 267. Knutsen, E., O. Ween, and L. S. Havarstein. 2004. Two separate quorum-sensing systems upregulate

transcription of the same ABC transporter in Streptococcus pneumoniae. J. Bacteriol. 186:3078-3085.

268. Kobayashi, N., K. Nishino, and A. Yamaguchi. 2001. Novel macrolide-specific ABC-type efflux

transporter in Escherichia coli. J. Bacteriol. 183:5639-5644. 269. Koczulla, A. R. and R. Bals. 2003. Antimicrobial peptides: current status and therapeutic potential.

Drugs 63:389-406.

270. Kolodkin-Gal, I. and H. Engelberg-Kulka. 2006. Induction of Escherichia coli chromosomal mazEF by stressful conditions causes an irreversible loss of viability. J. Bacteriol. 188:3420-3423.

References

151

271. Konz, D., A. Klens, K. Schorgendorfer, and M. A. Marahiel. 1997. The bacitracin biosynthesis

operon of Bacillus licheniformis ATCC 10716: molecular characterization of three multi-modular

peptide synthetases. Chem. Biol. 4:927-937. 272. Koponen, O., T. M. Takala, U. Saarela, M. Qiao, and P. E. Saris. 2004. Distribution of the NisI

immunity protein and enhancement of nisin activity by the lipid-free NisI. FEMS Microbiol Lett.

231:85-90. 273. Kotel'nikova, E. A. and M. S. Gel'fand. 2002. Regulation of transcription in the system of genes

responsible for bacteriocins production in Streptococcus equi. Genetika 38:911-915.

274. Kovacs, M., A. Halfmann, I. Fedtke, M. Heintz, A. Peschel, W. Vollmer, R. Hakenbeck, and R. Bruckner. 2006. A functional dlt operon, encoding proteins required for incorporation of d-alanine in

teichoic acids in gram-positive bacteria, confers resistance to cationic antimicrobial peptides in

Streptococcus pneumoniae. J. Bacteriol. 188:5797-5805. 275. Kramer, N. E., S. A. van Hijum, J. Knol, J. Kok, and O. P. Kuipers. 2006. Transcriptome analysis

reveals mechanisms by which Lactococcus lactis acquires nisin resistance. Antimicrob. Agents

Chemother. 50:1753-1761. 276. Kreth, J., D. C. Hung, J. Merritt, J. Perry, L. Zhu, S. D. Goodman, D. G. Cvitkovitch, W. Shi, and F.

Qi. 2007. The response regulator ComE in Streptococcus mutans functions both as a transcription

activator of mutacin production and repressor of CSP biosynthesis. Microbiology 153:1799-1807. 277. Kreth, J., J. Merritt, C. Bordador, W. Shi, and F. Qi. 2004. Transcriptional analysis of mutacin I

(mutA) gene expression in planktonic and biofilm cells of Streptococcus mutans using fluorescent

protein and glucuronidase reporters. Oral Microbiol. Immunol. 19:252-256. 278. Kreth, J., J. Merritt, W. Shi, and F. Qi. 2005. Co-ordinated bacteriocin production and competence

development: a possible mechanism for taking up DNA from neighbouring species. Mol. Microbiol

57:392-404. 279. Kreth, J., J. Merritt, L. Zhu, W. Shi, and F. Qi. 2006. Cell density- and ComE-dependent expression

of a group of mutacin and mutacin-like genes in Streptococcus mutans. FEMS Microbiol Lett.

265:11-17.

280. Krull, R. E., P. Chen, J. Novak, M. Kirk, S. Barnes, J. Baker, N. R. Krishna, and P. W. Caufield.

2000. Biochemical structural analysis of the lantibiotic mutacin II. J. Biol. Chem. 275:15845-15850.

281. Kruszewska, D., H. G. Sahl, G. Bierbaum, U. Pag, S. O. Hynes, and A. Ljungh. 2004. Mersacidin eradicates methicillin-resistant Staphylococcus aureus (MRSA) in a mouse rhinitis model. J.

Antimicrob. Chemother. 54:648-653.

282. Kuipers, A., E. de Boef, R. Rink, S. Fekken, L. D. Kluskens, A. J. Driessen, K. Leenhouts, O. P. Kuipers, and G. N. Moll. 2004. NisT, the transporter of the lantibiotic nisin, can transport fully

modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic

peptides. J. Biol. Chem. 279:22176-22182. 283. Kuipers, A., J. Meijer-Wierenga, R. Rink, L. D. Kluskens, and G. N. Moll. 2008. Mechanistic

dissection of the enzyme complexes involved in biosynthesis of lacticin 3147 and nisin. Appl

Environ. Microbiol 74:6591-6597. 284. Kuipers, O. P., M. M. Beerthuyzen, P. G. de Ruyter, E. J. Luesink, and W. M. de Vos. 1995.

Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction. J. Biol. Chem.

270:27299-27304. 285. Kuipers, O. P., M. M. Beerthuyzen, R. J. Siezen, and W. M. de Vos. 1993. Characterization of the

nisin gene cluster nisABTCIPR of Lactococcus lactis. Requirement of expression of the nisA and

nisI genes for development of immunity. Eur. J. Biochem. 216:281-291. 286. Kuipers, O. P., M. M. Beerthuyzen, R. J. Siezen, and W. M. de Vos. 1993. Characterization of the

nisin gene cluster nisABTCIPR of Lactococcus lactis. Requirement of expression of the nisA and

nisI genes for development of immunity. Eur. J. Biochem. 216:281-291. 287. Kuipers, O. P., G. Bierbaum, B. Ottenwalder, H. M. Dodd, N. Horn, J. Metzger, T. Kupke, V. Gnau,

R. Bongers, P. van den Bogaard, H. Kosters, H. S. Rollema, W. M. de Vos, R. J. Siezen, G. Jung, F.

Götz, H. G. Sahl, and M. J. Gasson. 1996. Protein engineering of lantibiotics. Antonie Van Leeuwenhoek 69:161-169.

288. Kuipers, O. P., A. de Jong, R. J. Baerends, S. A. van Hijum, A. L. Zomer, H. A. Karsens, C. D. den Hengst, N. E. Kramer, G. Buist, and J. Kok. 2002. Transcriptome analysis and related databases of

Lactococcus lactis. Antonie Van Leeuwenhoek 82:113-122.

289. Kuipers, O. P., H. S. Rollema, W. M. de Vos, and R. J. Siezen. 1993. Biosynthesis and secretion of a precursor of nisin Z by Lactococcus lactis, directed by the leader peptide of the homologous

lantibiotic subtilin from Bacillus subtilis. FEBS Lett. 330:23-27.

290. Kuipers, O. P., Ruyter, P.G., M. Kleerebezem, and W. M. de Vos. 1998. Quorum sensing controlled gene expression in lactic acid bacteria. J. Biotechnol 64:15-21.

References

152

291. Kupke, T. and F. Götz. 1996. Post-translational modifications of lantibiotics. Antonie Van

Leeuwenhoek 69:139-150.

292. Kupke, T. and F. Götz. 1997. In vivo reaction of affinity-tag-labelled epidermin precursor peptide with flavoenzyme EpiD. FEMS Microbiol Lett. 153:25-32.

293. Kupke, T. and F. Götz. 1997. The enethiolate anion reaction products of EpiD. Pka value of the

enethiol side chain is lower than that of the thiol side chain of peptides. J. Biol. Chem. 272:4759-4762.

294. Kupke, T., C. Kempter, V. Gnau, G. Jung, and F. Götz. 1994. Mass spectroscopic analysis of a novel

enzymatic reaction. Oxidative decarboxylation of the lantibiotic precursor peptide EpiA catalyzed by the flavoprotein EpiD. J. Biol. Chem. 269:5653-5659.

295. Kupke, T., C. Kempter, G. Jung, and F. Götz. 1995. Oxidative decarboxylation of peptides catalyzed

by flavoprotein EpiD. Determination of substrate specificity using peptide libraries and neutral loss mass spectrometry. J. Biol. Chem. 270:11282-11289.

296. Kupke, T., S. Stevanovic, H. G. Sahl, and F. Götz. 1992. Purification and characterization of EpiD, a

flavoprotein involved in the biosynthesis of the lantibiotic epidermin. J. Bacteriol. 174:5354-5361. 297. Kuroda, M., H. Kuroda, T. Oshima, F. Takeuchi, H. Mori, and K. Hiramatsu. 2003. Two-component

system VraSR positively modulates the regulation of cell-wall biosynthesis pathway in

Staphylococcus aureus. Mol. Microbiol 49:807-821. 298. Kuttner, A. G. 1966. Production of bacteriocines by group A streptococci with special reference to

the nephritogenic types. J. Exp. Med. 124:279-291.

299. Landsberg, H. 1949. Prelude to the Discovery of Penicillin. Isis 40. 300. Lange, R., C. Wagner, A. de Saizieu, N. Flint, J. Molnos, M. Stieger, P. Caspers, M. Kamber, W.

Keck, and K. E. Amrein. 1999. Domain organization and molecular characterization of 13 two-

component systems identified by genome sequencing of Streptococcus pneumoniae. Gene 237:223-234.

301. Lanie, J. A., W. L. Ng, K. M. Kazmierczak, T. M. Andrzejewski, T. M. Davidsen, K. J. Wayne, H.

Tettelin, J. I. Glass, and M. E. Winkler. 2007. Genome sequence of Avery's virulent serotype 2

strain D39 of Streptococcus pneumoniae and comparison with that of unencapsulated laboratory

strain R6. J. Bacteriol. 189:38-51.

302. Larkin, M. A., G. Blackshields, N. P. Brown, R. Chenna, P. A. McGettigan, H. McWilliam, F. Valentin, I. M. Wallace, A. Wilm, R. Lopez, J. D. Thompson, T. J. Gibson, and D. G. Higgins. 2007.

Clustal W and Clustal X version 2.0. Bioinformatics. 23:2947-2948.

303. Leenhouts, K., G. Buist, A. Bolhuis, B. A. ten, J. Kiel, I. Mierau, M. Dabrowska, G. Venema, and J. Kok. 1996. A general system for generating unlabelled gene replacements in bacterial chromosomes.

Mol. Gen. Genet. 253:217-224.

304. Lu, Y. J. and C. O. Rock. 2006. Transcriptional regulation of fatty acid biosynthesis in Streptococcus pneumoniae. Mol. Microbiol 59:551-566.

305. Lubelski, J., W. Overkamp, L. D. Kluskens, G. N. Moll, and O. P. Kuipers. 2008. Influence of

shifting positions of Ser, Thr, and Cys residues in prenisin on the efficiency of modification reactions and on the antimicrobial activities of the modified prepeptides. Appl Environ. Microbiol

74:4680-4685.

306. Lubelski, J., R. Rink, R. Khusainov, G. N. Moll, and O. P. Kuipers. 2008. Biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin. Cell Mol. Life Sci. 65:455-

476.

307. Lux, T., M. Nuhn, R. Hakenbeck, and P. Reichmann. 2007. Diversity of bacteriocins and activity spectrum in Streptococcus pneumoniae. J. Bacteriol. 189:7741-7751.

308. Lyon, G. J. and T. W. Muir. 2003. Chemical signaling among bacteria and its inhibition. Chem.

Biol. 10:1007-1021. 309. Lysenko, E. S., A. J. Ratner, A. L. Nelson, and J. N. Weiser. 2005. The role of innate immune

responses in the outcome of interspecies competition for colonization of mucosal surfaces. PLoS.

Pathog. 1:e1. 310. Majchrzykiewicz, J. A., J. Lubelski, G. N. Moll, A. Kuipers, J. J. Bijlsma, O. P. Kuipers, and R.

Rink. 2010. Production of a class II two-component lantibiotic of Streptococcus pneumoniae using the class I nisin synthetic machinery and leader sequence. Antimicrob. Agents Chemother. 54:1498-

1505.

311. Majer, F., D. G. Schmid, K. Altena, G. Bierbaum, and T. Kupke. 2002. The flavoprotein MrsD catalyzes the oxidative decarboxylation reaction involved in formation of the peptidoglycan

biosynthesis inhibitor mersacidin. J. Bacteriol. 184:1234-1243.

References

153

312. Malke, H., K. Steiner, W. M. McShan, and J. J. Ferretti. 2006. Linking the nutritional status of

Streptococcus pyogenes to alteration of transcriptional gene expression: the action of CodY and

RelA. Int. J. Med. Microbiol. 296:259-275. 313. Manco, S., F. Hernon, H. Yesilkaya, J. C. Paton, P. W. Andrew, and A. Kadioglu. 2006.

Pneumococcal neuraminidases A and B both have essential roles during infection of the respiratory

tract and sepsis. Infect. Immun. 74:4014-4020. 314. Marahiel, M. A. 2009. Working outside the protein-synthesis rules: insights into non-ribosomal

peptide synthesis. J. Pept. Sci. 15:799-807.

315. Marahiel, M. A., T. Stachelhaus, and H. D. Mootz. 1997. Modular Peptide Synthetases Involved in Nonribosomal Peptide Synthesis. Chem. Rev 97:2651-2674.

316. Marciset, O., M. C. Jeronimus-Stratingh, B. Mollet, and B. Poolman. 1997. Thermophilin 13, a

nontypical antilisterial poration complex bacteriocin, that functions without a receptor. J. Biol. Chem. 272:14277-14284.

317. Margolis, E. 2009. Hydrogen peroxide-mediated interference competition by Streptococcus

pneumoniae has no significant effect on Staphylococcus aureus nasal colonization of neonatal rats. J. Bacteriol. 191:571-575.

318. Margolis, E., A. Yates, and B. R. Levin. 2010. The ecology of nasal colonization of Streptococcus

pneumoniae, Haemophilus influenzae and Staphylococcus aureus: the role of competition and interactions with host's immune response. BMC. Microbiol 10:59.

319. Marra, A., J. Asundi, M. Bartilson, S. Lawson, F. Fang, J. Christine, C. Wiesner, D. Brigham, W. P.

Schneider, and A. E. Hromockyj. 2002. Differential fluorescence induction analysis of Streptococcus pneumoniae identifies genes involved in pathogenesis. Infect. Immun. 70:1422-1433.

320. Marra, A., S. Lawson, J. S. Asundi, D. Brigham, and A. E. Hromockyj. 2002. In vivo

characterization of the psa genes from Streptococcus pneumoniae in multiple models of infection. Microbiology 148:1483-1491.

321. Marrer, E., A. T. Satoh, M. M. Johnson, L. J. Piddock, and M. G. Page. 2006. Global transcriptome

analysis of the responses of a fluoroquinolone-resistant Streptococcus pneumoniae mutant and its

parent to ciprofloxacin. Antimicrob. Agents Chemother. 50:269-278.

322. Marrer, E., K. Schad, A. T. Satoh, M. G. Page, M. M. Johnson, and L. J. Piddock. 2006.

Involvement of the putative ATP-dependent efflux proteins PatA and PatB in fluoroquinolone resistance of a multidrug-resistant mutant of Streptococcus pneumoniae. Antimicrob. Agents

Chemother. 50:685-693.

323. Martin, M., J. Gutierrez, R. Criado, C. Herranz, L. M. Cintas, and P. E. Hernandez. 2007. Cloning, production and expression of the bacteriocin enterocin A produced by Enterococcus faecium

PLBC21 in Lactococcus lactis. Appl Microbiol Biotechnol 76:667-675.

324. Martin, M., J. Gutierrez, R. Criado, C. Herranz, L. M. Cintas, and P. E. Hernandez. 2007. Chimeras of mature pediocin PA-1 fused to the signal peptide of enterocin P permits the cloning, production,

and expression of pediocin PA-1 in Lactococcus lactis. J. Food Prot. 70:2792-2798.

325. Martin, N. I., T. Sprules, M. R. Carpenter, P. D. Cotter, C. Hill, R. P. Ross, and J. C. Vederas. 2004. Structural characterization of lacticin 3147, a two-peptide lantibiotic with synergistic activity.

Biochemistry 43:3049-3056.

326. Martinez, B., T. Bottiger, T. Schneider, A. Rodriguez, H. G. Sahl, and I. Wiedemann. 2008. Specific interaction of the unmodified bacteriocin lactococcin 972 with the cell wall precursor lipid II. Appl.

Environ. Microbiol. 74:4666-4670.

327. Martinez, B., M. Fernandez, J. E. Suarez, and A. Rodriguez. 1999. Synthesis of lactococcin 972, a bacteriocin produced by Lactococcus lactis IPLA 972, depends on the expression of a plasmid-

encoded bicistronic operon. Microbiology 145 ( Pt 11):3155-3161.

328. Martinez, B., A. Rodriguez, and J. E. Suarez. 2000. Lactococcin 972, a bacteriocin that inhibits septum formation in lactococci. Microbiology 146 ( Pt 4):949-955.

329. Martínez, B., J. E. Suárez, and A. Rodríguez. 1996. Lactococcin 972: a homodimeric lactococcal

bacteriocin whose primary target is not the plasma membrane. Microbiology 142:2393-2398. 330. Martinez, B., A. L. Zomer, A. Rodriguez, J. Kok, and O. P. Kuipers. 2007. Cell envelope stress

induced by the bacteriocin Lcn972 is sensed by the lactococcal two-component system CesSR. Mol. Microbiol. 64:473-486.

331. Martner, A., S. Skovbjerg, J. C. Paton, and A. E. Wold. 2009. Streptococcus pneumoniae autolysis

prevents phagocytosis and production of phagocyte-activating cytokines. Infect. Immun. 77:3826-3837.

332. Marugg, J. D., C. F. Gonzalez, B. S. Kunka, A. M. Ledeboer, M. J. Pucci, M. Y. Toonen, S. A.

Walker, L. C. Zoetmulder, and P. A. Vandenbergh. 1992. Cloning, expression, and nucleotide

References

154

sequence of genes involved in production of pediocin PA-1, and bacteriocin from Pediococcus

acidilactici PAC1.0. Appl. Environ. Microbiol 58:2360-2367.

333. Mascher, T., N. G. Margulis, T. Wang, R. W. Ye, and J. D. Helmann. 2003. Cell wall stress responses in Bacillus subtilis: the regulatory network of the bacitracin stimulon. Mol. Microbiol.

50:1591-1604.

334. Mascher, T., D. Zahner, M. Merai, N. Balmelle, A. B. de Saizieu, and R. Hakenbeck. 2003. The Streptococcus pneumoniae cia regulon: CiaR target sites and transcription profile analysis. J.

Bacteriol. 185:60-70.

335. Mascher, T., S. L. Zimmer, T. A. Smith, and J. D. Helmann. 2004. Antibiotic-inducible promoter regulated by the cell envelope stress-sensing two-component system LiaRS of Bacillus subtilis.

Antimicrob. Agents Chemother. 48:2888-2896.

336. Mattick, A. and A. Hirsch. 1947. Further observations on an inhibitory substance (nisin) from lactic streptococci. Lancet 2:5-8.

337. Mattick, T. R. and A. Hirsch. 1944. A Powerful Inhibitory Substance Produced by Group N

Streptococci. Nature 154:551. 338. McAuliffe, O., C. Hill, and R. P. Ross. 1999. Inhibition of Listeria monocytogenes in cottage cheese

manufactured with a lacticin 3147-producing starter culture. J. Appl Microbiol 86:251-256.

339. McAuliffe, O., C. Hill, and R. P. Ross. 2000. Each peptide of the two-component lantibiotic lacticin 3147 requires a separate modification enzyme for activity. Microbiology 146 ( Pt 9):2147-2154.

340. McAuliffe, O., T. O'Keeffe, C. Hill, and R. P. Ross. 2001. Regulation of immunity to the two-

component lantibiotic, lacticin 3147, by the transcriptional repressor LtnR. Mol. Microbiol 39:982-993.

341. McClerren, A. L., L. E. Cooper, C. Quan, P. M. Thomas, N. L. Kelleher, and W. A. van der Donk.

2006. Discovery and in vitro biosynthesis of haloduracin, a two-component lantibiotic. Proc. Natl. Acad. Sci. U. S. A 103:17243-17248.

342. McClerren, A. L., L. E. Cooper, C. Quan, P. M. Thomas, N. L. Kelleher, and W. A. van der Donk.

2006. Discovery and in vitro biosynthesis of haloduracin, a two-component lantibiotic. Proc. Natl.

Acad. Sci. U. S. A 103:17243-17248.

343. McCormick, J. K., R. W. Worobo, and M. E. Stiles. 1996. Expression of the antimicrobial peptide

carnobacteriocin B2 by a signal peptide-dependent general secretory pathway. Appl. Environ. Microbiol 62:4095-4099.

344. McGee, L., L. McDougal, J. Zhou, B. G. Spratt, F. C. Tenover, R. George, R. Hakenbeck, W.

Hryniewicz, J. C. Lefevre, A. Tomasz, and K. P. Klugman. 2001. Nomenclature of major antimicrobial-resistant clones of Streptococcus pneumoniae defined by the pneumococcal molecular

epidemiology network. J. Clin. Microbiol. 39:2565-2571.

345. Medzhitov, R. 2007. Recognition of microorganisms and activation of the immune response. Nature 449:819-826.

346. Meyer, C., G. Bierbaum, C. Heidrich, M. Reis, J. Suling, M. I. Iglesias-Wind, C. Kempter, E.

Molitor, and H. G. Sahl. 1995. Nucleotide sequence of the lantibiotic Pep5 biosynthetic gene cluster and functional analysis of PepP and PepC. Evidence for a role of PepC in thioether formation. Eur.

J. Biochem. 232:478-489.

347. Miller, L. M., C. Chatterjee, W. A. van der Donk, and N. L. Kelleher. 2006. The dehydratase activity of lacticin 481 synthetase is highly processive. J. Am. Chem. Soc. 128:1420-1421.

348. Millette, M., G. Cornut, C. Dupont, F. Shareck, D. Archambault, and M. Lacroix. 2008. Capacity of

human nisin- and pediocin-producing lactic Acid bacteria to reduce intestinal colonization by vancomycin-resistant enterococci. Appl. Environ. Microbiol 74:1997-2003.

349. Mindich, L. 1966. Bacteriocins of Diplococcus pneumoniae. I. Antagonistic relationships and

genetic transformations. J. Bacteriol. 92:1090-1098. 350. Mitchell, T. J. 2003. The pathogenesis of streptococcal infections: from tooth decay to meningitis.

Nat. Rev. Microbiol 1:219-230.

351. Mohedano, M. L., K. Overweg, A. de la Fuente, M. Reuter, S. Altabe, F. Mulholland, D. de Mendoza, P. Lopez, and J. M. Wells. 2005. Evidence that the essential response regulator YycF in

Streptococcus pneumoniae modulates expression of fatty acid biosynthesis genes and alters membrane composition. J. Bacteriol. 187:2357-2367.

352. Moll, G. N., W. N. Konings, and A. J. Driessen. 1999. Bacteriocins: mechanism of membrane

insertion and pore formation. Antonie Van Leeuwenhoek 76:185-198. 353. Moll, G. N., E. van den Akker, H. H. Hauge, J. Nissen-Meyer, I. F. Nes, W. N. Konings, and A. J.

Driessen. 1999. Complementary and overlapping selectivity of the two-peptide bacteriocins

plantaricin EF and JK. J. Bacteriol. 181:4848-4852.

References

155

354. Molle, V., Y. Nakaura, R. P. Shivers, H. Yamaguchi, R. Losick, Y. Fujita, and A. L. Sonenshein.

2003. Additional targets of the Bacillus subtilis global regulator CodY identified by chromatin

immunoprecipitation and genome-wide transcript analysis. J. Bacteriol. 185:1911-1922. 355. Morgan, S., R. P. Ross, and C. Hill. 1995. Bacteriolytic activity caused by the presence of a novel

lactococcal plasmid encoding lactococcins A, B, and M. Appl. Environ. Microbiol 61:2995-3001.

356. Morona, J. K., D. C. Miller, R. Morona, and J. C. Paton. 2004. The effect that mutations in the conserved capsular polysaccharide biosynthesis genes cpsA, cpsB, and cpsD have on virulence of

Streptococcus pneumoniae. J. Infect. Dis. 189:1905-1913.

357. Morrison, D. A. and M. S. Lee. 2000. Regulation of competence for genetic transformation in Streptococcus pneumoniae: a link between quorum sensing and DNA processing genes. Res.

Microbiol 151:445-451.

358. Mota-Meira, M., C. Lacroix, G. LaPointe, and M. C. Lavoie. 1997. Purification and structure of mutacin B-Ny266: a new lantibiotic produced by Streptococcus mutans. FEBS Lett. 410:275-279.

359. Mulders, J. W., I. J. Boerrigter, H. S. Rollema, R. J. Siezen, and W. M. de Vos. 1991. Identification

and characterization of the lantibiotic nisin Z, a natural nisin variant. Eur. J. Biochem. 201:581-584. 360. Muriana, P. M. and T. R. Klaenhammer. 1991. Purification and partial characterization of lactacin F,

a bacteriocin produced by Lactobacillus acidophilus 11088. Appl. Environ. Microbiol 57:114-121.

361. Muriana, P. M. and T. R. Klaenhammer. 1991. Cloning, phenotypic expression, and DNA sequence of the gene for lactacin F, an antimicrobial peptide produced by Lactobacillus spp. J. Bacteriol.

173:1779-1788.

362. Murphy, T. F., L. O. Bakaletz, and P. R. Smeesters. 2009. Microbial interactions in the respiratory tract. Pediatr. Infect. Dis. J. 28:S121-S126.

363. Nakamura, S. and E. Racker. 1984. Inhibitory effect of duramycin on partial reactions catalyzed by

(Na+,K+)-adenosinetriphosphatase from dog kidney. Biochemistry 23:385-389. 364. Navaratna, M. A., H. G. Sahl, and J. R. Tagg. 1998. Two-component anti-Staphylococcus aureus

lantibiotic activity produced by Staphylococcus aureus C55. Appl. Environ. Microbiol 64:4803-

4808.

365. Navarro, J., J. Chabot, K. Sherrill, R. Aneja, S. A. Zahler, and E. Racker. 1985. Interaction of

duramycin with artificial and natural membranes. Biochemistry 24:4645-4650.

366. Nelson, A. L., J. Ries, F. Bagnoli, S. Dahlberg, S. Falker, S. Rounioja, J. Tschop, E. Morfeldt, I. Ferlenghi, M. Hilleringmann, D. W. Holden, R. Rappuoli, S. Normark, M. A. Barocchi, and B.

Henriques-Normark. 2007. RrgA is a pilus-associated adhesin in Streptococcus pneumoniae. Mol.

Microbiol 66:329-340. 367. Nelson, A. L., A. M. Roche, J. M. Gould, K. Chim, A. J. Ratner, and J. N. Weiser. 2007. Capsule

enhances pneumococcal colonization by limiting mucus-mediated clearance. Infect. Immun. 75:83-

90. 368. Nes, I. F. and H. Holo. 2000. Class II antimicrobial peptides from lactic acid bacteria. Biopolymers

55:50-61.

369. Netz, D. J., H. G. Sahl, R. Marcelino, N. J. dos Santos, S. S. de Oliveira, M. B. Soares, and do Carmo de Freire Bastos. 2001. Molecular characterisation of aureocin A70, a multi-peptide

bacteriocin isolated from Staphylococcus aureus. J. Mol. Biol. 311:939-949.

370. Ng, W. L., H. C. Tsui, and M. E. Winkler. 2005. Regulation of the pspA virulence factor and essential pcsB murein biosynthetic genes by the phosphorylated VicR (YycF) response regulator in

Streptococcus pneumoniae. J. Bacteriol. 187:7444-7459.

371. Nicolas, G., H. Morency, G. LaPointe, and M. C. Lavoie. 2006. Mutacin H-29B is identical to mutacin II (J-T8). BMC. Microbiol 6:36.

372. Nissen-Meyer, J., L. S. Havarstein, H. Holo, K. Sletten, and I. F. Nes. 1993. Association of the

lactococcin A immunity factor with the cell membrane: purification and characterization of the immunity factor. J. Gen. Microbiol 139:1503-1509.

373. Nissen-Meyer, J., H. Holo, L. S. Havarstein, K. Sletten, and I. F. Nes. 1992. A novel lactococcal

bacteriocin whose activity depends on the complementary action of two peptides. J. Bacteriol. 174:5686-5692.

374. Nissen-Meyer, J., P. Rogne, C. Oppegard, H. S. Haugen, and P. E. Kristiansen. 2009. Structure-function relationships of the non-lanthionine-containing peptide (class II) bacteriocins produced by

gram-positive bacteria. Curr. Pharm. Biotechnol. 10:19-37.

375. Niyonsaba, F., I. Nagaoka, and H. Ogawa. 2006. Human defensins and cathelicidins in the skin: beyond direct antimicrobial properties. Crit Rev. Immunol. 26:545-576.

376. Niyonsaba, F., I. Nagaoka, H. Ogawa, and K. Okumura. 2009. Multifunctional antimicrobial

proteins and peptides: natural activators of immune systems. Curr. Pharm. Des 15:2393-2413.

References

156

377. Nizet, V., B. Beall, D. J. Bast, V. Datta, L. Kilburn, D. E. Low, and J. C. De Azavedo. 2000. Genetic

locus for streptolysin S production by group A streptococcus. Infect. Immun. 68:4245-4254.

378. Noske, N., U. Kammerer, M. Rohde, and S. Hammerschmidt. 2009. Pneumococcal interaction with human dendritic cells: phagocytosis, survival, and induced adaptive immune response are

manipulated by PavA. J. Immunol. 183:1952-1963.

379. O'Connor, L., A. Coffey, C. Daly, and G. F. Fitzgerald. 1996. AbiG, a genotypically novel abortive infection mechanism encoded by plasmid pCI750 of Lactococcus lactis subsp. cremoris UC653.

Appl. Environ. Microbiol 62:3075-3082.

380. Obert, C., J. Sublett, D. Kaushal, E. Hinojosa, T. Barton, E. I. Tuomanen, and C. J. Orihuela. 2006. Identification of a Candidate Streptococcus pneumoniae core genome and regions of diversity

correlated with invasive pneumococcal disease. Infect. Immun. 74:4766-4777.

381. Ogunniyi, A. D., M. Grabowicz, D. E. Briles, J. Cook, and J. C. Paton. 2007. Development of a vaccine against invasive pneumococcal disease based on combinations of virulence proteins of

Streptococcus pneumoniae. Infect. Immun. 75:350-357.

382. Ogunniyi, A. D., M. Grabowicz, L. K. Mahdi, J. Cook, D. L. Gordon, T. A. Sadlon, and J. C. Paton. 2009. Pneumococcal histidine triad proteins are regulated by the Zn2+-dependent repressor AdcR

and inhibit complement deposition through the recruitment of complement factor H. FASEB J.

23:731-738. 383. Ogunniyi, A. D., K. S. LeMessurier, R. M. Graham, J. M. Watt, D. E. Briles, U. H. Stroeher, and J.

C. Paton. 2007. Contributions of pneumolysin, pneumococcal surface protein A (PspA), and PspC to

pathogenicity of Streptococcus pneumoniae D39 in a mouse model. Infect. Immun. 75:1843-1851. 384. Okuda, K., Y. Aso, J. Nagao, K. Shioya, Y. Kanemasa, J. Nakayama, and K. Sonomoto. 2005.

Characterization of functional domains of lantibiotic-binding immunity protein, NukH, from

Staphylococcus warneri ISK-1. FEMS Microbiol Lett. 250:19-25. 385. Okuda, K., Y. Aso, J. Nakayama, and K. Sonomoto. 2008. Cooperative transport between NukFEG

and NukH in immunity against the lantibiotic nukacin ISK-1 produced by Staphylococcus warneri

ISK-1. J. Bacteriol. 190:356-362.

386. Okuda, K., S. Yanagihara, K. Shioya, Y. Harada, J. Nagao, Y. Aso, T. Zendo, J. Nakayama, and K.

Sonomoto. 2008. Binding specificity of the lantibiotic-binding immunity protein NukH. Appl.

Environ. Microbiol 74:7613-7619. 387. Oppegard, C., L. Emanuelsen, L. Thorbek, G. Fimland, and J. Nissen-Meyer. 2010. The lactococcin

G immunity protein recognizes specific regions in both peptides constituting the two-peptide

bacteriocin lactococcin G. Appl. Environ. Microbiol 76:1267-1273. 388. Oren, Z., J. C. Lerman, G. H. Gudmundsson, B. Agerberth, and Y. Shai. 1999. Structure and

organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the

molecular basis for its non-cell-selective activity. Biochem. J. 341 ( Pt 3):501-513. 389. Orihuela, C. J., G. Gao, K. P. Francis, J. Yu, and E. I. Tuomanen. 2004. Tissue-specific

contributions of pneumococcal virulence factors to pathogenesis. J. Infect. Dis. 190:1661-1669.

390. Orihuela, C. J., J. N. Radin, J. E. Sublett, G. Gao, D. Kaushal, and E. I. Tuomanen. 2004. Microarray analysis of pneumococcal gene expression during invasive disease. Infect. Immun. 72:5582-5596.

391. Otto, M., A. Peschel, and F. Götz. 1998. Producer self-protection against the lantibiotic epidermin

by the ABC transporter EpiFEG of Staphylococcus epidermidis Tu3298. FEMS Microbiol Lett. 166:203-211.

392. Overbeek, R., N. Larsen, T. Walunas, M. D'Souza, G. Pusch, Selkov E Jr, K. Liolios, V. Joukov, D.

Kaznadzey, I. Anderson, A. Bhattacharyya, H. Burd, W. Gardner, P. Hanke, V. Kapatral, N. Mikhailova, O. Vasieva, A. Osterman, V. Vonstein, M. Fonstein, N. Ivanova, and N. Kyrpides.

2003. The ERGO genome analysis and discovery system. Nucleic Acids Res. 31:164-171.

393. Pag, U. and H. G. Sahl. 2002. Multiple activities in lantibiotics--models for the design of novel antibiotics? Curr. Pharm. Des 8:815-833.

394. Paik, S. H., A. Chakicherla, and J. N. Hansen. 1998. Identification and characterization of the

structural and transporter genes for, and the chemical and biological properties of, sublancin 168, a novel lantibiotic produced by Bacillus subtilis 168. J. Biol. Chem. 273:23134-23142.

395. Pao, S. S., I. T. Paulsen, and M. H. Saier, Jr. 1998. Major facilitator superfamily. Microbiol. Mol. Biol. Rev. 62:1-34.

396. Papadelli, M., A. Karsioti, R. Anastasiou, M. Georgalaki, and E. Tsakalidou. 2007. Characterization

of the gene cluster involved in the biosynthesis of macedocin, the lantibiotic produced by Streptococcus macedonicus. FEMS Microbiol. Lett. 272:75-82.

397. Papathanasopoulos, M. A., G. A. Dykes, A. M. Revol-Junelles, A. Delfour, A. von Holy, and J. W.

Hastings. 1998. Sequence and structural relationships of leucocins A-, B- and C-TA33a from Leuconostoc mesenteroides TA33a. Microbiology 144 ( Pt 5):1343-1348.

References

157

398. Parente, E. and A. Ricciardi. 1999. Production, recovery and purification of bacteriocins from lactic

acid bacteria. Appl. Microbiol Biotechnol. 52:628-638.

399. Park, I. H., D. G. Pritchard, R. Cartee, A. Brandao, M. C. Brandileone, and M. H. Nahm. 2007. Discovery of a new capsular serotype (6C) within serogroup 6 of Streptococcus pneumoniae. J. Clin.

Microbiol. 45:1225-1233.

400. Patton, G. C., M. Paul, L. E. Cooper, C. Chatterjee, and W. A. van der Donk. 2008. The importance of the leader sequence for directing lanthionine formation in lacticin 481. Biochemistry 47:7342-

7351.

401. Pei, J. and N. V. Grishin. 2001. Type II CAAX prenyl endopeptidases belong to a novel superfamily of putative membrane-bound metalloproteases. Trends Biochem. Sci. 26:275-277.

402. Pericone, C. D., K. Overweg, P. W. Hermans, and J. N. Weiser. 2000. Inhibitory and bactericidal

effects of hydrogen peroxide production by Streptococcus pneumoniae on other inhabitants of the upper respiratory tract. Infect. Immun. 68:3990-3997.

403. Perry, J. A., M. B. Jones, S. N. Peterson, D. G. Cvitkovitch, and C. M. Levesque. 2009. Peptide

alarmone signalling triggers an auto-active bacteriocin necessary for genetic competence. Mol. Microbiol 72:905-917.

404. Peschel, A., J. Augustin, T. Kupke, S. Stevanovic, and F. Götz. 1993. Regulation of epidermin

biosynthetic genes by EpiQ. Mol. Microbiol 9:31-39. 405. Peschel, A. and F. Götz. 1996. Analysis of the Staphylococcus epidermidis genes epiF, -E, and -G

involved in epidermin immunity. J. Bacteriol. 178:531-536.

406. Peschel, A., M. Otto, R. W. Jack, H. Kalbacher, G. Jung, and F. Götz. 1999. Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial

peptides. J. Biol. Chem. 274:8405-8410.

407. Peschel, A. and H. G. Sahl. 2006. The co-evolution of host cationic antimicrobial peptides and microbial resistance. Nat. Rev. Microbiol 4:529-536.

408. Peschel, A., N. Schnell, M. Hille, K. D. Entian, and F. Götz. 1997. Secretion of the lantibiotics

epidermin and gallidermin: sequence analysis of the genes gdmT and gdmH, their influence on

epidermin production and their regulation by EpiQ. Mol. Gen. Genet. 254:312-318.

409. Petersen, F. C., G. Fimland, and A. A. Scheie. 2006. Purification and functional studies of a potent

modified quorum-sensing peptide and a two-peptide bacteriocin in Streptococcus mutans. Mol. Microbiol 61:1322-1334.

410. Peterson, S. N., C. K. Sung, R. Cline, B. V. Desai, E. C. Snesrud, P. Luo, J. Walling, H. Li, M.

Mintz, G. Tsegaye, P. C. Burr, Y. Do, S. Ahn, J. Gilbert, R. D. Fleischmann, and D. A. Morrison. 2004. Identification of competence pheromone responsive genes in Streptococcus pneumoniae by

use of DNA microarrays. Mol. Microbiol 51:1051-1070.

411. Petranovic, D., E. Guedon, B. Sperandio, C. Delorme, D. Ehrlich, and P. Renault. 2004. Intracellular effectors regulating the activity of the Lactococcus lactis CodY pleiotropic transcription regulator.

Mol. Microbiol. 53:613-621.

412. Pettigrew, M. M., J. F. Gent, K. Revai, J. A. Patel, and T. Chonmaitree. 2008. Microbial interactions during upper respiratory tract infections. Emerg. Infect. Dis. 14:1584-1591.

413. Piard, J. C., P. M. Muriana, M. J. Desmazeaud, and T. R. Klaenhammer. 1992. Purification and

Partial Characterization of Lacticin 481, a Lanthionine-Containing Bacteriocin Produced by Lactococcus lactis subsp. lactis CNRZ 481. Appl. Environ. Microbiol 58:279-284.

414. Pietiainen, M., M. Gardemeister, M. Mecklin, S. Leskela, M. Sarvas, and V. P. Kontinen. 2005.

Cationic antimicrobial peptides elicit a complex stress response in Bacillus subtilis that involves ECF-type sigma factors and two-component signal transduction systems. Microbiology 151:1577-

1592.

415. Piper, C., P. D. Cotter, R. P. Ross, and C. Hill. 2009. Discovery of medically significant lantibiotics. Curr. Drug Discov. Technol. 6:1-18.

416. Podbielski, A. and B. Kreikemeyer. 2004. Cell density--dependent regulation: basic principles and

effects on the virulence of Gram-positive cocci. Int. J. Infect. Dis. 8:81-95. 417. Pohl, K., P. Francois, L. Stenz, F. Schlink, T. Geiger, S. Herbert, C. Goerke, J. Schrenzel, and C.

Wolz. 2009. CodY in Staphylococcus aureus: a regulatory link between metabolism and virulence gene expression. J. Bacteriol. 191:2953-2963.

418. Pozzi, G., L. Masala, F. Iannelli, R. Manganelli, L. S. Havarstein, L. Piccoli, D. Simon, and D. A.

Morrison. 1996. Competence for genetic transformation in encapsulated strains of Streptococcus pneumoniae: two allelic variants of the peptide pheromone. J. Bacteriol. 178:6087-6090.

419. Pracht, D., C. Elm, J. Gerber, S. Bergmann, M. Rohde, M. Seiler, K. S. Kim, H. F. Jenkinson, R.

Nau, and S. Hammerschmidt. 2005. PavA of Streptococcus pneumoniae modulates adherence, invasion, and meningeal inflammation. Infect. Immun. 73:2680-2689.

References

158

420. Pridmore, D., N. Rekhif, A. C. Pittet, B. Suri, and B. Mollet. 1996. Variacin, a new lanthionine-

containing bacteriocin produced by Micrococcus varians: comparison to lacticin 481 of Lactococcus

lactis. Appl. Environ. Microbiol 62:1799-1802. 421. Qi, F., P. Chen, and P. W. Caufield. 1999. Functional analyses of the promoters in the lantibiotic

mutacin II biosynthetic locus in Streptococcus mutans. Appl. Environ. Microbiol. 65:652-658.

422. Qi, F., P. Chen, and P. W. Caufield. 1999. Purification of mutacin III from group III Streptococcus mutans UA787 and genetic analyses of mutacin III biosynthesis genes. Appl. Environ. Microbiol.

65:3880-3887.

423. Qi, F., P. Chen, and P. W. Caufield. 2000. Purification and biochemical characterization of mutacin I from the group I strain of Streptococcus mutans, CH43, and genetic analysis of mutacin I

biosynthesis genes. Appl. Environ. Microbiol. 66:3221-3229.

424. Qi, F., P. Chen, and P. W. Caufield. 2001. The group I strain of Streptococcus mutans, UA140, produces both the lantibiotic mutacin I and a nonlantibiotic bacteriocin, mutacin IV. Appl. Environ.

Microbiol. 67:15-21.

425. Quadri, L. E., M. Sailer, M. R. Terebiznik, K. L. Roy, J. C. Vederas, and M. E. Stiles. 1995. Characterization of the protein conferring immunity to the antimicrobial peptide carnobacteriocin B2

and expression of carnobacteriocins B2 and BM1. J. Bacteriol. 177:1144-1151.

426. Ra, R., M. M. Beerthuyzen, W. M. de Vos, P. E. Saris, and O. P. Kuipers. 1999. Effects of gene disruptions in the nisin gene cluster of Lactococcus lactis on nisin production and producer

immunity. Microbiology 145 ( Pt 5):1227-1233.

427. Ra, S. R., M. Qiao, T. Immonen, I. Pujana, and E. J. Saris. 1996. Genes responsible for nisin synthesis, regulation and immunity form a regulon of two operons and are induced by nisin in

Lactoccocus lactis N8. Microbiology 142 ( Pt 5):1281-1288.

428. Radek, K. and R. Gallo. 2007. Antimicrobial peptides: natural effectors of the innate immune system. Semin. Immunopathol. 29:27-43.

429. Ramnath, M., S. Arous, A. Gravesen, J. W. Hastings, and Y. Hechard. 2004. Expression of mptC of

Listeria monocytogenes induces sensitivity to class IIa bacteriocins in Lactococcus lactis.

Microbiology 150:2663-2668.

430. Ramnath, M., M. Beukes, K. Tamura, and J. W. Hastings. 2000. Absence of a putative mannose-

specific phosphotransferase system enzyme IIAB component in a leucocin A-resistant strain of Listeria monocytogenes, as shown by two-dimensional sodium dodecyl sulfate-polyacrylamide gel

electrophoresis. Appl. Environ. Microbiol 66:3098-3101.

431. Ramos-Montanez, S., H. C. Tsui, K. J. Wayne, J. L. Morris, L. E. Peters, F. Zhang, K. M. Kazmierczak, L. T. Sham, and M. E. Winkler. 2008. Polymorphism and regulation of the spxB

(pyruvate oxidase) virulence factor gene by a CBS-HotDog domain protein (SpxR) in serotype 2

Streptococcus pneumoniae. Mol. Microbiol. 67:729-746. 432. Ratnayake-Lecamwasam, M., P. Serror, K. W. Wong, and A. L. Sonenshein. 2001. Bacillus subtilis

CodY represses early-stationary-phase genes by sensing GTP levels. Genes Dev. 15:1093-1103.

433. Rayman, M. K., B. Aris, and A. Hurst. 1981. Nisin: a possible alternative or adjunct to nitrite in the preservation of meats. Appl Environ. Microbiol 41:375-380.

434. Regev-Yochay, G., M. Lipsitch, A. Basset, E. Rubinstein, R. Dagan, M. Raz, and R. Malley. 2009.

The pneumococcal pilus predicts the absence of Staphylococcus aureus co-colonization in pneumococcal carriers. Clin. Infect. Dis. 48:760-763.

435. Regev-Yochay, G., K. Trzcinski, C. M. Thompson, R. Malley, and M. Lipsitch. 2006. Interference

between Streptococcus pneumoniae and Staphylococcus aureus: In vitro hydrogen peroxide-mediated killing by Streptococcus pneumoniae. J. Bacteriol. 188:4996-5001.

436. Reichmann, P. and R. Hakenbeck. 2000. Allelic variation in a peptide-inducible two-component

system of Streptococcus pneumoniae. FEMS Microbiol. Lett. 190:231-236. 437. Riley, M. A. and D. M. Gordon. 1999. The ecological role of bacteriocins in bacterial competition.

Trends Microbiol 7:129-133.

438. Rink, R., L. D. Kluskens, A. Kuipers, A. J. Driessen, O. P. Kuipers, and G. N. Moll. 2007. NisC, the cyclase of the lantibiotic nisin, can catalyze cyclization of designed nonlantibiotic peptides.

Biochemistry 46:13179-13189. 439. Rink, R., A. Kuipers, E. de Boef, K. J. Leenhouts, A. J. Driessen, G. N. Moll, and O. P. Kuipers.

2005. Lantibiotic structures as guidelines for the design of peptides that can be modified by

lantibiotic enzymes. Biochemistry 44:8873-8882. 440. Risoen, P. A., O. Johnsborg, D. B. Diep, L. Hamoen, G. Venema, and I. F. Nes. 2001. Regulation of

bacteriocin production in Lactobacillus plantarum depends on a conserved promoter arrangement

with consensus binding sequence. Mol. Genet. Genomics 265:198-206.

References

159

441. Robson, C. L., P. A. Wescombe, N. A. Klesse, and J. R. Tagg. 2007. Isolation and partial

characterization of the Streptococcus mutans type AII lantibiotic mutacin K8. Microbiology

153:1631-1641. 442. Rodgers, G. L., A. Arguedas, R. Cohen, and R. Dagan. 2009. Global serotype distribution among

Streptococcus pneumoniae isolates causing otitis media in children: potential implications for

pneumococcal conjugate vaccines. Vaccine 27:3802-3810. 443. Rogers, A. H. 1972. Effect of the medium on bacteriocin production among strains of Streptococcus

mutans. Appl. Microbiol 24:294-295.

444. Rogers, L. A. 1928. The inhibiting effect of Streptococcus lactis on Lactobacillus bulgaricus. J Bacteriol. 16:321-325.

445. Rogers, P. D., T. T. Liu, K. S. Barker, G. M. Hilliard, B. K. English, J. Thornton, E. Swiatlo, and L.

S. McDaniel. 2007. Gene expression profiling of the response of Streptococcus pneumoniae to penicillin. J. Antimicrob. Chemother. 59:616-626.

446. Rollema, H. S., O. P. Kuipers, P. Both, W. M. de Vos, and R. J. Siezen. 1995. Improvement of

solubility and stability of the antimicrobial peptide nisin by protein engineering. Appl Environ. Microbiol 61:2873-2878.

447. Rose, L., P. Shivshankar, E. Hinojosa, A. Rodriguez, C. J. Sanchez, and C. J. Orihuela. 2008.

Antibodies against PsrP, a novel Streptococcus pneumoniae adhesin, block adhesion and protect mice against pneumococcal challenge. J. Infect. Dis. 198:375-383.

448. Rosenow, C., P. Ryan, J. N. Weiser, S. Johnson, P. Fontan, A. Ortqvist, and H. R. Masure. 1997.

Contribution of novel choline-binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae. Mol. Microbiol. 25:819-829.

449. Ross, K. F., C. W. Ronson, and J. R. Tagg. 1993. Isolation and characterization of the lantibiotic

salivaricin A and its structural gene salA from Streptococcus salivarius 20P3. Appl. Environ. Microbiol 59:2014-2021.

450. Ross, R. P., M. Galvin, O. McAuliffe, S. M. Morgan, M. P. Ryan, D. P. Twomey, W. J. Meaney,

and C. Hill. 1999. Developing applications for lactococcal bacteriocins. Antonie Van Leeuwenhoek

76:337-346.

451. Ryan, M. P., J. Flynn, C. Hill, R. P. Ross, and W. J. Meaney. 1999. The natural food grade inhibitor,

lacticin 3147, reduced the incidence of mastitis after experimental challenge with Streptococcus dysgalactiae in nonlactating dairy cows. J Dairy Sci. 82:2625-2631.

452. Ryan, M. P., W. J. Meaney, R. P. Ross, and C. Hill. 1998. Evaluation of lacticin 3147 and a teat seal

containing this bacteriocin for inhibition of mastitis pathogens. Appl. Environ. Microbiol 64:2287-2290.

453. Ryan, M. P., M. C. Rea, C. Hill, and R. P. Ross. 1996. An application in cheddar cheese

manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147. Appl. Environ. Microbiol 62:612-619.

454. Sa-Nogueira, I. and L. J. Mota. 1997. Negative regulation of L-arabinose metabolism in Bacillus

subtilis: characterization of the araR (araC) gene. J. Bacteriol. 179:1598-1608. 455. Sahm, D. F., J. Kissinger, M. S. Gilmore, P. R. Murray, R. Mulder, J. Solliday, and B. Clarke. 1989.

In vitro susceptibility studies of vancomycin-resistant Enterococcus faecalis. Antimicrob. Agents

Chemother. 33:1588-1591. 456. Saier, M. H., Jr., J. T. Beatty, A. Goffeau, K. T. Harley, W. H. Heijne, S. C. Huang, D. L. Jack, P. S.

Jahn, K. Lew, J. Liu, S. S. Pao, I. T. Paulsen, T. T. Tseng, and P. S. Virk. 1999. The major facilitator

superfamily. J. Mol. Microbiol. Biotechnol. 1:257-279. 457. Sambrook, J., E. F. Fritsch, and A. Maniatis. 1989. Molecular cloning: a laboratory manual, In .

Cold Spring Laboratory Press, Cold Spring Harbor, NY.

458. Sandgren, A., K. Sjostrom, B. Olsson-Liljequist, B. Christensson, A. Samuelsson, G. Kronvall, and N. B. Henriques. 2004. Effect of clonal and serotype-specific properties on the invasive capacity of

Streptococcus pneumoniae. J. Infect. Dis. 189:785-796.

459. Sashihara, T., M. Dan, H. Kimura, H. Matsusaki, K. Sonomoto, and A. Ishizaki. 2001. The effect of osmotic stress on the production of nukacin ISK-1 from Staphylococcus warneri ISK-1. Appl.

Microbiol Biotechnol. 56:496-501. 460. Sashihara, T., H. Kimura, T. Higuchi, A. Adachi, H. Matsusaki, K. Sonomoto, and A. Ishizaki.

2000. A novel lantibiotic, nukacin ISK-1, of Staphylococcus warneri ISK-1: cloning of the structural

gene and identification of the structure. Biosci. Biotechnol. Biochem. 64:2420-2428. 461. Schägger, H. and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel

electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem.

462. Schlegel, R. and H. D. Slade. 1973. Properties of a Streptococcus sanguis (group H) bacteriocin and its separation from the competence factor of transformation. J. Bacteriol. 115:655-661.

References

160

463. Schnell, N., G. Engelke, J. Augustin, R. Rosenstein, V. Ungermann, F. Götz, and K. D. Entian.

1992. Analysis of genes involved in the biosynthesis of lantibiotic epidermin. Eur. J. Biochem.

204:57-68. 464. Schwarzer, D., R. Finking, and M. A. Marahiel. 2003. Nonribosomal peptides: from genes to

products. Nat Prod. Rep. 20:275-287.

465. Scott, J. A. 2007. The preventable burden of pneumococcal disease in the developing world. Vaccine 25:2398-2405.

466. Sebert, M. E., L. M. Palmer, M. Rosenberg, and J. N. Weiser. 2002. Microarray-based identification

of htrA, a Streptococcus pneumoniae gene that is regulated by the CiaRH two-component system and contributes to nasopharyngeal colonization. Infect Immun 70:4059-4067.

467. Semedo, T., S. M. Almeida, P. Martins, M. F. Silva Lopes, J. J. Figueiredo Marques, R. Tenreiro,

and M. T. Barreto Crespo. 2003. Comparative study using type strains and clinical and food isolates to examine hemolytic activity and occurrence of the cyl operon in enterococci. J. Clin. Microbiol.

41:2569-2576.

468. Semedo, T., S. M. Almeida, P. Martins, M. F. Silva Lopes, J. J. Figueiredo Marques, R. Tenreiro, and M. T. Barreto Crespo. 2003. Comparative study using type strains and clinical and food isolates

to examine hemolytic activity and occurrence of the cyl operon in enterococci. J. Clin. Microbiol.

41:2569-2576. 469. Serror, P. and A. L. Sonenshein. 1996. Interaction of CodY, a novel Bacillus subtilis DNA-binding

protein, with the dpp promoter region. Mol. Microbiol. 20:843-852.

470. Severina, E., A. Severin, and A. Tomasz. 1998. Antibacterial efficacy of nisin against multidrug-resistant Gram-positive pathogens. J Antimicrob. Chemother. 41:341-347.

471. Shaper, M., S. K. Hollingshead, W. H. Benjamin, Jr., and D. E. Briles. 2004. PspA protects

Streptococcus pneumoniae from killing by apolactoferrin, and antibody to PspA enhances killing of pneumococci by apolactoferrin [corrected]. Infect. Immun. 72:5031-5040.

472. Shen, K., J. Gladitz, P. Antalis, B. Dice, B. Janto, R. Keefe, J. Hayes, A. Ahmed, R. Dopico, N.

Ehrlich, J. Jocz, L. Kropp, S. Yu, L. Nistico, D. P. Greenberg, K. Barbadora, R. A. Preston, J. C.

Post, G. D. Ehrlich, and F. Z. Hu. 2006. Characterization, distribution, and expression of novel

genes among eight clinical isolates of Streptococcus pneumoniae. Infect. Immun. 74:321-330.

473. Shivers, R. P., S. S. Dineen, and A. L. Sonenshein. 2006. Positive regulation of Bacillus subtilis ackA by CodY and CcpA: establishing a potential hierarchy in carbon flow. Mol. Microbiol. 62:811-

822.

474. Shivshankar, P., C. Sanchez, L. F. Rose, and C. J. Orihuela. 2009. The Streptococcus pneumoniae adhesin PsrP binds to Keratin 10 on lung cells. Mol. Microbiol 73:663-679.

475. Siegers, K. and K. D. Entian. 1995. Genes involved in immunity to the lantibiotic nisin produced by

Lactococcus lactis 6F3. Appl. Environ. Microbiol 61:1082-1089. 476. Siezen, R. J., O. P. Kuipers, and W. M. de Vos. 1996. Comparison of lantibiotic gene clusters and

encoded proteins. Antonie Van Leeuwenhoek 69:171-184.

477. Siezen, R. J., H. S. Rollema, O. P. Kuipers, and W. M. de Vos. 1995. Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic

nisin. Protein Eng 8:117-125.

478. Sinensky, M. 2000. Recent advances in the study of prenylated proteins. Biochim. Biophys. Acta 1484:93-106.

479. Siragusa, G. R. and C. N. Cutter. 1993. Brochocin-C, a new bacteriocin produced by Brochothrix

campestris. Appl. Environ. Microbiol 59:2326-2328. 480. Skaugen, M., C. I. Abildgaard, and I. F. Nes. 1997. Organization and expression of a gene cluster

involved in the biosynthesis of the lantibiotic lactocin S. Mol. Gen. Genet. 253:674-686.

481. Skaugen, M. and I. F. Nes. 2000. Transposition in Lactobacillus sakei: inactivation of a second lactocin S operon by the insertion of IS1520, a new member of the IS3 family of insertion

sequences. Microbiology 146 ( Pt 5):1163-1169.

482. Slamti, L. and D. Lereclus. 2002. A cell-cell signaling peptide activates the PlcR virulence regulon in bacteria of the Bacillus cereus group. EMBO J. 21:4550-4559.

483. Smith, L. and J. Hillman. 2008. Therapeutic potential of type A (I) lantibiotics, a group of cationic peptide antibiotics. Curr. Opin. Microbiol 11:401-408.

484. Sonenshein, A. L. 2005. CodY, a global regulator of stationary phase and virulence in Gram-positive

bacteria. Curr. Opin. Microbiol. 8:203-207. 485. Song, J. H., K. S. Ko, J. Y. Lee, J. Y. Baek, W. S. Oh, H. S. Yoon, J. Y. Jeong, and J. Chun. 2005.

Identification of essential genes in Streptococcus pneumoniae by allelic replacement mutagenesis.

Mol. Cells 19:365-374.

References

161

486. Sorensen, O. E., P. Follin, A. H. Johnsen, J. Calafat, G. S. Tjabringa, P. S. Hiemstra, and N.

Borregaard. 2001. Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by

extracellular cleavage with proteinase 3. Blood 97:3951-3959. 487. Stein, T., S. Borchert, P. Kiesau, S. Heinzmann, S. Kloss, C. Klein, M. Helfrich, and K. D. Entian.

2002. Dual control of subtilin biosynthesis and immunity in Bacillus subtilis. Mol. Microbiol

44:403-416. 488. Stein, T., S. Heinzmann, S. Dusterhus, S. Borchert, and K. D. Entian. 2005. Expression and

functional analysis of the subtilin immunity genes spaIFEG in the subtilin-sensitive host Bacillus

subtilis MO1099. J. Bacteriol. 187:822-828. 489. Stein, T., S. Heinzmann, I. Solovieva, and K. D. Entian. 2003. Function of Lactococcus lactis nisin

immunity genes nisI and nisFEG after coordinated expression in the surrogate host Bacillus subtilis.

J. Biol. Chem. 278:89-94. 490. Stone, D. K., X. S. Xie, and E. Racker. 1984. Inhibition of clathrin-coated vesicle acidification by

duramycin. J. Biol. Chem. 259:2701-2703.

491. Stone, K. J. and J. L. Strominger. 1971. Mechanism of action of bacitracin: complexation with metal ion and C 55 -isoprenyl pyrophosphate. Proc. Natl. Acad. Sci. U. S. A 68:3223-3227.

492. Storm, D. R. and J. L. Strominger. 1973. Complex formation between bacitracin peptides and

isoprenyl pyrophosphates. The specificity of lipid-peptide interactions. J. Biol. Chem. 248:3940-3945.

493. Stothard, P., D. G. Van, S. Shrivastava, A. Guo, B. O'Neill, J. Cruz, M. Ellison, and D. S. Wishart.

2005. BacMap: an interactive picture atlas of annotated bacterial genomes. Nucleic Acids Res. 33:D317-D320.

494. Subbash Chandra Parija. 2009. Textbook of Microbiology & Immunology, p. 648-650. In Shabina

Nasim (ed.). ELSEVIER. 495. Sulavik, M. C. and D. B. Clewell. 1996. Rgg is a positive transcriptional regulator of the

Streptococcus gordonii gtfG gene. J. Bacteriol. 178:5826-5830.

496. Sulavik, M. C., G. Tardif, and D. B. Clewell. 1992. Identification of a gene, rgg, which regulates

expression of glucosyltransferase and influences the Spp phenotype of Streptococcus gordonii

Challis. J. Bacteriol. 174:3577-3586.

497. Tahara, T. and K. Kanatani. 1996. Isolation, partial characterization and mode of action of acidocin J1229, a bacteriocin produced by Lactobacillus acidophilus JCM 1229. J. Appl. Bacteriol. 81:669-

677.

498. Tahara, T., M. Oshimura, C. Umezawa, and K. Kanatani. 1996. Isolation, partial characterization, and mode of action of Acidocin J1132, a two-component bacteriocin produced by Lactobacillus

acidophilus JCM 1132. Appl. Environ. Microbiol 62:892-897.

499. Tai, S. S. 2006. Streptococcus pneumoniae protein vaccine candidates: properties, activities and animal studies. Crit Rev. Microbiol. 32:139-153.

500. Takala, T. M. and P. E. Saris. 2006. C terminus of NisI provides specificity to nisin. Microbiology

152:3543-3549. 501. Tamura, G. S., D. S. Bratt, H. H. Yim, and A. Nittayajarn. 2005. Use of glnQ as a counterselectable

marker for creation of allelic exchange mutations in group B streptococci. Appl Environ. Microbiol

71:587-590. 502. Tamura, G. S., A. Nittayajarn, and D. L. Schoentag. 2002. A glutamine transport gene, glnQ, is

required for fibronectin adherence and virulence of group B streptococci. Infect Immun 70:2877-

2885. 503. Tano, K., C. Olofsson, E. Grahn-Hakansson, and S. E. Holm. 1999. In vitro inhibition of S.

pneumoniae, nontypable H. influenzae and M. catharralis by alpha-hemolytic streptococci from

healthy children. Int. J. Pediatr. Otorhinolaryngol. 47:49-56. 504. Terzaghi, B. E. and W. E. Sandine. 1975. Improved medium for lactic streptococci and their

bacteriophages. Appl Microbiol 29:807-813.

505. Tettelin, H., K. E. Nelson, I. T. Paulsen, J. A. Eisen, T. D. Read, S. Peterson, J. Heidelberg, R. T. DeBoy, D. H. Haft, R. J. Dodson, A. S. Durkin, M. Gwinn, J. F. Kolonay, W. C. Nelson, J. D.

Peterson, L. A. Umayam, O. White, S. L. Salzberg, M. R. Lewis, D. Radune, E. Holtzapple, H. Khouri, A. M. Wolf, T. R. Utterback, C. L. Hansen, L. A. McDonald, T. V. Feldblyum, S. Angiuoli,

T. Dickinson, E. K. Hickey, I. E. Holt, B. J. Loftus, F. Yang, H. O. Smith, J. C. Venter, B. A.

Dougherty, D. A. Morrison, S. K. Hollingshead, and C. M. Fraser. 2001. Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293:498-506.

506. Thijs, G., M. Lescot, K. Marchal, S. Rombauts, B. De Moor, P. Rouze, and Y. Moreau. 2001. A

higher-order background model improves the detection of promoter regulatory elements by Gibbs sampling. Bioinformatics. 17:1113-1122.

References

162

507. Thijs, G., K. Marchal, M. Lescot, S. Rombauts, B. De Moor, P. Rouze, and Y. Moreau. 2002. A

Gibbs sampling method to detect overrepresented motifs in the upstream regions of coexpressed

genes. J. Comput. Biol. 9:447-464. 508. Thompson, W., E. C. Rouchka, and C. E. Lawrence. 2003. Gibbs Recursive Sampler: finding

transcription factor binding sites. Nucleic Acids Res. 31:3580-3585.

509. Throup, J. P., K. K. Koretke, A. P. Bryant, K. A. Ingraham, A. F. Chalker, Y. Ge, A. Marra, N. G. Wallis, J. R. Brown, D. J. Holmes, M. Rosenberg, and M. K. Burnham. 2000. A genomic analysis of

two-component signal transduction in Streptococcus pneumoniae. Mol. Microbiol 35:566-576.

510. Tichaczek, P. S., R. F. Vogel, and W. P. Hammes. 1993. Cloning and sequencing of curA encoding curvacin A, the bacteriocin produced by Lactobacillus curvatus LTH1174. Arch. Microbiol

160:279-283.

511. Tichaczek, P. S., R. F. Vogel, and W. P. Hammes. 1994. Cloning and sequencing of sakP encoding sakacin P, the bacteriocin produced by Lactobacillus sake LTH 673. Microbiology 140 ( Pt 2):361-

367.

512. Tjalsma, H., A. Bolhuis, J. D. Jongbloed, S. Bron, and J. M. van Dijl. 2000. Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol

Mol. Biol. Rev 64:515-547.

513. Todar, K. 2008. Todar's Online Textbook of Bacteriology, In . 514. Tomita, H., S. Fujimoto, K. Tanimoto, and Y. Ike. 1996. Cloning and genetic organization of the

bacteriocin 31 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative

plasmid pYI17. J. Bacteriol. 178:3585-3593. 515. Tomita, H., S. Fujimoto, K. Tanimoto, and Y. Ike. 1997. Cloning and genetic and sequence analyses

of the bacteriocin 21 determinant encoded on the Enterococcus faecalis pheromone-responsive

conjugative plasmid pPD1. J. Bacteriol. 179:7843-7855. 516. Truong-Bolduc, Q. C. and D. C. Hooper. 2007. The transcriptional regulators NorG and MgrA

modulate resistance to both quinolones and beta-lactams in Staphylococcus aureus. J. Bacteriol.

189:2996-3005.

517. Tseng, H. J., A. G. McEwan, J. C. Paton, and M. P. Jennings. 2002. Virulence of Streptococcus

pneumoniae: PsaA mutants are hypersensitive to oxidative stress. Infect. Immun. 70:1635-1639.

518. Tsuda, H., Y. Yamashita, Y. Shibata, Y. Nakano, and T. Koga. 2002. Genes involved in bacitracin resistance in Streptococcus mutans. Antimicrob. Agents Chemother. 46:3756-3764.

519. Tu, A. H., R. L. Fulgham, M. A. McCrory, D. E. Briles, and A. J. Szalai. 1999. Pneumococcal

surface protein A inhibits complement activation by Streptococcus pneumoniae. Infect. Immun. 67:4720-4724.

520. Tullius, M. V., G. Harth, and M. A. Horwitz. 2003. Glutamine synthetase GlnA1 is essential for

growth of Mycobacterium tuberculosis in human THP-1 macrophages and guinea pigs. Infect Immun 71:3927-3936.

521. Turner, D. L., L. Brennan, H. E. Meyer, C. Lohaus, C. Siethoff, H. S. Costa, B. Gonzalez, H. Santos,

and J. E. Suarez. 1999. Solution structure of plantaricin C, a novel lantibiotic. Eur. J. Biochem. 264:833-839.

522. Turner, J., Y. Cho, N. N. Dinh, A. J. Waring, and R. I. Lehrer. 1998. Activities of LL-37, a cathelin-

associated antimicrobial peptide of human neutrophils. Antimicrob. Agents Chemother. 42:2206-2214.

523. Uguen, P., J. P. Le Pennec, and A. Dufour. 2000. Lantibiotic biosynthesis: interactions between

prelacticin 481 and its putative modification enzyme, LctM. J. Bacteriol. 182:5262-5266. 524. Upton, M., J. R. Tagg, P. Wescombe, and H. F. Jenkinson. 2001. Intra- and interspecies signaling

between Streptococcus salivarius and Streptococcus pyogenes mediated by SalA and SalA1

lantibiotic peptides. J. Bacteriol. 183:3931-3938. 525. Utaida, S., P. M. Dunman, D. Macapagal, E. Murphy, S. J. Projan, V. K. Singh, R. K. Jayaswal, and

B. J. Wilkinson. 2003. Genome-wide transcriptional profiling of the response of Staphylococcus

aureus to cell-wall-active antibiotics reveals a cell-wall-stress stimulon. Microbiology 149:2719-2732.

526. Valsesia, G., G. Medaglia, M. Held, W. Minas, and S. Panke. 2007. Circumventing the effect of product toxicity: development of a novel two-stage production process for the lantibiotic

gallidermin. Appl. Environ. Microbiol. 73:1635-1645.

527. van Belkum, M. J., J. Kok, and G. Venema. 1992. Cloning, sequencing, and expression in Escherichia coli of lcnB, a third bacteriocin determinant from the lactococcal bacteriocin plasmid

p9B4-6. Appl. Environ. Microbiol 58:572-577.

References

163

528. van Belkum, M. J., J. Kok, G. Venema, H. Holo, I. F. Nes, W. N. Konings, and T. Abee. 1991. The

bacteriocin lactococcin A specifically increases permeability of lactococcal cytoplasmic membranes

in a voltage-independent, protein-mediated manner. J. Bacteriol. 173:7934-7941. 529. van Belkum, M. J. and M. E. Stiles. 2000. Nonlantibiotic antibacterial peptides from lactic acid

bacteria. Nat. Prod. Rep. 17:323-335.

530. van Belkum, M. J. and M. E. Stiles. 1995. Molecular characterization of genes involved in the production of the bacteriocin leucocin A from Leuconostoc gelidum. Appl. Environ. Microbiol

61:3573-3579.

531. van der Ploeg, Jr. 2005. Regulation of bacteriocin production in Streptococcus mutans by the quorum-sensing system required for development of genetic competence. J. Bacteriol. 187:3980-

3989.

532. van der Ploeg, Jr. 2005. Regulation of bacteriocin production in Streptococcus mutans by the quorum-sensing system required for development of genetic competence. J. Bacteriol. 187:3980-

3989.

533. van Heijenoort, J. 2007. Lipid intermediates in the biosynthesis of bacterial peptidoglycan. Microbiol Mol. Biol. Rev 71:620-635.

534. van Hijum, S. A., A. de Jong, R. J. Baerends, H. A. Karsens, N. E. Kramer, R. Larsen, C. D. den

Hengst, C. J. Albers, J. Kok, and O. P. Kuipers. 2005. A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data. BMC.

Genomics 6:77.

535. van Hijum, S. A., J. Garcia de la Nava, O. Trelles, J. Kok, and O. P. Kuipers. 2003. MicroPreP: a cDNA microarray data pre-processing framework. Appl Bioinformatics. 2:241-244.

536. Venema, K., R. E. Haverkort, T. Abee, A. J. Haandrikman, K. J. Leenhouts, L. de Leij, G. Venema,

and J. Kok. 1994. Mode of action of LciA, the lactococcin A immunity protein. Mol. Microbiol 14:521-532.

537. Wani, J. H., J. V. Gilbert, A. G. Plaut, and J. N. Weiser. 1996. Identification, cloning, and

sequencing of the immunoglobulin A1 protease gene of Streptococcus pneumoniae. Infect. Immun.

64:3967-3974.

538. Ware, D., Y. Jiang, W. Lin, and E. Swiatlo. 2006. Involvement of potD in Streptococcus

pneumoniae polyamine transport and pathogenesis. Infect. Immun. 74:352-361. 539. Wartha, F., K. Beiter, B. Albiger, J. Fernebro, A. Zychlinsky, S. Normark, and B. Henriques-

Normark. 2007. Capsule and D-alanylated lipoteichoic acids protect Streptococcus pneumoniae

against neutrophil extracellular traps. Cell Microbiol 9:1162-1171. 540. Watson, D. A., D. M. Musher, J. W. Jacobson, and J. Verhoef. 1993. A brief history of the

pneumococcus in biomedical research: a panoply of scientific discovery. Clin. Infect. Dis. 17:913-

924. 541. Weiser, J. N., R. Austrian, P. K. Sreenivasan, and H. R. Masure. 1994. Phase variation in

pneumococcal opacity: relationship between colonial morphology and nasopharyngeal colonization.

Infect. Immun. 62:2582-2589. 542. Wescombe, P. A. and J. R. Tagg. 2003. Purification and characterization of streptin, a type A1

lantibiotic produced by Streptococcus pyogenes. Appl. Environ. Microbiol 69:2737-2747.

543. Wescombe, P. A., M. Upton, K. P. Dierksen, N. L. Ragland, S. Sivabalan, R. E. Wirawan, M. A. Inglis, C. J. Moore, G. V. Walker, C. N. Chilcott, H. F. Jenkinson, and J. R. Tagg. 2006. Production

of the lantibiotic salivaricin A and its variants by oral streptococci and use of a specific induction

assay to detect their presence in human saliva. Appl. Environ. Microbiol. 72:1459-1466. 544. Whitehead, H. R. 1933. A substance inhibiting bacterial growth, produced by certain strains of lactic

streptococci. Biochem. J 27:1793-1800.

545. Whitford, M. F., M. A. McPherson, R. J. Forster, and R. M. Teather. 2001. Identification of bacteriocin-like inhibitors from rumen Streptococcus spp. and isolation and characterization of

bovicin 255. Appl. Environ. Microbiol 67:569-574.

546. WHO, D. o. I. V. a. B. 2007. Weekly epidemiological record, In . 547. WHO, D. o. I. V. a. B. 2008. Weekly epidemiological record, In .

548. Widdick, D. A., H. M. Dodd, P. Barraille, J. White, T. H. Stein, K. F. Chater, M. J. Gasson, and M. J. Bibb. 2003. Cloning and engineering of the cinnamycin biosynthetic gene cluster from

Streptomyces cinnamoneus cinnamoneus DSM 40005. Proc. Natl. Acad. Sci. U. S. A 100:4316-

4321. 549. Wiedemann, I., R. Benz, and H. G. Sahl. 2004. Lipid II-mediated pore formation by the peptide

antibiotic nisin: a black lipid membrane study. J. Bacteriol. 186:3259-3261.

550. Wiedemann, I., T. Bottiger, R. R. Bonelli, T. Schneider, H. G. Sahl, and B. Martinez. 2006. Lipid II-based antimicrobial activity of the lantibiotic plantaricin C. Appl. Environ. Microbiol 72:2809-2814.

References

164

551. Wiedemann, I., T. Bottiger, R. R. Bonelli, A. Wiese, S. O. Hagge, T. Gutsmann, U. Seydel, L.

Deegan, C. Hill, P. Ross, and H. G. Sahl. 2006. The mode of action of the lantibiotic lacticin 3147--a

complex mechanism involving specific interaction of two peptides and the cell wall precursor lipid II. Mol. Microbiol 61:285-296.

552. Wiedemann, I., E. Breukink, C. van Kraaij, O. P. Kuipers, G. Bierbaum, B. de Kruijff, and H. G.

Sahl. 2001. Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. J. Biol. Chem. 276:1772-1779.

553. Willey, J. M. and W. A. van der Donk. 2007. Lantibiotics: peptides of diverse structure and function.

Annu. Rev. Microbiol 61:477-501. 554. Willey, J. M. and W. A. van der Donk. 2007. Lantibiotics: peptides of diverse structure and function.

Annu. Rev. Microbiol 61:477-501.

555. Wirawan, R. E., N. A. Klesse, R. W. Jack, and J. R. Tagg. 2006. Molecular and genetic characterization of a novel nisin variant produced by Streptococcus uberis. Appl. Environ.

Microbiol. 72:1148-1156.

556. Woodruff, W. A., J. Novak, and P. W. Caufield. 1998. Sequence analysis of mutA and mutM genes involved in the biosynthesis of the lantibiotic mutacin II in Streptococcus mutans. Gene 206:37-43.

557. Worobo, R. W., T. Henkel, M. Sailer, K. L. Roy, J. C. Vederas, and M. E. Stiles. 1994.

Characteristics and genetic determinant of a hydrophobic peptide bacteriocin, carnobacteriocin A, produced by Carnobacterium piscicola LV17A. Microbiology 140 ( Pt 3):517-526.

558. Worobo, R. W., M. J. van Belkum, M. Sailer, K. L. Roy, J. C. Vederas, and M. E. Stiles. 1995. A

signal peptide secretion-dependent bacteriocin from Carnobacterium divergens. J. Bacteriol. 177:3143-3149.

559. Wysocki, J., A. Galaj, F. Omeñaca, and et al. 2008. Immunogenicity of the new 10-valent

pneumococcal non-typeable Haemophilus influenza protein D conjugate (PHiD-CV) in infants after 3-dose priming before 6 months of age., In ISPPD 2008.

560. Xie, X. S., D. K. Stone, and E. Racker. 1983. Determinants of clathrin-coated vesicle acidification.

J. Biol. Chem. 258:14834-14838.

561. Yan, L. Z., A. C. Gibbs, M. E. Stiles, D. S. Wishart, and J. C. Vederas. 2000. Analogues of

bacteriocins: antimicrobial specificity and interactions of leucocin A with its enantiomer,

carnobacteriocin B2, and truncated derivatives. J. Med. Chem. 43:4579-4581. 562. Yeaman, M. R. and N. Y. Yount. 2003. Mechanisms of antimicrobial peptide action and resistance.

Pharmacol. Rev. 55:27-55.

563. Yoneyama, F., Y. Imura, S. Ichimasa, K. Fujita, T. Zendo, J. Nakayama, K. Matsuzaki, and K. Sonomoto. 2009. Lacticin Q, a lactococcal bacteriocin, causes high-level membrane permeability in

the absence of specific receptors. Appl. Environ. Microbiol 75:538-541.

564. Yoneyama, F., Y. Imura, K. Ohno, T. Zendo, J. Nakayama, K. Matsuzaki, and K. Sonomoto. 2009. Peptide-lipid huge toroidal pore, a new antimicrobial mechanism mediated by a lactococcal

bacteriocin, lacticin Q. Antimicrob. Agents Chemother. 53:3211-3217.

565. Yoneyama, F., K. Shioya, T. Zendo, J. Nakayama, and K. Sonomoto. 2010. Effect of a negatively charged lipid on membrane-lacticin Q interaction and resulting pore formation. Biosci. Biotechnol.

Biochem. 74:218-221.

566. Yonezawa, H. and H. K. Kuramitsu. 2005. Genetic analysis of a unique bacteriocin, Smb, produced by Streptococcus mutans GS5. Antimicrob. Agents Chemother. 49:541-548.

567. Yuste, J., M. Botto, J. C. Paton, D. W. Holden, and J. S. Brown. 2005. Additive inhibition of

complement deposition by pneumolysin and PspA facilitates Streptococcus pneumoniae septicemia. J. Immunol. 175:1813-1819.

568. Zendo, T., M. Fukao, K. Ueda, T. Higuchi, J. Nakayama, and K. Sonomoto. 2003. Identification of

the lantibiotic nisin Q, a new natural nisin variant produced by Lactococcus lactis 61-14 isolated from a river in Japan. Biosci. Biotechnol. Biochem. 67:1616-1619.

569. Zendo, T., S. Koga, Y. Shigeri, J. Nakayama, and K. Sonomoto. 2006. Lactococcin Q, a novel two-

peptide bacteriocin produced by Lactococcus lactis QU 4. Appl. Environ. Microbiol 72:3383-3389. 570. Zhang, Y., A. W. Masi, V. Barniak, K. Mountzouros, M. K. Hostetter, and B. A. Green. 2001.

Recombinant PhpA protein, a unique histidine motif-containing protein from Streptococcus pneumoniae, protects mice against intranasal pneumococcal challenge. Infect. Immun. 69:3827-

3836.

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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.

Samenvatting voor de leek

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).

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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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