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Study of the physiological and molecular mechanisms underlying
peptide-induced cell death and biofilm formation in
Streptococcus mutans
JULIE ANN PERRY
A thesis submitted in conformity with the requirements for the
bacteriocins ex. enterocin AS48), and subclass IId (non-pediocin single linear peptides ex.
lactococcin A) (Cotter et al., 2005). The third class of bacteriocins is represented by non-
bacteriocin lytic proteins called bacteriolysins. This class includes large, heat-labile proteins
(often murein hydrolases), like lysostaphin and enterolysin A (Cotter et al., 2005). Most
bacteriocins from streptococci belong to the lantibiotic class (Nes et al., 2007). However,
among the bacteriocins classified for S. mutans are both traditional class I bacteriocins (mutacin
I, mutacin II, mutacin III/mutacin 1140, mutacin N and mutacin B-Ny266), two-peptide class I
lantibiotics (SmbA and SmbB), a class IIb two-peptide bacteriocin (mutacin IV) and the
bacteriocin mutacin V, which has structural homology to class IIa pediocin-like bacteriocins.
1.3.4.3.2 Biosynthesis and export of class IIa bacteriocins
Mutacin V is a class IIa bacteriocin, and deserves specific mention since it is the focus of
Chapter 2 of this dissertation. Expression of class II bacteriocins like mutacin V typically
requires an inducer peptide pheromone and a TCS (van der Ploeg, 2005). Interestingly, the
majority of putative bacteriocins and their accessory genes identified in the UA159 genome are
located in a 13.5kb island which also harbours the comC, comD and comE genes (van der
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Ploeg, 2005) (Table 1.5). Some of these bacteriocins-encoding genes including mutacin V were
also found preceded by a putative ComE binding site, implicating this regulator in their
expression. Furthermore, the gene encoding mutacin V (SMU.1914, also known as nlmC) is
located immediately upstream of comC itself (Figure 1.4), although the two are transcribed
divergently.
Table 1.5: Putative and known bacteriocins encoded by S. mutans strain UA159 located in the
genomic island that also includes CSP-ComDE (adapted from (van der Ploeg, 2005))
Gene ID Size of pre- bacteriocin
(amino acids)
Putative ComE
binding site
Characteristics
SMU.1889 87 - SMU.1889 and SMU.1892 are
located adjacent to each other, may form a two-peptide bacateriocin SMU.1892 61 -
SMU.1895 53 - Separated from SMU.1889/1892 by
an insertion element. SMU.1895/1896 may also form a two
peptide bacteriocin. SMU.1896 83 -
SMU.1902 47 - Single peptide bacteriocin?
SMU.1905 62 + SMU.1905 and SMU.1906 are located adjacent to each other, may
form a two-peptide bacateriocin SMU.1906 70 +
SMU.1914 76 + Transcribed divergently from comC. Single peptide bacteriocin mutacin V
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At least four genes are required for production of class IIa bacteriocins like mutacin V: 1) the
structural gene, encoding the „prebacteriocin‟ with its leader peptide; 2) a gene encoding the
immunity protein, which is usually co-transcribed with the bacteriocin structural gene; 3) a gene
encoding an ABC transporter necessary for secretion of the bacteriocin; and 4) a gene encoding
an accessory protein of unknown function (Drider et al., 2006). These four required gene
elements are not necessarily found in a single operon, and may be found transcribed as three
separate units where one operon encodes the bacteriocin and immunity protein, a second
operon carries genes for secretion, and a third operon encodes genes involved in the regulation
of bacteriocin expression (Drider et al., 2006). Interestingly, with few exceptions (including
mutacin V), most class IIa bacteriocins are plasmid encoded (Drider et al., 2006).
The class IIa bacteriocins are translated as „prebacteriocins‟, having an N-terminal extension.
This presequence is removed during export by site-specific cleavage following a conserved
double glycine motif. This leader sequence may serve as an export signal to direct bacteriocins
to the correct ABC transporter, but is also thought to play a protective role in preventing the
insertion of the bacteriocin into the membrane of the producing cell (Drider et al., 2006).
1.3.4.3.3 Mode of action
The majority of bacteriocins have a net positive charge and contain sequences of
hydrophobic and/or amphiphilic nature, allowing them to insert into the negatively charged
Gram-positive cytoplasmic membrane, creating pores in target cells (Hechard and Sahl, 2002).
The result is a disruption of proton motive force, ATP depletion and leakage of nutrients and
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Figure 1.7: Cartoon representation of the mechanism of action of bacteriocins, divided
according to classification. Class I bacteriocins both inhibit cell wall synthesis by binding to lipid
II (to prevent translocation of peptidoglycan precursors across the cell membrane), and create
pores in target cell membranes. Class II bacteriocins act by pore formation and disruption of the
cell‟s PMF. Bacteriolysins actively degrade the cell wall, and are considered murein hydrolases
rather than true bacteriocins (figure adapted from (Cotter et al., 2005)).
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metabolites from the target cell, and eventual cell death (Hechard and Sahl, 2002). The killing
spectrum of bacteriocins is narrow, and typically includes only closely related target organisms
(Drider et al., 2006). However, with few exceptions, little is known about how bacteriocins
specifically recognize their target cells. The well characterized type I lantibiotic bacteriocin nisin
employs the cell-wall precursor lipid II as a docking molecule, and subsequently kills cells by
simultaneously inhibiting peptidoglycan biosynthesis and creating pores in the cytoplasmic
membrane (Linnett and Strominger, 1973). The receptor for lactococcin A (and similar pediocin-
like type IIa bacteriocins) is also known. This class of bacteriocins act by binding to the proteins
IIC and IID of the mannose phosphotransferase system and permeabilizing the cytoplasmic
membrane (Diep et al., 2007). Immunity is conferred to the producing cell via binding of the
cognate immunity protein to the bacteriocin-receptor complex, thereby preventing the further
action of the bacteriocin (Diep et al., 2007).
1.3.4.4 CSP and the stress response
Bacteriocins are often produced by bacteria to inhibit competitors at high cell density, during
which nutrient, oxidative, and acid end-product stresses abound. Importantly, recent work by
the Claverys lab on S. pneumoniae indicates that the fourth CSP-regulated phenotype in
streptococci may be the coordination of the general stress response. Prudhomme et al. (2006)
demonstrated that antibiotic and DNA-damage stress could induce competence in pneumococci
via up-regulation of expression of the CSP-ComDE circuit (Prudhomme et al., 2006). Induction
of competence in these stressed cells was proposed to be a survival strategy designed to
enhance the fitness of the organism by allowing it to scavenge the environment for potential
resistance genes. These authors suggested that CSP is not simply an indicator of cell density in
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pneumococci, but may also signal the presence of environmental stress as an inducible peptide
„alarmone‟. Furthermore, Pinas et al. (2008) found that acid stress could induce autolysis
independently from competence in S. pneumoniae in a CSP-independent ComE-mediated
pathway. These authors proposed that ComE is a principal player in a global stress response
that includes the uptake of fitness enhancing DNA via the competence cascade (Pinas et al.,
2008). Finally, competence has been shown to respond to the presence of alkaline conditions
also in a cell-density-independent manner. These results imply that competence is linked to the
stress in the environment more closely than to cell density.
In S. mutans, evidence has implicated the CSP-ComDE pathway in the acid tolerance
response (Li et al., 2001a), arguably one of the most important environmental stresses that this
organism encounters. Furthermore, the coordinated regulation of competence and bacteriocin
production through CSP-ComDE has been shown to result in DNA exchange between S.
mutans and its neighbour S. gordonii (Kreth et al., 2005). Even in the most traditional definition
of function for CSP, it can be argued that the CSP-ComDE circuit is involved in adaptation to the
significant competitive stresses that occur at high cell density. Since the oral biofilm
environment is rife with stresses, could the S. mutans CSP-ComDE system be functioning as a
mediator of the stress response in that environment? Biofilm formation may represent the
combined effects of all CSP-mediated phenotypes described thus far.
1.3.4.5. Biofilm formation
The formation of a densely packed biofilm provides an efficient milieu for chemical signalling,
which breeds cooperative „multi-cellular‟-type behaviours (described in detail in sections 1.1 and
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1.2). Environmental stresses accumulate when cells are located in such close proximity,
demanding efficient stress response mechanisms. The autolytic sacrifice of individual cells
damaged by toxic factors, viral infection or during nutritional starvation, the production of
secreted bacteriocins, and/or the scavenging of fitness-enhancing genes from the environment
through competence induction may provide solutions to the problems associated with growth at
high cell density. Given the nature of the high cell density biofilm lifestyle, it is not surprising
that biofilm formation represents the final phenotype we describe which has been linked to CSP
signalling in S. pneumoniae (Oggioni et al., 2006) and S. mutans (Li et al., 2001b; Li et al.,
2002b).
In vitro, S. pneumoniae forms biofilms only in the presence of exogenously added CSP, and
does not form biofilms in the absence of the ComD receptor (Oggioni et al., 2006). These
authors also reported that ComD-deficient mutants were less virulent in a murine model of
pneumoniae, which they describe as a biofilm-like infection. While the study of CSP‟s influence
on biofilm formation in S. pneumoniae has received less attention than its role in competence
development, a well established connection exists between CSP and biofilm formation in S.
mutans.
Evidence supporting a role for CSP signalling in S. mtuans biofilms was first presented by Li
et al., who found that a comC- mutant of S. mutans formed a biofilm with an altered structure,
which could be restored to wild-type architecture using exogenously added CSP or plasmid-
based complementation (Li et al., 2002b). Interestingly, biofilm formation by mutants defective
in the ComD and ComE TCS components formed biofilms with reduced biomass which could
not be fully complemented with exogenous CSP (Li et al., 2002b). The different biofilm
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phenotypes associated with inactivation of the signal and sensory components of the QS
system indicate that CSP may impact on more than one TCS in the biofilm. Alternatively,
signals other than CSP may be sensed by ComDE in the biofilm. The expression of ComX has
also been monitored in S. mutans biofilms using a green fluorescent protein (GFP)-promoter
fusion, revealing CSP signalling occurs in areas of high cell density (Aspiras et al., 2004).
Although similar results linking CSP signalling to biofilm formation and architecture in S. mtuans
have since been reported by others (Petersen et al., 2005; Zhang et al., 2009), no mechanism
has been proposed to explain the role of CSP-ComDE in biofilm formation.
This dissertation aims to address the role of the CSP peptide pheromone in the physiology of
S. mutans, with specific focus on its role in autolysis, competence, and biofilm formation. We
attempt to show that the overriding theme common to all CSP-responsive phenotypes in S.
mutans is their involvement in a global CSP-mediated stress response, which is necessary to
the proper formation and maintenance of the high density biofilm community.
1.4. Statement of the problem
As the major etiological agent of human dental caries, the naturally transformable oral
bacterium Streptococcus mutans is well studied at the genetic and physiological level. The
regulatory system that governs genetic competence in this species is similar to the system in
S. pneumoniae, and is composed of the CSP peptide pheromone, the ComDE two-component
signal transduction system, and the alternate sigma factor ComX. In S. mutans, the CSP
pheromone has been linked to the induction of genetic competence, autolysis, bacteriocin
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production and acid tolerance. While streptococcal CSP was originally thought to act as a
classical quorum sensing signal, recent data in S. pneumoniae has shown that comCDE
expression can be altered under certain environmental stress conditions. Differences exist in the
timing and regulation of competence in S. pneumoniae and S. mutans. However, direct and
indirect evidence has tied CSP to the S. mutans stress response in the past. Although many
phenotypes have been attributed to CSP signalling in S. mutans, little is known about the
genetic pathways downstream of ComDE, nor about how the seemingly diverse CSP-regulated
phenotypes are connected. The general aim of this dissertation was to examine the
physiological and molecular response to the CSP pheromone in S. mutans, and determine the
contribution of phenotypes regulated by this pathway to the evolutionary fitness of the organism.
General hypothesis: S. mutans co-ordinates genetic competence and autolysis with its stress
response through the CSP peptide pheromone to acquire fitness-enhancing genes under stress
and build a stronger biofilm.
Primary objective: To understand how and when CSP-induced autolysis occurs in S. mutans,
and what role this process plays in the growth and genetic adaptability of the organism.
Rationale: Recent studies have suggested that competence may play a role in the stress
response in S. pneumoniae, and that fratricide may contribute DNA for the exchange of fitness
enhancing genes under stress. Although the competence cascades of S. pneumoniae and S.
mutans are physiologically divergent, S. mutans has been shown to regulate autolysis through
CSP accumulation. Moreover, the tightly packed oral biofilm community provides an excellent
environment for gene exchange, since spatial proximity, a „multi-cellular‟ altruistic lifestyle, and
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constant selective pressure from environmental stress would appear to favour the evolution of
such a strategy. Alternatively, the release of DNA into the biofilm environment via autolysis may
contribute to stress tolerance by adding to the extracellular matrix of the biofilm. Given that
biofilm formation and stress tolerance are vital to the virulence of S. mutans, understanding the
contribution of the primary intracellular communication system to their regulation is of utmost
importance. To attempt to elucidate the physiological and molecular mechanisms underlying
the CSP response in S. mutans, the goal of this dissertation is to investigate the following
specific aims:
Specific Aim 1: Determine if and how the CSP peptide pheromone participates in the
S. mutans stress response.
Specific Aim 2: Characterize the physiological response to CSP and determine the
signaling pathways involved in this response.
Specific Aim 3: Determine the role of autolysis in the physiology of S. mutans, with
emphasis on the competence cascade and biofilm formation.
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Chapter 2: Peptide alarmone signalling triggers an auto-active
bacteriocin necessary for genetic competence
JA Perry, MB Jones, SN Peterson, DG Cvitkovitch, and CM Lévesque. 2009. Mol
Micro. 72: 905-917
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2.1 Abstract
The induction of genetic competence is a strategy used by bacteria to increase their genetic
repertoire under stressful environmental conditions. Recently, Streptococcus pneumoniae has
been shown to coordinate the uptake of transforming DNA with fratricide via increased
expression of the peptide pheromone responsible for competence induction. Here we document
that environmental stress induced expression of the peptide pheromone CSP in the oral
pathogen Streptococcus mutans. We showed that CSP is involved in the stress response, and
determined the CSP-induced regulon in S. mutans by microarray analysis. Contrary to
pneumococcus, S. mutans responds to increased concentrations of CSP by cell lysis in only a
fraction of the population. We have focused on the mechanism of cell lysis, and have identified
a novel bacteriocin as the „death effector‟. Most importantly, we showed that this bacteriocin
causes cell death via a novel mechanism of action: intracellular action against self. We have
also identified the cognate bacteriocin immunity protein, which resides in a separate unlinked
genetic locus to allow its differential regulation. The role of the lytic response in S. mutans
competence is also discussed. Together, these findings reveal a novel autolytic pathway in
S. mutans which may be involved in the dissemination of fitness-enhancing genes in the oral
biofilm.
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2.2 Introduction
Free-living bacteria are at the mercy of a variety of environmental stress conditions that
impose constant selective pressure on the microorganism. To compete or simply survive in their
ecological niche, bacteria must rely on an ability to sense and respond to stress. Often, the
response to stress involves the induction of a transient state of hyper-mutability, which is argued
to increase the probability of generating adaptive variants in the bacterial population (Bjedov et
al., 2003). Although some debate exists as to whether mutagenesis is an inductive strategy or
simply a by-product of the accumulation of DNA lesions, stress-induced mutations certainly
participate in the adaptive evolution of bacteria (Bjedov et al., 2003).
Although an increased mutation rate may lead to the chance development of a fitness-
enhancing phenotype, the probability of such an event occurring is limited to the available DNA
sequence in an organism‟s own genome. However, naturally transformable bacteria are able to
sample the DNA pool of an entire community during stress, and acquire fitness enhancing
genes across species barriers. The major human pathogen Streptococcus pneumoniae and the
soil-dweller Bacillus subtilis are the best-characterized naturally transformable Gram-positive
bacteria. Although they employ different mechanisms to achieve the competent state, both
organisms turn on their competence regulons in response to specific environmental stresses,
which may improve fitness by generating genetic diversity through natural transformation
(Claverys et al., 2006).
As the major etiological agent of human dental caries (Mitchell, 2003), the naturally
transformable oral bacterium Streptococcus mutans is well studied at the genetic and
physiological level. The regulatory system that governs genetic competence in this species is
homologous to the system in S. pneumoniae ((Håvarstein et al., 1996)), and is composed of a
peptide pheromone (competence stimulating peptide, or CSP), the ComDE two-component
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signal transduction system, and the alternate sigma factor ComX (Li et al., 2001b). Although
competence is regulated by similar signaling systems in both streptococcal species, important
differences separate the two species‟ response to the pheromone. Firstly, while competence
develops uniformly across a population of S. pneumoniae (Håvarstein et al., 2006), it is well
established that only a fraction of the S. mutans population (~1%) ever becomes CSP-
responsive (Aspiras et al., 2004; Li et al., 2001b; Qi et al., 2005). Moreover, the competence
cascade in S. mutans is known to incorporate inputs from additional two-component systems
(Ahn et al., 2006; Perry et al., 2008; Qi et al., 2004; Senadheera et al., 2005). Finally, while
S. pneumoniae controls expression of its bacteriocins through the dedicated BlpRH system (de
Saizieu et al., 2000), S. mutans controls the expression of many of its bacteriocins through
ComDE (Hale et al., 2005a; Kreth et al., 2005; Kreth et al., 2006a; Kreth et al., 2007; van der
Ploeg, 2005). The co-ordination of bacteriocin production and competence suggests that
S. mutans can generate DNA for uptake from lysis of neighboring species (Kreth et al., 2005), in
what may be an evolutionary adaptation to the multi-species oral biofilm environment.
Streptococcal CSP pheromone was originally thought to accumulate passively in proportion
to population density, and act as a classical quorum sensing signal to activate the competence
regulon at a specific cell density (Håvarstein et al., 1995; Håvarstein et al., 1996; Li et al.,
2001b). However, early work done in S. mutans (Li et al., 2001a; Li et al., 2002a) suggested an
intimate link between the competence cascade and the organism‟s response to acid stress. A
link between competence and oxidative stress has also been made in S. mutans (Senadheera
et al., 2006; Wen et al., 2005), but a mechanistic explanation for these phenotypes has
remained elusive. Evidence for stress-induced genetic plasticity has also accumulated in regard
to S. pneumoniae (Chastanet et al., 2001; Claverys et al., 2000; Prudhomme et al., 2006),
where it has been suggested that pneumococcal CSP may act as a secreted stress-induced
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pheromone (or „alarmone‟) that triggers expression of stress-responsive genes (Claverys et al.,
2006).
Here we present evidence that S. mutans integrates its response to specific environmental
stresses with its competence cascade via the CSP pheromone, and describe for the first time
the global transcriptome analyses of CSP-regulated genes in S. mutans. Our most important
finding was that in the presence of high concentrations of CSP pheromone, the unprocessed
form of mutacin V acted as an intracellular auto-active bacteriocin causing S. mutans autolysis.
To our knowledge, this is a completely novel mechanism of action for a bacteriocin. Moreover,
the impaired ability of S. mutans cells lacking mutacin V to become competent indicates that
stress-induced lysis in a subpopulation may be required for the acquisition of diversity through
genetic transformation in the surviving cells.
2.3 Experimental procedures
Culture conditions
The S. mutans strains used in this study are listed in Table 2.1. Mutants were constructed in
S. mutans UA159 wild-type as described previously (Lau et al., 2002). Strains were grown in
Todd-Hewitt–Yeast Extract (THYE) broth at 37ºC with 5% CO2. Growth was monitored using a
microbiology workstation (Bioscreen C Labsystems, Finland). Co-culture experiments were
conducted by adding equal volumes of each strain, and CFUs were enumerated by plating.
Viability staining was performed using the LIVE/DEAD BacLight kit (Invitrogen) according to the
manufacturer‟s directions. Lysis was assessed by harvesting the supernatant of cultures
expressing a -glucuronidase (GUS) reporter gene cloned into a theta-replicating plasmid
(Biswas et al., 2008) in the absence and presence of 2 μM sCSP. Supernatants were combined
in equal parts with 2 GUS buffer (100 mM Na2HPO4, 20 mM -mercaptoethanol, 2 mM EDTA,
69
0.2% Triton-X, 1 mM PNPG substrate (Sigma)). Absorbance at 420 nm was measured after 15
min of color development. GUS activity was expressed as [1000 A420]/[time (min) OD600] in
Miller units (MU).
Table 2.1. Bacterial strains used in this study
Strain Description* Reference
S. mutans UA159 Wild-type ATCC ∆comC ∆smu.1915; Emr This work ∆comC complemented ∆smu.1915(pcomC); Emr, Spcr This work ∆comE ∆smu.1917; Emr This work ∆comDE ∆smu.1916-smu.1917; Emr This work ∆comX ∆smu.1997; Emr This work ∆mutacin IV ∆smu.150-smu.151; Emr This work ∆cipB ∆smu.1914; Emr This work ∆cipI ∆smu.925; Emr This work ∆1913 ∆smu.1913; Emr This work ∆423 ∆smu.423; Emr This work ∆1906 ∆smu.1906; Emr This work ∆nlmTE ∆smu.286-smu.287; Emr Hale et al., 2005 ∆luxS ∆smu.474; Emr Sztajer et al., 2008 UA159(pIB187) Plasmid with the gusA reporter gene under
the constitutive control Biswas et al., 2008
UA159(PcomX–gfp) PcomX–gfp fusion into pDL277; Spcr Aspiras et al., 2004 UA159(pDL277) pDL277; Spcr This work UA159(Pmsm–1914) Pmsm–1914 into pDL277; Spcr This work ∆cipI(Pmsm–1914) Pmsm–1914 into pDL277; Emr, Spcr This work UA159(p925) Smu.925 into pDL277; Spcr This work
E. coli BL21(pET28a(+)) T7 expression vector, non-expression
BL21(His6-fullCipB) CipB precursor cloned into pET28a(+), non-expression host; Kanr
This work
BL21(His6-GGCipB) Mature form of CipB cloned into pET28a(+),non-expression host; Kanr
This work
BL21DE3(His6-fullCipB) CipB precursor cloned into pET28a(+), expression host; Kanr
This work
BL21DE3(His6-GGCipB) Mature form of CipB cloned into pET28a(+), expression host; Kanr
This work
L. lactis I6 Indicator strain, susceptible to CipB Hale et al., 2005 S. salivarius 25975 Wild-type M. Frenette, U. Laval S. thermophilus LMG18311 Wild-type S. Moineau, U. Laval
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Static biofilms were developed on polystyrene microtiter plates in semi-defined minimal medium
(SDM) (Li et al., 2002a) supplemented with 44 mmol l-1 glucose. Plates were incubated at 37°C
in air with 5% CO2 for 16 h (standard assay for biofilm quantification), or for 8 h before the
addition of SDM-glucose supplemented with either 0.5 mmol l-1 H2O2, 2.5% NaCl, or SDM-
glucose adjusted to pH5 with HCl for a further 8 h (stress assay). Planktonic cells were removed
after incubation, and the biofilms were air dried overnight before quantification as described
previously (Levesque et al., 2005).
Scanning electron microscopy
Scanning electron microscopy (SEM) was performed on 16 h biofilms grown on glass discs
according to the standard assay. Biofilms were washed with sterile phosphate-buffered saline
(PBS, pH 7.2), dehydrated through ethanol rinses, critical point dried with liquid CO2, mounted,
and sputter coated with gold. Samples were then examined using a scanning electron
microscope (model S-2500; Hitachi Instruments, San Jose, CA).
DNA microarrays
Biofilms of UA159 and SMRR11 were grown for 16 h in SDM-glucose as described in the
standard assay. Both planktonic and biofilm cell pools were harvested, washed once in PBS,
resuspended in Trizol reagent (Invitrogen), and processed with the Bio101 Fast Prep system
(Qbiogene). DNA-free RNA samples were labeled and prepared for hybridization according to
the PFGRC protocol (http://pfgrc.tigr.org/protocols/M007.pdf). Microarray chips were scanned
using a Gene Pix 4000B (Axon). The software package TM4 Microarray Software Suite
(http://www.tm4.org/) was used for data analysis. Microarray assays was performed on three
independent RNA isolations, and validated by quantitative real-time RT-PCR using the
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QuantiTech SYBR Green PCR kit in a Mx3000P QPCR system (Stratagene). Statistical
significance was determined using Student‟s t-test and a P value of <0.05.
Transformation experiments
One µg of plasmid pDL289 was added to growing cultures at an OD600 of 0.1 both in the
presence and absence of 0.4μM synthetic CSP, and incubated at 37°C for 2.5 h. Cultures were
then gently sonicated, and spread on THYE agar plates. Transformation efficiency was
expressed as the percentage of kanamycin resistant transformants over the total number of
recipient cells.
4.4 Results
4.4.1 Phenotypic characterization of Δrr11 defective mutant
The RR11-encoding gene of the S. mutans HK/RR11 TCS was successfully inactivated by
deletion-insertion mutagenesis. No significant difference in growth kinetics was observed during
planktonic growth. Li et al. (2002) reported that their S. mutans strain NG8 rr11–defective
mutant formed significantly less biofilm than the parent strain. In our experiments using
background strain UA159, SMRR11 mutant cells formed stable and reproducible biofilms with a
biomass of 12.2 %± 4.9% less than the wild-type. A closer examination by SEM revealed that
biofilms formed by the SMRR11 mutant exhibited an altered structure, with larger channels
visible at low magnifications and a difference in cell morphology observed higher magnifications
(Figure 4.1).
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Figure 4.1. S. mutans UA159 and RR11- mutant biofilm formation. Scanning electron
micrographs of UA159 and SMRR11 biofilms accumulated on the surface of glass
discs. Magnifications, 1 K (top panels), and 60 K
4.4.2 Microarray identification of RR11-regulated genes involved in the stress
response.
To investigate the morphological differences observed in SMRR11 biofilms, gene expression
profiles of the UA159 wild-type and SMRR11 were analyzed using DNA microarrays.
Expression data comparing biofilm and planktonic growth phases in the wild-type and mutant
strains suggested that RR11 directly and/or indirectly regulated 174 genes (~9% of the genome)
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in S. mutans biofilms. Of these, several genes encoding proteins involved in general stress
response were found differentially regulated (Table 4.2).
The microarray results also identified other TCSs whose expression was altered in SMRR11
mutant biofilms. Among these genes was comD, the receptor for the CSP pheromone. ComDE
was originally characterized for its role in the development of genetic competence in S.
pneumoniae (Håvarstein et al., 1995; Håvarstein et al., 1996) and S. mutans(Li et al., 2002a),
but has recently been implicated in the control of the stress-responsive autolysis pathway in
pneumococci (Guiral et al., 2005). The gene encoding the RR of the CiaHR TCS also showed
altered expression in our microarray. CiaHR is involved in stress tolerance and competence
development in S. mutans (Ahn et al., 2006). Finally, the gene encoding RR9 of the S. mutans
TCS HK/RR9 was also upregulated. This TCS has recently been shown to be involved in S.
mutans acid survival (Levesque et al., 2007).
4.4.3 SMRR11 biofilms under oxidative, osmotic and acid stresses
During the preparation of this manuscript, RR11 was shown to be involved in oxidative and
thermal stress responses in planktonic cultures (Biswas et al., 2007). Since changes in
expression of the above mentioned RR11-regulated stress response genes could impact the
formation of S. mutans biofilms through a reduced ability to respond to stress in the biofilm
environment, we examined the ability of SMRR11 biofilms to grow in the presence of oxidative,
osmotic, and acid stress conditions. A significant reduction in biofilm biomass (37.8 ± 17.1%,
P = 0.002) was observed when SMRR11 biofilms were grown in the presence of H2O2
compared to UA159 grown under the same conditions. We found no statistically significant
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Table 4.2. Genes potentially regulated by RR11 in S. mutans growing in biofilms
Locus
(NCBI)
Description/putative function Relative fold-
change
UA159 SMRR11
SMU.91 peptidyl-prolyl isomerase RopA
(trigger factor)
n
c
+2.2
SMU.228 alkaline-shock protein homolog nc +3.8
SMU.403 DNA-damage-inducible protein P nc +2.5
SMU.949 ATP-dependent protease Clp,
ATPase subunit ClpX
–2.2 nc
SMU.1063 ABC transporter, ATP-binding,
proline/glycine betaine
nc +2.5
SMU.1129 response regulator CiaR +2.6 nc
SMU.1672 ATP-dependent Clp protease,
proteolytic subunit
nc +2.0
SMU.1916 histidine kinase of the
competence regulon
–2.1 nc
SMU.1964 response regulator nc –2.0
SMU.2030 transcriptional regulator CtsR nc +2.0
SMU.2116 osmoprotectant amino acid ABC
transporter, ATP-binding
nc +5.1
nc= no change in gene expression
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difference between the growth of the wild-type and RR11- biofilms under osmotic and acid
stress. Under all three stress conditions assayed, planktonic cultures of UA159 and
SMRR11grew with identical kinetics, indicating that the defect in biofilm formation under
oxidative stress was due to true biofilm-specific growth impairments, and not simply slower
growth of the culture in both phases.
4.4.4 Regulatory role for RR11 in competence development
Our microarray demonstrated that the gene encoding the ComD receptor for the CSP
pheromone was likely regulated by RR11. Because competence and stress response in S.
pneumonie are linked through ComDE, we also examined the competence phenotype of the
SMRR11 mutant. Ahn et al. (2006) suggested that more than one TCS may be involved in
triggering competence induction, and proposed that CiaHR, and possibly some other
unidentified regulators, integrate CSP signals (Ahn et al., 2006). We hypothesized that RR11
may be one such additional regulator, that could be responsible for integrating stress signals in
the biofilm to trigger competence. We investigated the role of RR11 in the development of
genetic competence by evaluating the ability of comDE- (SMComDE), rr11- (SMRR11) and
rr11/comDE (SMDE11) mutants to be transformed with plasmid DNA (Fig. 4.2). Our results
demonstrated that SMRR11 had a ~20-fold reduction in transformation efficiency vs. the wild-
type strain in the absence of CSP. As expected, inactivation of comDE diminished the
transformation efficiency by several-fold. Surprisingly, the SMDE11 double mutant behaved like
the wild-type strain in the absence of CSP, regardless of whether CSP was added. This finding
led us to hypothesize that both ComE and RR11 may negatively regulate a third regulator in the
CSP-independent pathway. To test whether CiaR could be involved in CSP-independent
competence induction, transformation efficiency was measured in the ciaHR– deficient mutant.
However, inactivation of ciaHR had no impact on competence (results not shown).
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Figure 4.2. Transformation efficiency of S. mutans wild-type and mutant strains. The
transformation of S. mutans strains with plasmid pDL289 is plotted with and without the addition
of synthetic CSP. Transformation efficiency is expressed as the percentage of viable cells
transformed to kanamycin resistance. The results are expressed as the mean + standard
deviation of at least two independent experiments.
4.5 Discussion
Environmental conditions in the oral biofilm are highly variable with respect to pH, oxygen
and osmotic balance. Shifts from neutral pH to as low as 3.0 occur during host ingestion of
dietary carbohydrates, oxygen gradients occur in the oral biofilm, and salts may accumulate
from tooth demineralization. Thus, the ability of S. mutans to adapt to its environment is vital to
its fitness. The involvement of two-component signal transduction systems in environmental
stress response has been characterized during planktonic growth of S. mutans, but few studies
have examined the role of these systems in the stress response in the biofilm environment. Our
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aim was to characterize the response regulator component of the HK11/RR11 TCS with respect
to its role in biofilm development. Towards this goal, we examined RR11- biofilms for qualitative
differences by SEM, and for gene expression differences by DNA microarray. We discovered
phenotypic differences between the wild-type and SMRR11 via stress tolerance and
competence assays.
Although only a small difference in biofilm biomass resulted from deletion of RR11 in S.
mutans strain UA159, SEM data indicated that both biofilm structure and cellular morphology
were altered in its absence. To investigate the transcriptome underlying these changes in
morphology, a DNA microarray was performed. Several stress response genes were identified
in our microarray analysis, including ropA and clpP. RopA is a molecular chaperone that
functions in protein biogenesis and stress survival (Hesterkamp and Bukau, 1996). ClpP is the
proteolytic subunit of the ATP-dependent Clp protease, which performs protein reactivation and
degradation (Porankiewicz et al., 1999). Interestingly, Wen et al. (2005) found that an S. mutans
ropA mutant also showed longer chain length in broth and altered biofilm architecture, which
they attributed to alterations in protein trafficking. clpP null mutants are also defective in genetic
competence and biofilm formation, and are more susceptible to stress (Lemos and Burne, 2002;
Wen et al., 2005). These results suggested that the changes in ropA and/or clp gene expression
through RR11 may be responsible for the altered cell morphology and biofilm structure seen in
the SMRR11 mutant.
Specific stress response proteins like the osmoprotectant ABC transporters encoded by
SMU.2116 and SMU.1063 were also up-regulated in RR11- biofilms. SMU.2116 is highly
homologous to OpuCA of Streptococcus agalactiae, while SMU.1063 shares high identity with a
proline/glycine betaine transporter found in Lactococcus lactis. Both these transporters have
been shown to be upregulated under osmotic stress conditions in planktonically grown S.
mutans, and implicated in the survival of the organism under those stress conditions (Abranches
135
et al., 2006) (Abranches et al., 2006). The possible regulatory role for RR11 in the general
stress response prompted us to examine the growth of RR11-deficient biofilms under stress.
Biswas et al. (2007) have recently shown that RR11 is involved in the oxidative stress response
in the planktonic phase, and our results indicated that RR11 may also be important in the
response to oxidative stress in the biofilm. Combining our microarray analysis with our
physiological data suggests that the increased susceptibility to oxidative stress may occur due
to RR11‟s role in regulating the turnover of damaged proteins via RopA and ClpP, which may
result in the abnormal biofilm architecture and cell morphology observed by SEM.
Exciting evidence has recently emerged linking the competence cascade to the general
stress response program in pneumococci (Claverys and Havarstein, 2007; Guiral et al., 2005).
These authors have shown that a population under antibiotic stress triggers the death of
damaged cells via the CSP signaling molecule and ComDE. A similar link between competence
and cell death exists in S. mutans (Leblanc et al., 1992; Perry et al., 2009). Due to microarray
evidence linking RR11 and ComD, and physiological evidence of a role for RR11 in stress
response, we hypothesized that the competence phenotype would also be affected in SMRR11.
Indeed, competence was reduced ~20-fold in the absence of CSP in SMRR11. Based on our
competence data, we have proposed a model for the S. mutans CSP-dependent and CSP-
independent competence regulatory networks (Figure 4.3). In this model, the ComDE TCS is
the primary circuit sensing CSP, and induces a high level of transformation at high levels of
CSP. At low levels of CSP, the major competence system remain inactive, and
unphosphorylated ComD may cross-regulate RR11 to induce a basal level of genetic
competence. Investigations are ongoing in our lab to elucidate the role of stress in the
development of genetic competence, including the role of RR11 in this pathway.
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Figure 4.3 Model for competence development in S. mutans. This model integrates the CSP-
dependent and CSP-independent pathways (see text for details).
4.6 Acknowledgements
This study was supported by National Institute of Dental and Craniofacial Research
grant R01 DE013230. DGC is supported by a Canada Research Chair. JAP and PS
are both supported by CIHR Cell Signals Fellowships. JAP, PS and MB performed
experiments; JAP, CL, RM, RC, SP and DGC contributed to experimental design, data
analysis and/or to the writing of this manuscript. The authors thank Robert Chernecky
for technical services.
137
4.7 References
Abranches, J., J. A. Lemos, and R. A. Burne. 2006. Osmotic stress responses of Streptococcus mutans UA159. FEMS Microbiology Letters 255:240-246. Ahn, S. J., Z. T. Wen, and R. A. Burne. 2006. Multilevel control of competence development and stress tolerance in Streptococcus mutans UA159. Infect Immun 74:1631-1642. Biemans-Oldehinkel, E., M. K. Doeven, and B. Poolman. 2006. ABC transporter architecture and regulatory roles of accessory domains. FEBS Lett 580:1023-1035. Biswas, I., L. Drake, D. Erkina, and S. Biswas. 2007. Involvement of Sensor Kinases in the Stress Tolerance Response of Streptococcus mutans. J. Bacteriol.:JB.00990-00907. Claverys, J. P., and L. S. Havarstein. 2007. Cannibalism and fratricide: mechanisms and raisons d'etre. Nat Rev Microbiol 5:219-229. 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. Havarstein, L. S., G. Coomaraswamy, and D. A. Morrison. 1995. An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae. Proc Natl Acad Sci U S A 92:11140-11144. Havarstein, L. S., P. Gaustad, I. F. Nes, and D. A. Morrison. 1996. Identification of the streptococcal competence-pheromone receptor. Mol Microbiol 21:863-869. Hesterkamp, T., and B. Bukau. 1996. The Escherichia coli trigger factor. FEBS Lett 389:32-34. Lemos, J. A., and R. A. Burne. 2002. Regulation and Physiological Significance of ClpC and ClpP in Streptococcus mutans. J Bacteriol 184:6357-6366. Levesque, C. M., R. W. Mair, J. A. Perry, P. C. Y. Lau, Y.-H. Li, and D. G. Cvitkovitch. 2007. Systemic inactivation and phenotypic characterization of two-component systems in expression of Streptococcus mutans virulence properties. Letters in Applied Microbiology 45:398-404. Li, Y. H., N. Tang, M. B. Aspiras, P. C. Lau, J. H. Lee, R. P. Ellen, and D. G. Cvitkovitch. 2002. A quorum-sensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation. J Bacteriol 184:2699-2708. Porankiewicz, J., J. Wang, and A. K. Clarke. 1999. New insights into the ATP-dependent Clp protease: Escherichia coli and beyond. Mol Microbiol 32:449-458. Prudhomme, M., L. Attaiech, G. Sanchez, B. Martin, and J.-P. Claverys. 2006. Antibiotic Stress Induces Genetic Transformability in the Human Pathogen Streptococcus pneumoniae. Science 313:89-92. Wen, Z. T., P. Suntharaligham, D. G. Cvitkovitch, and R. A. Burne. 2005. Trigger factor in Streptococcus mutans is involved in stress tolerance, competence development, and biofilm formation. Infect Immun 73:219-225.
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Chapter 5: Summary and Conclusions
139
5.1 Summary of Dissertation
This dissertation examines the biological role of cell death and lysis in the biofilm-forming
organism Streptococcus mutans. We have shown that the CSP peptide pheromone is an
inducible signal in S. mutans, which may communicate stress in the population through ComDE.
The result of CSP upregulation is the induction of cell death and lysis in a fraction of the
population through intracellular accumulation of the auto-active bacteriocin CipB. The finding
that a bacteriocin may induce death through intracellular action is a novel finding in the field. We
have also characterized the mechanism of immunity to CSP-induced cell death, which occurs
through the differential regulation of the CipI immunity protein at low cell density via the LiaFSR
(formerly HK/RR11) regulatory system. This regulation is made possible due to the physical
separation of cipB and cipI on the chromosome, and allows for S. mutans survival at low cell
density. Further work also elucidated the LiaFSR regulon in the biofilm (which includes
regulation of cipI expression), and demonstrated a role for this signalling system in the
regulation of oxidative stress tolerance in the biofilm.
In the high cell density biofilm environment, the CipB/CipI cell death pathway contributes to
release of DNA into the extracellular matrix through cell lysis. This eDNA contributes to the
stability of the biofilm. Finally, we also provide evidence that the CipB/CipI death pathway is
involved in genetic competence, which we suggest may contribute to exchange of fitness
enhancing genes under stress and contribute to the evolutionary fitness of the organism.
140
5.2 General Discussion
5.2.1 Peptide pheromone-induced cell death
Previous reports in both S. pneumoniae and S. mutans suggested that the CSP pheromone
was involved in inducing bacterial cell death (Dagkessamanskaia et al., 2004; Guiral et al.,
2005; Qi et al., 2005). We set out to characterize the response to CSP in S. mutans, framed in
the physiological context of elevated concentrations of the peptide induced by stress. Although
streptococcal CSP has traditionally been defined as a quorum sensing signal, our study and
those in S. pneumoniae have expanded that view by suggesting that CSP may also be an
inducible „alarmone‟-type molecule, capable of signalling stress in the population. However, we
suggest that in fact high cell density is a stress itself, and that quorum sensing is (and always
has been) a stress response system.
Our results showed that high concentrations of CSP induce lysis in a fraction of the S.
mutans population by intracellular accumulation of the auto-active bacteriocin CipB. This
mechanism is in contrast to that observed in S. pneumoniae, in which cell lysis is accomplished
in trans by competent cells expressing the CbpD protease or the two-peptide bacteriocin CibAB
on their cell surface. However, the intracellular action of CipB makes sense in the broader
context of biofilm growth, since expression of an auto-active death peptide on the cell surface
has destructive potential in the tightly packed biofilm environment. In fact, we suggest that
export of CipB is a detoxification mechanism, since no susceptibility to the exported form of the
bacteriocin has been noted for S. mutans or any of its normal target organisms in the oral
biofilm. The intracellular mechanism of action also prevents the lysis of the entire population,
141
since S. mutans has only a single pherotype of CSP. While S. pneumoniae has the capacity to
differentially express immunity to cell death through the production of multiple CSP pherotypes,
S. mutans safe-guards against lysis of the whole population by triggering cell death
intracellularly.
We found that cell death in the biofilm may contribute extracellular DNA (eDNA) to
strengthen the biofilm matrix. Bayles has proposed a role for autolysis in biofilm formation by S.
aureus (Bayles, 2007). He argued that the biofilm lifestyle is akin to a multi-cellular organism in
its specialization of function, and that death of a sub-population is a natural extension of that
lifestyle. We propose a similar role for CSP-induced cell death in S. mutans. In the high density
oral biofilm where environmental stresses abound, the ability to form a biofilm and survive as a
sessile community is a strong evolutionary pressure. The act of cellular suicide under stress (or
high CSP concentration) provides both nutrients for continued growth and added protection in
the form of an enhanced extracellular matrix containing eDNA.
5.2.2 Immunity to peptide induced cell death
Although most bacteriocins are co-transcribed with their cognate immunity genes, it is not
without precedent to find an immunity gene un-linked on the chromosome (Diep et al., 2007).
We found that the CipI immunity protein-encoding gene was differentially regulated from the
CipB bacteriocin, and suggested that this differential expression necessitated the duplication of
immunity elsewhere in the genome. The up-regulation of CipI expression (by either plasmid-
based over-expression or through pre-growth of cultures prior to sCSP exposure) was shown to
142
be protective in cultures at low cell density. In the context of stress and biofilm formation, this
result makes intuitive sense. If CSP (and autolysis) is high during stress and at high cell density
in the biofilm, low CSP (or density) should signal the absence of stress, and trigger survival and
proliferation. It is tempting to speculate that such conditions are met in the planktonic
population that departs the biofilm in the last stages of its lifecycle.
We found that the LiaFSR signalling system governs the up-regulation of CipI at low cell
density, and that LiaR (formerly RR11) is responsible for the down-regulation of cipI in biofilm
cells. We (and others (Li et al., 2002a)) found the LiaR mutant formed biofilms with aberrant
architecture. In the absence of LiaR in the biofilm, the cell death pathway would show
diminished activity due to an inability to down-regulate cipI. Could this imbalance lead to the
altered architecture we observed? Although it is impossible to say conclusively, the results of
our biofilm experiments suggest that the opposite is certainly true: a biofilm formed in the
absence of CipI has more biomass than the wild-type through the release of eDNA. Our
experiments with LiaR/RR11 biofilms also suggested potential cross-talk between ComD and
LiaR, albeit in the context of genetic competence. It has been previously suggested that these
two signalling systems can both respond to the CSP pheromone (Li et al., 2002a). Results
presented in both Chapters 3 and 4 of this dissertation support a link between these two
signalling systems.
5.2.3 Peptide-induced cell death in genetic competence
The release of DNA into the extracellular matrix has been shown to be essential for proper
biofilm architecture (Whitchurch et al., 2002). However, co-ordinated DNA release from the oral
143
biofilm dweller S. gordonii and competence induction by S. mutans has been shown to allow the
transmission of usable genetic material from one species to the other (Kreth et al., 2005). Could
the eDNA released into biofilm by CSP-induced lysis serve both to physically strengthen the
biofilm and provide a reservoir of fitness-enhancing DNA under stress? Extensive horizontal
gene transfer has likely occurred between streptococcal species throughout evolution
(Cvitkovitch, 2001). However, the likelihood of productive recombination decreases with
evolutionary distance due to chromosomal divergence. Therefore, the exchange of DNA
between different strains within the same species offers the greatest opportunity for acquisition
of functional genes. We have shown that the induction of CipB-mediated cell death in a culture
is somehow tied to induction of competence, and suggested that the presence of cellular debris
may serve as an additional signal to induce CSP-independent DNA uptake. In the biofilm,
cellular debris released during CSP-induced lysis may trigger CSP-independent competence in
the surviving population, to allow for the uptake of fitness-enhancing genes under stress.
In summary, we have provided a detailed examination of the CSP-induced cell death
pathway in S. mutans, and have attempted to show a physiological role for this pathway in the
stress response, genetic competence and biofilm formation. Our data provides a mechanistic
link between phenotypes previously ascribed to CSP-ComDE signalling.
5.3 Future Directions
While significant progress has been made towards understanding the CSP-induced signalling
cascade, several important questions remain unanswered. Although the CipB-mediated cell
144
death pathway appears to be able to induce genetic competence in a CSP-independent
manner, it is not clear how death in a population is able to trigger DNA uptake by the surviving
population. Moreover, although we suggest that CSP responsiveness vs. unresponsiveness in
a population of S. mutans is due to bi-stability, further investigation into these dual responses is
warranted. Questions also remain as to the role of CSP-induced genes not required for
competence, and surrounding the shut-off of the CSP response in S. mutans. Finally, although
our results further corroborate past evidence that the LiaFSR TCS may also respond to CSP,
definitive proof of this interaction remains elusive.
5.4 Significance
The ability of S. mutans to form biofilms and tolerate the fluctuating environmental conditions
within those environments is vital to its virulence. We have made significant progress towards
understanding the CSP-induced signalling pathway, which controls its ability to form biofilms
and is central to its stress response. S. mutans itself is of importance as one of the primary
causative agent of the most prevalent human infections, dental caries. However, it is also a
member of the medically important genus Streptococcus and a biofilm-forming organism. As
such, understanding the pathways involved in its stress response and biofilm formation may
provide clues to help combat infections beyond the oral cavity.
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5.5 References
Bayles, K.W. (2007) The biological role of death and lysis in biofilm development. Nat Rev Microbiol 5: 721-726.
Cvitkovitch, D.G. (2001) Genetic competence and transformation in oral streptococci. Crit Rev Oral Biol Med 12: 217-243.
Dagkessamanskaia, A., Moscoso, M., Henard, V., Guiral, S., Overweg, K., Reuter, M., Martin, B., Wells, J., and Claverys, J.P. (2004) Interconnection of competence, stress and CiaR regulons in Streptococcus pneumoniae: competence triggers stationary phase autolysis of ciaR mutant cells. Mol Microbiol 51: 1071-1086.
Diep, D.B., Skaugen, M., Salehian, Z., Holo, H., and Nes, I.F. (2007) Common mechanisms of target cell recognition and immunity for class II bacteriocins. Proc Natl Acad Sci 104: 2384-2389.
Guiral, S., Mitchell, T.J., Martin, B., and Claverys, J.P. (2005) Competence-programmed predation of noncompetent cells in the human pathogen Streptococcus pneumoniae: genetic requirements. Proc Natl Acad Sci 102: 8710-8715.
Kreth, J., Merritt, J., Shi, W., and Qi, F. (2005) Co-ordinated bacteriocin production and competence development: a possible mechanism for taking up DNA from neighbouring species. Mol Microbiol 57: 392-404.
Li, Y.H., Lau, P.C., Tang, N., Svensater, G., Ellen, R.P., and Cvitkovitch, D.G. (2002a) Novel two-component regulatory system involved in biofilm formation and acid resistance in Streptococcus mutans. J Bacteriol 184: 6333-6342.
Qi, F., Kreth, J., Levesque, C.M., Kay, O., Mair, R.W., Shi, W., Cvitkovitch, D.G., and Goodman, S.D. (2005) Peptide pheromone induced cell death of Streptococcus mutans. FEMS Microbiol Lett 251: 321-326.
Whitchurch, C.B., Tolker-Nielsen, T., Ragas, P.C., and Mattick, J.S. (2002) Extracellular DNA required for bacterial biofilm formation. Science 295: 1487.
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Appendix A: Supplementary Information
147
SI Table S1. Genes showing a minimum ± 2-fold difference in expression when S. mutans
UA159 cells were exposed to 2 μM sCSP
Gene ID Putative or assigned function Fold Amino acid biosynthesis