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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
Degree of Doctor of Philosophy
Graduate Department of Dentistry
University of Toronto
Copyright by Julie A. Perry 2009 ©
ii
Study of the physiological and molecular mechanisms underlying peptide-
induced cell death and biofilm formation in Streptococcus mutans
Julie Ann Perry
Doctor of Philosophy, Faculty of Dentistry, University of Toronto
2009
Abstract
Biofilms are complex and highly adapted communities of microorganisms found attached to
surfaces. Among the best characterized infectious multi-cellular biofilms is the oral community
known as dental plaque. Streptococcus mutans resides in the oral biofilm, and is one of the
main causative agents of dental caries. Streptococci are known to monitor their population
density using a peptide pheromone (CSP)/two component signalling system (ComDE) in a
process classically known as quorum sensing (QS). Previous work in S. mutans has implicated
the QS system in genetic competence, the stress response, bacteriocin production and biofilm
formation. Our objective in this work was to thoroughly characterize the transcriptional and
phenotypic response to CSP in S. mutans, and determine its role in biofilm formation. We have
shown that the CSP pheromone is more than simply a QS signal, and is also an inducible
„alarmone‟ capable of communicating stress in the population. We have demonstrated that
elevated concentrations of CSP such as those that occur during stress trigger autolysis in a
fraction of the population. Importantly, we have shown that autolysis in S. mutans occurs via a
novel mechanism of action: intracellular accumulation of a self-acting bacteriocin. We have also
identified and characterized the autolysis immunity protein, which is differentially regulated from
the bacteriocin to allow survival at low cell density. A second regulatory system was shown to
govern expression of autolysis immunity in the absence of CSP signaling, and also contribute to
the oxidative stress response in the biofilm. Finally, we present evidence that autolysis is
involved in the release of DNA in the biofilm, which contributes to the architecture of the
extracellular matrix and may provide a mechanism for the dissemination of fitness-enhancing
genes under stress. Together, our data provides a mechanistic link between phenotypes
previously ascribed to the CSP pheromone in S. mutans.
iii
Acknowledgements
I extend sincere thanks to both my supervisors, Dr. Celine Levesque and Dr. Dennis Cvitkovitch
for their patience, mentorship and support over the years. I am particularly grateful to Dr.
Levesque, who took me on as her first student, and trusted me with this project. I truly
appreciate the opportunities, challenges, advice and friendship you have given me. I would like
to thank our collaborators Dr. Marcus Jones and Dr. Scott Peterson at the J. Craig Venter
Institute for their help performing the DNA microarrays presented in Chapters two and four. I
am also grateful to Dr. Roslyn Devlin, who gave me my first taste of science and set me on this
path. I sincerely thank Dr. Stanley Holt for having such confidence in me, and for always
treating me like a fellow scientist. Finally, I am grateful to the members of my advisory
committee, Dr. Debora Foster, Dr. Chris McCulloch and Dr. Martin McGavin for their invaluable
critiques and expert guidance.
Thank you also to the members of the Levesque and Cvitkovitch labs, particularly Kirsten,
Timmy, Elena and Marie-Christine. Your friendship and support has been much appreciated.
Beyond the lab, I would like to thank my friends for putting up with my moments of „intensity‟,
and always listening when I needed it.
Finally, I thank my family for their unwavering support. To Mom, Dad, Ted, and of course to
Patrick, I am unendingly grateful for the opportunities your love has provided me, and for the
peace you bring to my life. Thank you.
Julie Ann Perry
2009
iv
Table of Contents
Abstract....................................................................................................................................... ii
Acknowledgements .................................................................................................................... iii
Table of Contents ....................................................................................................................... iv
List of Tables ........................................................................................................................... viii
List of Figures ............................................................................................................................ ix
Preface ...................................................................................................................................... xi
Publications reproduced as dissertation chapters ..................................................................... xii
Awards ..................................................................................................................................... xiii
Abbreviations ........................................................................................................................... xiv
Quote ....................................................................................................................................... xvi
Chapter 1: Literature Review ............................................................................................. 1
1.1. The biofilm mode of growth ................................................................................................. 2
1.1.1. The lifecycle of a biofilm ........................................................................................... 2
1.1.2. Structure of a mature biofilm..................................................................................... 4
1.1.3. Bacterial biofilm infections ........................................................................................ 6
1.2. Streptococcus mutans: a model for infectious biofilm formation ................................... 8
1.2.1. S. mutans in the oral biofilm ..................................................................................... 9
1.2.1.1. Attachment to the tooth: formation of the oral biofilm ......................................... 9
1.2.1.2. Acidogenicity and aciduricity ............................................................................13
1.2.1.3 Interspecies competition in the oral biofilm: production of mutacins ..................14
1.2.1.4. Two-component signal transduction systems ...................................................16
1.3. Quorum sensing.................................................................................................................19
1.3.1. A brief history of quorum sensing ............................................................................20
1.3.2. Quorum sensing mechanisms .................................................................................22
1.3.3. Quorum Sensing in streptococci ..............................................................................25
1.3.3.1. Overview of the CSP-ComDE system in S. pneumoniae ..................................25
1.3.3.2 CSP-ComDE in S. mutans ............................................................................28
v
1.3.4 Phenotypes controlled by CSP-ComDE signalling in streptococci ........................31
1.3.4.1 Genetic competence ..........................................................................................31
1.3.4.2.1 Mechanism of DNA uptake in streptococci .................................................31
1.3.4.2.2 Purpose of DNA uptake .............................................................................33
1.3.4.2 Autolysis and pneumococcal fratricide ..........................................................35
1.3.4.2.1 Autolysins and autolysis ............................................................................36
1.3.4.2.2 The holin/antiholin system in S. aureus......................................................37
1.3.4.2.3 Streptococcal fratricide ..............................................................................38
1.3.4.3 Bacteriocin production ..................................................................................43
1.3.4.3.1 Classification of bacteriocins ......................................................................43
1.3.4.3.2 Biosynthesis and export of class IIa bacteriocins .......................................44
1.3.4.3.3 Mode of action ...........................................................................................46
1.3.4.4 CSP and the stress response .......................................................................48
1.3.4.5. Biofilm formation ...........................................................................................49
1.4. Statement of the problem ...................................................................................................51
General hypothesis: ...............................................................................................................52
Primary objective: ..................................................................................................................52
Rationale: ..............................................................................................................................52
1.5 References ........................................................................................................................54
Chapter 2: Peptide alarmone signalling triggers an auto-active bacteriocin
necessary for genetic competence ..................................................................................64
2.1 Abstract .............................................................................................................................65
2.2 Introduction ........................................................................................................................66
2.3 Experimental procedures ...................................................................................................68
Culture conditions ..................................................................................................................68
Gene expression analysis......................................................................................................70
DNA microarrays ...................................................................................................................70
Recombinant peptides ...........................................................................................................71
vi
Bacteriocin overlay assays ....................................................................................................72
Transformation assays ..........................................................................................................72
2.4 Results ...............................................................................................................................72
2.4.1 Stress induces expression of the CSP pheromone ..................................................72
2.4.2 CSP pheromone triggers autolysis in a fraction of the population ............................75
2.4.3 Genome-wide expression response to CSP: identification of mutacin V ..................78
2.4.4 CipB bacteriocin likely acts intracellularly ................................................................84
2.4.5 The small protein CipI (SMU.925) confers immunity ................................................89
2.4.6 Role of CSP-induced lysis in genetic competence ...................................................91
2.5 Discussion .........................................................................................................................96
2.6 Acknowledgements ............................................................................................................99
2.7 References ...................................................................................................................... 100
Chapter 3: Cell Death in Streptococcus mutans Biofilms: a Link Between CSP and
Extracellular DNA ............................................................................................................... 104
3.1 Abstract ........................................................................................................................... 105
3.2 Introduction ...................................................................................................................... 106
3.3 Materials and methods ..................................................................................................... 107
3.4 Results and discussion .................................................................................................... 110
3.4.1 Low cell density-dependent expression of cipI is regulated by LiaR....................... 110
3.4.2 CipI is protective at low cell density, while CipB is lethal at high cell density .......... 111
3.4.3 Cell death participates in biofilm formation through eDNA release......................... 114
3.5 Conclusions ..................................................................................................................... 118
3.6 Acknowledgements .......................................................................................................... 119
3.7 References ...................................................................................................................... 120
Chapter 4: Involvement of Streptococcus mutans regulator RR11 in oxidative
stress response during biofilm growth and in the development of genetic
competence ......................................................................................................................... 123
4.1 Abstract ........................................................................................................................... 124
vii
4.2 Introduction ...................................................................................................................... 125
4.3 Materials and methods ..................................................................................................... 126
Bacterial strains and growth conditions................................................................................ 126
In vitro model for growing biofilms ....................................................................................... 127
Scanning electron microscopy ............................................................................................. 127
DNA microarrays ................................................................................................................. 127
Transformation experiments ................................................................................................ 128
4.4 Results ............................................................................................................................. 128
4.4.1 Phenotypic characterization of Δrr11 defective mutant .......................................... 128
4.4.2 Microarray identification of RR11-regulated genes involved in the stress response.
129
4.4.3 SMRR11 biofilms under oxidative, osmotic and acid stresses ............................... 130
4.4.4 Regulatory role for RR11 in competence development .......................................... 132
4.5 Discussion ....................................................................................................................... 133
4.6 Acknowledgements .......................................................................................................... 136
4.7 References ...................................................................................................................... 137
Chapter 5: Summary and Conclusions .......................................................................... 138
5.1 Summary of Dissertation .................................................................................................. 139
5.2 General Discussion .......................................................................................................... 140
5.2.1 Peptide pheromone-induced cell death ................................................................. 140
5.2.2 Immunity to peptide induced cell death .................................................................. 141
5.2.3 Peptide-induced cell death in genetic competence ................................................ 142
5.3 Future Directions .............................................................................................................. 143
5.4 Significance ..................................................................................................................... 144
5.5 References ...................................................................................................................... 145
Appendix A: Supplementary Information ...................................................................... 146
viii
List of Tables
Table 1.1: Common biofilm-mediated infections in humans........................................................ 7
Table 1.2: S. mutans two-component signal transduction systems ............................................18
Table 1.3: Comparison of the CSP-ComDE systems in S. pneumoniae and S. mutans ............29
Table 1.4: Autolysin and fratricidal effector genes and their homologs in S. mutans UA159 ......42
Table 1.5: Bacteriocins encoded by S. mutans..........................................................................45
Table 2.1: Bacterial strains used in this study ...........................................................................69
Table 2.2: Relative expression levels of highly-expressed CSP-induced S. mutans genes
encoding putative and known bacteriocins and their accessory genes ..................................79
Table 3.1: Bacterial strains used in this study ......................................................................... 106
Table 4.1: Bacterial strains used in this study ......................................................................... 125
Table 4.2: Genes potentially regulated by RR11 in S. mutans growing in biofilms ................... 130
Table S1: Genes showing a minimum ± 2-fold difference in expression when S. mutans UA159
cells were exposed to 2 μM sCSP ………………………………………………………………. 147
Table S2: S. mutans genes showing a minimum ± 2-fold difference in expression when
S. mutans ∆comX cells were exposed to 2 μM sCSP ………………………………………… 153
ix
List of Figures
Figure 1.1: Summary of factors influencing the survival and virulence of S. mutans in the oral
biofilm. ......................................................................................................................................10
Figure 1.2: Formation of dental plaque ......................................................................................12
Figure 1.3: Simplified schematic representation of quorum sensing systems in bacteria ...........24
Figure 1.4: Mechanistic and genomic representation of the CSP-ComDE circuit controlling
competence development in S. pneumoniae and S. mutans. ..................................................30
Figure 1.5: Representation of the holin/anti-holin system in S. aureus ......................................39
Figure 1.6: Proposed mechanism of action of the CSP-induced fratricidal pathway in S.
pneumoniae ..............................................................................................................................41
Figure 1.7: Cartoon representation of the mechanism of action of bacteriocins .........................47
Figure 2.1. Recovery of S. mutans from stress. .........................................................................74
Figure 2.2. S. mutans growth kinetics .......................................................................................76
Figure 2.3. Effect of sCSP on culture density and cell lysis .......................................................77
Figure 2.4: A sub-population of cells is always resistant to sCSP-mediated cell lysis. ...............78
Figure 2.5. Growth of Streptococcus thermophilus and Streptococcus salivarius in increasing
concentrations of their species-specific signaling peptides ........................................................81
Figure 2.6. Effect of 2 µM sCSP pheromone on S. mutans wild-type and mutants defective in
the bacteriocin CipB (SMU.1914) and its putative immunity factors SMU.1913 and CipI
(SMU.925). ...............................................................................................................................82
Figure 2.7. Agar overlay assays showing extracellular bacteriocin ............................................83
Figure 2.8. Growth kinetics of S. mutans UA159 strain containing CipB under the control of a
raffinose-inducible promoter .....................................................................................................85
Figure 2.9. RT-PCR gene expression profiles of cipB and cipI ..................................................86
Figure 2.10. Quantitative real-time RT-PCR gene expression profiles of cipB, comC and cipI at
following dilution from an overnight culture ................................................................................86
Figure 2.11. CipB may act intracellularly ...................................................................................88
Figure 2.12. Growth of the wild-type and a wild-type strain over-expressing cipI .......................91
Figure 2.13. Transformation efficiency of S. mutans wild-type strain and its mutants deficient in
the CipB bacteriocin and CipI immunity protein .........................................................................93
x
Figure 2.14. Competence and lysis in cultures of S. mutans .....................................................94
Figure 2.15. Summary of data ...................................................................................................95
Figure 3.1: Growth of S. mutans TCS mutants in the presence of 2 µM sCSP ........................ 111
Figure 3.2: Growth kinetics of S. mutans UA159 wild-type strain in the presence of 2µM sCSP
............................................................................................................................................... 113
Figure 3.3: Fold-change in quantity of eDNA in ΔCipI and ΔCipB mutant biofilms ................... 115
Figure 3.4: Biofilm biomass of S. mutans UA159 wild-type strain, ΔCipB and ΔCipI mutants .. 117
Figure 4.1. S. mutans UA159 and RR11- mutant biofilm formation ......................................... 129
Figure 4.2. Transformation efficiency of S. mutans wild-type and mutant strains. .................... 133
Figure 4.3 Model for competence development in S. mutans ................................................. 136
xi
Preface
Dissertation format
This dissertation is presented in the „Publishable Style‟. Chapter 1 presents a general
introduction to the subject, and serves to provide context for the following chapters. It includes
portions of an invited book chapter appearing in the ASM Press publication „Genomic Inquiries
Into Oral Bacterial Communities‟ (P.E. Kolenbrander, Ed.). Chapters 2 to 4 describe
experimental data that have either been published or submitted for publication. They are
presented in their published form, other than minor changes made to improve readability and
reduce repetition. Chapter 5 serves a brief discussion of all experimental data. Written
permission for reproduction of all publications has been obtained, and is held on file.
xii
Publications reproduced as dissertation chapters
Perry JA, Cvikovitch DG. 2009. Autoinducer-2-regulated genes in Streptococcus mutans and
impact on oral bacterial communities. Invited book chapter in Genomic Inquiries Into Oral
Bacterial Communities, Paul E. Kolenbrander (Ed.). ASM Press.
Perry JA, Jones MB, Peterson SN, Cvitkovitch DG, Lévesque CM. 2009. Peptide alarmone
signaling triggers an auto-active bacteriocin necessary for genetic competence. Mol Microbiol.
72: 905-917.
Perry JA, Cvitkovitch DG, Levesque CM. 2009. Cell death in Streptococcus mutansbiofilms: a
link between CSP and extracellular DNA. FEMS Microbiol. Lett. 299:261-6
Perry JA, Lévesque CM, Suntharaligam P, Mair RW, Bu M, Cline RT, Peterson SN, Cvitkovitch
DG. 2008. Involvement of Streptococcus mutans Regulator RR11 in Oxidative Stress Response
in the Biofilm and in the Development of Genetic Competence. Lett Appl Microbiol. 47: 439-44.
Additional publications
Kreth J, Merritt J, Huang D, Perry J, Zhu L, Goodman S, Cvitkovitch DG, Shi W, Qi F. 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-
807.
Lévesque CM, Mair RW, Perry JA, Lau P, Li Y-H, Cvitkovitch DG. 2007. Systemic inactivation
and phenotypic characterization of two-component systems in expression of Streptococcus
mutans virulence properties. Lett Appl Microbiol. 45: 398-404.
Perry J, Ho M, Viero S, Zheng K, Jacobs R, Thorner P. 2007. The intermediate filament nestin
is highly expressed in normal human podocytes and podocytes in glomerular disease. Pediatr
Dev Pathol.10: 369-82.
Harvey S, Perry J, Zheng K, Chen D, Sado Y, Jefferson B, Ninomiya Y, Jacobs R, Hudson B,
Thorner, P. 2006. Sequential expression of type IV collagen networks: testis as a model and
relevance to spermatogenesis. Am J Pathol. 168: 1587-97
Perry J, Tam S, Zheng K, Harvey S, Sado Y, Jefferson B, Jacobs R, Thorner P. 2006. Type IV
collagen induces podocytic features in bone marrow stromal stem cells in vitro. J Am Soc
Nephrol. 17: 66-76.
Zheng K, Perry J, Harvey S, Sado Y, Ninomiya Y, Jefferson B, Jacobs R, Hudson B, Thorner P.
2005. Regulation of collagen type IV genes is organ-specific: evidence from a canine model of
Alport syndrome.Kidney Int 68: 2121-2130.
xiii
Awards
2007 2nd place poster award, Faculty of Dentistry Research Day
2007-2009 Ontario Graduate Scholarship
2006-2009 CIHR Strategic Training Program (“Cell Signals”) Fellowship
2005-2009 Harron Scholarship, Faculty of Dentistry, University of Toronto
2005 1st place poster award, Hospital for Sick Children Research Day
2003-2005 University of Toronto Open Fellowship
2002-2003 Ontario Thoracic Society Summer Studentship
1999 Univeristy of Guelph Entrance Scholarship
xiv
Abbreviations
aa amino acid
ABC ATP-binding casette
AHL acylhomoserine lactone
AI-2 auto-inducer 2
Ala alanine
ATP adenosine triphosphate
ATR acid tolerance response
BIP bacteriocin-inducing peptide
CSP competence stimulating peptide
DPD (S)-4,5-dihydroxy-2,3-pentanedione
ds double-stranded
eDNA extracellular DNA
EDTA ethylenediaminetetraacetic acid
EPS exopolymeric substances
FTF fructosyltransferase
GTF glucosytransferase
GUS β-glucuronidase
h hour
His histidine
HK histidine kinase
IPTG isopropyl-beta-D-thiogalactopyranoside
LB Luria-Bertani
min minute
MU Miller units
xv
PBS phosphate-buffered saline
PCR polymerase chain reaction
PMF proton motive force
PNPG p-nitrophenyl glucuronide
RR response regulator
s second
ss single-stranded
SAH S-adenosylhomocysteine
SAM S-adenosylmethionine
sCSP synthetic CSP
SDM semi-defined minimal medium
SEM scanning electron microscopy
TCS two-component signal transduction system
THYE Todd-Hewitt-Yeast extract
WT wild-type
xvi
Quote
“It is not the strongest of the species that survives, nor the most intelligent. It is the one
that is the most adaptable to change.”
– Charles Darwin
1
Chapter 1: Literature Review
2
1.1. The biofilm mode of growth
Bacteria have long been studied as single-celled, primitive organisms, free-floating in
laboratory culture. However, bacterial species in nature have a strong tendency to colonize
surfaces and form complex, multi-species communities referred to as biofilms (Costerton et al.,
1994). This behaviour has been likened to the human condition as described by the
philosopher John Locke (1632-1704), who argued that the default state of human existence is
independent and solitary, but that individuals may voluntarily form communities for mutual
protection and economic prosperity. In nature, biofilms are found on rocks in streams, in
industrial bioreactors, and in animal host environments like the oropharyngeal, gastrointestinal
and vaginal tracts, and on medical prostheses (Costerton et al., 1994; Costerton et al., 1999).
The ubiquitous presence of biofilms in such diverse environments suggests a strong
evolutionary advantage for surface dwellers over their free-floating counterparts (Dunne, 2002).
The obvious explanation of the sessile lifestyle is that nutrients in an aqueous environment tend
to concentrate at solid surfaces. However, surface-associated community living offers many
more advantages to biofilm dwellers, including protection from antimicrobial agents and host
defences and division of the metabolic burden between neighbours.
1.1.1. The lifecycle of a biofilm
A biofilm in its simplest form is composed of a surface (or „substratum‟), surface attached
cells, and a surrounding extracellular matrix of biopolymers (Dunne, 2002). The lifecycle of a
biofilm begins with the attachment of free-floating (or „planktonic‟) cells to a living or an abiotic
substratum, followed by the formation of discreet microcolonies, the subsequent maturation of
3
the biofilm including the development of a complex three dimensional architecture, and finally
production of a planktonic subpopulation for dispersal and re-colonization.
Primary attachment of a bacterium to a substratum is often considered to be a two step
process: an initial, reversible, non-specific interaction (e.g hydrophobic interaction), is followed
by a secondary, permanent interaction mediated by specific adhesins (Dunne, 2002). During
the initial attachment phase, the adherent cells are not considered „committed‟ to biofilm
differentiation, and may leave the surface to resume their planktonic lifestyle (reviewed in
(Stoodley et al., 2002)). In the well-characterized Gram-negative biofilm-forming organism
Pseudomonas aeruginosa, the reversible attachment phase ends when cells begin to up-
regulate the gene responsible for production of the exopolysaccharide alginate, within 15
minutes of initial contact with the substratum (Davies et al., 1993). Indeed, production of
exopolymeric substances (EPS) is considered to be the hallmark of the transition to biofilm
growth. Once biofilm formation is initiated by the primary colonizers, other bacteria may adhere
to the surface-attached primary colonizers through specific receptor-mediated interactions (for a
review of this process during the formation of the dental plaque biofilm, see section 1.2). The
receptor-mediated nature of these interactions results in an organized succession of bacterial
attachment in a multi-species biofilm. Often, metabolically complementary species are located
in spatial proximity in a multi-species biofilm, to benefit from metabolic substrate exchange and
waste removal within the microcolony. These types of reciprocal relationships are only possible
during dense, surface-associated growth. Once attached, cells begin to divide (and to die),
forming dense microcolonies within the larger biofilm. The lifecycle of a biofilm is complete once
the biofilm reaches a critical mass: at this point, the outermost layers of growth begin to
generate planktonic cells which depart the biofilm and colonize other surfaces (Dunne, 2002).
4
Recent evidence has suggested that the stages in the maturation of a biofilm are controlled by
cell-density monitoring and cell-cell communication systems in a process known as quorum
sensing (reviewed in (Davies et al., 1998; Parsek and Greenberg, 2005) and discussed in
section 1.3), and that each stage in the biofilm lifecycle is distinct from the others (Stoodley et
al., 2002). At maturity, biofilms formed by P. aeruginosa show changes in as much as 50% of
the detectable proteome compared with their planktonic counterparts (Sauer et al., 2002).
1.1.2. Structure of a mature biofilm
While the biofilm lifestyle provides the opportunity for nutrient and waste-sharing, the obvious
disadvantage to permanent surface attachment is the inevitable over-crowding and nutrient and
oxygen shortages within each micro-colony. The three-dimensional structure of a biofilm is
therefore essential to its function. By microscopic analysis, a biofilm appears to be a highly
hydrated and open structure, composed mainly of non-cellular material including water channels
and EPS (Lawrence et al., 1991). The EPS is composed of a hydrated, anionic mesh of
bacterial exopolymers and trapped environmental molecules, and forms the outermost layer of
the biofilm (Branda et al., 2005). Despite the universal presence of EPS in all biofilms, there is
variation in the composition and timing of EPS production in biofilms from different species
(Branda et al., 2005). The most extensively studied components of the EPS are carbohydrate-
rich polymers (like alginate) and proteins, but extracellular DNA (eDNA) is now also widely
recognized as a major constituent of the matrix (Allesen-Holm et al., 2006; Hall-Stoodley et al.,
2008; Thomas et al., 2009; Whitchurch et al., 2002). Whatever its composition, the extracellular
matrix functions as a permeability barrier to limit both the diffusion of beneficial nutrients away
from the biofilm, and prevent or slow the diffusion of harmful substances like antibiotics and
5
predatory cells of the immune system from accessing matrix-embedded cells (Costerton et al.,
1999).
Within the extracellular matrix, individual cells occupy a distinct niche environment that is
connected to the rest of the biofilm and to the external environment by a network of water-filled
channels, which behave like a primitive circulatory system (Costerton et al., 1994). These
channels have been shown to permit the penetration of large molecules (up to 2,000-kDa) into
the environment, and to transport dissolved oxygen into the biofilm by convective flow
(Costerton et al., 1994). However, nutrients and dissolved oxygen can only penetrate into the
outer-most cells of each microcolony, and the growth of the biofilm is limited by the availability of
nutrients to deeply embedded cells. The result of this limited diffusion is that the centre of each
microcolony is essentially anaerobic, and may experience nutrient deficiencies and waste build-
up. Likewise, oxygen decreases through the depth of the biofilm, such that the surface is more
aerobic while cells located close to the colonized surface are anaerobic (Costerton et al., 1994).
The spatial separation of sessile cells combined with nutrient/waste and oxygen gradients
within the biofilm results in a heterogeneous population of cells, distinct from their planktonic
counterparts in gene expression patterns and behaviours. The metabolic task sharing,
communication and heterogeneity within a biofilm lead to the success of the group where
individual bacteria would have failed (Parsek and Greenberg, 2005). In essence, the combined
metabolic repertoire of multiple bacterial genomes acting in concert is able to accomplish the
function of a multi-cellular organism. However, the advantage to sharing metabolic
responsibilities among many small prokaryotic genomes instead of one large eukaryotic
genome is the phenotypic plasticity possible in a prokaryotic multi-species biofilm (Stoodley et
al., 2002). The result is that biofilms may take advantage of periods of nutrient bounty (during
which they may even release fast-growing planktonic cells), but may also survive periods of
6
nutrient shortage through the increased adaptation potential present in multiple genomes
located in close proximity (Stoodley et al., 2002). This ability to adapt and persist in the face of
adversity has led to the ubiquitous nature of biofilms in the environment and in infectious
disease.
1.1.3. Bacterial biofilm infections
What may be a highly beneficial lifestyle for bacteria has become highly problematic for their
human hosts. Although acute bacterial infections were historically caused by planktonic
populations of specialized pathogens like Vibrio cholerae and Yersinia pestis, more than 80% of
modern-day infections in the developed world are thought to involve biofilms (Costerton et al.,
1999; Fux et al., 2005) (Table 1.1). Infections caused by biofilms are often slow-growing, slow
to produce overt symptoms, and are rarely resolved by either the host immune system or
conventional antibiotic therapy. These characteristics are common to all biofilm-mediated
infections, no matter the location or the causative agent, and thought to result from the matrix-
enclosed and sequestered nature of the biofilm lifestyle. Antibodies and cells of the immune
system cannot access biofilm-embedded cells due to the extracellular matrix, often resulting in
immune complex damage to surrounding tissues (Stewart and Costerton, 2001). The increased
antibiotic tolerance of biofilms is not due to traditional mechanisms (efflux, modifying enzymes
or target mutations), but is instead due to the combined effects of diffusion limitation by the
extracellular matrix, the slow growth rate of matrix-enclosed cells and the formation of dormant
and highly resistant „persister‟ cells in the biofilm (for excellent reviews see (Fux et al., 2005;
Lewis, 2005; Stewart and Costerton, 2001)). As a result, antibiotic therapy often results in
short-term relief of symptoms through the elimination of planktonic cells released from the
biofilm, but fails to kill the surface-associated infectious centre. Biofilms are therefore thought to
7
act as reservoirs for recurrent infections that persist until the colonised surface is removed from
the body by physical means (Stewart and Costerton, 2001). Due to their prevalence and
persistence, understanding the processes leading to the formation and maturation of a biofilm is
critical to both our understanding of ecology and of pathogenesis.
Table 1.1: Common bacterial biofilm-mediated infections in humans (adapted from (Costerton
et al., 1999).
Site Infection or disease Bacterial species involved
Oral cavity Dental caries
Acid-producing Gram-positive cocci (e.g. Streptococcus)
Periodontitis Gram-negative anaerobes
Ear, nose, throat Otitis media
Non-typeable Haemophilus influenzae
Chronic tonsillitis Various species
Lung Cystic fibrosis pneumoniae Pseudomonas aeruginosa, Burkholderia cepacia
Heart Endocarditis Viridans group streptococci, staphylococci
Bone Musculosketetal infections Gram-positive cocci
Osteomyelitis Various species
Genitourinary tract Bacterial prostatitis
Escherichia coli and other Gram-negative rods
Infectious kidney stones Gram-negative rods
Medical device-related
Contact lens P. aeruginosa, Gram-positive cocci
Ventilation-associated pneumoniae
Gram-negative rods
Mechanical heart valves Staphylococci
Urinary catheter infections E. coli, Gram-negative rods
Orthopaedic prosthesis Staphylococci
8
1.2. Streptococcus mutans: a model for infectious biofilm formation
The genus Streptococcus comprises Gram positive, non-spore forming, non-motile cocci,
which characteristically grow as pairs or chains. Streptococci are carried in the nasopharynx of
man as members of the commensal microflora. However, streptococci may also cause disease
ranging in severity and prevalence, including pharyngitis, necrotizing faciitis, meningitis, and the
most common infectious diseases affecting humans: dental caries (Mitchell, 2003).
First isolated in 1924, Streptococcus mutans is the causative agent of one of the most benign
(although the most prevalent) streptococcal disease: human dental caries. In rare cases, S.
mutans has also been shown to cause infective endocarditis (Nomura et al., 2006). In both
cases, S. mutans colonizes the host as a biofilm. The transmission of S. mutans is thought to
occur from mother to child via salivary transfer, although there is typically a high degree of
homology between strains recovered from members of the same family, indicating both vertical
and horizontal routes of transmission (Napimoga et al., 2005). Studies of initial colonization by
S. mutans have shown that these bacteria require erupted tooth surfaces to become established
in the oral cavity, but are among the first colonizers of those surfaces. Interestingly, the same
initially colonizing strain may persist until young adulthood (Napimoga et al., 2005). S. mutans
has several virulence factors that contribute to its pathogenicity, foremost of which is its ability to
adhere to the tooth surface as part of the multi-species oral biofilm community.
9
1.2.1. S. mutans in the oral biofilm
The oral biofilm comprises more than 700 other species (approximately 20% of which are
streptococci) (Aas et al., 2005). It is now generally recognized that the etiology of dental caries
is influenced by the metabolism of the entire biofilm, and that disease results from the combined
interactions of multiple acid-producing organisms in the oral biofilm (Kuramitsu et al., 2007).
However, S. mutans is the only organism that has been conclusively linked to the causation of
dental caries (Aas et al., 2008; Loesche, 1986). What characteristics of S. mutans separate it
from the other residents of dental plaque as particularly cariogenic? S. mutans has the ability to
attach tenaciously to the tooth, to both produce and withstand highly acidic conditions, to out-
compete its neighbors through the production of antimicrobial peptides called bacteriocins, and
to finely tune all these virulence factors through the coordinated efforts of fourteen two-
component signal transduction systems that monitor the environment and population density.
The factors influencing the survival and virulence of S. mutans in the oral biofilm are discussed
in detail below, and summarized in Figure 1.1.
1.2.1.1. Attachment to the tooth: formation of the oral biofilm
The formation of the oral biofilm known as dental plaque is a well characterized process,
involving a defined sequence of adhesion and maturation events. In the oral cavity, the tooth
surface is bathed in saliva, which rapidly coats the tooth in salivary proteins, glycoproteins and
polysaccharides like mucins, salivary agglutinins, amylases and statherin (reviewed in (Rickard,
2008a)). These polymers, in addition to bacterially-derived proteins, form the so-called
„acquired„ or „salivary-pellicle‟, which is bound by „early colonizing‟ bacteria such as oxygen-
tolerant Gram-positive bacteria like streptococci (Kolenbrander et al., 2006). Over time, an
10
Figure 1.1: Summary of factors influencing the survival and virulence of S. mutans in the oral
biofilm. S. mutans is able to adhere robustly to the tooth surface via the production of glucans
and glucan binding proteins in the presence of sucrose. In addition to sucrose, the organism
can metabolize a wide variety of sugars, which result in the production of acid that desolves the
enamel of the tooth surface and causes dental caries. The ability of S. mutans to tolerate that
acidic environment allows it to persist in the oral biofilm. Additonal environmental conditions are
sensed using two-component signal transduction systems (TCSs). S. mutans is also able to
monitor its population density using the quorum sensing system composed of the CSP peptide
pheromone and the ComDE TCS. Phenotypes regulated by CSP-ComDE include the
production of bacteriocins (important to interspecies competition) and the development of
genetic competence, which allows S. mutans to take up DNA from the environment and further
adapt to its surroundings.
11
increasingly complex population of bacteria develops through binding of „secondary-‟ or „late-
colonizers‟ to the early colonizers. The attachment of each progressive cell type presents a new
surface for the subsequent attachment of different species of bacteria, resulting in a progression
of nascent surfaces and changes in species diversity that culminates in non-random bacterial
succession in the oral biofilm (Figure 1.2). The adhesive interactions between members of the
same or different species can occur through non-specific physico-chemical interactions, or
through specific adhesion-receptor interactions known as auto-aggregation or co-aggregation
(reviewed in (Rickard, 2008a)). Auto-aggregation refers to the propensity for cells of the same
species to aggregate, while co-aggregations are non-random interactions between genetically
distinct microorganisms (Kinder and Holt, 1994; Rickard, 2008a). Both types of aggregation are
mediated by specific adhesion/receptor interactions, and imply that the formation of dental
plaque is a co-ordinated developmental process involving complex interactions between
bacteria and host, and between interacting bacterial species (Kolenbrander et al., 2006;
Kuramitsu et al., 2007).
S. mutans is among the early colonizers, and uses two methods to attach to the tooth surface
depending on the availability of sucrose in the growth environment. In the absence of sucrose,
the bacterium expresses several main adhesins including streptococcal protein antigen P
(SpaP, also known as antigen I/II), which mediates binding to the salivary component salivary
agglutinin glycoprotein (SAG) (Jenkinson and Demuth, 1997). SpaP is a multi-functional
adhesin that can also mediate binding of the bacterium to collagen and other host proteins
(Demuth and Irvine, 2002). S. mutans may also adhere to other bacteria, the extracellular
matrix and epithelial cell surface receptors in the absence of sucrose using ionic and lectin-like
interactions (Mitchell, 2003). The adhesion of S. mutans to the tooth surface is greatly improved
when sucrose is present. In the presence of sucrose, cell-wall-associated enzymes called
12
Figure 1.2: Formation of dental plaque. Early colonizing streptococci (in blue) include
Streptococcus sanguinis (formerly S.sanguis), Streptococcus mitis, Streptococcus mutans,
Streptococcus oralis and Streptococcus gordonii. Other early colonizers shown are
Actinomyces naeslundii, Haemophilus parainfluenzae, Capnocytophaga ochracea, Eikenella
corrodens, Prevotella denticola, Prevotella loescheii, Propionibacterium acnes, and Veillonella
atypica. Fusobacterium nucleatum is considered the „bridge‟ between the early colonizers and
the late colonizers, which include Actinobacillus actinomycetemcomitans, Prevotella intermedia,
Treponema denticola and Porphyromonas gingivalis. Figure adapted from (Kolenbrander et al.,
2006).
13
glucosyltransferases (GTFs) mediate the synthesis of D-glucose polysaccharides known as
glucans, which are in turn bound by the surface associated glucan-binding proteins to promote
cell-cell aggregation. At least three glucan binding proteins (Gbp) have been identified in S.
mutans:GbpA, GbpB and GbpC (reviewed in (Banas and Vickerman, 2003)). Mutation of the
genes encoding either the glucosyltransferases or glucan-binding proteins can alter dental
plaque structure and cariogenesis (Munro et al., 1991; Yamashita et al., 1993). In addition to
the GTFs, S. mutans also produces a fructosyltransferase (FTF) enzyme which synthesizes
fructan polymers (Birkhed et al., 1979). However, the role of fructan in S. mutans is believed to
be in nutrient storage, not in adhesion (Burne et al., 1996). Other adhesins produced by S.
mutans are wall-associated protein A (WapA), SloC, and a protein with similarity to the
fibronectin-binding protein PavA from Streptococcus pneumoniae (reviewed in (Mitchell, 2003).
Once adhered to the tooth surface, S. mutans causes dental caries through its ability to produce
acid from the fermentation of host dietary carbohydrates, and withstand the acidic conditions
better than many other inhabitants of the oral biofilm.
1.2.1.2. Acidogenicity and aciduricity
While other bacterial diseases are caused by the production of virulence factors like secreted
proteases or toxins, the main pathology of dental caries is due to the broad metabolic repertoire
of S. mutans (Lemos and Burne, 2008). Genome sequencing indicates that S. mutans has
genes for the transport and metabolism of glucose, fructose, sucrose, lactose, galactose,
mannose, cellobiose, -glucosides, trehalose, maltose, raffinose, ribulose, mellobiose, starch,
isomaltosaccharides and possibly sorbose (Ajdic et al., 2002)- more metabolic diversity than
any other Gram-positive organism sequenced thus far (Mitchell, 2003). The metabolism of
14
sugars provides both the principal source of energy for S. mutans, and the acid that causes the
erosion of tooth enamel leading to dental caries. Sugar levels can increase up to 1,000-fold
following intake of heavily sweetened food, which results in a decrease in the pH of the oral
biofilm to values around pH 4 (Lemos et al., 2005). Sustained acidification of the biofilm can
lead to population shifts that favor acid tolerant species. The ability of S. mutans to tolerate the
acidic conditions caused by its own metabolism is central to its survival in the oral biofilm, and
means that stress tolerance by the bacterium is vital to its virulence (Lemos and Burne, 2008).
The production of acid via sugar fermentation lowers the pH of the oral biofilm such that
many species can no longer grow. However, S. mutans has developed a sophisticated series of
both constitutive and inducible mechanisms to cope with the acidification of the environment
resulting from its own metabolism. Central to the constitutive response is a membrane-bound,
acid-stable, proton-translocating F1F0 ATPase that operates to maintain a ΔpH of approximately
1.0 (Quivey et al., 2001). The inductive mechanisms S. mutans uses to adapt to low pH are
collectively referred to as the acid tolerance response (ATR). As part of its ATR, S. mutans
alters its catabolic pathways to attempt to alkalinize the cytoplasm, alters its cell envelope to
decrease proton permeability, and induces the expression of protein chaperones and DNA
repair pathways (for excellent reviews of these processes, see (Lemos et al., 2005; Lemos and
Burne, 2008)).
1.2.1.3 Interspecies competition in the oral biofilm: production of mutacins
The exceptional ability of S. mutans to withstand the acidic conditions in the oral biofilm
allows this microorganism to out-compete many of its co-inhabitants, and become the
15
numerically dominant species in carious lesions (Loesche, 1986). However, the intense
selective pressures found in the tightly packed oral biofilm community (where nutritional and
oxidative stresses compound with acid stress) may have necessitated the development of
additional competitive strategies. As is often the case, disease causing microorganisms like S.
mutans are also present under healthy conditions, and shifts in the population which favor the
outgrowth of the pathogenic species are part of what lead to disease. Although the
mechanisms leading to changes in the species composition of the dental biofilm are not well
understood, S. mutans gains a competitive advantage in the oral biofilm via the production of
small antimicrobial peptides called bacteriocins.
S. mutans is known to produce a repertoire of ribosomally synthesized antimicrobial peptides
belonging to the bacteriocin family (known as mutacins in the case of S. mutans), which are
secreted into the environment and are active against closely related competitors in the dental
biofilm. Mutacins are important in this dissertation, and are discussed here only briefly from an
ecological perspective in the context of interspecies competition. A more comprehensive
discussion of the structure, regulation and mechanism of action of mutacins in found in section
1.3.4.3).
Mutacins are assumed to confer an ecological advantage to the producing strain in vivo by
eliminating closely related organisms. Mutacin production is therefore advantageous in
situations of high cell density, were closely related organisms are in direct competition for limited
(and metabolically overlapping) nutrients. While Gronroos et al. found a link between mutacin
production and the ability of S. mutans to establish itself in the nacent biofilm following
transmission from mother to child (Gronroos et al., 1998), others have found no correlation
between mutacin expression and transmission (Alaluusua et al., 1991; Longo et al., 2003).
16
However, Kamiya and colleagues showed distinct mutacin production profiles between
individuals with active carious lesions and those without (Kamiya et al., 2005). The authors
postulated that mutacin production enabled S. mutans to prevail numerically in the biofilm of
caries-active individuals, resulting in more acid production and more tooth destruction. Although
mutacin production is potentially highly advantageous to S. mutans, the production of secreted
proteins is costly for the cell, particularly under conditions of nutrient stress. To ensure that
mutacins are produced only when competitors are present, their expression is linked to
situations of high population density via the quorum sensing system discussed in detail in
section 1.3.3. Quorum sensing in Gram-positive organisms occurs via specialized
sensor/receptor systems known as two-component signal transduction systems. Like the ATR
and the production of mutacins, the stress-responses mediated by two-component signal
transduction systems allow S. mutans to adapt to environmental shifts in the biofilm, and out-
compete its neighbors.
1.2.1.4. Two-component signal transduction systems
The S. mutans reference strain UA159 has only one alternative sigma factors in its genome
(Ajdic et al., 2002). As a result, this microorganism relies on regulatory systems to integrate
various chemical and physical signals to coordinate gene expression in response to stress.
Two Component Signal transduction systems (TCSs) are the systems used by bacteria,
archaea, protozoa, fungi and plants to sense and respond to environmental stimuli (Stock et al.,
2000). They are composed of a transmembrane sensor kinase that detects environmental
changes, and a cytosolic regulatory protein which binds to DNA when activated to modulate
gene expression. Activation of the TCS occurs when a stimulus triggers the
17
autophosphorylation of the sensor on a conserved histidine residue (giving this class of
receptors the designation histidine kinases, or HKs), which subsequently transfers the
phosphoryl group to a conserved aspartate residue on the cognate response regulator (RR) to
activate it (Stock et al., 2000). Analysis of the UA159 genome sequence revealed the presence
of 13 TCSs (Ajdic et al., 2002), while a fourteenth was subsequently identified experimentally
(Biswas et al., 2008).
Since subverting the ability of S. mutans to sense and respond to stress could potentially
attenuate its ability to cause caries, TCSs are viewed as desirable targets for new antimicrobial
therapies (Lemos and Burne, 2008). Several studies have therefore been conducted to
evaluate the role of each TCS in the stress response (Bhagwat et al., 2001; Kawada-Matsuo et
al., 2009; Levesque et al., 2007) (Summarized in Table 1.2). We preformed one such broad-
ranging study, and found that the TCS-2 (CiaRH) is involved in biofilm formation and tolerance
to environmental stresses, the TCS-3 (ScnRK-like) participates in the survival of cells at acidic
pH, and the TCS-9 affects the acid tolerance response and the process of streptococcal
competence development. Further work was also done to determine the role of the response
regulatory component of the TCS LiaFSR (formerly known as HK/RR11) in biofilm formation
(Chapter 4). Other TCSs have been linked to the oxidative stress response (VicRK,
(Senadheera et al., 2006)), to the stringent response (HK/RR4,(Lemos et al., 2007)), and in
tolerance to acid and DNA damage-induced stress (CiaHR, (Ahn et al., 2006; Biswas et al.,
2008; Qi et al., 2004)) (Table 1.2). The most well studied of the S. mutans TCSs is the ComDE
system, which senses the competence stimulating peptide (CSP) pheromone in a form of
intraspecies communication known as quorum sensing (Li et al., 2001b).
18
Table 1.2: S. mutans two-component signal transduction systems identified by bioinformatic
analysis of the UA159 genome sequence (modified from (Levesque et al., 2007)).
TCS ORF
(NCBI) Best match
a
(% aa identity) Known Phenotypes
TCS-1 (VicKR)
SMU.1516 VicK S. pyogenes (73%) Oxidative stress, biofilm
formation, competence SMU.1517 VicR S. pyogenes (84%)
TCS-2 (CiaHR)
SMU.1128 CiaH S. agalactiae (60%)
Mutacin production, genetic competence, biofilm formation, oxidative and osmotic stress tolerance, sensitivity to DNA
damage SMU.1129 CiaR S. pneumoniae (89%)
TCS-3 SMU.1145
ScnK S. pyogenes (26%) Acid stress
SMU.1146 ScnR S. pyogenes (41%)
TCS-4 SMU.928
Putative HK S. pyogenes (56%) (p)ppGpp regulation?
SMU.927 Putative RR S. agalactiae (55%)
TCS-5
SMU.1814 ScnK S. pyogenes (66%)
Oxidative stress tolerance, susceptibility to phagocyte-
mediated killing SMU.1815 ScnR S. pyogenes (83%)
TCS-6 SMU.660
SpaK S. thermophilus (44%) Uncharacterized
SMU.659 SpaR S. thermophilus (62%)
TCS-7 SMU.1037
Putative HK C. acetobutylicum (28%) Uncharacterized
SMU.1038 Putative RR L. casei (45%)
TCS-8 SMU.1009
Putative HK S. agalactiae (45%)
Uncharacterized SMU.1008 Putative RR S. suis (71%)
TCS-9
SMU.1965 Putative HK L. johnsonii (39%)
Acid stress, competence
SMU.1964 Putative RR C. acetobutylicum (67%)
19
TCS-10 SMU.577
LytS S. aureus (39%) Uncharacterized
SMU.576 Putative RR S. agalactiae (77%)
TCS-11
SMU.486 Putative HK S. pyogenes (61%) Cell envelope stress; oxidative
stress in the biofilm, genetic competence
SMU.487 YvqC S. pyogenes (85%)
TCS-12 SMU.1548 Putative HK S. agalactiae (46%)
Uncharacterized
SMU.1547 Putative RR S. thermophilus (78%)
TCS-13 (ComDE)
SMU.1916 ComD S. pneumoniae (36%) Genetic competence, mutacin production, autolysis, biofilm
formation, acid stress tolerance SMU.1917 ComE S. pyogenes (52%)
TCS-14
SMU.45 BaeS Microscilla marina
Uncharacterized
SMU.46 LuxR
1.3. Quorum sensing
Communication amongst cells in higher order organisms like eukaryotes permits the
development and differentiation of highly specialized cell types and complex networks of
interacting systems. It is now widely recognized that prokaryotes also use cell-cell signalling
systems to regulate a diverse set of behavioural and virulence traits between individuals in a
clonal population, resulting in coordinated population-level behaviour (Keller and Surette, 2006).
For true communication to occur, one (or several) individuals must produce a signal that can be
detected by other individuals, and that signal must alter the behaviour of the perceivers (Keller
20
and Surette, 2006). Most importantly, communication amongst microorganisms will only remain
stable across evolution if both parties benefit from the transfer of information conveyed by the
signal (Keller and Surette, 2006). Communication between different members of prokaryotic
multi-species biofilm community occurs either via physical cell-cell adhesion events mediated by
specific surface-attached protein adhesins and polysaccharide receptors on complementary cell
types, or by diffusible chemical signalling molecules produced by the cells expressly for the
purpose of communication (Rickard, 2008a). We refer to the regulation of bacterial gene
expression in response to cell population density as „quorum sensing‟. Quorum sensing occurs
when bacteria produce, sense, and respond to small extracellular molecules called
autoinducers, that are produced (usually) constitutively as the population grows. As a result, the
extracellular concentration of autoinducer mimics the population density of autoinducer-
producing bacteria. When a threshold autoinducer concentration is reached, it triggers a change
in gene expression of the population as a whole. As a result, bacteria can behave in a co-
ordinated manner akin to that observed with multi-cellular organisms.
1.3.1. A brief history of quorum sensing
The view that bacteria were more than independently acting single celled organisms was
first challenged in the 1960s and 1970s in work done by Tomasz and Hotchchkiss (Tomasz and
Hotchkiss, 1964) on the nasopharyngeal inhabitant S. pneumoniae, and by Nealson and
Hastings on the bioluminescent marine bacteria Vibrio fischeri and Vibrio harveyi ((Nealson et
al., 1970), and reviewed in (Bassler and Losick, 2006)). Tomasz and Hotchkiss reported that
the ability of cultures of S. pneumoniae to co-ordinate DNA uptake from the environment (a
process known as „genetic competence‟) was governed by an extracellular factor produced by
the bacteria themselves (Tomasz and Hotchkiss, 1964). Further work by Pakula and Walczak
21
identified this competence „activator‟ as a protein-like macromolecule (Pakula and Walczak,
1963), which we now know as competence stimulating peptide, or CSP (Håvarstein et al.,
1995). Nealson‟s work on the bioluminescence of marine vibrios is perhaps the better known
example of the discovery of co-ordinated behaviour in prokaryotes. Nealson and Hastings‟ work
described the ability of V. fischeri and V. harveyi to produce light at high cell density or in cell-
free „conditioned medium‟ from dense cultures. This work was the first to use the term
„autoinduction‟, and suggest that co-ordinated behaviour might be beneficial to a bacterial
population (Nealson et al., 1970). The „autoinducer‟ responsible for biolumniscence was later
identified as an acyl-homoserine lactone molecule (Eberhard et al., 1981). These discoveries
were thought to be isolated incidences of species-specific behaviours, and were largely ignored
for the following 20 years. However, a virtual explosion has occurred in the field of bacterial
cell-cell communication since the early 1990s, during which over two thousand articles have
been published documenting the wide-spread nature of this phenomenon. Bacterial density
dependent cell-cell communication has now been linked to the control of processes including
virulence factor secretion, antibiotic production, sporulation, and biofilm formation (reviewed in
(Waters and Bassler, 2005)) in addition to genetic competence and bioluminescence, and exists
in both Gram-negative and Gram-positive organisms. Intriguingly, recent evidence indicates
bacteria may also be able to communicate reciprocally with their hosts through hormone and
hormone-like signals (reviewed in (Hughes and Sperandio, 2008)). Although beyond the scope
of this review, the possibility of inter-kingdom signalling adds a new level of complexity to the
story of host/microbe co-evolution.
22
1.3.2. Quorum sensing mechanisms
The mechanics of chemical quorum sensing can be divided into three categories
(summarized in Figure 1.3), which range from broadly-ranging to highly-specific. The first
category (and least specific) is the interspecies quorum sensing system typically referred to as
LuxS/AI-2. The gene required for AI-2 production encodes the S-ribosylhomocysteinase LuxS
(Surette et al., 1999), which functions in the S-adenosylmethionine (SAM) utilization pathway.
SAM is an essential methyl donor in DNA, RNA and other methylation reactions. Its use as a
methyl donor yields the toxic intermediate S-adenosylhomocysteine (SAH), which is detoxified
through several intermediates before eventual cleavage by LuxS into homocystine and 4,5-
dihydroxy-2,3-pentanedione (DPD) (reviewed in (Vendeville et al., 2005)). DPD spontaneously
forms the cyclic pro-AI-2 molecule, which reacts with borate to form a stable cyclic furanosyl
borate diester in equilibrium with several compounds (Thiel et al., 2009), some (or perhaps all)
or which encode information. The biosynthetic pathways, chemical intermediates, and possibly
even the autoinducer signal AI-2 itself are identical in all AI-2 producing bacteria (Xavier and
Bassler, 2003). These similarities mean that the AI-2 signal may be recognized by any AI-2
producing bacteria, and may function in interspecies communication. However, few instances
of true AI-2 mediated communication have been documented in naturally occurring bacterial
pairings (with the exception of the oral biofilm (Rickard et al., 2006; Rickard et al., 2008b)),
leaving some debate as to whether the conservation of the LuxS enzyme across the bacterial
kingdom is due to its role in the activated methyl cycle and not in inter-species signalling.
The second category of quorum sensing is found in Gram-negative bacteria, which use N-
acylated homoserine lactone (AHL) molecules for signalling. Most AHLs in Gram-negative
bacteria are all synthesized via similar pathways, which are homologuous to the V. fisheri
23
pathway composed of the AHL synthase LuxI and the sensor/response regulator LuxR (Miller
and Bassler, 2001). The binding of an AHL autoinducer by LuxR results in transcriptional
activation of target genes. AHL molecules are species-specific due to variations in acyl-chain
length and substitutions (typically oxo- or hydroxy- groups), although the LuxR sensor may
show some cross-reactivity with AHLs from other species (Keller and Surette, 2006). While the
prototypical interspecies quorum sensing system (AI-2) was initially discovered in V. harveyi
(Surette et al., 1999), this organism also possesses an intraspecies quorum sensing system
which operates via a slightly different mechanism from other Gram-negative organisms. Two
variants of the species-specific AHL signal are produced, called HA-1 and CAI-1. HAI-1 is
synthesized by LuxLM, and sensed by LuxN, while CAI-1 is synthesized by CqsA and sensed
by CqsS. The intraspecies pathway converges with the universal AI-2 pathway at the shared
LuxU phosphorelay protein, and LuxO activates transcription of target genes (Keller and
Surette, 2006) (Figure 1.3).
The most species-specific signals are represented by the peptide quorum sensing systems of
Gram-positive bacteria (Sturme et al., 2002). Typically, the peptide signal is synthesized as a
propeptide, and cleaved upon export by a dedicated ABC-type transporter to generate the
mature signal. This signal is sensed by a cognate receptor protein of a dedicated two-
component signal transduction system. These Gram-positive signals are so specific that
signalling can sometimes discriminate between different strains of the same species (Keller and
Surette, 2006). The best characterized streptococcal quorum sensing system belongs to the
human pathogen S. pneumoniae.
24
Figure 1.3: Simplified schematic representation of quorum sensing systems in bacteria. A)
LuxI/R quorum sensing in Gram-negative bacteria. Acyl-homoserine lactone molecules are
synthesized by a homologue of the LuxI autoinducer synthase, and freely diffuse out of the cell.
At a specific concentration, AHL binds a homologue of the LuxR response regulator which then
activates transcription of target genes. (B) Peptide quorum sensing system of Gram-positive
bacteria. The quorum sensing signal is synthesized as a propeptide, which is processed upon
export by an ABC transporter to generate the mature signal. The peptide binds to its cognate
receptor protein, which autophosphorylates on a conserved histidine residue. The phosphoryl
group is subsequently transferred to a response regulator protein, which regulates transcription
of target genes. (C, D) Quorum sensing in V. harveyi. Two species-specific systems operate in
Vibrio species: HAI-1 is synthesized by LuxLM, and sensed by LuxN, while CAI-1 is synthesized
by CqsA and sensed by CqsS. The universal quorum sensing system operates via the AI-2
furanone signal (synthesized via LuxS), which is detected at the cell surface by the LuxP
periplasmic binding protein and the receptor LuxQ. Both pathways converge at the shared
LuxU phosphorelay protein, and LuxO activates transcription of target genes.
25
1.3.3. Quorum Sensing in Streptococci
1.3.3.1. Overview of the CSP-ComDE system in S. pneumoniae
The pioneering work of Pakula and Walczak (described above) identified a secreted signal in
S. pneumoniae which induced genetic competence (Pakula and Walczak, 1963). Subsequent
studies by the Morrison and Håvarstein labs (Håvarstein et al., 1995; Håvarstein et al., 1996;
Pestova et al., 1996) identified this signal as an unmodified peptide of 17 amino acids (ComC,
also known as CSP), which is translated as a 41-amino acid precursor peptide and cleaved
upon export by the dedicated ABC-transporter ComAB. Activation of CSP was found to occur
concomitant to cleavage of the propeptide at a Gly-Gly residue in a cleavage reaction typical of
peptide pheromones and bacteriocins (reviewed in (Senadheera, 2005)). The mature form of
CSP is sensed by the histidine kinase receptor protein ComD, which consists of an N-terminal
membrane-spanning domain and a C-terminal kinase domain (Håvarstein et al., 1996; Stock et
al., 2000). In general, streptococcal species respond only to their own peptide signal and not to
heterologous signals. However, S. pneumoniae is known to harbour 6 distinct CSP variants,
although the majority of strains produce one of two variants: CSP-1 or CSP-2 (Pozzi et al.,
1996; Whatmore et al., 1999). This auto-dependency functions to induce competence in the
presence of genetically identical or highly similar bacteria (and bacterial DNA) are present,
optimizing the chance of donor DNA being similar enough for homologous recombination
(Winans and Zhu, 2000).
Upon CSP binding, ComD autophosphorylates and transfers the phosphoryl group to the
cognate response regulator ComE. Like other response regulators, ComE is composed of an
N-terminal regulatory domain that interacts with the ComD receptor, and a C-terminal DNA-
26
binding domain (Stock et al., 2000). The phosphorylation of ComE enhances its binding to
promoters containing two 9 bp imperfect direct repeats separated by 12 bp (aCAtTTct(a/g)G ---
12 bp--- ACA(t/g)TtgAG) (Ween et al., 1999). Genes that are transcribed due to direct
ComE/promoter binding are known as „early genes‟, and include the genes encoding the CSP
signal and two-component signal transduction system themselves (transcribed as a single
transcript comCDE) and the ABC transporter responsible for CSP maturation and export
(comAB). This auto-catalytic expression of the CSP-ComDE circuit leads to a high degree of
synchrony in pneumococcal cultures, resulting in induction of competence in almost the entire
population simultaneously. However, expression of the early genes is brief (peaking at 5
minutes after CSP exposure and declining to baseline levels by 20 min post-exposure), and is
followed by a long period in which previously responsive cells become unresponsive to CSP
(Alloing et al., 1998). In total, 23 „early genes‟ have been identified, encoding transporters,
bacteriocin-related genes and regulators (Peterson et al., 2004).
Two early genes transcribed via direct ComE binding are absolutely required for competence
induction: comX (present in duplicate in the genome, and encoding the alternate sigma factor
ComX) (Lee and Morrison, 1999; Luo and Morrison, 2003) and comW, encoding a protein
whose function is less well characterized (Luo et al., 2004). ComX (also known as Sigma X or
σx) shows high homology to the σ70 family of RNA polymerase sigma subunits, which in complex
with RNA polymerase binds to the so-called „cin-box‟ (TACGAATA) upstream of target genes
(Luo and Morrison, 2003). ComX is essential for the transcription of the CSP-induced „late
genes‟, which are expressed maximally after 10-12 minutes (Lee and Morrison, 1999)
(Summarized in Figure 1.4). Eighty-one genes were found to belong to the late class, including
all genes previously identified as necessary for the uptake and processing of DNA for
transformation (Peterson et al., 2004). While ComX controls the competence transcriptome, S.
27
pneumoniae also controls its competence cascade post-transcriptionally via ComW. ComW
both activates ComX by an unknown mechanism, and acts to prevent its proteolysis by the ClpE
and ClpP proteases (Sung and Morrison, 2005). It was once thought that the proteolytic
degradation of ComX lead to the eventual shut-off of competence (Lee and Morrison, 1999).
However, a rapid decrease of late competence gene transcripts has been observed even when
ComX is still present (Luo and Morrison, 2003). While the ClpE and ClpP proteases appear to
be responsible for the degradation of ComX after cells escape from the competent state, it has
recently been shown that termination of ComX activity is not due to proteolysis of the sigma
factor or of ComW (Piotrowski et al., 2009). Instead, it has been proposed that competence
shut-off could occur due to the presence of a ComE-specific phosphatase (Alloing et al., 1998),
through the modification/sequestration of ComX and/or ComW, through inhibition of translation
of late gene products, or through the accumulation of an inhibitor (Piotrowski et al., 2009).
However, evidence for a ComE-specific phosphatase or inhibitor has remained elusive. Given
the importance of the competence cascade to the physiology of S. pneumoniae, elucidating the
mechanism of its shut-off remains an important goal.
In addition to the well characterized „early‟ and „late‟ genes, a third group of 19 „delayed
genes‟ was identified in the CSP regulon. Expression of this cluster of genes continued to
accumulate after the peak in expression of both the early and late classes, and showed varying
dependence on comX for expression. Genes in this class include stress-reponse genes and
chaperones (Peterson et al., 2004). Interestingly, the competence cascade has been
subsequently linked to the stress reponse in S. pneumoniae (Prudhomme et al., 2006),
discussed in detail in section 1.3.4.4.
28
1.3.3.2 CSP-ComDE in S. mutans
Pioneering work done in S. pneumoniae has led to the discovery of similar quorum-sensing
regulated competence systems in other streptococci, including those in the oral cavity (for
reviews, see (Martin et al., 2006; Senadheera, 2005)). In the pre-genome sequence era, many
of these competence systems were identified by PCR using primers complementary to the Arg-
and Glu-tRNA genes that flank the comCDE operon in S. pneumoniae (Håvarstein et al., 1997).
However, the genes encoding the competence system in S. mutans are not flanked by tRNA
genes. Instead, the signal peptide is transcribed divergently from the sensor/receptor gene pair
(Figure 1.4; (Li et al., 2001b; Li et al., 2002a)) like the pneumococcal CSP-ComDE paralog BIP-
BlpHR (de Saizieu et al., 2000). In S. mutans, the CSP precursor peptide is 46 amino acids
long, and is thought to be cleaved upon export to generate the mature 21 amino acid-long signal
peptide. Structure analysis of the mature S. mutans CSP indicated that the C-terminal portion
of the peptide is essential for receptor activation (Syvitski et al., 2007). In contrast to the
multiple pneumococcal CSP pherotypes, S. mutans produces only one CSP pherotype despite
the presence of minor genetic variation in sequence among strains (Allan et al., 2007). When
the peptide reaches a critical concentration (at early- to mid-exponential phase in S. mutans), it
activates the ComDE TCS leading to competence induction through a homologue of the
alternate sigma factor ComX. In contrast to the pneumococcal model, only a small percentage
(≤ 1%) of the S. mutans population ever becomes competent in the presence of CSP when
grown planktonically. In addition, a residual level of transformation is present in S. mutans
mutants lacking the com genes (reviewed in (Senadheera, 2005)). Moreover, while competence
develops between 5-7 minutes following exogenous CSP addition in S. pneumoniae, a ~2 hr
delay in competence is common following exogenous CSP addition in S. mutans. The highest
levels of transformation in S. mutans are observed during biofilm growth, during which
transformation efficiencies can approach levels 10- 600-fold higher than their planktonic
29
counterparts (Li et al., 2001b). While this increase in transformation efficiency may simply
represent more efficient CSP signalling between cells in close spatial proximity, the known
phenotypic differences between biofilm and planktonic cells means that additional biofilm-
specific factors cannot be ruled out. Together, these results indicate that multiple inputs and/or
shut-off mechanisms many exist for the S. mutans competence cascade, and that significant
differences exist between the prototypical competence cascade of S. pneumoniae and that of S.
mutans (summarized in Table 1.3). However, most of the phenotypes regulated by this system
in pneumococcus appear to be also regulated by its homologue in S. mutans.
Table 1.3: Comparison of the CSP-ComDE systems in S. pneumoniae and S. mutans.
S. pneumoniae S. mutans
Peptide Pheromone Induction Signal CSP, 17 aa CSP, 21 aa
CSP Pherotypes? 6 known, 2 main 1
Other Induction Stress (antibiotics,
DNA damage) ??
Receptor/Effectors ComDE, ComX ComDE, ComX
Accessory proteins ComW ??
Paralogous system? BIP-BlpHR No
Timing of Induction after CSP addition 5-7 minutes ~2 hours
Percent of culture induced Approaching 100% ≤1%
Phenotypes controlled
Competence,fratricide, biofilm formation, stress
response
Competence, bacteriocin production, biofilm formation, autolysis, stress response.
30
Figure 1.4: Mechanistic and genomic representation of the CSP-ComDE circuit controlling
competence development in S. pneumoniae and S. mutans. Entries in parentheses are unique
to S. pneumoniae.For simplicity‟s sake, only genes involved in competence development are
shown. The role of the pneumococcal CSP-ComDE circuit in stress tolerance and fratricide is
discussed in section 1.3.4, and a more detailed representation of the fratricidal pathway is
presented in Figure 1.5.
31
1.3.4 Phenotypes controlled by CSP-ComDE signalling in streptococci
1.3.4.1 Genetic competence
Genetic competence is a transient physiological state during which bacteria take up DNA
from the environment and integrate it into their genomes (Cvitkovitch, 2001). For successful
gene transfer to occur, recipient cells must be in a metabolically active, competent state, and
DNA must be present in the environment. For recombination to occur, the foreign DNA
sequence must share between 70-100% identity with the sequence in the recipient strain‟s
genome (Dowson et al., 1997). Considerable energy is used during the transition to
competence, which also requires the complex genetic machinery described in the preceeding
section. Competence therefore plays an important role in the physiology of the organism to
remain evolutionarily conserved in such varying species. A brief overview of the processes
involved are discussed below.
1.3.4.2.1 Mechanism of DNA uptake in streptococci
In Gram-positive bacteria, DNA must pass through the cell wall and the cytoplasmic
membrane before integration into the genome for transformation. In streptococci, this process
is initiated when exogenous double-stranded DNA binds to the surface of a competent cell,
which has been induced to express DNA uptake and processing machinery by CSP. This
binding is sequence-independent in streptococci, meaning that both autologous and foreign
DNA can be incorporated (Berge et al., 2002). The first hurdle in DNA uptake is traversing the
cell wall. To take up DNA, the cell expresses a pseudopilus composed of the ComGC (major
pilin-like protein), ComGD, ComGE and ComGG (minor pilin-like) proteins (Dubnau, 1999). The
32
retraction of the pseudopilus (by disassembly of the structure) allows the DNA to traverse the
cell wall peptidoglycan (Craig and Li, 2008). Once across the cell wall, double stranded DNA
binds to the C-terminal domain of the membrane-anchored DNA binding protein ComEA. Single
stranded breaks are then introduced into the bound DNA molecule by an unknown
endonuclease, and one strand of the double-stranded DNA molecule is degraded by the
endonuclease EndA concomitant with its import in a 3‟ to 5‟direction (Mejean and Claverys,
1993). This reaction has been shown to be energy dependent, and to occur through an
aqueous channel in the membrane composed of ComEC, ComFA and possibly EndA itself
(Dubnau, 1999). Following uptake, DNA recovered from transformed pneumococci is no longer
able to transform other cultures of competent cells. This transient loss of transforming activity is
called eclipse (Ephrussi-Taylor, 1960), and reflects the fact that ssDNA has less transforming
activity than dsDNA (Miao and Guild, 1970). Investigation of eclipse DNA revealed that ssDNA
isolated after transformation was bound to protein, forming the „eclipse complex‟ (Morrison,
1977, 1978). This protein component has been subsequently identified as SsbB (Morrison et
al., 2007), and renders ssDNA in eclipse complex 50-1000-fold more resistant to exogenous
nuclease activity (Morrison and Mannarelli, 1979). Incoming ssDNA is also protected via the
recombination mediator protein DprA and the recombinase RecA (Berge et al., 2003). DprA is
involved in recruiting RecA to the ssDNA, which can be then wholly or partially integrated into
the recipient‟s genome (depending on homology) via RecA-mediated recombination (Mortier-
Barriere et al., 2007). Although beyond the scope of this dissertation, a detailed description of
the steps involved in DNA uptake, processing and recombination can be found in (Claverys et
al., 2009).
33
1.3.4.2.2 Purpose of DNA uptake
There are now over 70 known naturally transformable bacterial species in nature (Johnsborg
et al., 2007). Competence requires the expression of more than a dozen specialized proteins,
which are all highly controlled in terms of expression. The widespread and complicated nature
of the competence phenotype begs the question “What use is competence?” to a bacterial cell?
Three hypotheses are generally put forward to explain the evolutionary conservation of the
competence phenotype: DNA for food, DNA for repair, and DNA for genetic diversity.
The fact that competence induction is generally induced by „quorum sensing‟-type systems
implies that it is a phenotype associated with high cell density. Since nutritional starvation is a
feature of high cell density, it has been proposed that DNA uptake may serve as a source of
nucleotides for a starving cell. In fact, DNA may serve as the sole carbon and energy source for
Escherichia coli at stationary phase (Finkel and Kolter, 2001). While the DNA-for-food
hypothesis is certainly valid, several points argue against it as the sole role for the DNA uptake
machinery. Firstly, although the CSP-ComDE circuit has been shown to be necessary for
transformation in S. mutans, competence naturally develops in liquid cultures at very low cell
density (OD600 ~0.1) where nutrients are abundant and nucleotide scavenging is not necessary.
More generally, the degradation of one strand of the incoming DNA molecule suggests that the
uptake of nucleotides for food is not the main goal of transformation- why throw away half your
food if you are hungry? Moreover, B. subtilis is known to secrete a powerful nonspecific
nuclease, and express uptake systems for the nucleolytic products. This would seem to be a far
simpler mechanism for scavenging nucleotides from the environment (Dubnau, 1999). Finally,
the naturally competent organisms Haemophilus influenzae and Neisseria gonorrhoeae exhibit
sequence specificity in their DNA uptake systems to restrict incoming DNA to their own species.
34
This presumably acts to increase the chances of homologous recombination, and strongly
suggests the DNA for repair or transformation hypotheses.
Evidence supports both the DNA-for-repair and the DNA-for-genetic diversity hypotheses,
and may indicate that the two are not mutually exclusive. It is now known that DNA damaging
agents can induce the competence regulon in S. pneumoniae (Prudhomme et al., 2006), and it
is widely recognized that DNA repair machinery is induced during competence in many Gram-
positive organisms (reviewed in (Dubnau, 1999)). However, supporting the DNA-for-repair
hypothesis, transformation of UV-irradiated H. influenzae with a cloned fragment had the same
effect on survival as transformation with total chromosomal DNA (while foreign DNA had no
effect), indicating that recombination is important, but not necessary at the site of repair
(Mongold, 1992). An alternative explanation to the induction of competence in stress is therefore
that cells are scavenging the environment for fitness-enhancing genes. It is widely recognized
that non-competent bacteria become hypermutable under stressful conditions, which has been
argued to be an attempt to generate genetic diversity under stress (Bjedov et al., 2003). While
this strategy is efficient enough to be evolutionarily conserved, it would be an obvious
advantage for competent bacteria to also co-ordinate the uptake of intact and functional DNA
from the environment during stress. It has been proposed that S. pneumoniae induces its
competence regulon as part of its general stress response for just such a purpose (Claverys et
al., 2006). Since instances of adaptation mediated by horizontal gene transfer are known to
exist in nature, this hypothesis has gained acceptance in the literature, and is widely recognized
as one of the principle functions for competence.
35
If one accepts the DNA-for-genetic diversity hypothesis, however, the obvious next question
becomes “What is the source of transforming DNA?” Interestingly, recent studies have shown
that the majority of CSP-responsive genes in S. pneumoniae (~70 of 105-124 genes) are
dispensable for transformation (Dagkessamanskaia et al., 2004; Peterson et al., 2000; Peterson
et al., 2004). What is the function of these other CSP-induced genes? The answer may lie in
the newly discovered link between the competence cascade and autolysis.
1.3.4.2 Autolysis and pneumococcal fratricide
Nowhere is the concept of quorum sensing-mediated multi-cellular behaviour more apparent
than in the field of bacterial cell death and lysis. First observed in S. pneumoniae in the early
1900s, the seemingly counterproductive habit of some bacterial cells to commit „cellular suicide‟
and self-lyse has received considerable attention in the past decade. In the context of multi-
cellular biofilm growth, programmed cell death-like processes may benefit the community by
sacrificing individual cells damaged by toxic factors, viral infection or during nutritional
starvation. The lysis phenomenon has been documented in several developmental processes,
and is often linked to quorum sensing, high population density, or environmental stress. A
distinction is made between the phenomenon of autolysis, observed during biofilm formation in
Staphylococcus aureus and preceding sporulation in Bacillus subtilis and the fratricidal pathway
of S. pneumoniae, which involves the killing of non-competent sibling cells by CSP-induced but
(otherwise clonal) competent cells.
36
1.3.4.2.1 Autolysins and autolysis
Autolysis is the self-digestion of the cell wall by bacterial peptidoglycan (or murein)
hydrolases called autolysins (Shockman et al., 1996). It is thought that these cell wall
degrading enzymes are involved in normal cell wall turnover, and have been shown to be
required for daughter cell separation after completion of the newly formed septum during cell
division (Rice and Bayles, 2008). Additional roles for autolysins have been shown in antibiotic
resistance (Groicher et al., 2000), cell-to-surface adhesion (Heilmann et al., 1997; Heilmann et
al., 2005), genetic competence (Moscoso and Claverys, 2004), protein secretion and
pathogenicity (Ahn and Burne, 2007). The activity of murein hydrolases has been studied
extensively in Gram-negative bacteria, and regulatory mechanisms such as sequestration within
lipid membranes, controlled transport across the cytoplasmic membrane, and topographical
control within the peptidoglycan have been reported (reviewed in (Holtje and Tuomanen, 1991)).
In Gram-positive organisms, the control of autolysin activity may also be regulated by the family
of carbohydrates referred to as teichoic acids. Two basic forms of teichoic acids are present in
the cell wall of Gram-positive bacteria: wall teichoic acids, which are covalently attached to the
peptidoglycan, and lipoteichoic acids, which are anchored in the cytoplasmic membrane. These
sugar molecules combine with the peptidoglycan to form a polyanionic gel surrounding the cell,
which acts as a semi-permeable buffer zone between the external environment and the
cytoplasmic membrane (Rice and Bayles, 2008). Since they are negatively charged, teichoic
acids are susceptible to cationic peptide antibiotics and bacteriocins. However their primary
function may be to control the activity of autolysins during normal cell growth.
The autolysins produced by many bacteria contain elements that target them to the cell wall.
In the case of S. pneumoniae, the primary autolysin LytA contains repeated choline-binding
37
domains which associate with choline-substituted teichoic acids in the cell wall. The autolysin is
activated in vitro by this association in a process known as „conversion‟ (reviewed in (Rice and
Bayles, 2008)). Teichoic acid modification has also been implicated in regulation of murein
hydrolase activity. D-alanylation of teichoic acids plays a vital role in modulating surface charge,
which seems to have a negative impact on autolysin activity. The extent of cell wall modification
with D-ala is thought to be dependent on the charged state of the membrane, or its proton
motive force (PMF), since protonation is thought to stabilize D-alanyl ester linkages within the
teichoic acid. Therefore, in a metabolically active cell, the pH gradient that is established
moving away from the respiring membrane results in less D-ala modification farther away from
the cell membrane than close to it. As a result, cell-wall associated autolysins positioned farther
away from the membrane are most active, accounting for the increased peptidoglycan turnover
in the outer layers of the cell wall (Figure 1.5). This model also accounts for data showing that
autolysis is inhibited by growth at low pH (where D-ala substitution of the teichoic acids would
increase), and that PMF was critical in controlling this process. The processes described up to
this point are part of the normal control of cell wall re-modelling during growth. However,
induction of autolysin activity has also been described during nutrient starvation and biofilm
growth in the major Gram-positive human pathogen Staphylococcus aureus and in response to
CSP accumulation in S. pneumoniae.
1.3.4.2.2 The holin/antiholin system in S. aureus
The role of PMF in control of autolysis is best illustrated in the autolysis cascade of S.
aureus. Autolysis in this organism is mediated by the balance of gene expression between two
regulatory systems: 1) LytSR, which controls the expression of a bicistronic operon designated
lrgA and lrgB and 2) CidR, controlling expression of a second bicistronic operon designated
38
cidA and cidB. The gene products of lrgAB and cidAB are predicted to be extremely
hydrophobic, and are likely membrane proteins. Interestingly, all four proteins showed high
homology with bacteriophage membrane permeabilizing proteins called holins. Holins function
by inserting into the cell membrane causing pores, leading to the gradual dissipation of the
proton gradient. They are also thought to control phage-encoded murein hydrolases by either
controlling their transport or mediating their activation (Bayles, 2007). In S. aureus, CidA is
predicted to function as the „holin‟ component, causing PMF dispersal and activation of
bacterially encoded murein hydrolases in the cell wall. The presence of LrgA is thought to
inhibit the action of CidA, earning it the designation „anti-holin‟ (Figure 1.5). The CidAB/LrgAB
cell death pathway has been implicated in biofilm development in S. aureus, due to the release
of genomic DNA for stabilization of the extracellular matrix. While cell death mediated by the
holin/antiholin system is induced by changes in carbohydrate metabolism, not by a CSP-like
peptide, the functional similarity between the membrane-bound pore-forming holin-like peptides
and the pore-forming membrane bound bacteriocins induced by CSP and involved in fratricide
deserves mention.
1.3.4.2.3 Streptococcal fratricide
While all cells in a population of S. aureus are potentially susceptible to autolysis by the
CidAB holin system, the process of streptococcal fratricide is characterized by lysis of the non-
competent fraction of the population by competent sister cells. One constitutively expressed
autolysin, encoded by lytC, and the products of six CSP-induced genes, cbpD, cibA, cibB, cibC,
comM and lytA have been implicated in CSP-induced cell death in S. pneumoniae (reviewed in
(Claverys et al., 2007)) (Figure 1.6). In liquid culture, the key component of the lytic pathway is
the late competence gene cbpD. CbpD is a putative amidase/peptidase consisting of a cysteine
39
Figure 1.5: Representation of the holin/anti-holin system in S. aureus. In this model, the proton
gradient that naturally exists in a polarized, respiring cell causes the D-ala-D-ala linkages on
teichoic acids and lipoteichoic acids close to the cell surface to become protonated (red
crosses). This local area of protonation keeps murein hydrolases present in the cell wall in an
inactive form. Further away from the cell membrane, the localized area of acidity is reduced,
and the inhibition of murein hydrolases is lifted, allowing for peptidoglycan degradation in the
outermost leaflet of the cell wall. The holin/anti-holin system composed of CidAB and LrgAB
(respectively) maintains the polarized state of the membrane when both components are
present. If lysis is triggered, the LrgAB antiholin component is degraded, and the holin
component causes membrane depolarization, de-protonation of the teichoic acids, and
subsequent activation of the murein hydrolases in the cell wall (Figure adapted from (Rice and
Bayles, 2008)).
40
protease (CHAP) domain, two 3(SH3b) domains with Src homology, and four choline-binding
domains (Guiral et al., 2005). CHAP domains are present in bacterial and phage autolysins,
and are known to act as murein hydrolases. SH3 domains in eukaryotes function in protein-
protein interactions involving proline-rich motifs, but in bacteria may mediate protein binding to
the cell wall (Guiral et al., 2005). The choline binding domains likely fulfill a similar function by
mediating binding of CbpD to the teichoic acid residues to position the CHAP domain relative to
its substrate. It has been suggested that CbpD is tethered to the surface of competent cells,
where it cleaves the peptide bonds within murein stem peptides of target cells via cell-to-cell
contact. This cleavage is thought to trigger the activation of the autolysins LytA and LytC,
resulting in cell lysis (Guiral et al., 2005). While CbpD is expressed only in competent predatory
cells, recent work has shown that LytA and LytC are more effective when expressed by the
target cells themselves (Eldholm et al., 2009).
While CbpD is absolutely required for cell lysis in liquid culture, the products of cibA and cibB
form a putative two- peptide bacteriocin that is necessary for allolysis in S. pneumoniae grown
on solid surfaces (Guiral et al., 2005; Kausmally et al., 2005). Although it has been proposed
that CibAB are also tethered to the cell surface of competent cells and function via cell contact-
dependent activation of LytA and LytC, the mechanism of action is not known (Claverys et al.,
2007; Guiral et al., 2005). Competent cells protect themselves from lysis through the action of
ComM and CibC, which are protective against the autolysins (CbpD, LytA and LytC) and the
bacteriocin (CibAB), respectively (Guiral et al., 2005; Håvarstein et al., 2006)(Figure 1.6).
Pneumococcal fratricide is thought to occur to provide DNA for uptake during natural
transformation, and has been suggested to represent a means for S. pneumoniae to increase its
genetic repertoire through DNA exchange under stress. CSP is also known to induce cell death
41
at high concentrations in S. mutans(Qi et al., 2005). Homologues of several autolysin- and
fractridical effector-encoding genes exist in S. mutans (Table 1.4), although their involvement in
CSP-induced cell death in this organism is not known.
Figure 1.6: Proposed mechanism of action of the CSP-induced fratricidal pathway in S.
pneumoniae. A pre-competent cell may differentiate into either a competent lineage if it
responds to CSP, or remain non-competent if it expresses and responds to a different
pherotype than what is present in the environment. CSP-responsive cells express the CbpD
amidase or the CibAB bacteriocins on their cell surface (in liquid culture or on solid surfaces,
respectively), and are protected via the ComM or CibC immunity proteins. Non-competent
sibling cells are then killed via cell-to-cell contact, which requires the presence of the LytA and
LytC autolysins. Figure adapted from (Rice and Bayles, 2008)
42
Table 1.4: Autolysin and fratricidal effector genes and their homologs in S. mutans UA159
Genea
(GenBank accession)
Description Homologue in S. mutans UA159 (% aa identity)b
S.
au
reu
s
lytR (L42945) Two-component response regulator
LytR, controls lrgAB SMU.576 (38%; 96/249)
lytS (L42945) Two-component sensor histidine
kinase LytS, controls lrgAB SMU.577 (42%; 245/582)
lrgA (U52961) Antiholin-like protein LrgA SMU.575 (38%; 46/118)
lrgB (U52961) LytSR-regulated gene SMU.574 (48%; 105/215)
cidA (AY581892) Holin-like protein CidA SMU.1701 (40%; 35/87)
cidB (AY581892) Hydrophobic protein CidB SMU.1700 (30%; 63/207)
cidC (AY581892) Pyruvate oxidase SMU.231 (29%; 165/560)
cidR (AY581892) LysR-type transcriptional regulator
(LTTR) SMU.2060 (22%; 61/273)
S.
pn
eu
mo
nia
e
lytA (SPD1737) autolysin/N-acetylmuramoyl-L-
alanine amidase SMU.704 (25%; 32/128)
cbpD (SPD2028) choline binding protein D SMU.609 (32%; 47/144)
lytC (SPD1403) 1,4-beta-N-acetylmuramidase,
putative SMU.689 (24%; 47/192)
cibA (SPG0129) competence induced bacteriocin A SMU.150 (35%; 20/57)
cibB (SPG0128) competence induced bacteriocin A no significant homologies
aNCBI annotation.
bGenomic BLAST Search (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) was used
for sequence comparison.
43
1.3.4.3 Bacteriocin production
In addition to competence and fratricide, a third CSP-regulated phenotype in streptococci is
the production of bacteriocins. Bacteriocins are small ribosomally synthesized peptides or
proteins with antibacterial activity. As mentioned above in section 1.2.1.3, bacteriocins are
assumed to confer an ecological advantage to the producing strain in vivo by eliminating closely
related organisms. Mutacin production is therefore advantageous in situations of high cell
density, were closely related organisms are in direct competition for limited (and metabolically
overlapping) nutrients.The field of bacteriocin research has been directed mainly towards those
produced by lactic acid bacteria in the food industry, as a means of preventing food spoilage. In
the age of increasing antibiotic resistance, bacteriocins have been receiving increasing attention
as an alternative means of preventing infection by pathogens (reviewed in (Nes et al., 2007)). A
survey of 143 strains of S. mutans revealed that 70% produce one or more bacteriocins in vitro
(van der Ploeg, 2005), although their ecological significance in dental plaque is still a matter of
debate (see section 1.2.1.3, above). Studies by van der Ploeg and Kreth have shown that the
expression of mutacin IV is regulated via CSP-ComDE, and is co-ordinated with expression of
the competence system which can lead to exchange of DNA in co-culture with the mutacin-
susceptible strains (Kreth et al., 2005; van der Ploeg, 2005). Bacteriocins therefore appear to
play important roles in both the ecology and the genetics of S. mutans.
1.3.4.3.1 Classification of bacteriocins
Several classifications schemes have been proposed for bacteriocins from Gram-positive
bacteria, based on their structure and mode of action. For the purpose of this dissertation, we
44
adopt the classification scheme proposed by Cotter et al. (2005) (Figure 1.7). Accordingly,
Class I bacteriocins, or lantibiotics, contain the amino acids lanthionine or β-methyllanthionine,
and are post-translationallly modified (Cotter et al., 2005). Up to 11 subclasses have been
proposed for the lantibiotics, according to structural features. Well known bacteriocins including
nisin, mersacidin and cytolysin are characterized as class I (lantibiotics). Class II bacteriocins
include small non-modified, heat-stable peptides that do not contain lanthionine modifications.
They are a heterogeneous class, and are divided into subclass IIa (pediocin-like bacteriocins ex.
leucocin A), subclass IIb (two-peptide bacteriocins ex. mutacin IV), subclass IIc (cyclic
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
45
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
46
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
47
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)).
48
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
49
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
50
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
51
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
52
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
53
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.
54
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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,
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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
host; Kanr Novagen
BL213DE3 (pET28a(+)) T7 expression vector, expression host; Kanr
Novagen
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
*Emr, erythromycin resistance; Spcr, spectinomycin resistance; Kanr, kanamycin resistance
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Gene expression analysis
Transcriptional analysis of comC in environmentally stress-induced UA159 cells was conducted
by real-time reverse transcription (RT)-PCR. Cells were grown in THYE until an OD600 of ~ 0.4
(mid-log phase) was reached, and an aliquot was reserved (pre-stress control). Cells were then
re-suspended in fresh THYE and exposed for 30 min at 37ºC to the following stresses: acid
shock (THYE acidified to pH 5.0 by the addition of HCl), amino acid starvation (100 µg ml-1
serine hydroxamate), DNA damage (50 ng ml-1 mitomycin C), and inhibition of RNA synthesis
(erythromycin and spectinomycin at sub-MIC of 0.5 µg ml-1 and 50 µg ml-1, respectively). UA159
cells were processed with the Bio101 Fast Prep System (Qbiogen), and total RNA was
extracted using Trizol reagent (Invitrogen). DNA-free RNA samples were subjected to reverse
transcription using the First-Strand cDNA Synthesis Kit (MBI Fermentas). Real-time RT-PCR
reactions were carried out using the QuantiTech SYBR Green PCR master mix (Qiagen) in an
MX3000P System (Stratagene). A standard curve was plotted with cycle threshold (Ct) values
obtained from amplification of known quantities of cDNAs. The standard curve was used to
determine the efficiency (E) of comC primer set binding and amplification: E=10-1/slope.
Comparison of the expression of comC gene between its control and stress was determined
using the formula: Ratio=(EcomC)∆Ct(control-stress)/(E16SrRNA)∆Ct(control-stress). The 16S rRNA gene was
used as internal reference as we found the expression of this gene to be stable under the test
conditions. All assays were performed in triplicate with RNA isolated from three independent
experiments and using a P <0.01.
DNA microarrays
S. mutans UA159 and ΔcomX cells were grown with 2 µM sCSP or without (uninduced control)
to mid-log phase. Total RNA was extracted as described above. The cDNAs were prepared for
hybridization using the PFGRC protocol (http://pfgrc.tigr.org/protocols/M007.pdf). Microarray
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chips were scanned using a Gene Pix 4000B (Axon) and analyzed using the TM4 Microarray
Software Suite (http://www.tm4.org/). Transcripts levels were measured by cDNA hybridized to
a fourthfold redundant S. mutans microarray and averaged for three replicated hybridization.
Differential gene expression was based on a post-normalization cutoff of ± >2-fold. Significance
was determined using a one-class t-test.
Recombinant peptides
Recombinant CipB fusion peptides (precursor and mature form) were generated using the T7
expression vector pET28a(+) (Novagen). The full-length coding region of CipB (His6-fullCipB)
and its mature form (His6-GGCipB) were PCR amplified using forward and reverse primers
containing an NheI and XhoI restriction site, respectively. The amplicons were cloned in-frame
downstream from the hexa-His sequence in the T7 expression vector pET-28a(+) precut by the
same enzymes and transformed into E. coli BL21 (non-expression host) competent cells. The
nucleotide sequences of the inserts were confirmed by DNA sequencing. Recombinant
plasmids were then transformed into E. coli BL21(DE3) competent cells. E. coli transformant
cells were incubated aerobically at 37ºC in 100-ml LB-kanamycin 30 µg ml-1 supplemented with
1% glucose until the culture reached an OD600 of 0.6. IPTG was then added at a final
concentration of 1 mM to induce expression of recombinant fusion proteins and the incubation
was continued for a further 3 h at 37ºC. The cells were collected by centrifugation and
resuspended in 1× binding buffer (Novagen) and disrupted on ice by sonication. The soluble
fractions of the disrupted cells were recovered by centrifugation and the hexa-His-tagged
recombinant CipB proteins, His6-fullCipB and His6-GGCipB, were then purified by affinity
chromatography on Ni2+-nitrilotriacetic acid (Ni-NTA) resin (Novagen) as described previously
(Levesque et al., 2004). A total cell protein extract was prepared from IPTG-induced vectorless
E. coli BL21(DE3) and used as a negative control.
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Bacteriocin overlay assays
Twenty microlitres of S. mutans cultures were spotted onto THYE-agar plates directly from
overnight cultures (control) or after growth to mid-log phase in the presence of 2 µM sCSP.
Spots were allowed to dry, and were then over-laid with a 1/100 dilution of the indicator strain
L. lactis I6 suspended in 3 ml of THYE top agar. Plates were allowed to set, and were incubated
overnight before analysis.
Transformation assays
S. mutans cells were exposed to stress at mid-log phase as described for gene expression
analysis. To test the role of cell lysis in competence, cells were grown to OD600 of ~ 0.1 and
divided into three aliquots: i) no sCSP; ii) 0.2 µM sCSP; iii) 2 µM sCSP. Ten micrograms of
streptococcal genomic DNA containing a spectinomycin resistance marker was added to the
cultures, which were grown for a further 2.5 h before differential plating.
2.4 Results
2.4.1 Stress induces expression of the CSP pheromone
We asked whether environmental stress could activate the S. mutans competence regulon
by monitoring the expression of the CSP pheromone-encoding gene (comC) under stress.
Levels of comC transcript were significantly induced by acidic conditions at pH 5.0 (4.8 ± 0.8-
fold) and in the presence of a sub-inhibitory concentration of the protein synthesis-inhibitor
antibiotic spectinomycin (8.9 ± 3.8-fold). Levels of comC transcript were, however, unchanged in
the presence of the DNA damaging agent mitomycin C, the anti-metabolite serine hydroximate,
73
and the antibiotic erythromycin under the conditions assayed. These data suggest that up-
regulation of expression of the CSP pheromone may link the competence cascade and the
stress response in S. mutans under some conditions. To test the impact of CSP on the stress
response directly, cells of a mutant defective in the CSP pheromone-encoding gene (∆comC)
were exposed to antibiotic and acid stress, harvested, and re-suspended in fresh medium for
growth analysis. The same experiment was carried out with the ∆comC mutant complemented
with a functional comC gene in trans. When exposed to a sub-inhibitory concentration of
spectinomycin, ∆comC mutant showed a significant increase of lag phase before recovering
from the antibiotic stress, while the complemented strain showed better recovery (Figure 2.1A).
In keeping with the less pronounced induction of comC expression under low pH stress
condition, the lag phase of ∆comC mutant was significantly shorter in acid stress (Figure. 2.1B)
than in antibiotic stress (Figure 2.1A). As observed under spectinomycin-stressed condition,
complementation of the ∆comC mutant under acid stress resulted in an initial increase in growth
rate followed by an eventual decline in cellular yield compared with the wild-type UA159 strain.
These results suggested to us that the up-regulation of CSP expression that occurs under
stress actively contributes to the initial stages of recovery as indicated by resumed cell growth,
but is detrimental if it is allowed to continue accumulating in the culture and may even cause cell
death if over-produced.
74
Figure 2.1. Recovery of S. mutans from stress. To test the contribution of endogenous CSP to
stress recovery, early-log phase cells of the UA159 wild-type, a ∆comC mutant (no CSP
production) and an ∆comC complemented strain (overproduction of CSP) were grown in THYE
(control), in THYE containing spectinomycin (A), or in THYE at pH 5.0 (B) for 2.5 h. Cells were
harvested, and re-suspended in fresh THYE at 1/100 dilution. Absorbance of the growing
cultures was then automatically recorded for 16 h. Cells that recovered from stress in the
absence of CSP showed a growth defect, while the CSP over-producing cells recovered from
stress more quickly but attained lower growth yields than the wild-type. These results imply that
the S. mutans CSP pheromone is important in the stress response to acid and the antibiotic
spectinomycin.
75
2.4.2 CSP pheromone triggers autolysis in a fraction of the population
These results suggest a mechanistic link for competence induction under stress in
S. mutans, and broaden the implications of previous work done by Claverys‟ group
(Dagkessamanskaia et al., 2004; Prudhomme et al., 2006) in S. pneumoniae, by demonstrating
a new role for the CSP pheromone in the stress response of more than one streptococcal
species. However, since important differences exist between the CSP-induced competence
cascade in S. mutans and S. pneumoniae (Martin et al., 2006), we examined the phenotypic
effect of increasing the concentration of CSP in cultures of S. mutans. We used exogenously
added synthetic CSP (sCSP) to study the effect of the CSP pheromone reproducibly, without
secondary complications from the stress itself (e.g., protein synthesis inhibition from
spectinomycin). We routinely use 0.2 μM sCSP to induce competence in S. mutans (Li et al.,
2001b), and based our choice of sCSP concentrations on our stress experiments (which
showed up to a 15-fold increase in comC transcript levels). It is possible that even higher
localized endogenous CSP concentrations exist in the biofilm environment, but we attempted to
avoid potential artifacts associated with overloading a signaling system by restricting the amount
of exogenously added sCSP to concentrations close to those which induce competence in vitro.
By monitoring the growth kinetics of planktonic cultures of S. mutans in the presence of
increasing concentrations of sCSP, we found a ComX-dependent decrease in the growth rate
and a ComDE-dependent decrease in the final growth yield of S. mutans proportional to the
concentration of sCSP (Fig. 2. 2). This phenotype differs from autolysis in S. pneumoniae, in
which the whole population lyses in the presence of CSP (Ronda et al., 1987).
76
Figure 2.2. S. mutans growth kinetics. (A) Growth of S. mutans in the presence of sCSP. To
artificially mimic the high CSP concentrations induced by stress, cells were diluted into media
supplemented with increasing concentrations of sCSP or without (control) and grown for 16 h.
Cultures responded to sCSP with a slower growth rate and lower growth yield proportional to
the increase in sCSP concentration. To ensure the specificity of the phenotype, a peptide was
synthesized with the same amino acid composition as S. mutans CSP but with the order
randomized. The randomized peptide did not have any effect on S. mutans growth (not shown).
(B) Growth of S. mutans wild-type (WT), ∆comDE mutant and ∆comX mutant in the absence (–)
and presence (+) of 2 µM CSP pheromone. While eliminating ComDE restores both the growth
rate and yield of the culture in the presence of sCSP, the ∆comX mutant shows a defect in
growth yield both in the absence and presence of sCSP. We infer from this data that the altered
growth yield of the culture in the presence of sCSP is due to ComE-controlled genes.
Our cultures responded to high sCSP concentrations with a decreased growth rate but
stable plateau, which we attributed to either the average of two distinct populations of CSP-
induced (lysing) and uninduced (resistant) cells (known as all-or-none induction, reviewed in
(Davidson and Surette, 2008)), or to a uniform state of bacteriostasis in the whole population.
To distinguish between these two phenomena, we monitored the supernatants of S. mutans
cultures constitutively expressing a -glucuronidase (GUS) reporter gene for release to the
culture medium of intracellular GUS by cell lysis. GUS activity was significantly increased in the
supernatant of cultures grown with sCSP, indicating cell lysis occured at high sCSP
77
concentrations (Figure 2.3). We next quantified the extent of cell death in mid-log phase
cultures grown in the presence of 2 µM sCSP using fluorescent staining for viability, and found
that <10% of the S. mutans UA159 population stains with propidium iodide due to cell death
(Figure 2.4). This result is in agreement with previous work done on S. mutans strain UA140
using fluorescence viability staining, in which only a sub-population of cells lysed no matter what
the concentration of exogenous sCSP (Qi et al., 2005). Finally, we restored the growth of the
culture to wild-type levels by sub-culturing sCSP-exposed cells into fresh medium (Figure 2.4),
confirming the existence of a CSP-resistant subpopulation even at concentrations of sCSP up to
200 μM. We conclude from these results that the CSP pheromone induces a state of population-
level stasis in S. mutans and invokes lysis in a fraction of the population while sparing the
remainder.
Figure 2.3. Effect of sCSP on culture density and cell lysis. The culture-wide graded response
to sCSP could be due to an identical bacteriostatic response in the whole population, or to an
„all-or-none‟ induction of lysis in a fraction of the population. To distinguish between the two, the
release of the cytoplasmic enzyme -glucuronidase (GUS) into the supernatant of growing
cultures was quantified. The observed increase in GUS activity in sCSP indicates lysis in a
fraction of the population. „Control‟ cultures were grown in THYE without sCSP, while „+ sCSP‟
cultures were supplemented with 2 μM sCSP. GUS activity is normalized to the optical density
of the culture at each time point, and is expressed in Miller units (MU).
78
Figure 2.4: A sub-set of the population undergoes cells lysis, while the majority remains sCSP-
unresponsive. Viability staining using propidium iodide indicates less than 10% of the population
is dead in the presence of 2 μM sCSP (left). Growth of cells when re-suspended in fresh THYE
after overnight exposure to sCSP at the concentrations indicated. Growth resumes after sCSP
exposure even at 200 μM, suggesting that a sub-population of cells is always resistant to sCSP-
mediated cell lysis (right).
2.4.3 Genome-wide expression response to CSP: identification of mutacin V
Microarray-based expression profiling showed that 2 μM of sCSP altered the expression
(± ≥2-fold) of 277 genes in the S. mutans UA159 genome (Appendix A; Supplementary
Information SI Table S1). Since CSP induces gene expression both directly through ComE
signaling and secondarily through ComX, we also determined the expression response to sCSP
in the absence of ComX by microarray (Appendix A; SI Table S2). We found that ComE controls
the expression of 37 genes, among which are the S. mutans bacteriocins and comX itself (Table
2.2). Like S. pneumoniae, S. mutans ComX is a competence-specific sigma factor responsible
for expression of the entire competence regulon, including the CSP-encoding gene itself
(Peterson et al., 2004).
79
Table 2.2. Relative expression levels of highly-expressed CSP-induced S. mutans genes
encoding putative and known bacteriocins and their accessory genes.
Locus* Common name, putative function Fold†
SMU.150 NlmA mutacin IV +11.19
SMU.151 NlmB mutacin IV +12.40
SMU.423 Putative bacteriocin +14.55
SMU.925 Putative immunity factor +18.20
SMU.1897 ABC transporter, ATP-binding; ComA +9.19
SMU.1898 Putative ABC transporter, ATP-binding and permease +4.18
SMU.1899 Putative ABC transporter fragment +5.22
SMU.1900 ABC transporter; ComB +5.92
SMU.1902 GG-motif-containing peptide +9.76
SMU.1903 Hypothetical protein +15.97
SMU.1904 Hypothetical protein +12.25
SMU.1905 GG-motif-containing peptide +10.11
SMU.1906 Putative bacteriocin +11.36
SMU.1907 Hypothetical protein +8.93
SMU.1908 Hypothetical protein +18.27
SMU.1909 Hypothetical protein +19.61
SMU.1910 Hypothetical protein +18.26
SMU.1912 Hypothetical protein +22.23
SMU.1913 Putative immunity protein +15.23
SMU.1914 Bacteriocin, mutacin V +20.41
SMU.1915 Precursor of CSP pheromone +3.76
SMU.1916 Histidine kinase ComD +10.50
SMU.1917 Response regulator ComE +11.32
SMU.1997 Sigma factor ComX +14.27
*Results for selected genes were ordered based on their position in the UA159
chromosome. The grey highlighted area indicates the genes located in a 13.5-kb
bacteriocin-related genomic island. Putative bacteriocin-encoding genes are in bold.
†Transcripts levels were measured by cDNA hybridized to a fourfold redundant S. mutans
microarray and averaged for three replicates hybridization. Quantitative real-time RT-PCR
was performed on selected genes to confirm the results obtained using microarray.
80
The products of six CSP-responsive genes (cbpD, lytA, comM, cibA, cibB, cibC) have been
directly implicated in S. pneumoniae autolysis (Guiral et al., 2005; Håvarstein et al., 2006). Our
search for homologous effectors among the CSP-induced genes identified by microarray yielded
no significant sequence homologies, but suggested the involvement of a bacteriocin in
S. mutans CSP-induced lysis. Our search was also guided by experiments performed in
Streptococcus thermophilus and Streptococcus salivarius, showing similar dose-dependent
growth inhibition in the presence of the species-specific CSP paralogue BIP (Figure 2.5), a
signaling peptide known to induce bacteriocin expression (Fontaine et al., 2007). S. mutans
isogenic mutants were generated to be defective in mutacin IV (SMU.150, SMU.151), mutacin V
(SMU.1914), and GG-motif-containing peptides (SMU.423, SMU.1906). Importantly, inactivation
of SMU.1914, encoding the nonlantibiotic peptide bacteriocin mutacin V (Hale et al., 2005a),
drastically attenuated the response to sCSP in S. mutans (Fig. 2.6). To ensure that the
observed phenotype was not due to comC repression, we performed transcriptional analysis by
quantitative real-time PCR and found that the expression of the CSP-encoding gene was not
affected in the ∆1914 mutant (data not shown).
To confirm the bacteriocin-like activity of SMU.1914, we performed a series of agar overlay
assays using the indicator strain Lactococcus lactis I6. As observed by Hale et al., 2005, the
∆1914 mutant had a reduced zone of inhibition that was not observed for the mutacin IV mutant
against L. lactis I6 (Figure 2.7). Since some bacteriocins produced by Gram-positive bacteria
require two peptides for activity, we also investigated whether mutacin V (SMU.1914) caused
cell lysis alone or in combination with CSP itself (SMU.1915), given that the two peptide-
encoding genes share an overlapping (but divergent) promoter region (Kreth et al., 2007). We
constructed a CSP-independent raffinose-inducible expression system to express mutacin V
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Figure 2.5. Growth of Streptococcus thermophilus (A) and Streptococcus salivarius (B) in
increasing concentrations of their species-specific signaling peptides. In these two non-
competence species of streptococci, the peptide signaling system is composed of CSP-ComDE
paralogues BIP (bacteriocin-inducing peptide) and BlpRH, known to induce expression of
bacteriocins. The similarity between these BIP-induced growth curves and the CSP-induced
growth curves in S. mutans strongly suggests the involvement of a bacteriocin in the S. mutans
cell death cascade.
82
Figure 2.6. Effect of 2 µM sCSP pheromone („+‟) on S. mutans wild-type (WT) and mutants
defective in the bacteriocin CipB (SMU.1914) and its putative immunity factors SMU.1913 and
CipI (SMU.925). Growth of the wild-type strain in THYE alone provided a baseline for
comparison (control).
83
Figure 2.7. Agar overlay assays showing extracellular bacteriocin concentration in the UA159
wild-type and mutants defective in the CipB bacteriocin, the CipI immunity protein, the
bacteriocin mutacin IV and the bacteriocin transporter NlmTE. Extracellular bacteriocin
concentration was monitored by spoting mid-log phase cultures of the test strain grown in the
absence (control) and presence of sCSP.on agar plates. The test strains were then
subsequently overlayed with a 1/100 dilution of the indicator strain Lactococcus lactis I6
suspended in 1% top agar. Plates were then grown overnight at 37°C, and the amount of
extracellular bacteriocin was assessed according to the width of the zone of inhibition of L. lactis
surrounding the test strain.
84
alone in S. mutans from a multi-copy plasmid, bypassing the need for exogenous sCSP
addition. This system allowed us to rule out the contribution of sCSP itself to our experimental
system, given that excesses of signaling peptide have been documented to function as
bacteriocin-like peptides at extremely high concentrations (Anderssen et al., 1998). Using this
CSP-independent inducible expression system, we found that mutacin V gene expression was
induced 5.0 ± 1.6-fold in raffinose (inducer) vs. in glucose (repressor). Although this level falls
short of the 20-fold induction of mutacin V we observed with sCSP (Table 2.1), we were still
able to induce a growth defect similar to sCSP using the raffinose-inducible system (Fig. 2.8).
This result confirms that mutacin V is necessary for the observed autolytic phenotype in sCSP.
We confirmed that SMU.1914 was regulated directly by ComE (Fig. 2.9), and was transcribed in
direct proportion to the amount of sCSP (data not shown) or the concentration of naturally
accumulating CSP in a growing culture of S. mutans (2.10). Although (Hale et al., 2005a)
proposed that SMU.1914 be designated nlmC (gene product mutacin V), we instead suggest
the three-letter prefix cip for CSP-induced peptide, and propose the name CipB for this self-
acting bacteriocin.
2.4.4 CipB bacteriocin likely acts intracellularly
We reasoned that CipB‟s action against self would occur either from 1) a position tethered to
the cell wall via cell-to-cell contact, 2) the extracellular environment, or 3) intracellularly via
membrane insertion from the cytoplasmic side. The Cib bacteriocins of S. pneumoniae are
tethered to the cell wall after export, and able to kill neighboring cells via cell-to-cell contact
(Guiral et al., 2005). We co-cultured sCSP-induced wild-type cells with ∆cipB mutant cells to
provide cell contact between CipB-expressing wild-type cells and the CipB-deficient mutant, but
did not observe any functional complementation of the mutant (results not shown). This result
85
Figure 2.8. Growth kinetics of S. mutans UA159 strain containing the shuttle plasmid pDL277
(Leblanc et al., 1992) harboring the CipB-encoding gene under the control of the raffinose-
inducible promoter (notation „pMSM‟) of the S. mutans multiple-sugar metabolism operon
(McLaughlin and Ferretti, 1996). Growth was monitored by following OD600 for 16 h in TYE
containing either 0.5% raffinose (inducer, notation „raf‟) or 0.5% glucose (repressor, notation
„glc‟). The vector alone (no insert) was used as control. Also plotted is the growth of a mutant
defective in the putative immunity gene SMU.925 carrying the pMSM construct following
induction with 0.5% raffinose.
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Figure 2.9. RT-PCR gene expression profiles of cipB and cipI, encoding the bacteriocin and
immunity protein respectively, in S. mutans wild-type strain and mutants defective in the
alternate sigma factor ComX and the response regulator ComE. In addition to the lack of
detectable transcript in the ΔcomE mutant, both cipB and cipI have putative ComE binding sites
in their promoter regions (van der Ploeg, 2005). The constitutively expressed 16S rRNA gene
served as a loading control.
Figure 2.10. Quantitative real-time RT-PCR gene expression profiles of SMU.1914 (encoding
CipB), comC (encoding CSP) and SMU.925 (encoding CipI) at 1-h, 2-h, and 3-h intervals
following 1:100 dilution from an overnight culture. Gene expression was normalized to the
expression of the constitutively expressed 16S rRNA gene.
87
indicated that CipB is not likely to act from a position tethered to the cell wall via cell-cell contact,
reinforcing the notion that differences exist between the autolytic pathways of S. mutans and S.
pneumoniae. Since bacteriocins traditionally act from the extracellular environment, we
investigated extracellular activity by growing the ∆cipB mutant in cell-free supernatant from a
sCSP-induced wild-type culture, and in medium supplemented with purified recombinant CipB
peptides (precursor and mature form). We found no effect on the growth of the S. mutans ∆cipB
mutant in conditioned medium or at concentrations of recombinant CipB peptides that
completely inhibited the indicator strain L. lactis I6 (Figure 2.11), indicating that extracellular
concentrations of the bacteriocin had no effect on the producing strain. Importantly, we found
that the recombinant bacteriocin precursor peptide (which includes the leader peptide required
for export) was equally effective at killing L. lactis. The function of the leader peptide is thought
to be two-fold: its presence keeps the bacteriocin inactive during translation, and secondly
traffics the bacteriocin to the correct ABC transporter for export (Drider et al. 2006). Since the
full-length peptide was equally effective against L. lactis, export-dependent processing does not
appear to be required for activation of the CipB bacteriocin. This result suggests that an
intracellular accumulation of the unprocessed bacteriocin may be lethal to the producing cell. To
further test if intracellular accumulation of CipB could cause lysis, we inactivated the dedicated
ABC transporter NlmTE, required for the export of S. mutans nonlantibiotic bacteriocins
including CipB (Hale et al., 2005b). We confirmed that the ∆nlmTE mutant was unable to
secrete CipB using an agar overlay assay with L. lactis indicator cells (Figure 2.7). We found a
significant growth defect in the ∆nlmTE mutant compared to the wild-type strain at all sCSP
concentrations tested (Figure 2.11), which we attribute to an increased intracellular
accumulation of unprocessed CipB. Interestingly, we also observed a longer lag phase in the
∆nlmTE mutant in the absence of sCSP following dilution from an overnight culture. Since the
CipB bacteriocin was highly expressed in overnight cultures (Figure 2.10), this initial growth
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Figure 2.11. CipB may act intracellularly. (A) Growth kinetics of S. mutans (WT) and L. lactis I6
in the presence of recombinant CipB (precursor and mature form). The full-length precursor
peptide represents the intracellular form of the bacteriocin, including its leader sequence. The
mature peptide represents the extracellular form of the bacteriocin, having been cleaved at the
GG-motif. Importantly, the precursor peptide is equally effective against L. lactis, implying that
export-dependent processing is not necessary for activity. (B) Effect of 2 μM sCSP („+‟) on the
wild-type strain and a mutant defective in NlmTE, the ABC transporter responsible for export of
CipB. The ΔnlmTE mutant was assayed over a range of sCSP concentrations, and showed
growth defects compared to the wild-type strain at all concentrations assayed (not shown), likely
due to the intracellular accumulation of CipB. The ΔnlmTE mutant has a growth defect even in
the absence of sCSP, possibly due to the accumulation of intracellular CipB induced by the high
endogenous CSP concentration found in the overnight culture from which it was diluted.
89
defect supports the hypothesis that intracellular CipB accumulation was lethal to the producing
cell.
Of final note, the expression of nlmTE decreased approximately 2-fold in the presence of
2 µM sCSP, which was reflected in decreased zones of inhibition by agar overlay (Figure 2.7).
Given that the expression of cipB was increased 20-fold in these cells (Table 2.1), it appears
that CipB accumulated to high intracellular levels in the presence of high concentrations of
sCSP. This result was important in ruling out the notion that excess sCSP could have simply
overwhelmed the normal CipB export pathway. Instead, an active decrease in expression of the
transporter coupled with increased expression of the bacteriocin suggested that intracellular
accumulation of CipB may be an active process. Together, these data strongly suggested a
completely novel mechanism of action for a bacteriocin: intracellular action against self.
2.4.5 The small protein CipI (SMU.925) confers immunity
Bacteriocin-encoding genes are usually co-transcribed with their cognate immunity genes,
which are protective against the action of the bacteriocin (Abee, 1995; Diep et al., 2007).
Synthetic CSP exposure induced two candidate immunity genes, SMU.925 and SMU.1913
(Table 2.1), which share 82% a.a. identity and have putative ComE binding sites in their
promoter regions (van der Ploeg, 2005). Unexpectedly, the growth of a ∆1913 mutant was
identical to the wild-type strain in the presence of sCSP (Figure 2.6), indicating that although
SMU.1913 was co-transcribed with cipB (data not shown), it did not prevent CSP-induced lysis.
In contrast, inactivation of SMU.925 resulted in almost complete growth arrest at high sCSP
concentrations (Figure 2.6). To confirm the role of SMU.925 in immunity to cell death, wild-type
cells expressing SMU.925 from its own native promoter on a multicopy plasmid were exposed to
sCSP. The cells over-expressing SMU.925 were significantly more resistant to sCSP than the
90
wild-type (Figure 2.12). We also transformed the Δ925 mutant with the same CSP-independent
raffinose-inducible cipB expression construct used above. Cells deficient in the immunity gene
were more susceptible than the wild-type to high levels of cipB expressed in presence of
raffinose (Figure 2.8), consistent with the proposed role for CipI as the immunity gene. We thus
propose the designation cipI (CSP-induced peptide Immunity) for SMU.925.
As for cipB, cipI was found to be dependent on ComE for its transcription (Figure 2.9), and was
transcribed in proportion to the amount of sCSP added (data not shown). However, unlike cipB,
the expression of cipI showed an additional level of density-dependent control, since dilution
from an overnight culture (high to low cell density) caused an increase in its expression (2.10).
We considered that a second mechanism controlling cipI expression might exist to prevent cell
lysis at low cell density, and tested whether autoinducer-2 (AI-2) might be the signaling
molecule regulating this second system. No growth defect was observed when a ∆luxS mutant
deficient in the LuxI-type autoinducer synthetase (enzyme responsible for the synthesis of AI-2
(Bassler and Losick, 2006) was grown in the presence of increasing sCSP concentrations
(results not shown), indicating that AI-2 is likely not the second signal molecule.
91
Figure 2.12. Growth of the wild-type and a wild-type strain over-expressing SMU.925 from a
multi-copy plasmid in the absence and presence of 2 μM sCSP. The sCSP-resistant CipB
mutant was used as a control.
2.4.6 Role of CSP-induced lysis in genetic competence
In addition to its known role in competence (Li et al., 2001b), we have shown that S. mutans
CSP is involved in the stress response and can trigger autolysis. Bacteria are unicellular
organisms, and would seem incapable of altruistic behaviors like cellular suicide. For an
altruistic trait like autolysis to be preserved by evolution, it must be linked to a behavior that
benefits genetically identical sibling cells (Keller and Surette, 2006). We reasoned that the
linkage of the stress response, competence induction and autolysis through the CSP
pheromone could serve to facilitate the exchange of fitness-enhancing DNA under stress, and
maintain the autolysis pathway through evolution. If our hypothesis is correct, S. mutans cells
must be equally transformable at the high sCSP concentrations in which we observe cell lysis.
Indeed, we found cells exposed to 2 μM sCSP to be transformed at frequencies similar to those
92
obtained using 0.2 μM sCSP (Figure 2.13). We next examined whether lysis and competence
development could occur simultaneously in a culture using fluorescent staining for viability and a
GFP reporter fused to the promoter of the gene encoding the sigma factor responsible for
induction of the CSP-dependent competence regulon, comX. We observed sporadic expression
of PcomX–gfp throughout the culture, consistent with the hypothesis that only a fraction of the
S. mutans population is sCSP-induced (and competent) at any one time (Figure 2.14A).
However, when sCSP-treated cultures were counter-stained with propidium iodide, we observed
overlap between PcomX–gfp expression and cell death in the majority of cases (Figure 2.14B-
C). This result was not surprising, since both cipB and comX are regulated directly via CSP-
ComDE signaling and suggests that sCSP-induced competent cells continue to accumulate
CipB under continuous sCSP stimulation, resulting in cell death. We therefore tested the
transformation efficiency of the ∆cipB mutant in the presence of sCSP to determine the
transformation efficiency at high sCSP concentrations in the absence of cell death. When cell
death in the culture was prevented by inactivation of cipB, the transformation efficiency in the
presence of sCSP could not be induced beyond sCSP-independent levels (no sCSP added)
(Figure 2.13). Conversely, when cell death in the culture was promoted by removal of the
immunity protein, ΔcipI cultures reached levels of transformation comparable to wild-type sCSP-
induced levels in the absence of exogenously added sCSP (Figure 2.13). These results
indicated that the sCSP-induced competence cascade is connected to CipB-mediated autolysis
in S. mutans. We are currently investigating whether factors released via lysis of a sub-
population of cells contribute to the development of competence in the surviving population.
93
Figure 2.13. Transformation efficiency of S. mutans wild-type strain and its mutants deficient in
the CipB bacteriocin and CipI immunity protein. Sheared genomic DNA carrying an antibiotic
resistance gene was added alone or with 0.2 μM or 2 μM sCSP to growing cultures. Cells were
grown for a further 2.5 h before differential plating. Results obtained for the wild-type strain
showed that transformation is possible and equally efficient at the concentrations of sCSP that
induce cell lysis. Mutants unable to undergo cell lysis (CipB–) showed no increase in
transformation frequency in the presence of sCSP, while mutants with increased lysis potential
(CipI–) showed increased transformation in the absence of sCSP. Transformation efficiencies
are expressed as the number of antibiotic-resistant CFUs divided by the total number of CFUs.
A ComE-deficient strain served as a transformation-deficient control.
94
Figure 2.14. Competence and lysis in cultures of S. mutans. The alternate sigma factor ComX
is induced by ComE, and is responsible for induction of the CSP-dependent competence
regulon. A PcomX–gfp reporter fusion was used to monitor the development of competence in
the presence of 2 μM sCSP (A). Cultures were then counter-stained with propidium iodide (B) to
determine cell death. When images were merged (C), it was apparent that cells expressing
comX were also undergoing cell lysis.
The results obtained with PcomX-gfp suggest that the sCSP-dependent competence
pathway does not contribute to the uptake of fitness-enhancing genes in a stressed population,
since sCSP-induced cells are killed due to CipB accumulation. However, we examined whether
stress alone (in the absence of sCSP) could induce transformation in S. mutans using sub-
inhibitory concentrations of spectinomycin known to increase comC expression. We found a
4.0 ± 1.3-fold increase in transformation efficiency in stressed cultures vs. control cultures.
Importantly, the transformation efficiency of a ∆comC mutant strain was identical to the wild-type
in presence of sub-inhibitory concentration of spectinomycin, indicating that the increase in
transformation we observed was through the CSP-independent competence pathway. Together,
these data suggest that the increase in CSP pheromone production under stress causes
S. mutans autolysis through the ComDE signal transduction system, which releases DNA for
uptake via the CSP-independent competence pathway (Figure 2.15).
95
Figure 2.15. Summary of data. The sensor histidine kinase ComD is activated either directly by
stress or by an accumulation of the CSP pheromone, and activates its cognate response
regulator ComE by phosphorylation. Activated ComE then directly regulates the expression of
37 genes, including the alternate sigma factor ComX, the CipB bacteriocin and its immunity
protein CipI. CipB causes cell lysis in a fraction of the population, which potentially contributes
DNA for uptake and other secondary signals to trigger genetic competence in the surviving
population. Expression of 240 genes, including the competence cascade and the CSP molecule
itself, are directly controlled by ComX. Open grey arrows: direct genetic regulation. Solid black
arrow: phenotype caused.
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2.5 Discussion
Experimental evidence has long supported a link between the competence cascade and
stress response in S. mutans (Ahn et al., 2006; Li et al., 2001a; Li et al., 2002a; Qi et al., 2004),
but a mechanistic explanation for these phenotypes has remained elusive. In this study, we
report the characterization of a novel self-acting intracellular bacteriocin which functions as a
mediator of autolysis in S. mutans. We have characterized this autolytic response in the
physiological context of an elevated concentration of the CSP pheromone under stress, which
provides the proverbial „missing link‟ between stress and competence in S. mutans. Moreover,
our results broaden the implications of previous work in pneumococci (Claverys et al., 2006) by
demonstrating the presence of a peptide „alarmone‟ in phylogenetic groups of streptococci
beyond the mitis group. However, like in pneumococci (Prudhomme et al., 2006), not all
stressful conditions assayed with S. mutans triggered CSP up-regulation. While we have
focused on elucidating the downstream pathways triggered by CSP up-regulation in S. mutans,
future work directed towards understanding why some stresses trigger CSP up-regulation while
others do not will complete our understanding of the stress response in this organism.
We focused on the effect of the stress-induced up-regulation of the CSP pheromone in the
absence of the stress itself using exogenously added sCSP. We demonstrated a slower growth
rate and a reduction in growth yield at high concentrations of sCSP due to a balance between
cell growth and autolysis in S. mutans. This response differs from the uniform autolytic response
mounted by S. pneumoniae in the presence of high CSP. S. pneumoniae achieves both a
death-susceptible and a death-resistant population by expressing two different CSP pherotypes
(Claverys et al., 2006). However, S. mutans has been shown to encode a single CSP pherotype
(Allan et al., 2007). The nature of cell lysis (and competence) responses in S. mutans is
therefore different than the response in S. pneumoniae, and may be closer to the other well-
characterized naturally transformable Gram-positive organism, B. subtilis, whose competence
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response occurs in only a fraction of the population due to bistability (Smits et al., 2005). The
bistable response occurs when positive feedback into a system occurs on a fast timescale and
negative feedback occurs on a slower timescale (Davidson and Surette, 2008). The result is an
amplification of transcriptional “noise” in a system, which manifests as two transient but distinct
populations of induced and uninduced cells (Davidson and Surette, 2008; Dubnau and Losick,
2006). We envision a situation in S. mutans in which stress-induced CSP upregulation serves to
„prime the pump‟ of bistability in the CSP-ComDE circuit, causing some cells to respond with a
rapid and extreme up-regulation of CSP-controlled genes (including comC itself), due to positive
feedback. What is unique about the genetic organization of comC in S. mutans is its divergent
regulation from mutacin V (Kreth et al., 2006b; Kreth et al., 2007). Kreth and colleagues showed
that this genetic organization results in repression of comC transcription when SMU.1914
expression is activated by ComE binding to their common intergenic region (Kreth et al., 2007).
When SMU.1914 expression was high, a built-in „safety mechanism‟ prevented the further
accumulation of CSP in the culture by repressing comC transcription, which eventually feeds
back into the loop to prevent autolysis of the whole population.
We identified the type II bacteriocin mutacin V (CipB) as a major factor in CSP-induced lysis
using genome-wide gene expression analysis and mutagenesis. Importantly, we have
demonstrated that its activity against self is due to intracellular accumulation in the producing
strain. We have presented evidence that CipB acts alone to cause cell death, that the
unprocessed form of the bacteriocin is active against an indicator strain, and that inactivation of
the NlmTE transporter causes cell death. This intracellular mechanism makes sense when
examined from an environmental context – a secreted or surface-located „death peptide‟ would
have the potential to wreak havoc in the tightly packed oral biofilm environment. Bacteriocins
generally act by creating channels or pores in the cell membrane that destroy the membrane
potential and cause cell death by cellular energy depletion (Nes et al., 2007). CipB likely causes
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cell death initially via a similar mechanism, and cell lysis secondarily by activating murein
hydrolases and/or lytic enzymes (Galvez et al., 1990). Alternatively, intracellular accumulation of
CipB may cause cell lysis in a manner analogous to the holin/anti-holin system characterized in
Staphylococcus aureus, in which a pore-forming peptide (the holin) inserts into the cytoplasmic
membrane to allow degradative enzymes to access the cell wall (Rice et al., 2003). The
antimicrobial activity of CipB is directed mainly against non-streptococcal species, and not
against traditional mutacin target organisms like Streptococcus gordonii and
Streptococcus salivarius (Hale et al., 2005a). Given our data showing that CipB acts
intracellularly, we suggest that the main function of this peptide is CSP-mediated autolysis, and
not as an exported bacteriocin. It is possible that the export of CipB outside S. mutans by
NlmTE transporter is part of a detoxification mechanism, and that the few bacterial species that
are susceptible to it are bystanders rather than primary targets. Data showing a significant
growth defect in S. mutans NlmTE-deficient cells compared with the parental strain supports this
argument. We also show the potential for a similar auto-active bacteriocin in members of the
salivarius group of streptococci.
Unexpectedly, inactivation of SMU.1913 excluded its involvement in CSP-mediated
autolysis. Instead, the product of the cipI gene encodes the immunity factor involved in
S. mutans autolysis. Although it is unexpected to find genes paired in function unlinked on the
genome, the need for redundant controls over this autolytic pathway may have necessitated this
duplication. Our view is supported by evidence showing that in addition to ComE regulation at
high CSP concentrations, an additional (and currently unknown) regulatory pathway triggered
activation of cipI expression at low cell density. This safety mechanism would be impossible if
the immunity protein was co-transcribed with the bacteriocin itself.
We hypothesized that competence and lysis would allow the exchange of fitness-enhancing
DNA under stress. However, when we monitored the induction of cell death and competence on
99
a cell-by-cell basis using a transcriptional GFP reporter gene fusion, we found that the same
population became competent and lysed. This finding was not entirely surprising since both
competence and lysis are triggered by the CSP-ComDE circuit. Instead, we showed that
transformation can occur in a CSP-independent manner in spectinomycin stress, providing an
alternative pathway for the acquisition of fitness-enhancing genes. The lack of transformation in
the ∆cipB mutant is somewhat paradoxical, given that these cells are the survivors in a CSP-
induced population, and would be the expected recipients of transforming DNA. However, the
contrary result with ∆cipI implies that cell death in the CSP-responsive members of a population
may trigger genetic competence in the CSP-unresponsive survivors, and suggests that cellular
factors released via lysis could provide secondary signals to induce competence via a CSP-
ComDE-independent pathway. The ability to sense cell lysis would permit naturally competent
bacteria to turn on their uptake machinery when DNA is available in their environment. We are
currently exploring this fascinating possibility.
2.6 Acknowledgements
We thank John Tagg and Nicholas Heng for providing mutants related to mutacin V transport,
Indranil Biswas for providing shuttle expression plasmids for S. mutans, and Elena Voronejskaia
for assisting with the mutant constructions. This work was supported by CIHR-Priority
Announcement IMHA Grant FRN-90114 (to C.M.L.) and by NIDCR Grant R01 DE013230-08 (to
D.G.C.). DNA microarrays were supported through NIDCR via NIAID contract number N01-
AI15447 to JCVI. J.A.P. is the recipient of a CIHR Strategic Training Fellowship in Cell Signaling
in Mucosal Inflammation and Pain.
100
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Chapter 3: Cell Death in Streptococcus mutans Biofilms: a Link
Between CSP and Extracellular DNA
JA Perry, DG Cvitkovitch and CM Levesque. 2009. FEMS Micro Lett. 299:261-6
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3.1 Abstract
Streptococcal competence stimulating peptides were once thought to passively communicate
population density in a process known classically as quorum sensing. However, recent evidence
has shown that these peptides may also be inducible „alarmones‟, capable of conveying
sophisticated messages in a population including the induction of altruistic cellular suicide under
stressful conditions. We have previously characterized the alarmone response in
Streptococcus mutans, a cariogenic resident of the oral flora, in which a novel bacteriocin-like
peptide causes cell death in a sub-set of the population. Our objective in this work was to
characterize the mechanism of immunity to cell death in S. mutans. Towards this goal, we have
identified the conditions under which immunity is induced, and identified the regulatory system
responsible for differential (and protective) expression of immunity. We also showed that CSP-
induced death contributes to S. mutans biofilm formation through the release of chromosomal
DNA into the extracellular matrix, providing a long sought-after mechanistic explanation for the
role of CSP in S. mutans biofilm formation.
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3.2 Introduction
Bacteria have long been studied as single-celled, primitive organisms, free-floating in
laboratory culture. However, bacterial species in nature have a strong tendency to colonize
surfaces and form complex, multi-species communities referred to as biofilms (Costerton et al.,
1994). In nature, biofilms are found on rocks in streams, in industrial bioreactors, and in animal
host environments like the oropharyngeal, gastrointestinal and vaginal tracts, and on medical
devices. A biofilm in its simplest form is composed of a surface (or „substratum‟), surface-
attached cells, and a surrounding extracellular matrix of biopolymers (Dunne, 2002). By
microscopic analysis, a biofilm appears to be a highly hydrated and open structure, composed
mainly of non-cellular material including water channels and exopolymeric substances (EPS)
which form the extracellular matrix (Lawrence et al., 1991). The EPS forms the outermost layer
of the biofilm, and is composed of a hydrated, anionic mesh of bacterial exopolymers and
trapped environmental molecules (Branda et al., 2005). EPS comprise a wide variety of
polysaccharides, proteins, glycoproteins, glycolipids, and in some cases, large amounts of
extracellular DNA (eDNA). DNA was first shown to be present in the extracellular matrix of
biofilms formed by Pseudomonas aeruginosa (Whitchurch et al., 2002), and is now widely
recognized as a major constituent of the matrix (Flemming et al., 2007). The matrix functions as
a permeability barrier to limit both the diffusion of beneficial nutrients away from the biofilm, and
prevent or slow the diffusion of harmful substances like antibiotics and predatory cells of the
immune system from accessing matrix-embedded cells (Costerton et al., 1999).
The spatial separation of sessile cells combined with nutrient/waste and oxygen gradients
within the biofilm results in a heterogeneous population of cells, distinct from their planktonic
counterparts in gene expression patterns and „behaviours‟ (Stoodley et al., 2002; Beloin &
Ghigo, 2005). The metabolic task sharing, communication and phenotypic heterogeneity within
107
a biofilm have led to their being likened to multicellular-type organisms, since cooperation
results in the success of the group (Shapiro, 1998; Parsek & Greenberg, 2005). The
coordinated behaviour of single-celled bacteria is accomplished using different classes of small
diffusible signalling molecules in a process called quorum sensing (reviewed in Waters &
Bassler, 2005).
Streptococcus mutans is a well-characterized resident of the oral biofilm, and is thought to be
the main causative agent of the most common human infectious disease, dental caries
(Loesche, 1986). The S. mutans quorum sensing system is composed of the competence
stimulating peptide (CSP) pheromone and the ComDE two-component signal transduction
system (TCS). The S. mutans CSP-ComDE system regulates several phenotypes including
genetic competence (Li et al., 2001a), biofilm formation (Li et al., 2002b), acid tolerance (Li et
al., 2001b), and bacteriocin production (Kreth et al., 2006). We have recently shown that the
CSP pheromone is also stress-inducible, and triggers autolysis in a fraction of the S. mutans
population at high concentrations (Perry et al., 2009). Autolysis in S. mutans occurs through the
intracellular accumulation of a self-acting bacteriocin, CipB, and is prevented by the action of
the bacteriocin immunity protein CipI. Previously, we showed that CipI was differentially
regulated from CipB at low cell density (Perry et al., 2009). Here we report the characterization
of the CipI immunity protein, detailing its expression and regulation, and propose a role for CSP-
induced autolysis in the release of eDNA in the S. mutans biofilm.
3.3 Materials and methods
Bacterial strains and culture conditions
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S. mutans UA159 wild-type strain and its mutants were grown in Todd-Hewitt–Yeast Extract
(THYE) broth at 37ºC with 5% CO2 without agitation. Growth kinetics were assessed using a
Bioscreen microbiology workstation (Bioscreen C Labsystems, Finland) as previously described
(Hasona et al., 2005). Results represent an average of five technical replicates, and two to four
independent experiments.
Table 3.1: Bacterial strains used in this study.
Strain Properties Reference
UA159 S. mutans wild-type ATCC
ΔcipI ∆smu.925; Emr (Perry et al., 2009)
ΔcipB ∆smu.1914; Emr (Perry et al., 2009)
ΔliaS ∆smu.486; Emr (Levesque et al., 2007)
ΔliaR ∆smu.487; Emr (Perry et al., 2008)
CipI expression studies
The expression of CipI-encoding gene in UA159 wild-type, ΔLiaS and ΔLiaR strains was
quantified by real-time reverse-transcription (RT)-PCR. Briefly, cells were harvested and total
RNA was extracted using the Bio101 Fast Prep System (Qbiogen) and Trizol reagent
(Invitrogen). DNA-free RNA samples were then reverse-transcribed using the First-Strand cDNA
Synthesis Kit (MBI Fermentas) in preparation for real-time RT-PCR using the QuantiTech SYBR
Green PCR kit in an Mx3000P QPCR system (Stratagene). Gene expression was determined
using the following formula: Ratio = (EcipI)∆Ct(control-test)/(E16SrRNA)∆Ct(control-test), where E = (10-1/slope)
109
represents the efficiency of gene amplification. The 16S rRNA gene was used as internal
reference as we found the expression of this gene to be stable under the test conditions. All
assays were performed in triplicate with RNA isolated from three independent experiments and
using a P ≤0.01.
Biofilm assays
Static biofilms were developed in polystyrene microtiter plates at 37°C with 5% CO2 using semi-
defined minimal medium (SDM) containing 1% sucrose as previously described (Perry et al.,
2008). After 16 h of growth, the planktonic phase was carefully removed, and fresh SDM-
sucrose alone (control) or containing 2 µM synthetic CSP (sCSP) pheromone was overlayed
onto established biofilms and the plates were incubated for a further 5 h. The same experiment
was also repeated with SDM-sucrose or SDM-sucrose + sCSP supplemented with 50 U/ml
DNase I (Fermentas). The upper phase was then removed, and biofilms were allowed to dry
overnight before staining with crystal violet for biomass quantification. Purification and
quantification of eDNA in 16-h biofilms were performed according to (Rice et al., 2007). The
amount of eDNA was determined using real-time RT-PCR, using four sets of primers designed
to amplify genes randomly distributed across the S. mutans genome. CT expression values
were averaged, and normalized to the expression in the wild-type strain. All assays were
performed in triplicate from three independent experiments.
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3.4 Results and discussion
3.4.1 Low cell density-dependent expression of cipI is regulated by LiaR
In this work, we sought to characterize the regulation of the cell death immunity protein CipI.
Since previous findings indicated that CipI was differentially regulated from the death effector
protein CipB at low cell density, we first attempted to identify the regulator responsible for its
differential expression. We hypothesized that the regulator responsible for cipI expression at low
cell density would be one of the thirteen known TCSs in the S. mutans UA159 genome. TCSs
are typically composed of a membrane-bound histidine kinase sensor, which phosphorylates a
cytoplasmic response regulator when triggered by environmental stimuli (Stock et al., 2000). We
screened mutants defective in each TCS (Lévesque et al., 2007) for differential growth kinetics
under high concentrations of sCSP pheromone, conditions known to increase gene expression
of both the CipB bacteriocin and the CipI immunity protein (Perry et al., 2009). Inactivation of the
response regulator LiaR (originally referred to as RR11 (Li et al., 2002a; Perry et al., 2008;
Suntharalingam et al., 2009) resulted in an increased sensitivity to sCSP, while inactivation of its
cognate histidine kinase sensor LiaS (formerly known as HK11) showed an increased
resistance to sCSP compared to the wild-type (Fig. 3.1). These results implicate the LiaSR TCS
in regulation of cipI expression. To prove the direct regulation of cipI by LiaSR at low cell
density, we compared the expression of cipI between high and low cell density in the wild-type
strain, a ΔLiaS mutant and a ΔLiaR mutant. While the wild-type strain showed a highly
significant increase in cipI gene expression upon dilution from high to low cell density (120.6 ±
24.3-fold), little- to no-change in cipI expression was found in ΔLiaS and ΔLiaR mutant strains
(7.3 ± 7.6-fold and 17.3 ± 3.8-fold, respectively). Moreover, we found a 64.6 ± 4.4-fold increase
in liaR itself upon dilution of the wild-type strain from an overnight culture. Together, our results
111
suggest that the LiaSR TCS is responsible for regulation of cipI gene expression at low cell
density.
Figure 3.1: Growth of S. mutans TCS mutants in the presence of 2 µM sCSP. All thirteen
TCSs in the UA159 genome were inactivated (Levesque et al., 2007) and assayed for their
growth kinetics in the presence of sCSP. The TCS composed of LiaS and LiaR showed
differential sensitivity to sCSP compared to the wild-type strain, with the histidine kinase sensor
deficient mutant (LiaS) showing increased resistance and the response regulator deficient
mutant (LiaR) showing increased sensitivity to sCSP.
3.4.2 CipI is protective at low cell density, while CipB is lethal at high cell density
Having now shown that cipI is differentially regulated at low cell density through the LiaSR TCS,
we next sought to determine if the up-regulation of cipI expression at low cell density protected
112
S. mutans cells from CSP-induced autolysis via the action of CipB bacteriocin. To test this
hypothesis, we pre-grew aliquots of a UA159 overnight culture in THYE broth without sCSP for
up to 2.5 hr to induce expression of CipI at low cell density, before exposing them to 2 µM
sCSP. We found that inducing CipI expression before sCSP exposure had a protective effect on
the culture (Fig. 3.2).
Although CipI up-regulation at low cell density has a protective effect, cultures begin to
experience significant levels of autolysis as growth progresses in presence of sCSP,
culminating at stationary phase (Perry et al., 2009). How is the transition to autolysis
accomplished in these cultures? We hypothesized that death due to the CipB/CipI system would
occur at stationary phase if the CipB bacteriocin was expressed at higher levels than its CipI
immunity factor at high cell density. Indeed, the gene expression of CipB is induced 60.5 ± 1.2-
fold at stationary phase vs. early log phase, while the gene expression of CipI remains
unchanged (-0.6 ± 2.3-fold change in expression) as determined by real-time RT-PCR analysis.
It is tempting to speculate on the implications of these results in the physiological context of
cell death. Autolysis is often found linked to signals that convey high population density, since
the purpose of cell death in a unicellular organism is to provide benefits (e.g., provide nutrients,
space or transforming DNA; eliminate competition) to sibling cells (Claverys & Havarstein,
2007). Lysis of a sparse population serves no purpose since sibling cells are unlikely to benefit,
and is therefore counter-selected by evolution. The unlinked transcriptional units encoding CipB
and CipI may therefore serve to protect cells from lysis at low cell density to allow expansion of
the clonal population. Conversely, when nutrients become scarce at high cell density, autolysis
is triggered through CipB up-regulation to provide a competitive growth advantage to surviving
siblings. Nowhere is the above high-cell density-mediated altruistic process more likely to occur
than in a biofilm. In a previous report, we found that the expression of cipI is down-regulated
2.1-fold through LiaR in biofilm cells compared to their planktonic counterparts (Perry et al.,
113
2008). In addition, others have reported the up-regulation of cipB expression in the biofilm
phase (Shemesh et al., 2007). We therefore hypothesized that the CipB/CipI autolysis cascade
is inhibited during rapid growth in planktonic cultures, but is induced during the high cell density
biofilm growth mode of S. mutans.
Figure 3.2: Growth kinetics of S. mutans UA159 wild-type strain in the presence of 2µM sCSP.
Overnight cultures were pre-grown for 2.5 hr before sCSP addition, to induce expression of the
CipI immunity protein-encoding gene at low cell density. Control cultures were exposed to sCSP
directly upon dilution from an overnight culture („no pre-growth‟), or were grown in the absence
of sCSP („control‟). The induction of cipI expression before sCSP addition confers a protective
effect on the culture, reflected in an increase in initial growth rate compared to the no pre-growth
curve.
114
3.4.3 Cell death participates in biofilm formation through eDNA release
From the outset, both the CSP-ComDE and the LiaSR systems have been implicated in
S. mutans biofilm formation (Li et al., 2002a; Li et al., 2002b). Recently, Zhang and coll.
reported that exogenous CSP addition to S. mutans resulted in increased in both cell death and
biofilm biomass (Zhang et al., 2009). These authors also reported that scanning electron
microscopy of a mutant unable to synthesize the CSP signal molecule produced biofilms
composed of loosely attached, single cells, but that complementing the mutant with exogenous
sCSP resulted in formation of large aggregates with abundant extracellular matrix. Reports have
also suggested that autolysis is necessary for S. mutans biofilm formation (Wen & Burne, 2002),
and that CSP induces eDNA release (Petersen et al., 2005). However, none of these studies
has provided a mechanistic explanation for CSP-induced release of eDNA. We first sought to
link cell lysis in the biofilm to the CipB/CipI system by measuring the amount of eDNA in 16-hr
biofilms formed by the wild-type strain, ΔCipB and ΔCipI mutants. Using real-time RT-PCR, we
found a significant decrease in eDNA in the ΔCipB mutant biofilm and a significant increase in
eDNA in the ΔCipI mutant biofilm compared to the wild-type (Fig. 3.3). We conclude from this
result that death due to the CipB/CipI system can influence the amount of eDNA in the biofilm
matrix via autolysis.
To test whether the modulation of eDNA release via the CipB/CipI autolysis cascade affects
S. mutans biofilm biomass, 16-hr biofilms were treated with DNase I and the biomass quantified
by crystal violet staining. DNase treatment of biofilms formed by the wild-type strain decreased
their biomass by more than 20% (Fig. 4). In either case, this result confirms previous reports
that eDNA plays an important role in S. mutans biofilm formation (Petersen et al., 2005). We
next assayed the ΔCipB and ΔCipI mutant strains for their ability to form biofilms. Importantly,
ΔCipI formed biofilms with greater biomass than the wild-type strain even in the absence of
115
Figure 3.3: Fold-change in quantity of eDNA in ΔCipI and ΔCipB mutant biofilms compared to
the UA159 wild-type control. Quantitative real-time RT-PCR was used to amplify four randomly
selected chromosomal genes from DNA extracted from the extracellular matrix of UA159, ΔCipI
and ΔCipB biofilms. Amplification values for UA159 were arbitrarily set at one, and results are
expressed as a fold-change relative to the UA159 wild-type. Standard deviation represents the
variation in amplification across the four chromosomal genes selected. Both CipI and CipB
results represent significant differences (P value ≤ 0.01) from the UA159 wild-type control.
116
sCSP, as observed by an increased biomass of ~ 30% (Fig. 3.4). The ΔcipI mutant cells
possess an intact CSP-encoding gene, and are able to produce endogenous CSP pheromone
during biofilm formation. When grown planktonically, the ΔCipI mutant is approximately 10-times
more sensitive to sCSP than the wild-type (unpublished data), and is likely succumbing to the
accumulation of endogenous CSP in the biofilm. The increase in biomass is due solely to eDNA
release, since treatment with DNase I restored the biofilm biomass to wild-type DNase-treated
levels (Fig. 3.4). Interestingly, the ΔCipB mutant also formed more biofilm biomass than the
wild-type. However, when the ΔCipB mutant biofilms were treated with DNase I, the biofilm
biomass was unchanged (Fig. 3.4). This result indicates that ΔCipB has a growth advantage in
the biofilm due to its resistance to autolysis, and the increase in biofilm biomass appears to be
mostly due to increasing cell number.
Finally, we added sCSP to established biofilms to induce CipB-mediated autolysis. As
expected, adding sCSP to UA159 and ΔCipI mutant biofilms increased the biofilm biomass
beyond CSP-uninduced conditions (Fig. 3.4). The increase in biomass was again due to eDNA
in the extracellular matrix, since DNase I restored the biomass to wild-type levels. These results
suggest that cell death has a positive impact on biofilm biomass through the release of eDNA
into the extracellular matrix, through the endogenous CSP-induced CipB/CipI-mediated cell
death pathway.
117
Figure 3.4: Biofilm biomass of S. mutans UA159 wild-type strain, ΔCipB and ΔCipI mutants.
Biofilms were allowed to develop for 16 h in SDM-sucrose. The planktonic phase was then
removed, and fresh SDM-sucrose alone (control) or supplemented with 2 µM sCSP was
overlayed onto the biofilms and incubated for a further 5 h. The same experiment was repeated
with SDM-sucrose supplemented with 50 U/ml DNase in the overlay. Biofilms were washed
once with sterile dH2O before quantification. Quantification of biomass was performed by optical
density (OD) of crystal violet-stained UA159, ΔCipB and ΔCipI biofilms. Results are expressed
as a % increase in biofilm biomass compared to the biofilm biomass of the UA159 wild-type
control, which was set at 100%. Significant (P <0.02) increase in biomass compared to the
UA159 wild-type control condition is denoted with a „*‟, while significant decrease in biomass
compared to the UA159 wild-type control is denoted with a „†‟.
118
3.5 Conclusions
The lifecycle of a biofilm includes periods of both exponential growth and of nutrient limitation
(Stoodley et al., 2002). The cooperative nature of biofilm growth has been likened to the task-
sharing behavior common in higher-order multicellular organisms. As such, the biofilm lifestyle
may permit altruistic behaviors like autolysis in unicellular prokaryotic organisms, which can
contribute nutrients for the continued survival of siblings in a stressed population. Our recent
findings led us to propose a mechanistic explanation implicating CSP pheromone during the
development of S. mutans biofilm. At low cell density, S. mutans up-regulates expression of the
CipI immunity protein through the LiaSR TCS. Up-regulation of CipI has a protective effect on
the cell, and allows the culture to proliferate under favorable environmental conditions.
Conversely, in the high cell density biofilm environment, the high concentrations of CSP
pheromone signal up-regulation of the CipB autolysis effector through the ComDE TCS.
Altruistic autolysis in the S. mutans biofilm contributes nutrients for the continued survival of the
population as a whole, as well as eDNA to the extracellular matrix.
119
3.6 Acknowledgements
This study was supported by CIHR-Priority Announcement IMHA Grant FRN-90114 (to
C.M.L.) and by National Institute of Dental and Craniofacial Research grant R01
DE013230-08 to D.G.C. DGC is supported by a Canada Research Chair. J.A.P is
supported by a CIHR Strategic Training Fellowship in Cell Signaling in Mucosal
Inflamation and Pain.
120
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Chapter 4: Involvement of Streptococcus mutans regulator RR11 in
oxidative stress response during biofilm growth and in the
development of genetic competence
JA Perry, CM Lévesque, P Suntharaligam, RW Mair, M Bu, RT Cline, SN Peterson and DG Cvitkovitch. 2008. Lett Appl Microbiol 47: 439-44.
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4.1 Abstract
Aims: To identify the genes regulated by RR11, the regulator component of the
Streptococcus mutans HK/RR11 two-component signal transduction system.
Methods and Results: The S. mutans RR11-encoding gene was inactivated and the effect of
gene disruption on the cell‟s ability to form biofilms was tested. DNA microarray used to
decipher RR11-regulated genes during biofilm growth showed that ~5% of the UA159 genome
underwent a significant change in gene expression. RR11 was found to regulate 174 genes,
including genes involved in competence development, stress-response, and cell division.
Phenotypic assessment of biofilms showed a reduction in biomass in cells lacking RR11
following exposure to oxidative stress. RR11 defective cells showed ~20-fold reduction in
transformation efficiency.
Conclusions: Target genes controlled by RR11during biofilm growth were identified by a
comparison of transcriptional profiles between an RR11 mutant and the parental strain. The
results demonstrated that RR11 is involved in the control of processes such as the formation of
biofilm under oxidative stress and development of genetic competence.
Significance and Impact of Study: Mapping the RR11 signal transduction pathway in S.
mutans biofilms has determined its involvement in stress tolerance. Targeting this pathway may
lead to future antimicrobial therapies designed specifically for biofilm infections.
125
4.2 Introduction
Streptococcus mutans causes dissolution of tooth enamel by producing acid from dietary
carbohydrates, and is considered one of the principal etiological agents of human dental
caries(Mitchell, 2003). It has evolved a biofilm lifestyle to survive and persist in the oral cavity,
where two-component systems are employed to sense and respond to environmental stimuli.
Changing environmental conditions are sensed by the membrane-bound histidine kinase (HK)
receptor, resulting in autophosphorylation. The resulting phosphoryl group is transferred to the
cognate cytosolic response regulator (RR), which acts as a transcriptional regulator (Stock et
al., 2000). One of the thirteen putative two-component systems found in S. mutans UA159
consists of the HK11 sensor and the regulator RR11(Ajdic et al., 2002; Levesque et al., 2007).
This system has a role in cell segregation, and in the response to thermal, oxidative, and acid
stresses (Biswas et al., 2007; Li et al., 2002a). Although HK/RR11 has been implicated in
S. mutans biofilm development, the genes controlled by RR11 during biofilm growth are still
unknown (Li et al., 2002a).
In the present study, DNA microarrays were used to determine gene expression under
biofilm and planktonic growth phases in wild-type S. mutans and its rr11– defective derivative to
determine the role of RR11 in biofilm development. Microarray data indicated that RR11 is
responsible for the induction of genes involved in general stress response in the biofilm. The
ability of an RR11-defective mutant to grow under stress in the biofilm and become competent
for genetic transformation was also investigated.
126
4.3 Materials and methods
Bacterial strains and growth conditions
Bacterial strains are found in Table 4.1. Antibiotics were added when required as follows: 10 µg
ml-1 erythromycin, 1000 µg ml-1 spectinomycin, or 500 µg ml-1 kanamycin. Mutants were
constructed in strain UA159 as described previously (Lau et al., 2002). The erythromycin and
spectinomycin resistance cassettes were amplified from plasmid pALN122 (Macrina et al.,
1983) and pDL277 (Leblanc et al., 1992) using the primer pairs Erm19/Erm20 (Levesque et al.,
2005) and SpecF/SpecR (Aspiras et al., 2004), respectively. Primer sequences for mutant
construction are available upon request.
Table 4.1. Bacterial strains used in this study. All strains were grown in Todd Hewitt Yeast
Extract (THYE) broth at 37ºC in air with 5% CO2.
Strain
Relevant characteristicsa
Source
UA159 Wild-type strain; Ems Kms Sps J. Ferretti, U. of
Oklahoma
SMRR11 UA159∆(rr11); Emr This work
SMComDE UA159∆(comDE); Emr This work
SMCiaHR UA159∆(ciaHR); Emr This work
SMDE11 UA1591∆(comDE, rr11); Emr Spr This work
aEm: erythromycin; Km: kanamycin; Sp: spectinomycin.
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In vitro model for growing biofilms
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
128
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).
129
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)
130
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).
133
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
134
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.
136
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.
138
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.
145
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.
146
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
SMU.1073 putative formyl-tetrahydrofolate synthetase -3.3
SMU.1265 putative phosphoribosyl formimino-5-aminoimidazole carboxamide ribonucleotide isomerase-2.0
SMU.1266 putative glutamine amidotransferase HisH -2.1
SMU.1268 putative imidazoleglycerol-phosphate dehydratase -2.2
SMU.1269 putative phosphoserine phosphatase -1.9
SMU.1270 putative histidinol dehydrogenase -2.3
SMU.1271 putative ATP phosphoribosyltransferase -2.1
SMU.1273 putative histidinol-phosphate aminotransferase -2.4
SMU.1877 putative PTS system, mannose-specific component IIAB -2.9
SMU.531 putative chorismate mutase 4.3
SMU.532 putative anthranilate synthase, alpha subunit 3.4
SMU.534 putative phosphoribosyl anthranilate transferase 3.4
SMU.535 putative indoleglycerol phosphate synthase 3.7
SMU.536 putative phosphoribosyl anthranilate isomerase 2.5
SMU.537 putative tryptophan synthase, beta subunit 2.5
SMU.538 putative tryptophan synthase, alpha subunit 2.8
Biosynthesis of cofactors, prosthetic groups, and carriers
SMU.1996 putative isopentenyl monophosphate kinase 3.3
SMU.353 conserved hypothetical protein 5.3
SMU.838 glutathione reductase 6.5
SMU.954 putative pyridoxal kinase 2.3
Cell envelope
SMU.109 conserved hypothetical protein; possible permease (efflux protein) 5.7
SMU.1196c conserved hypothetical protein 2.3
SMU.1677 putative UDP-N-acetylmuramoylananine-D-glutamate-2,6- diaminopimelate ligase; UDP-MurNac-tripeptide synthetase2.4
SMU.196c putative transfer protein 3.0
SMU.2075c conserved hypothetical protein 2.9
SMU.2081 hypothetical protein 7.5
SMU.539c signal peptidase type IV 22.4
SMU.610 cell surface antigen SpaP -2.5
SMU.627 conserved hypothetical protein 4.5
SMU.63c conserved hypothetical protein 5.3
SMU.67 putative acyltransferase 4.0
SMU.883 dextran glucosidase DexB -5.6
Cellular processes
SMU.1001 putative DNA processing Smf protein 19.0
SMU.1279c putative cell division protein (cell shape determining protein) 2.0
SMU.1343c putative polyketide synthase -3.4
SMU.1346 putative thioesterase BacT -2.5
SMU.150 hypothetical protein 11.2
SMU.1862 hypothetical protein 2.4
SMU.1897 putative ABC transporter, ATP-binding protein 9.2
SMU.1898 putative ABC transporter, ATP-binding and permease protein 4.2
SMU.1900 conserved hypothetical protein 5.9
SMU.1905c putative bacteriocin secretion protein 10.1
SMU.1906c hypothetical protein 11.4
148
SMU.1914c hypothetical protein 20.4
SMU.1916 putative histidine kinase of the competence regulon, ComD 10.5
SMU.1917 putative response regulator of the competence regulon, ComE 11.3
SMU.1983 putative competence protein ComYD 26.6
SMU.1984 putative competence protein ComYC 26.2
SMU.1985 putative ABC transporter ComYB; probably part of the DNA transport machinery 23.9
SMU.1987 putative ABC transporter, ATP-binding protein ComYA; late competence gene 17.5
SMU.1997 16S ribosomal RNA 14.3
SMU.2084c conserved hypothetical protein 5.6
SMU.400 putative secreted esterase 2.8
SMU.423 hypothetical protein 14.6
SMU.426 copper-transporting ATPase; P-type ATPase 7.3
SMU.499 putative late competence protein 18.0
SMU.54 putative amino acid recemase -3.5
SMU.625 putative competence protein 14.1
SMU.626 putative competence protein 25.6
SMU.629 putative manganese-type superoxide dismutase, Fe/Mn-SOD -2.2
SMU.632 putative transcriptional regulator 2.4
SMU.644 putative competence protein/transcription factor 27.2
SMU.655 putative MutE 3.9
SMU.753 conserved hypothetical protein 4.0
Central intermediary metabolism
SMU.636 putative N-acetylglucosamine-6-phosphate isomerase 3.1
DNA metabolism
SMU.1002 putative DNA topoisomerase I 8.2
SMU.1034c putative integrase/recombinase; XerC-like 2.2
SMU.1055 putative DNA repair protein RadC 14.1
SMU.1967 putative single-stranded DNA-binding protein 16.4
SMU.2085 recombination protein RecA 6.7
SMU.2086 putative competence and damage inducible protein CinA 10.2
SMU.327 putative DNA repair protein 3.2
SMU.505 putative adenine-specific DNA methylase 11.8
SMU.506 putative type II restriction endonuclease 9.0
SMU.64 Holliday junction DNA helicase RuvB 8.0
Energy metabolism
SMU.1004 glucosyltransferase-I 6.0
SMU.127 putative acetoin dehydrogenase (TPP-dependent), E1 component alpha subunit -2.2
SMU.128 putative acetoin dehydrogenase (TPP-dependent), E1 component beta subunit -2.5
SMU.129 putative dihydrolipoamide acetyltransferase -2.6
SMU.130 putative dihydrolipoamide dehydrogenase -2.7
SMU.1327c conserved hypothetical protein; possible 4Fe-4S ferredoxin 3.0
SMU.1424 putative dihydrolipoamide dehydrogenase 2.9
SMU.148 putative alcohol-acetaldehyde dehydrogenase 3.4
SMU.1978 putative acetate kinase 9.0
SMU.2037 putative trehalose-6-phosphate hydrolase TreA 6.7
SMU.352 putative ribulose-phosphate-3-epimerase 6.0
SMU.402 pyruvate formate-lyase -2.2
SMU.772 glucan-binding protein D with lipase activity; BglB-like protein 10.0
SMU.79 fructan hydrolase; exo-beta-D-fructosidase; FruB -4.5
149
SMU.877 alpha-galactosidase -3.3
SMU.881 sucrose phosphorylase, GtfA -5.8
SMU.886 galactokinase, GalK -2.6
SMU.887 galactose-1-P-uridyl transferase, GalT -2.6
SMU.888 UDP-galactose 4-epimerase, GalE -2.7
Fatty acid and phospholipid metabolism
SMU.1344c putative malonyl-CoA acyl-carrier-protein transacylase -2.9
SMU.1345c putative peptide synthetase similar to MycA -2.7
Hypothetical proteins
SMU.108 hypothetical protein 4.9
SMU.1197 tRNA-Arg 2.6
SMU.1267c hypothetical protein -2.1
SMU.1438c putative Zn-dependent protease 2.1
SMU.151 hypothetical protein 12.4
SMU.1651 putative arsenate reductase 2.4
SMU.167 hypothetical protein 10.0
SMU.1904c hypothetical protein 12.2
SMU.1915 competence stimulating peptide, precursor 3.8
SMU.1956c hypothetical protein -5.0
SMU.1979c conserved hypothetical protein 22.0
SMU.1980c conserved hypothetical protein 24.6
SMU.1982c conserved hypothetical protein 22.3
SMU.199c hypothetical protein 2.8
SMU.202c hypothetical protein 2.8
SMU.205c hypothetical protein 3.5
SMU.2076c hypothetical protein 10.1
SMU.2077c conserved hypothetical protein 2.7
SMU.2078c conserved hypothetical protein 2.4
SMU.2079c conserved hypothetical protein 2.8
SMU.2080 conserved hypothetical protein 3.3
SMU.209c hypothetical protein 3.8
SMU.212c hypothetical protein 3.8
SMU.217c hypothetical protein 2.5
SMU.326 conserved hypothetical protein 3.3
SMU.470 conserved hypothetical protein 2.4
SMU.503c hypothetical protein -2.2
SMU.53 conserved hypothetical protein -3.5
SMU.56 conserved hypothetical protein -3.1
SMU.649 conserved hypothetical protein -2.0
SMU.840c hypothetical protein 2.1
SMU.959c hypothetical protein -2.2
SMU.1047c hypothetical protein 6.2
SMU.1056 hypothetical protein 5.7
SMU.1069c hypothetical protein 3.7
SMU.1147c hypothetical protein 5.0
SMU.1250c hypothetical protein 1.9
SMU.152 hypothetical protein 16.1
SMU.153 hypothetical protein 12.2
SMU.166 hypothetical protein 9.1
SMU.1902c hypothetical protein 9.8
150
SMU.1903c hypothetical protein 16.0
SMU.1907 hypothetical protein 8.9
SMU.1908c hypothetical protein 18.3
SMU.1909c hypothetical protein 19.6
SMU.1910c hypothetical protein 18.3
SMU.1912c hypothetical protein 22.2
SMU.1913c putative immunity protein, BLpL-like 15.2
SMU.1976c hypothetical protein 3.6
SMU.200c hypothetical protein 2.8
SMU.204c hypothetical protein 3.0
SMU.2083c hypothetical protein 4.0
SMU.210c hypothetical protein 3.2
SMU.215c hypothetical protein 3.2
SMU.216c hypothetical protein 2.5
SMU.378 hypothetical protein 2.5
SMU.41 hypothetical protein 2.7
SMU.49 hypothetical protein -2.9
SMU.55 hypothetical protein -3.6
SMU.58 hypothetical protein -3.1
SMU.637c hypothetical protein 2.9
SMU.68 hypothetical protein 2.9
SMU.735 hypothetical protein 2.1
SMU.771c hypothetical protein 8.0
SMU.925 hypothetical protein 18.2
Mobile and extrachromosomal element functions
SMU.149 putative transposase 4.1
SMU.195c hypothetical protein; similar to ORF 5 of bacteriophage SPP1 3.3
SMU.198c putative conjugative transposon protein 2.6
SMU.2027 putative transcriptional regulator 4.7
Protein fate
SMU.131 putative lipoate-protein ligase -2.4
SMU.645 putative oligopeptidase 10.4
Protein synthesis
SMU.1044c putative pseudouridylate synthase 2.5
SMU.1512 putative phenylalanyl-tRNA synthetase (alpha subunit) -2.3
SMU.154 30S ribosomal protein S15 2.2
SMU.1886 putative seryl-tRNA synthetase -2.1
SMU.2000 50S ribosomal protein L17 -2.3
SMU.2002 30S ribosomal protein S11 -2.1
SMU.2012 30S ribosomal protein S8 -2.1
SMU.2014 30S ribosomal protein S14 -2.0
SMU.2015 50S ribosomal protein L5 -2.5
SMU.2016 50S ribosomal protein L24 -2.1
SMU.2017 50S ribosomal protein L14 -2.1
SMU.2022 50S ribosomal protein L22 -2.3
SMU.500 putative ribosome-associated protein 2.7
SMU.558 isoleucine-tRNA synthetase -2.1
Purines, pyrimidines, nucleosides, and nucleotides
SMU.30 putative phosphoribosylformylglycinamidine synthase, (FGAM synthase) -2.6
SMU.325 putative dUTPase 4.1
SMU.356 purine operon repressor 3.8
151
SMU.48 putative phosphoribosylamine-glycine ligase; phosphoribosyl glycinamide synthetase (GARS)-2.4
SMU.50 putative phosphoribosylaminoimidazole carboxylase, catalytic subunit -2.6
SMU.51 putative phosphoribosylaminoimidazole carboxylase, ATPase subunit -3.4
SMU.668c ribonucleotide reductase, large subunit -2.0
Regulatory functions
SMU.1048 conserved hypothetical protein 2.4
SMU.1145c putative histidine kinase; homolog of RumK and ScnK (HK3) 2.4
SMU.1193 putative transcriptional regulator 2.5
SMU.1409c putative transcriptional regulator 2.4
SMU.1509 putative transcriptional regulator 2.2
SMU.168 putative transcriptional regulator 8.7
SMU.1964c putative response regulator (RR9) 4.8
SMU.1977c putative transcriptional regulator 3.8
SMU.207c putative transposon protein 4.4
SMU.424 negative transcriptional regulator, CopY 8.4
SMU.507 putative transcriptional regulator (DeoR family) 6.0
SMU.61 putative transcriptional regulator 3.1
SMU.65 putative protein tyrosine-phosphatase 6.6
SMU.80 transcriptional regulator; repressor (HrcA) of class I heat shock genes 3.0
SMU.927 putative response regulator (RR4) 4.8
SMU.928 putative histidine kinase (HK4) 5.3
Signal transduction
SMU.1957 putative PTS system, mannose-specific IID component -5.1
SMU.1958c putative PTS system, mannose-specific IIC component -4.4
SMU.1960c putative PTS system, mannose-specific IIB component -3.4
SMU.1961c putative PTS system, sugar-specific enzyme IIA component -3.2
SMU.1965c putative histidine kinase (HK9) 5.5
Transcription
SMU.2001 DNA-directed RNA polymerase, alpha subunit -2.2
Transport and binding proteins
SMU.1006 putative ABC transporter, ATP-binding protein 2.9
SMU.1067c putative ABC transporter, permease protein 2.7
SMU.1068c putative ABC transporter, ATP-binding protein 3.0
SMU.1148 putative transporter, ATP-binding protein; bacteriocin immunity protein 2.4
SMU.1185 PTS system, mannitol-specific enzyme IIBC component 2.4
SMU.1194 putative ABC transporter, ATP-binding protein 2.2
SMU.1195 conserved hypothetical protein; possible permease 2.0
SMU.1848 hypothetical protein 2.1
SMU.1878 putative PTS system, mannose-specific component IIC -3.0
SMU.1879 putative PTS system, mannose-specific component IID -3.9
SMU.1899 putative ABC transporter, ATP-binding and permease protein (fragment) 5.2
SMU.1963c putative sugar-binding periplasmic protein 4.0
SMU.1966c putative periplasmic sugar-binding protein 7.1
SMU.2038 putative PTS system, trehalose-specific IIABC component 5.3
SMU.242c putative amino acid ABC transporter, permease protein, glutamine transport system -2.0
SMU.427 putative copper chaperone 6.5
SMU.862 conserved hypothetical protein; putative permease 2.1
SMU.863 putative ABC transporter, ATP-binding protein 1.9
SMU.864 putative ABC transporter, permease component 2.0
SMU.872 putative PTS system, fructose-specific enzyme IIABC component -2.1
152
SMU.878 multiple sugar-binding ABC transporter, sugar-binding protein precursor MsmE -4.4
SMU.879 multiple sugar-binding ABC transporter, permease protein MsmF -5.4
SMU.880 multiple sugar-binding ABC transporter, permease protein MsmG -5.6
SMU.882 multiple sugar-binding ABC transporter, ATP-binding protein, MsmK -5.9
Unclassified
SMU.1111c conserved hypothetical protein 2.0
SMU.1342 putative bacitracin synthetase 1; BacA -2.9
SMU.1372c hypothetical protein 2.2
SMU.193c conserved hypothetical protein 2.9
SMU.1981c conserved hypothetical protein 25.9
SMU.1988c putative DNA binding protein 2.0
SMU.2057c putative cadmium-transporting ATPase; P-type ATPase 4.0
SMU.214c hypothetical protein 2.2
SMU.219 hypothetical protein 2.0
SMU.52 conserved hypothetical protein -3.6
SMU.73 conserved hypothetical protein -2.7
SMU.758c conserved hypothetical protein 3.3
SMU.769 conserved hypothetical protein 9.5
SMU.78 fructan hydrolase; exo-beta-D-fructosidase; fructanase, FruA -5.5
SMU.836 hypothetical protein 18.3
Unknown function
SMU.1003 putative glucose-inhibited division protein 7.1
SMU.1046c putative GTP pyrophosphokinase 2.4
SMU.1053 conserved hypothetical protein 6.4
SMU.1054 putative glutamine amidotransferase 6.0
SMU.1070c conserved hypothetical protein 3.4
SMU.1322 putative acetoin dehydrogenase 2.0
SMU.1323 conserved hypothetical protein; possible hydrolase 2.0
SMU.1340 putative surfactin synthetase -2.0
SMU.1341c putative gramicidin S synthetase -2.4
SMU.1400c conserved hypothetical protein 4.7
SMU.1975c conserved hypothetical protein; possible membrane protein 2.5
SMU.208c putative transposon protein; possible DNA segregation ATPase 3.3
SMU.328 putative carbonic anhydrase 2.2
SMU.354 conserved hypothetical protein 6.2
SMU.355 putative CMP-binding factor 6.8
SMU.399 conserved hypothetical protein 2.5
SMU.401c conserved hypothetical protein 2.5
SMU.498 putative late competence protein 22.4
SMU.508 conserved hypothetical protein 7.2
SMU.641 putative oxidoreductase 2.1
SMU.646 putative phosphatase 9.5
SMU.647 putative methyltransferase 2.7
SMU.66 conserved hypothetical protein 5.0
SMU.72 conserved hypothetical protein -2.4
SMU.807 putative membrane protein 2.1
SMU.837 putative reductase 14.1
SMU.890 conserved hypothetical protein 2.0
SMU.926 conserved hypothetical protein; possible GTP-pyrophosphokinase 5.4
153
SI Table S2. S. mutans genes showing a minimum ± 2-fold difference in expression when
S. mutans ∆comX cells were exposed to 2 μM sCSP
Gene ID Putative or assigned function FoldSMU.925 hypothetical protein 2.7
SMU.150 hypothetical protein 5.3
SMU.151 hypothetical protein 4.9
SMU.152 hypothetical protein 5.4
SMU.153 hypothetical protein 5.2
SMU.1902c hypothetical protein 2.5
SMU.1903c hypothetical protein 5.0
SMU.1904c hypothetical protein 5.1
SMU.1905c putative bacteriocin secretion protein 5.1
SMU.1906c hypothetical protein 4.8
SMU.1908c hypothetical protein 4.9
SMU.1909c hypothetical protein 5.7
SMU.1910c hypothetical protein 5.6
SMU.1912c hypothetical protein 4.9
SMU.1913c putative immunity protein, BLpL-like 5.2
SMU.1914c hypothetical protein 4.5
SMU.2037 putative trehalose-6-phosphate hydrolase TreA 2.1
SMU.2038 putative PTS system, trehalose-specific IIABC component 2.3
SMU.41 hypothetical protein -3.3
SMU.423 hypothetical protein 5.8
SMU.424 negative transcriptional regulator, CopY 2.0
SMU.426 copper-transporting ATPase; P-type ATPase 2.5
SMU.427 putative copper chaperone 2.1
SMU.63c conserved hypothetical protein 2.7
SMU.64 Holliday junction DNA helicase RuvB 2.7
SMU.65 putative protein tyrosine-phosphatase 2.8
SMU.66 conserved hypothetical protein 2.1
SMU.78 fructan hydrolase; exo-beta-D-fructosidase; fructanase, FruA -2.0
SMU.799c conserved hypothetical protein 2.6
SMU.877 alpha-galactosidase -2.2
SMU.878 multiple sugar-binding ABC transporter, sugar-binding protein precursor MsmE -2.7
SMU.879 multiple sugar-binding ABC transporter, permease protein MsmF -2.6
SMU.880 multiple sugar-binding ABC transporter, permease protein MsmG -2.5
SMU.881 sucrose phosphorylase, GtfA -2.8
SMU.882 multiple sugar-binding ABC transporter, ATP-binding protein, MsmK -2.4
SMU.883 dextran glucosidase DexB -2.8
SMU.887 galactose-1-P-uridyl transferase, GalT -2.2
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