1

1

The formation of Streptococcus mutans persisters induced by the quorum-2

sensing peptide pheromone is affected by the LexA regulator 3

4

Vincent Leung,a Dragana Ajdic,

b Stephanie Koyanagi,

a Céline M. Lévesque

a* 5

6

Dental Research Institute, Faculty of Dentistry, University of Toronto, Toronto, Ontario, 7

Canadaa, Department of Dermatology and Cutaneous Surgery, University of Miami Miller 8

School of Medicine, Miami, Florida, USAb 9

10

*Corresponding author. Dental Research Institute, Faculty of Dentistry, University of Toronto, 11

124 Edward St., Room 454, Toronto, Ontario M5G 1G6, Canada. Phone: (416) 979-4917, ext. 12

4313. Fax: (416) 979-4936. 13

E-mail: celine.levesque@dentistry.utoronto.ca. 14

15

Running title: Contribution of LexA to CSP-induced persisters 16

17

JB Accepted Manuscript Posted Online 12 January 2015J. Bacteriol. doi:10.1128/JB.02496-14Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 18

The presence of multidrug tolerant persister cells within microbial populations has been 19

implicated with the resiliency of bacterial survival against antibiotic treatments, and is a major 20

contributing factor in chronic infections. The mechanisms by which these phenotypic variants 21

are formed have been linked to stress-response pathways in various bacterial species, but many 22

of these mechanisms remain unclear. We have previously shown that in the cariogenic organism 23

Streptococcus mutans, the quorum-sensing peptide CSP pheromone was a stress inducible 24

alarmone that triggered an increased formation of multidrug-tolerant persisters. In this study, we 25

characterized SMU.2027, a CSP-inducible gene encoding a LexA ortholog. We showed that in 26

addition to exogenous CSP exposure, stressors including heat shock, oxidative stress, and 27

ofloxacin antibiotic were capable of triggering expression of lexA in an auto-regulatory manner 28

akin to LexA-like transcriptional regulators. We demonstrated the role of LexA and its 29

importance in regulating tolerance towards DNA damage in a non-canonical SOS mechanism. 30

We showed its involvement and regulatory role in the formation of persisters induced by the 31

CSP-ComDE quorum-sensing regulatory system. We further identified key genes involved in 32

sugar and amino acid metabolism, CRISPR system, and autolysin from transcriptomic analyses 33

that contribute to the formation of quorum-sensing-induced persister cells. 34

35

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INTRODUCTION 36

The classical view of bacterial survival against antibiotic killing has usually been seen as the 37

expression of genetic resistance mechanisms that arise from mutations or gained through 38

horizontal gene transfer. These resistance mechanisms include host target modification, 39

degradation or modification of the antibiotic itself, and reduction in the permeability or increase 40

in the efflux of the drug (1). A major survival mechanism of bacteria is antibiotic tolerance, 41

whereby bacterial cells that have a slower or reduced growth rate become more tolerant towards 42

antibiotic killing (2, 3). This reduction in bacterial growth is prominently perceived as one of the 43

main survival mechanisms elicited in bacterial biofilms, by which the slower growth of biofilm 44

cells contributes towards the highly recalcitrant nature of biofilm infections, even in biofilm 45

populations that lack genetically encoded antibiotic resistance markers (4, 5). 46

Formation of persister cells is the main factor responsible for the tolerance of pathogens to 47

antibiotics. Persisters are non-growing dormant cells that are produced in a clonal population of 48

genetically identical cells. They constitute a small fraction of the bacterial population. Persisters 49

are not mutants but phenotypic variants of the wild-type population (6). In contrast to 50

aforementioned drug resistance mechanisms which allow for bacterial cells to actively grow and 51

divide unimpeded in the presence of specific antimicrobials, persisters are capable of surviving 52

lethal doses of multiple classes of bactericidal antibiotics at the expense of cellular growth in 53

both planktonic and biofilm populations (7). 54

Persister formation is governed by both stochastic and deterministic mechanisms (8, 9). 55

Persister cells are formed through random intrinsic fluctuations of protein levels, such as the 56

well-studied stable toxins expressed by chromosomal toxin-antitoxin (TA) modules (10-13). 57

Spontaneous gene expression and noise during transcription and translation, amplified by 58

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feedback regulatory processes, also give rise to fluctuating protein levels of persister genes that 59

result in a dormant multidrug-tolerant state (3, 14). Since the formation of persister cells is a 60

result of a variety of mechanisms, the subpopulation of persisters is heterogenous. The 61

redundancy in the formation of persisters hampers efforts in preventing tolerance towards 62

antimicrobial drugs through the targeting of a single mechanism (14). For example, it requires 63

genetic knockouts of at least ten TA modules to significantly reduce persister levels in 64

Escherichia coli (15). Recent studies have shown that the formation of dormant persister cells 65

can be induced by environmental and external stimuli (deterministic mechanisms), including 66

various stressors and signaling molecules. Studies done in E. coli have shown that DNA damage, 67

as a result of ciprofloxacin challenge, induced persisters through SOS-dependent expression of 68

the TisB toxin from the tisAB/istR TA locus (16). Another study showed that indole, an 69

intercellular signaling molecule expressed during stationary growth phase, was capable of 70

inducing the formation of persisters through the activation of stress response pathways (17). 71

While little work has been performed in streptococci, our lab showed – using 72

Streptococcus mutans as a model organism – that persisters were formed stochastically through 73

mild overexpression of chromosomal MazEF and RelBE TA modules (18). S. mutans is the main 74

causative agent of human dental caries (tooth decay) and a resident of the complex dental plaque 75

microflora (19, 20). As it inhabits and colonizes the dental biofilm, S. mutans is continuously 76

confronted to a variety of nutritional, chemical, and physical stresses (21, 22). We recently 77

demonstrated that challenge with an incoming stressor produced an increased numbers of 78

multidrug-tolerant persisters (18). More importantly, we discovered that S. mutans used its CSP-79

ComDE quorum-sensing system governing the development of genetic competence to produce 80

stress-inducible persisters (18). The CSP-ComDE system is composed of the CSP (competence 81

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stimulating peptide) pheromone acting through a membrane-bound histidine kinase receptor 82

(ComD) and its cognate cytoplasmic response regulator (ComE) (23). When the extracellular 83

level of CSP reaches a critical threshold, CSP interacts with the ComD sensor, resulting in its 84

autophosphorylation. This event initiates a phosphorelay cascade that results in the activation of 85

ComE. The phosphorylated form of ComE regulates transcription of several genes (24, 25). 86

Among the early genes is cipB encoding a small antimicrobial peptide that also functions as a 87

peptide regulator for the transcriptional control of the competence regulon via the alternative 88

sigma factor SigX (26). Recently, it has been demonstrated that the ComR/ComS system is the 89

proximal regulator of SigX in S. mutans (27, 28). 90

Many environmental stresses are capable of causing damage towards the chromosomal DNA, 91

leading to potential mutations and even the formation of DNA strand breaks that are lethal 92

during transcription and DNA replication (29-31). Streptococci lack a canonical SOS response 93

pathway to repair DNA damage and the induction of the SigX regulon has been proposed to act 94

as a general stress response (32). In this study, we continued our investigation of the CSP-95

ComDE pathway in regulating bacterial persistence in S. mutans. In particular, we characterized 96

the CSP-inducible gene SMU.2027 encoding a LexA-like transcriptional regulator. We showed 97

that this regulator was involved in initiating tolerance towards DNA-damaging agents through a 98

non-classical SOS pathway. More importantly, we demonstrated that the LexA pathway affected 99

the formation of CSP-induced persisters. 100

101

MATERIAL AND METHODS 102

Bacterial strains, plasmids, and growth conditions. A summary of the bacterial strains and 103

plasmids is provided in Table 1. Nonpolar insertion-deletion mutants were constructed in 104

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S. mutans UA159 wild-type (WT) strain by PCR ligation mutagenesis (33). Primers used for the 105

generation of PCR products are listed in Table S1. S. mutans strains were grown in Todd-Hewitt 106

medium supplemented with 0.3% yeast extract (THYE) and incubated statically at 37°C in air 107

with 5% CO2. E. coli strains were cultivated aerobically in Luria-Bertani (LB) medium at 37°C. 108

Plasmids were introduced into E. coli by transformation using electroporation or chemical 109

transformation. Plasmids were transferred to S. mutans by natural transformation as described 110

previously (26). When needed, antibiotics were added as follows: erythromycin (150 µg/ml), or 111

kanaymycin (50 µg/ml) for E. coli and kanaymycin (300 µg/ml), spectinomycin (1 mg/ml), or 112

erythromycin (10 µg/ml) for S. mutans. Cell growth was monitored by determining the optical 113

density at 600 nm (OD600). Cell viability was assessed by counting colony forming unit (CFU) 114

on replica agar plates. The minimum inhibitory concentration (MIC) test was performed 115

according to the broth microdilution method using THYE broth as described previously (34). 116

117

Construction of a non-cleavable LexA regulator. A non-cleavable LexA regulator was 118

generated by site-directed mutagenesis using the QuikChange II Site-Directed Mutagenesis Kit 119

(Agilent Technologies) following the manufacturer’s recommendations. Briefly, the full-length 120

coding region and promoter region of lexA gene (SMU.2027) was PCR amplified using UA159 121

genomic DNA as the template and the primer pair CMT-669/CMT-700. The PCR product was 122

purified, double digested with ApaI/BamHI, and then coned onto the shuttle vector pIB184 (35) 123

precut by the same enzymes. The recombinant plasmid pVL2 was confirmed by restriction 124

digestion and sequencing. Plasmid pVL2 was then used as the template for the replacement of 125

A115D using the two mutagenic PCR primers CMT-728 and CMT-729. The clone used to 126

produce LexA A115D mutant was designated pVL3 and confirmed by sequencing. The 127

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recombinant plasmid pVL3 was next used as a template for cloning into plasmid pIB107 (35) for 128

integration into the chromosome of S. mutans UA159, and the resulting plasmid was designated 129

pVL6. 130

131

Construction of expression vectors for induction in E. coli. To generate inducible 132

expression constructs for induction of LexA and LexA A115D mutation, fragment containing the 133

open reading frame for the lexA gene was PCR amplified using UA159 genomic DNA as the 134

template and the primer pair CMT-718/CMT-719. The PCR product was purified, cloned in-135

frame upstream from the His6 sequence under the control of araBAD promoter into pBAD202/D-136

TOPO vector (Invitrogen). The recombinant plasmid pVL4 was transferred into E. coli DH10B 137

and sequenced on both strands for confirmation. The LexA A115D mutation was achieved via 138

site-directed mutagenesis as described above using the primer pair CMT-728/CMT-729 and 139

pVL4 as the template. The mutation was confirmed by sequencing. The plasmids designated 140

pVL4 (lexA in pBAD) and pVL5 (lexA A115D in pBAD) were then transferred into 141

electrocompetent E. coli LMG194 cells for induction by arabinose. 142

143

Production and purification of recombinant fusion proteins. Overnight E. coli 144

LMG194(pVL4) and E. coli LMG194(pVL5) cultures were diluted (1:100) into fresh LB 145

medium supplemented with kanamycin and grown aerobically at 37°C until and OD600 of 0.5 146

was reached. Arabinose was then added at a final concentration of 0.2% (wt/vol) to induce the 147

expression of recombinant fusion proteins and the incubation was continued for another 3 h at 148

37°C with agitation. Protein expression was verified by SDS-PAGE using the Laemmli buffer 149

system and the protein bands were visualized by staining with Coomassie brilliant blue. In 150

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addition, the expression of recombinant proteins was confirmed with anti-His antibodies 151

(Sigma). The cells were collected by centrifugation, resuspended in 1 binding buffer 152

(Novagen), and disrupted on ice by sonication. The LexA-His6 and LexA A115D-His6 153

recombinant proteins were then purified by affinity chromatography on Ni2+

-nitrilotriacetic acid 154

(Ni-NTA) resin (Novagen) as described by the manufacturer. The Bradford protein assay was 155

used to determine the protein concentration of the purified samples. 156

157

Self-cleavage assay. Aliquots of purified recombinant LexA proteins (1 µg) were incubated 158

in 50 mM Tris-HCl buffer adjusted to pH values ranging from 6.0 to 10.0. LexA autodigestion 159

was performed at 37ºC for 16 h, and aliquots were stopped by adding SDS-PAGE loading buffer. 160

Samples were analyzed by SDS-PAGE using the buffer system of Laemmli at a constant voltage 161

(200 V) with gels containing 12% polyacrylamide in the separating gel and 4.5% polyacrylamide 162

in the stacking gel. The protein bands were visualized by staining with Coomassie brilliant blue. 163

164

Gene expression analysis following heat and DNA damage induction. Overnight cultures 165

of S. mutans strains were diluted (1:100) into fresh THYE broth and incubated at 37°C until an 166

OD600 of ~0.4 – 0.5 was reached. Cells were then exposed for 2 h at 37°C (except for heat shock) 167

to the following stresses: antibiotic stress (20 µg/ml of ofloxacin), DNA crosslinker (0.5 µg/ml 168

of mitomycin C), oxidative stress (0.5 mM of hydrogen peroxide), and heat shock (30 min at 169

50°C). Cells were processed with the Bio101 FastPrep System (Qbiogene) and total RNA was 170

extracted using TRIzol reagent (Invitrogen). Samples were DNase-treated with RQ1 DNAse 171

(Promega), and converted to cDNA using a RevertAid H Minus First-Strand cDNA Synthesis 172

Kit (Fermentas). Negative controls without reverse transcriptase were included in all 173

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experiments. Real-time quantitative PCR (QPCR) analysis was performed using the SsoFast 174

EvaGreen Supermix (Bio-Rad) and the CFX96 real-time PCR detection system (Bio-Rad). 175

QPCR assays were performed in triplicate with RNA isolated from three independent 176

experiments. Statistical significance was determined by using a Student’s t-test with the 177

parametric P value cutoff set at < 0.01. 178

179

Persistence assays. Overnight cultures of S. mutans strains were diluted (1:20) into fresh 180

THYE broth containing 20 µg/ml of ofloxacin (DNA damage), 15 µg/ml of oxacillin (inhibition 181

of cell wall synthesis), 50 µg/ml of rifampicin (inhibition of transcription) or 20 µg/ml of 182

vancomycin (inhibition of cell wall synthesis), followed by incubation for 24 h at 37°C. For the 183

quorum-sensing peptide pheromone assays, diluted S. mutans cells were incubated with 2 µM 184

synthetic competence stimulating peptide (CSP; Advanced Protein Technology Centre, Hospital 185

for Sick Children, Toronto, Ontario, Canada) pheromone for 2 h at 37°C before exposed to the 186

stresses (18). Samples were withdrawn at the indicated times, serially diluted, and plated on 187

THYE agar plates. The colonies were counted after 48 h of incubation. All assays were 188

performed in triplicate from three independent cultures. The statistical significance was 189

determined by using a Student’s t-test and a P value of <0.01. 190

191

DNA microarrays. DNA microarrays were performed as previously described (25). Briefly, 192

S. mutans WT strain and ∆lexA mutant cells were grown with 2 µM CSP (CSP-induced) or 193

without (uninduced) to mid-log phase. Cells were then collected by centrifugation, resuspended 194

in ice-cold RNAwiz (Ambion), and disrupted using 0.1-mm zirconia beads. Total RNA was 195

extracted using the Ambion RiboPure-Bacteria kit according to the provided protocol. Isolated 196

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RNA was treated twice with DNase I (Ambion) to remove traces of genomic DNA. After the 197

treatment, RNA samples were cleaned with the QIAGEN RNeasy MinElute cleanup kit. Aliquots 198

of the extracted total RNA were tested for quantity and quality. RNA concentration was 199

determined spectrophotometrically using the NaNodrop spectrophotometer and RNA integrity 200

was assessed by Agilent BioAnalyzer 6000 (ProkaryoteTotal RNA Nano assay). Inclusion 201

criteria considered total RNA with a ratio (A260/A280) ideally > 1.8 and absence of signs of RNA 202

degradation on the Agilent BioAnalyzer. 203

For cDNA synthesis, 15 µg of RNA was mixed with 1.25 µg of random primers and 204

incubated at 70°C for 10 min and then at 25°C for 10 min. The reverse transcription was 205

performed in 1 First Strand buffer supplemented with 10 mM dithiothreitol, 0.5 mM 206

deoxynucleoside triphosphate mix, 60 U of RNAseOut (Invitrogen), and 1,500 U of SuperScript 207

II reverse transcriptase (Invitrogen). The reaction mixture was incubated at 25°C for 10 min, 208

37°C for 1 h, 42°C for 1 h, and 70°C for 10 min. To remove residual RNA, the cDNA samples 209

were treated with 0.2 N NaOH at 65°C for 30 min and then neutralized with 0.2 N HCl before 210

being purified with the QIAGEN MinElute PCR purification kit. The cDNA was fragmented 211

using Roche DNase I (0.06 U per µg of cDNA) at 37°C for 10 min. Inactivation of the enzyme 212

was performed at 98°C for 10 min. Synthesized cDNA was quantified spectrophotometrically 213

and the integrity assessed by Agilent Bioanalyzer 6000. These processes were performed before 214

and after fragmentation. Fragmented samples represented by a smear of products falling between 215

50 and 200 bases were included and labelled with biotin-ddUTP using the BioArray terminal 216

labeling kit (Enzo). Microarrays were run using whole-genome custom GeneChips antisense 217

expression microarray previously designed in collaboration with Affymetrix (Santa Clara, CA) 218

(36). Hybridization, washing, and scanning of the microarray chips were performed at the 219

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Princess Margaret Genomics Centre (Toronto, Ontario, Canada) using Affymetrix protocol. 220

Results were validated by QPCR. 221

222

DNA transformation assays. Overnight cultures of S. mutans WT strain and its deletion 223

mutants were diluted (1:20) with fresh THYE broth and incubated at 37°C until an OD600 of 0.1 224

was reached. To test the effect of CSP on genetic transformation (CSP-induced conditions), 225

synthetic CSP was added at a final concentration of 0.2 µM (26). Ten micrograms of UA159 226

genomic DNA containing the spectinomycin resistance marker inserted into the rgp locus of the 227

UA159 strain, as inactivation of this locus has no effect on cell viability and transformation 228

efficiency (25), was added to the cultures (0.5 ml aliquots), which were grown for a further 2.5 h 229

at 37°C before differential plating. The transformation efficiency was calculated as the 230

percentage of spectinomycin resistant transformants divided by the total number of recipient 231

cells, which was determined by the number of CFU on antibiotic-free THYE agar plates. All 232

assays were performed in triplicate from three independent experiments. 233

234

RESULTS 235

An Intact CSP-Dependent Competence Pathway is Necessary for CSP-Inducible Formation 236

of Persisters. In the oral cavity, bacteria are constantly challenged by a wide variety of stressful 237

environmental conditions. We previously demonstrated that particular stresses activated CSP 238

production (25) and cells responded to high levels of CSP by inducing the formation of multi-239

drug tolerant persisters (18). In pursuit of determining the mechanism by which the CSP-ComDE 240

pathway regulates the inducible formation of persisters, several components of the competence 241

regulon necessary for the development of genetic competence in S. mutans were investigated. 242

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Individual knockout mutants in comE, cipB, comR, comS, and sigX genes were constructed and 243

tested for the CSP-inducible formation of persisters tolerant to ofloxacin. We used exogenously 244

added synthetic CSP to artificially mimic the high CSP concentrations induced by stress. Our 245

results showed that whereas the WT strain produced an increased number of persisters tolerant to 246

ofloxacin (~4-fold) in the presence of the CSP pheromone, the CSP-inducible persistence 247

phenotype was abolished for all mutants tested (Fig. 1). These results signified the importance of 248

an intact competence pathway initiating from CSP binding to the ComD membrane sensor to the 249

eventual activation of SigX, and suggests that putative persister genes are regulated by this 250

alternative sigma factor. 251

252

SMU.2027 gene induced by the quorum-sensing CSP pheromone possesses 253

characteristics of LexA-like transcriptional regulators. Work performed by some members of 254

our group showed that S. mutans employs its quorum-sensing CSP pheromone as a stress-255

inducible ‘alarmone’ to give an early warning signal of hostile conditions, and mount a specific 256

response to adapt and survive environmental assaults (reviewed in 37). SMU.2027 encoding a 257

putative LexA-like (or HdiR-like) regulator was found up-regulated ( 5-fold) under CSP 258

conditions used to mimic the high concentrations of CSP induced by stress (25). Although 259

SMU.2027 gene is induced by the CSP pheromone, it is unlikely that SigX directly regulates 260

SMU.2027 gene since no conserved CIN (competence-induced) box consensus sequence (23, 38) 261

has been found in its promoter region. A perfect inverted repeat (15 bp) was found in the 262

promoter region of SMU.2027 (Fig. S2) suggesting a putative binding site for autoregulatory 263

activity as observed in other LexA-like regulators (39). Bioinformatic analysis of S. mutans 264

SMU.2027 showed that it possesses the two conserved domains found in LexA-like regulators: i) 265

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a helix-turn-helix motif involved in sequence-specific DNA binding; ii) a signal peptidase-like 266

serine peptidase domain. A multiple sequence alignment of S. mutans SMU.2027 with the amino 267

acid sequences of various LexA and HdiR regulators revealed that the Ala-Gly bond, which is 268

involved in RecA-dependent autocleavage, was conserved in all sequences aligned (Fig. 2). 269

Under heat and DNA damage conditions, LexA regulators are cleaved and derepress 270

expression of genes involved in DNA damage tolerance and repair, and lexA gene itself (i.e., it is 271

autoregulatory) (39). The expression of S. mutans SMU.2027 gene in response to heat and DNA 272

damage conditions was then investigated. Our results showed that treatment with ofloxacin 273

antibiotic and heat shock, two stresses known to initiate self-cleavage of LexA repressors (39, 274

40), led to activation of S. mutans SMU.2027 gene (Table 2). Oxidative stress was also 275

investigated since reactive oxygen species (ROS) are known to cause DNA damage via oxidation 276

of nucleotide bases. Our results showed that hydrogen peroxide stress resulted in an increase in 277

SMU.2027 gene expression (Table 2). Consequently, we can hypothesize that SMU.2027 278

(denoted as LexA hereafter) belongs to the LexA-like (or HdiR-like) family based on domain 279

organization, sequence similarity, and induction in response to heat and DNA damage 280

conditions. 281

282

LexA undergoes self-cleavage and requires the ClpP proteolytic subunit for its 283

autoregulation. To further characterize the S. mutans LexA regulator, we produced a 284

recombinant LexA protein by cloning the full-length coding region of SMU.2027 into the pBAD 285

expression system for induction of gene expression using araBAD promoter dose-dependent 286

regulation. Purified recombinant protein was then utilized for determining if LexA undergoes 287

self-cleavage in vitro at alkaline pH, a characteristic feature of LexA-like repressors (41). In vitro 288

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cleavage requires RecA at neutral pH, but at alkaline pH, a spontaneous cleavage takes place. 289

This RecA-independent reaction is called autodigestion. As the purified recombinant LexA 290

protein was incubated in increasing pH, spontaneous self-cleavage occurred which resulted in 291

two cleavage products at pH ≥ 8.0 (Fig. 3A). A recombinant LexA regulator with a point 292

mutation at the conserved alanine residue at position 115 of the Ala-Gly bond to an asparatic 293

acid abolished the alkaline-induced self-cleavage (Fig. 3B). These results confirm that LexA is 294

capable of self-cleavage, specifically at the conserved A115 cleavage site. 295

After cleavage, the N-terminal domain of LexA-like repressors can remain bound to the DNA, 296

which can still repress gene expression (42). For this reason, the N-terminal cleavage products 297

are usually removed by Clp proteolytic complexes prior to derepression of target gene 298

expression. QPCR analysis of lexA gene was performed using WT strain and a mutant deficient 299

in the ClpP protease catalytic domain (ΔclpP). Results showed that derepression of lexA did not 300

occur in the ΔclpP mutant upon heat and DNA damage (Table 2). Together, these results suggest 301

that in S. mutans, Clp protease is required to degrade the N-terminal helix-turn-helix motif of 302

LexA that is predicted to remain bound to the DNA upon heat and DNA damage, similar to other 303

LexA-like repressors. 304

To confirm the role of S. mutans LexA in its autoregulation, we constructed a strain 305

expressing a mutated LexA protein unable to undergo autocleavage. A single-site mutation of 306

amino acid residue was introduced by PCR in the Ala-Gly bond of S. mutans LexA regulator. 307

The conserved alanine at position 115 was changed by aspartic acid (Fig. 2). The uncleavable 308

form of S. mutans lexA was then cloned under its own promoter into pIB107 vector (pVL6), and 309

integrated into the chromosome of an S. mutans ∆lexA mutant. The new mutant was designated 310

∆lexA::lexA A115D and was confirmed by PCR. The disruption of lexA gene had no effect upon 311

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S. mutans cell growth under normal growth conditions (data not shown). Moreover, no 312

discernable growth difference was observed between WT strain, ∆lexA mutant, and ∆lexA::lexA 313

A115D mutant under unstressed conditions (data not shown). As expected, no up-regulation of 314

lexA gene was observed in the S. mutans ∆lexA::lexA A115D uncleavable strain tested under the 315

same stress conditions (Table 2). Our results indicate that induction of lexA gene expression by 316

heat and DNA-damage is controlled by LexA-cleavable-mediated derepression of lexA gene. 317

Altogether, these results confirm that SMU.2027 encodes for a LexA-like repressor. 318

319

S. mutans LexA regulates tolerance towards DNA damage through a mechanism that is 320

different to the SOS-like response pathway in streptococci. In order to test the role of LexA 321

regulator on tolerance to DNA damage, S. mutans cells were incubated in the presence of a lethal 322

concentration of the fluoroquinolone ofloxacin, a broad-spectrum antibiotic. Fluoroquinolones 323

induce DNA damage by preventing the ligation reactions of DNA gyrase and topoisomerase, 324

resulting in double strand breaks (43). Cell survival assays showed that ∆lexA(pIB184) had a 325

significant decrease (3.7-fold) in survival after 24 h compared with WT strain expressing the 326

empty plasmid (Table 3). A more severe reduction (22.9-fold) was observed with strain 327

∆lexA(pVL3) expressing the uncleavable form of LexA. Similar results were obtained with 328

mitomycin C, a potent DNA crosslinker. Cell survival towards antibiotic oxacillin exhibiting a 329

different mechanism of action remained unaffected suggesting that LexA does not affect 330

tolerance towards non-DNA damaging agents (Table 3). Together, these results further indicate 331

that S. mutans lexA gene encodes for a regulator involved in tolerance towards DNA damage. 332

In determining if the route by which LexA initiates a DNA damage tolerance response via an 333

SOS-like pathway, we examined the role of UmuC homolog in S. mutans. UmuC is a subunit of 334

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the error-prone DNA polymerase V found in bacteria and is involved in bypassing DNA lesions 335

during DNA replication. In streptococci, this gene was found regulated by LexA-like repressors 336

in Streptococcus thermophilus (44) and Streptococcus uberis (45). In S. mutans UA159 strain, 337

SMU.403 encodes for an UmuC homolog exhibiting more than 85% amino acid sequence 338

similarity to UmuC of S. thermophilus and S. uberis. A strain disrupted in SMU.403 (∆umuC 339

mutant) was constructed in UA159 WT strain. The gene disruption had no effect upon cell 340

growth under normal growth conditions (data not shown). We then tested the effect of umuC 341

mutation on tolerance of S. mutans to DNA damage. Our results showed that in S. mutans, 342

UmuC homolog was dispensable in the tolerance and survival towards both ofloxacin 343

(concentrations varying between 2 µg/ml – 20 µg/ml) and mitomycin C (concentrations varying 344

between 0.18 µg/ml – 0.25 µg/ml). Furthermore, QPCR analysis showed that S. mutans umuC 345

gene was not differentially expressed in a ∆lexA mutant background (data not shown). We 346

conducted BLAST searches for homologous sequences using SMU.403 (umuC) as query 347

sequence. SMU.403 gene is highly conserved in all S. mutans strains, but SMU.403 sequence 348

does not share homology with any other sequences in S. mutans. Since knocking out SMU.403 349

failed to cause a change in the phenotype, our results suggest that other gene(s) can compensate 350

for the lost of SMU.403. Altogether, these results suggest that LexA regulates tolerance and 351

survival towards DNA damage through a mechanism that differs from the SOS-like response 352

pathway as described in S. thermophilus and S. uberis. 353

354

CSP-inducible formation of persisters in S. mutans is affected by the LexA pathway. 355

Persisters are dormant cells within an isogenic bacterial population that can tolerate lethal 356

concentrations of antibiotics (6, 46). The presence of dormant persisters has no effect on the MIC 357

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of antibiotics, since persisters are not growing and lack antibiotic resistance mechanisms. The 358

results presented above showed that although cell survival is decreased in ∆lexA mutant and 359

∆lexA(pVL3) strain expressing the uncleavable form of LexA (Table 3), antibiotic sensitivity 360

remained the same as the MIC values of ofloxacin against the mutants were not different from 361

those for the WT strain (data not shown). Consequently, we hypothesized that LexA could 362

promote the formation of persister cells. Our previous work showed that environmental stresses 363

such as heat and DNA damage, induce the formation of persister cells (18) reinforcing our 364

hypothesis. Moreover, we demonstrated that S. mutans integrates its response to specific 365

environmental stresses with its quorum-sensing system, the CSP-ComDE regulatory circuit, by 366

positively influencing the persister levels (18). Given the above, we tested the direct impact of 367

S. mutans LexA in the CSP-inducible development of persisters. S. mutans WT strain and its 368

∆lexA mutant were pre-exposed to the quorum-sensing CSP pheromone before being treated with 369

different drugs for 24 h. As expected, our data showed that the CSP pheromone significantly 370

increased the numbers of persister cells tolerant to ofloxacin, oxacillin, rifampicin, and 371

vancomycin for WT strain (Fig. 4). Interestingly, the CSP-inducible persistence phenotype was 372

abolished in the ∆lexA mutant for all classes of antibiotics tested except vancomycin, suggesting 373

that the LexA pathway affects the formation of CSP-inducible persisters towards several 374

different classes of antibiotics, and not just towards antibiotics inducing DNA damage. 375

It is most likely that the CSP conditions induce SigX factor to upregulate genes involved in 376

DNA repair and recombination leading to formation of single-stranded DNA (ssDNA) 377

intermediates. Based on work done in E. coli, we can hypothesize that the highly expressed 378

RecA proteins (recA is a SigX-regulated gene in S. mutans and showed ~7-fold increase under 379

CSP conditions; (25)) probably bind towards any exposed ssDNA within the cell to form RecA 380

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filaments, and this phenomenon is capable of serving as a SOS-inducing signal that leads to the 381

autocleavage of LexA and subsequent gene derepression (47). 382

383

The CSP-inducible LexA regulator is not necessary for the development of genetic 384

competence. It was recently shown that competence development and the “SOS pathway” in 385

S. thermophilus was antagonistic to one another (44). Since inactivation of lexA gene abolished 386

the formation of CSP-induced persister cells in S. mutans, whether competence is also hindered 387

was investigated. The transformation efficiency of the ∆lexA mutant was evaluated under CSP-388

induced and uninduced conditions. Our results showed that inactivation of lexA gene, and thus 389

constant activation of the LexA-regulated pathway, had no significant effect on transformation 390

efficiency under both conditions (Fig. 5). These results suggest that the S. mutans LexA pathway, 391

while preventing CSP-inducible formation of persisters, does not interfere with the development 392

of genetic competence. 393

394

Comparison of the gene expression profile between WT and ∆lexA mutant. To determine 395

the potential genes regulated by the LexA transcriptional regulator, transcriptional analysis of 396

both WT and its ∆lexA mutant were compared. RNA was extracted from cultures of both WT 397

and the deletion mutant grown to mid-exponential phase under unstressed conditions (see 398

Materials and Methods for microarray experiment details). The results from the microarray 399

identified scrA coding for enzyme II of the phosphoenolpyruvate-dependent sucrose 400

phosphotransferase system (48), as the only gene differentially regulated (3-fold reduction in the 401

∆lexA mutant). Confirming our earlier results, the transcriptome analyses showed that the genes 402

involved in DNA repair and recombination, including umuC, did not appear to be differentially 403

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expressed when LexA is inactivated (Table S2). These results confirmed that LexA regulates 404

tolerance towards DNA damage in a manner different from the classical SOS response. 405

406

Genome-wide expression response to CSP in the absence of LexA. Since our results 407

demonstrated that the CSP-inducible formation of persisters was affected by the LexA pathway, 408

we performed a comparison of the gene expression profiles between WT strain and its ∆lexA 409

mutant in the presence of the CSP pheromone (see Materials and Methods for microarray 410

experiment details). Our results showed that LexA regulated 11 genes (Table 4). Four genes were 411

selected for further analysis. Individual deletion mutants were constructed in the WT strain, and 412

each mutant was tested for its ability to produce CSP-induced persister cells. SMU.63 and 413

SMU.1402, two genes known to be differentially regulated under CSP conditions (25) were first 414

tested. SMU.1402 encodes for a CRISPR-associated Csn2 protein belonging to the CRISPR-1 415

region of S. mutans (49). CRISPR arrays with CRISPR-associated proteins are involved in 416

resistance to bacteriophage and resistance specificity is determined by the insertion of CRISPR 417

spacer-phage sequence similarity (50). Interestingly, inactivation of csn2 gene in S. mutans 418

completely abolished the increase in persister numbers observed for the WT strain following 419

pretreatment with CSP (Fig. 6). S. mutans Csn2 protein showed high similarity (81%; 154/187 420

aa) with Csn2 protein from S. thermophilus. In S. thermophilus, CRISPR systems provide 421

resistance against phages, and Csn2 protein is involved in spacer integration (50, 51). In 422

S. mutans, work previously done by van der Ploeg using strain OMZ 381, one of a few strains 423

sensitive to M102 phage attacking S. mutans, showed that deletion of CRISPR-1 region is 424

insufficient to render sensitivity to infection by the phage M102 (49). Natural phage resistance 425

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mechanisms in S. mutans have not been previously described mainly because phages and 426

sensitive strains are actually quite rare in S. mutans (52). 427

SMU.63 gene encodes for a conserved membrane protein of 613 amino acid residues of 428

unknown function. This gene is located immediately downstream the comRS locus involved in 429

CSP-inducible persister formation (Fig. 1) and genetic competence (27, 53). Our results showed 430

that ∆63 mutant was still able to increase the number of ofloxacin-tolerant persister cells 431

following treatment with the CSP pheromone (Fig. 6). Moreover, inactivation of SMU.63 did not 432

alter the number of persisters under no CSP conditions (Fig. S1) suggesting that SMU.63 is not 433

involved in the formation of persister cells. 434

SMU.984 gene encodes for a protein of 166 amino acid residues with no known function. The 435

protein possesses a C-terminal CHAP (cysteine- and histidine-dependent 436

aminohydrolase/peptidase) domain that functions in peptidoglycan hydrolysis. Pre-stressing cells 437

with the CSP pheromone followed by ofloxacin treatment revealed that a lack of SMU.984 438

abolished the CSP-inducible persistence phenotype (Fig. 6). Global transcriptome analysis 439

performed by Senadheera and coll. showed that SMU.984 gene was regulated by the two-440

component system VicRK (D. Senadheera, personal communication). In S. mutans, the VicRK 441

system modulates stress tolerance by direct modulation of the CSP pheromone (54). Altogether 442

these results suggest that SMU.984 could be involved in stress tolerance by modulating the CSP-443

inducible persistence phenotype. 444

The scrA gene (SMU.1841) is part of the scr regulon composed of the three genes – scrA, 445

scrB, and scrR – coding for a sucrose-specific IIABC phosphoenolpyruvate:carbohydrate 446

phosphate transferase system (PTS) component, a sucrose-6-phosphate hydrolase, and a sucrose 447

operon repressor, respectively (55). The scrA gene was found down-regulated in both 448

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transcriptome profile comparisons (CSP-induced and uninduced). The ∆scrA mutant was unable 449

to increase the number of ofloxacin-tolerant persister cells following treatment with the CSP 450

pheromone (Fig. 6). Moreover, a ∆scrA mutant had an approximately 4-fold decrease in persister 451

level after 24-h of ofloxacin treatment under no CSP condition (Fig. S1) suggesting that ScrA 452

also participates in the formation of persisters through quorum-sensing-independent 453

mechanism(s). 454

Using the online RSAT bioinformatic tool (http://rsat.ccb.sickkids.ca), we searched the 455

promoter region of SMU.984, SMU.1402 (csn2), and SMU.1841 (scrA) with the IR identified in 456

the promoter region of lexA (Fig. S2). No IR sequences were found, suggesting that these 457

persister genes are most probably indirectly regulated by LexA. 458

459

DISCUSSION 460

In this study, we have identified and characterized a LexA transcriptional regulator in S. mutans. 461

This LexA ortholog has many of the notable hallmarks of a typical LexA regulator such as auto-462

regulatory expression upon heat shock and DNA damage, and a conserved Ala-Gly self-cleavage 463

site. The observed full auto-regulatory expression is contingent on a functional ClpP subunit, 464

which is most likely required for the degradation of the N-terminal DNA binding domain after 465

LexA autocleavage to allow complete derepression of its own gene expression. Furthermore, the 466

S. mutans LexA regulator plays a role in a DNA damage response with regulating the formation 467

of persisters tolerant towards DNA damaging agents such as mitomycin C and fluoroquinolones. 468

Indeed, a ΔlexA(pVL3) strain overexpressing the mutated and uncleavable LexA A115D protein 469

showed a significant decreased survival towards ofloxacin and mitomycin C. However, the 470

LexA-regulated pathway in S. mutans differs greatly from the canonical SOS response seen in 471

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other bacteria, and even from other streptococcal species that have been identified with an SOS-472

like pathway via the LexA-like HdiR regulator. In S. thermophilus and S. uberis, HdiR functions 473

similarily as LexA in regulating an SOS-like response by directly upregulating the expression of 474

umuC upon DNA damage to bypass DNA lesions during DNA replication (44, 45). However, 475

umuC gene along with other genes with known or putative roles in DNA repair were not found 476

regulated by LexA in S. mutans (Table S2). In fact, a ΔumuC mutant did not affect tolerance and 477

survival towards DNA damage. 478

Most interestingly, S. mutans LexA was also identified in playing a significant role in the 479

formation of quorum-sensing-induced multidrug-tolerant persisters, in addition to its role in 480

forming persisters tolerant towards DNA-damaging agents. A lack of LexA completely abolished 481

the formation of CSP-induced persisters tolerant to multiple antibiotics suggesting that LexA 482

influences the deterministic formation of persisters mediated by the stress inducible alarmone. It 483

is interesting that a lack of LexA does not greatly affect survival and tolerance towards oxacillin 484

antibiotic, while under CSP conditions the lack of the LexA regulator prevents CSP-inducible 485

persistence. This suggests that there are at least two mechanisms regulated by LexA by which 486

S. mutans can respond to and tolerate DNA-damaging stress. One mechanism occurs under 487

normal growth, where LexA functions to provide an immediate response for tolerating DNA 488

damage conditions. The other mechanism occurs through activation of the CSP-ComDE quorum-489

sensing pathway most like responding to various environmental stressors. In this case, activation 490

of LexA regulator indirectly alters the expression profile of a group of genes that are required for 491

the formation of CSP-induced persisters tolerant not only to DNA damage but also to several 492

classes of antibiotics. 493

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In S. thermophilus, it was recently shown that the HdiR pathway functioned antagonistically 494

towards its own competence development (44). Activation of the SOS-like response via HdiR led 495

to the downregulation of competence genes and to the decrease in transformation efficiency, 496

whilst a SigX-deficient strain had an increased survivability towards both mitomycin C and 497

norfloxacin. Our results showed that the continuous activation of the LexA pathway in a LexA 498

deficient mutant acts negatively towards CSP-induced persisters, where the formation requires 499

the intact competence pathway from ComE to SigX. This may suggest that competence is also 500

negatively impacted in S. mutans, but this was not the case as we observed that the 501

transformation efficiency (Fig. 5) and the expression of competence genes (data not shown) were 502

not affected in a ∆lexA mutant treated with or without exogenous CSP. This difference is most 503

likely due to differing mechanism(s) and genes regulated by LexA in S. mutans as compared to 504

those regulated by HdiR in S. thermophilus. 505

Our transcriptomic analyses identified a number of genes with various cellular roles that 506

appear to contribute towards the formation of CSP-induced persisters. A few of these identified 507

genes are involved with metabolism, specifically amino acid biosynthesis. Although we did not 508

test individual knockout mutants for SMU.563 encoding the ornithine carbamoyltransferase 509

enzyme involved in arginine biosynthesis or the genes SMU.663 to SMU.666 belonging to the 510

glutamate family amino acid synthesis operon, it is likely that affecting amino acid biosynthesis 511

would greatly influence persister formation, as it has been recently shown in E. coli through the 512

use of a transposon library (56). 513

Our study led us to identify new genes involved in the formation of quorum-sensing-induced 514

persisters. These genes are most likely indirectly regulated through LexA. They include 515

SMU.984 encoding a hypothetical CHAP-containing protein. CHAP domains are typically 516

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associated with lytic proteins such as autolysins involved in peptidoglycan hydrolysis (57). Our 517

group had previously identified and characterized LytF-Sm, a SigX-regulated autolysin involved 518

in cell death induced by the CSP pheromone (58). Preliminary work done in our lab showed that 519

SMU.984 encodes a functional murein hydrolase but is not involved in the CSP-induced cell 520

death pathway. As similarities goes, SMU.984 may be a lytic protein that potentially functions 521

with reduced efficiency or potency in altering the cell wall integrity, leading to CSP-inducible 522

persister formation. 523

The CRISPR-associated csn2 gene was found to be important in the CSP-inducible 524

persistence phenotype. To the best of our knowledge, it is the first time a link is proposed 525

between a CRISPR-associated protein and persister formation. The CRISPR system is not well 526

characterized in S. mutans, and csn2 gene is only found in the type II-A systems. The Csn2 527

protein was recently characterized as forming a ring-shaped tetramer complex that binds to 528

double-strand DNA ends with the proposed function of assisting in the integration of new spacer 529

DNA fragments by stabilizing the DNA ends together, and as a potential accessory protein 530

recruiting DNA-repair proteins (59). Perhaps increased csn2 gene expression contributes to 531

ofloxacin tolerance by recruiting DNA repair proteins at the post-transcriptional level not 532

detected in our microarrays. How exactly Csn2 protein plays a role in the formation of CSP-533

induced persisters remains to be determined. 534

The scrA gene, encoding a PTS sucrose specific component, was the only gene found to be 535

significantly differentially regulated in a ∆lexA mutant cultivated under both CSP-induced and 536

non-induced conditions. The role of carbohydrate metabolism has been previously associated to 537

the formation of persisters (60, 61). Based on our results, we can hypothesize that a lack of LexA 538

derepresses gene(s) involved in the downregulation of scrA gene contributing to the reduced 539

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persister levels. Interestingly, inactivation of scrA gene led to decrease persister formation under 540

both CSP-induced and non-induced conditions, suggesting that scrA is involved in the formation 541

of persisters governed by both stochastic and deterministic mechanisms. ScrA is a sucrose 542

permease that is heavily involved in sucrose uptake. However, there are other systems that 543

contribute to uptake and/or catabolism of sucrose in S. mutans, specifically the 544

glucosyltransferases (GtfB, GtfC, GtfD), the fructosyltransferase (Ftf), and the multiple-sugar-545

metabolism pathway (62). This would likely eliminate the notion of sucrose metabolism being 546

the mechanism by which scrA contributes to persister formation. Additionally, our persistence 547

assays were performed in a nutrient-rich complex medium that contains glucose as the major 548

carbohydrate source. Interestingly, we have previously identified from an overexpression 549

genomic library that deletion of the sucrose operon repressor ScrR, which normally represses the 550

expression of scrA and scrB genes in S. mutans, led to ~10-fold increase in persister formation 551

(18) suggesting that a derepression of scrA may lead to an increase of persister formation. These 552

results also suggest that there are multiples layers of regulation on scrA. Regulation of scrA 553

could also occur from the IGR1445 intergenic region corresponding to the scrA/scrB promoter 554

region. Unfortunately efforts in identifying a regulatory anti-sense RNA in this intergenic region 555

have been unsuccessful thus far. 556

It is important to note that significantly upregulated genes were expected to be detected in 557

our comparative microarray analysis. Detection of up-regulated genes would signify that these 558

genes are most likely directly regulated by the LexA regulator in S. mutans. In contrast, all 559

significantly differentially regulated genes were downregulated. Our results suggest two possible 560

mechanisms by which LexA contributes to the persister phenotype in S. mutans. In the first 561

model, LexA acts as a transcriptional activator following its autocleavage. Under these 562

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conditions, deletion of lexA gene would cause downregulation of genes, and the loss of the CSP-563

inducible persistence phenotype. However, this would suggest that LexA regulator in S. mutans 564

possesses a different transcriptional function following its autocleavage, which would be in stark 565

contrast to the well-characterized LexA family of transcriptional regulators described in other 566

bacteria. In the second scenario, LexA acts as a repressor inhibiting another repressor of persister 567

genes, where LexA autocleavage leads to the indirect repression of these genes. The fact that the 568

identified IR located in the promoter region of S. mutans lexA gene was not found upstream the 569

downregulated genes identified in our arrays strongly supports this model. 570

It is also possible that upregulated genes were not detected because the binding affinity of 571

LexA towards the promoter regions of potential LexA-regulated genes is heterogeneous 572

depending on the strength and conservation of the consensus LexA-box motif, their relative 573

location in the target promoter, and even the promoter strength of the target gene itself, as it has 574

been well characterized in E. coli (63). This variation in LexA-binding results in a difference in 575

the expression level, timing and duration for different LexA-regulated genes. Some SOS-576

regulated genes in E. coli have low promoter activity in the absence of DNA-damaging agents 577

(64). Furthermore, genomic analyses in E. coli have shown that LexA is capable of binding and 578

regulating genes that do not contain a canonical LexA-box motif in their promoter regions (65). 579

All these factors make it challenging to identify genes that are directly regulated by LexA. 580

Moreover, it is possible that LexA-regulated genes are non-protein encoding regulatory RNAs 581

that would not be detected in our microarrays. In this case, transcriptome analyses using RNAseq 582

of S. mutans in the presence of DNA-damaging agents would need to be performed. 583

The results of this study presents two roles by which the LexA transcriptional regulator 584

partakes in regulating bacterial survival towards antibiotic challenge in S. mutans. One pathway 585

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involves DNA-damaging agents initiating LexA self-cleavage that indirectly leads to tolerance 586

through the regulation of scrA. The second pathway involves the stress-inducible CSP alarmone 587

that activates the competence regulon. Our results suggest that lexA gene is part of the sigX 588

regulon acting as a general stress response in streptococci. Once activated, the lexA pathway 589

leads to the expression of a group of genes including csn2, scrA, and SMU.984 that collectively 590

contribute to the stress-inducible multidrug-tolerant persister state. Although CSP-inducible 591

persister genes have been identified, their exact roles are unknown. Further experiments would 592

need to be performed to elucidate the exact mechanisms by which each identified gene 593

contributes towards persister formation. Nonetheless, disrupting the inducible persister pathway 594

at any point between the components of the bacterial quorum-sensing system to the effector 595

genes to prevent stress-inducible persister formation could lead to innovative drug designs and 596

strategies. 597

598

ACKNOWLEDGMENTS 599

We thank Delphine Dufour for careful reading of the manuscript. We thank Indranil Biswas for 600

the pIB107 and pIB184 plasmids. This study was supported by a Canadian Institutes of Health 601

Research (CIHR) grant MOP-93555 to C.M.L. and a Natural Sciences and Engineering Research 602

Council of Canada (NSERC) grant RGPIN 355968 to C.M.L. C.M.L. is a recipient of a Canada 603

Research Chair. 604

605

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41. Little JW. 1984. Autodigestion of lexA and phage lambda repressors. Proc. Natl. Acad. Sci. 701

U S A 81:1375–1379. 702

42. Neher SB, Flynn JM, Sauer RT, Baker TA. 2003. Latent ClpX-recognition signals ensure 703

LexA destruction after DNA damage. Genes Dev. 17:1084–1089. 704

43. Drlica K, Zhao X. 1997. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. 705

Mol. Biol. Rev. 61:377–392. 706

44. Boutry C, Delplace B, Clippe A, Fontaine L, Hols P. 2013. SOS response activation and 707

competence development are antagonistic mechanisms in Streptococcus thermophilus. 708

J. Bacteriol. 195:696–707. 709

45. Varhimo E, Savijoki K, Jalava J, Kuipers OP, Varmanen P. 2007. Identification of a 710

noval streptococcal gene cassette mediating SOS mutagenesis in Streptococcus uberis. 711

J. Bacteriol. 189:5210–5222. 712

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Appl. Environ. Microbiol. 79:7116–7121. 714

47. Kuzminov A. 1999. Recombinational repair of DNA damage in Escherichia coli and 715

bacteriophage lambda. Microbiol. Mol. Biol. Rev. 63:751–813. 716

48. Sato Y, Poy F, Jacobson GR, Kuramitsu HK. 1989. Characterization and sequence 717

analysis of the scrA gene encoding enzyme IIScr

of the Streptococcus mutans 718

phosphoenolpyruvate-dependent sucrose phosphotransferase system. J. Bacteriol. 171:263–719

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49. van der Ploeg JR. 2009. Analysis of CRISPR in Streptococcus mutans suggests frequent 721

occurrence of acquired immunity against infection by M102-like bacteriophages. 722

Microbiology 155:1966–1976. 723

50. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, 724

Horvath P. 2007. CRISPR provides acquired resistance against viruses in prokaryotes. 725

Science 315:1709–1712. 726

51. Deveau H, Barrangou R, Garneau JE, Labonte J, Fremaux C, Boyaval P, Romero DA, 727

Horvath P, Moineau S. 2008. Phage response to CRISPR-encoded resistance in 728

Streptococcus thermophilus. J. Bacteriol. 190:1390–1400. 729

52. Maruyama F, Kobata M, Kurokawa K, Nishida K, Sakurai A, Nakano K, Nomura R, 730

Kawabata S, Ooshima T, Nakai K, Hattori M, Hamada S, Nakagawa I. 2009. 731

Comparative genomic analyses of Streptococcus mutans provide insight into chromosomal 732

shuffling and species-specific content. BMC Genomics. 10:358. 733

53. Fontaine L, Goffin P, Dubout H, Delplace B, Baulard A, Lecat-Guillet N, Chambellon 734

E, Gardan P, Hols P. 2013. Mechanism of competence activation by the ComRS signalling 735

system in streptococci. Mol. Microbiol. 87:1113–1132. 736

54. Senadheera DB, Cordova M, Ayala EA, Chávez de Paz LE, Singh K, Downey JS, 737

Svensäter G, Goodman SD, Cvitkovitch DG. 2012. Regulation of bacteriocin production 738

and cell death by the VicRK signaling system in Streptococcus mutans. J. Bacteriol. 739

194:1307–1316. 740

55. Wang B, Kuramitsu HK. 2003. Control of enzyme IIscr

and sucrose-6-phosphate hydrolase 741

activities in Streptococcus mutans by transcriptional repressor ScrR binding to the cis-active 742

determinants of the scr regulon. J. Bacteriol. 185:5791–5799. 743

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56. Bernier SP, Lebeaux D, DeFrancesco AS, Valomon A, Soubigou G, Coppee JY, Ghigo 744

JM, Beloin C. 2013. Starvation, together with the SOS response, mediates high biofilm-745

specific tolerance to the fluoroquinolone ofloxacin. PLoS Genet. 9:e1003144. 746

57. Vollmer W, Joris B, Charlier P, Foster S. 2008. Bacterial peptidoglycan (murein) 747

hydrolases. FEMS Microbiol. Rev. 32:259–286. 748

58. Dufour D, Lévesque CM. 2013. Cell death of Streptococcus mutans induced by quorum-749

sensing peptide occurs via a conserved streptococcal autolysin. J. Bacteriol. 195:105–114. 750

59. Arslan Z, Wurm R, Brener O, Ellinger P, Nagel-Steger L, Oesterhelt F, Schmitt L, 751

Willbold D, Wagner R, Gohlke H, Smits SH, Pul U. 2013. Double-strand DNA end-752

binding and sliding of the toroidal CRISPR-associated protein Csn2. Nucleic Acids Res. 753

41:6347–6359. 754

60. Spoering AL, Vulic M, Lewis K. 2006. GlpD and PlsB participate in persister cell 755

formation in Escherichia coli. J. Bacteriol. 188:5136–5144. 756

61. Girgis HS, Harris K, Tavazoie S. 2012. Large mutational target size for rapid emergence 757

of bacterial persistence. Proc. Natl. Acad. Sci. U S A 109:12740–12745. 758

62. Zeng L, Choi SC, Danko CG, Siepel A, Stanhope MJ, and Burne RA. 2013. Gene 759

regulation by CcpA and catabolite repression explored by RNA-Seq in 760

Streptococcus mutans. PLoS ONE 8:e60465. 761

63. Butala M, Zgur-Bertok, Busby SJW. 2009. The bacterial LexA transcriptional repressor. 762

Cell Mol. Life Sci. 66:82–93. 763

64. Kamensek S, Podlesek Z, Gillor O, and Zgur-Bertok D. 2010. Genes regulated by the 764

Escherichia coli SOS repressor LexA exhibit heterogenous expression. BMC Microbiol. 765

10:283. 766

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65. Wade JT, Reppas NB, Church GM, and Struhl K. 2005. Genomic analysis of LexA 767

binding reveals the permissive nature of the Escherichia coli genome and identifies 768

unconventional target sites. Genes Dev. 19:2619–2630. 769

770

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FIGURE LEGENDS 771

FIG. 1. Role of essential competence genes in the formation of CSP-induced persister cells. 772

Overnight cultures of S. mutans WT strain and its ∆comD, ∆comE, ∆cipB, ∆comR, ∆comS, and 773

∆sigX mutants were diluted (1:100) into fresh THYE broth in the absence (no CSP) or presence 774

of CSP pheromone, and incubated at 37º for 2 h before being challenged with ofloxacin. Aliquots 775

of cells were removed at the introduction of the antibiotic and after the antibiotic treatment (24 h) 776

to determine cell survival by spot plating onto THYE agar plates. Results are expressed as the 777

log-fold change in cell survival normalized to non CSP pre-stress. The data are the averages and 778

standard errors of results from three independent cultures. 779

780

FIG. 2. Primary structures of LexA-like regulators. Multi-sequence alignment of amino acid 781

sequences of S. mutans SMU.2027 with LexA and HdiR of E. coli, Lactococcus lactis, 782

Staphylococcus aureus, Streptococcus uberis, Rhodobacter sphaeroides, and 783

Bifidobacterium longum. The helix-turn-helix motif is shown as a grey box at the N-terminal end 784

of SMU.2027. The C-terminal serine-peptidase domain (white box) is also shown. *Denotes 785

conserved amino acid residues involved in LexA autocleavage. The amino acid mutated in the 786

present study is in bold and underlined. 787

788

FIG. 3. LexA self-cleavage. LexA (A) and LexA A115D (B) recombinant proteins were 789

incubated in Tris-HCl buffer at pH values varying from 6.0 to 10.0. Autocleavage assay was 790

visualized on SDS-PAGE stained with Coomassie brilliant blue. Sizes in kilodaltons (kDa) of 791

Precision Plus Protein Dual Color Standards (Bio-Rad) are on the left. The arrows on the right 792

indicate the positions of breakdown products of LexA. 793

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FIG. 4. Effects of S. mutans LexA on the development of CSP-induced persister cells. 794

Overnight cultures of S. mutans WT strain and its ∆lexA mutant were diluted (1:100) into fresh 795

THYE broth in the absence or presence of CSP pheromone, and incubated at 37ºC for 2 h before 796

being challenged with ofloxacin, oxacillin, rifampicin, or vancomycin. Aliquots of cells were 797

removed at the introduction of the antibiotic and after the antibiotic treatment (24 h) to determine 798

cell survival by spot plating onto THYE agar plates. Results are expressed as the log-fold change 799

in cell survival normalized to non CSP pre-stress. The data are the averages and standard errors 800

of results from three independent cultures. 801

802

FIG. 5. Effect of inactivation of lexA gene on the development of genetic competence. 803

Overnight cultures of WT strain and its ∆lexA mutant were diluted (1:20) in fresh THYE broth 804

and grown until an OD600 of 0.1 was reached. Aliquoted cultures were cultivated in the absence 805

(no CSP) and presence of CSP, and 20 µg/ml of UA159 genomic DNA carrying the kanamycin 806

resistance marker. Cells were incubated at 37ºC for 2.5 h before differential plating. The 807

transformation efficiency (TE) was expressed as the percentage of kanamycin resistant 808

transformants divided by the total number of recipient cells. The data are the averages and 809

standard errors of results from three independent cultures. 810

811

FIG. 6. Role of selected LexA-regulated genes on the formation of CSP-induced persister 812

cells. Overnight cultures of S. mutans WT strain and its ∆63, ∆csn2, ∆scrA, and ∆984 mutants 813

were diluted (1:100) into fresh THYE broth in the absence or presence of CSP pheromone, and 814

incubated at 37º for 2 h before being challenged with ofloxacin. Aliquots of cells were removed 815

at the introduction of the antibiotic and after the antibiotic treatment (24 h) to determine cell 816

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survival by spot plating onto THYE agar plates. Results are expressed as the log-fold change in 817

cell survival normalized to non CSP pre-stress. The data are the averages and standard errors of 818

results from three independent cultures. 819

820

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TABLE 1. Bacterial strains and plasmids used in this study. 821

Strain or plasmid Relevant characteristic(s)a Source or

reference

Strains

S. mutans UA159 Wild-type reference strain

Lab stock

E. coli DH10B Host strain for cloning and plasmid production Lab stock

E. coli LMG194 Host strain for pBAD expression Invitrogen

E. coli XL10-Gold Host strain for site-directed mutagenesis Agilent

Technologies

ΔlexA mutant In-frame SMU.2027 deletion mutant derived from S. mutans

UA159; Spr

This study

UA159(pIB184) UA159 harbouring pIB184; Emr This study

ΔlexA::lexA A115D ΔlexA harbouring pVL6 chromosomally integrated; Spr, Km

r This study

ΔlexA(pVL2) ΔlexA harbouring pVL2; Spr, Em

r This study

ΔlexA(pVL3) ΔlexA harbouring pVL3; Spr, Em

r This study

LMG194(pVL4) LMG194 containing pVL4; Kmr This study

LMG194(pVL5) LMG194 containing pVL5; Kmr This study

ΔumuC mutant In-frame SMU.403 deletion mutant derived from S. mutans

UA159; Emr

This study

ΔclpP mutant In-frame SMU.1672 deletion mutant derived from S. mutans

UA159; Emr

This study

∆comE mutant In-frame SMU.1917 deletion mutant derived from S. mutans

UA159; Emr

(25)

∆cipB mutant In-frame SMU.1914 deletion mutant derived from S. mutans

UA159; Emr

(25)

∆comS mutant In-frame comS deletion mutant derived from S. mutans UA159;

Emr

This study

∆comR mutant In-frame SMU.61 deletion mutant derived from S. mutans

UA159; Emr

D. Morrison,

UIC

∆sigX mutant In-frame SMU.1997 deletion mutant derived from S. mutans

UA159; Emr

(25)

ΔscrA mutant In-frame SMU.1841 deletion mutant derived from S. mutans

UA159; Emr

This study

Δcsn2 mutant In-frame SMU.1402 deletion mutant derived from S. mutans

UA159; Emr

This study

Δ63 mutant In-frame SMU.63 deletion mutant derived from S. mutans

UA159; Emr

This study

Δ984 mutant In-frame SMU.984 deletion mutant derived from S. mutans

UA159; Spr

This study

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Plasmids

pBAD202/D-

TOPO

Expression vector linearized and topoisomerase-activated; Kmr Invitrogen

pIB184 Shuttle plasmid containing the constitutive P23 lactococcal

promoter; Emr

(35)

pIB107 Plasmid for chromosomal integration into S. mutans; Kmr (35)

pVL2 lexA cloned into pIB184 under its own promoter; Emr This study

pVL3 lexA A115D mutation into pVL2; Emr This study

pVL4 lexA cloned under the control of araBAD promoter into

pBAD202/D-TOPO vector; Kmr

This study

pVL5 lexA A115D mutation cloned under the control of araBAD

promoter into pBAD202/D-TOPO vector; Kmr

This study

pVL6 lexA A115D mutation cloned under its own promoter into

pIB107; Kmr

This study

a Em

r, erythromycin resistance; Km

r, kanamycin resistance; Sp

r, spectinomycin resistance. 822

823

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TABLE 2. Effects of heat and DNA damage stresses on lexA (SMU.2027) gene expression. 824

Change in S. mutansa

Stress WT ∆lexA::lexA A115D ∆clpP

50C +2.84 0.55 +1.18 0.17¶ -1.31 0.14

¶

Antibiotic ofloxacin +5.22 3.03 +1.09 0.11¶ +1.29 0.45

¶

Hydrogen peroxide +5.04 3.93 +1.32 0.25¶ +1.28 0.54

¶

aQPCR was performed to analyse lexA gene expression. The expression is presented as the 825

average fold change standard deviation compared with lexA gene expression in the absence of 826

stress. Statistical significance was determined by using a Student’s t-test with the parametric P 827

value cutoff set at < 0.01 (¶, mutant versus WT). 828

829

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TABLE 3. S. mutans cell survival towards different drugs. 830

Percentage of cell survival in S. mutans straina

Drugb WT(pIB184) ∆lexA(pIB184) ∆lexA(pVL3)

Ofloxacin 110 5.0 ( 10-3

) 30 20 ( 10-3

)¶ 4.8 1.2 ( 10

-3)

¶

Mitomycin C 67 1.0 ( 10-4

) 7 2.1 ( 10-4

)¶ 1.6 1.3 ( 10

-4)

¶

Oxacillin 2.2 1.0 1.6 0.8 1.6 0.7

aAliquots of cells were removed at the introduction of the drug and after the treatment (24 h) 831

to determine cell survival by spot plating onto THYE agar plates. The data are the averages 832

and standard errors of results from three independent cultures. Statistical significance was 833

determined by using a Student’s t-test with the parametric P value cutoff set at < 0.01 834

(¶, mutant versus WT(pIB184)). 835

bLethal concentrations of drugs were used. 836

837

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TABLE 4. DNA microarray data obtained by comparing the expression profiles of WT strain 838

and ∆lexA mutant in the presence of the CSP pheromone 839

Locusa Common name, putative function

Fold changeb

∆lexA mutant

SMU.63 Conserved membrane protein –1.8

SMU.503 Hypothetical protein –1.5

SMU.563 ornithine carbamoyltransferase –1.7

SMU.663-666 Glutamate family amino acid synthesis

operon

–1.5

SMU.984 Hypothetical protein; CHAP domain

containing protein

–1.5

SMU.1402 CRISPR-associated (CAS) protein –1.5

SMU.1841 ScrA; sucrose –specific IIABC

component

–2.2

SMU.1907 Hypothetical protein; bacteriocin-related

genomic island

–1.7

aResults for selected genes were ordered based on their position in the UA159 840

chromosome. 841

bDifferential gene expression was based on a post-normalization cut-off ± ≥1.5-842

fold. 843

844

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FIG. 1 845

846

847

848

4.07

1.08 0.96 0.83 0.82 0.71

0

1

2

3

4

5

6

UA159 ΔcomE ΔcipB ΔcomR ΔcomS ΔsigX

log

-fo

ld c

ha

ng

e in

cel

l su

rviv

al

(no

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no

CS

P p

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FIG. 2 849

850

>ECOLI_LexA --------------------------------------------------MKALTARQQEVFDLIRDHISQTGMPPTRAEIAQRLGFRSPNAAEEHLKALARKG 851 >LLACT_HdiR MNFGQNLKKLRKEAKLTQSQLADKLGMKQNAYVLWEQ-------KATNPTLELLE-KLADIYDLPIQELIK---NPDNGAEKQLID---------N-------- 852 >SMUTA_2027 MFSSQKLKERRKKLGLSQAQTADKLGISRPSYFNWEI-------GKTKPNQKNLD-KLAHLLKVDSA---------YFLSQHDIVE---------I-------- 853 >STAPH_REPR IIIAKNIRKFLNDSNMSQKKLAELINIKPSTLSDYLN-------LRSNPSHGVIQ-RIADVFEVGKSDIDT-----TYKDDNDITS---------I-------- 854 >SUBER_HDIR MFSGKQLKTIRQKHQMSQESLGQKLGVNKMTISNWEK-------GKNVPNQKHLN-QLLEIFHLDAD---------SFNPYQAIIL---------P-------- 855 >RSPHA_LexA ----------------------------------------------------MLTRKQMELLDFIKTRMDRDGVPPSFDEMKDALDLRSKSGIHRLITALEERG 856 >BLONG_LexA -------------------------------------MSTIPFSPKQKPDESTLTDRQRKVLDAIRTHIDEQGFAPSFREIGNAAGLKSPSSVKHQLQVLEDKG 857 858 >ECOLI_LexA VIEIVSGASRGIRLL------------------------------------------QEEE--EGLPLVGRVAAGEPLLAQQHIEGHYQVD-PSLFKPNADFLL 859 >LLACT_HdiR -------------YRSLTGEQQESVINFTDFLIEQNKADL---IDLKTY--------RRSSLQYAVVEDEALSAGFGQTANNTGG-HYRAY-TTENLGRYDGAA 860 >SMUTA_2027 -------------YTRLNESNKTKTLKYSQYLLEQQDKERNLM--------------KNKR--YPYRVYEKLSAGTGYSYFGDG--NFDTV-FYDEEIDHDFAS 861 >STAPH_REPR -------------YNKLTPPRQENVLNYANEQLEEQNSKGDNVVDINSY--------KQEK--TPVNVNGCVSAGVGERLHDET--LFTEM-VKGPIPTHDLAL 862 >SUBER_HDIR -------------YKQLTSLNQEKVVTYSKELLEEQNKIVQLS----QS--------QKKL--YVYRVYESLSAGTGFSYFGDG--NYDEV-FYDEQLDYDFAS 863 >RSPHA_LexA FIRRLAHRARAIEIVKLPEAMERAG--FSARAAK---------AAAAPLPKGAVTVETAGA--LDLPLMGRIAAGLPIEAINGGPQSVTVPGMMLSGRGQHYAL 864 >BLONG_LexA FIRMNANKGRAIEVVAGSAPNAEKPSQASEEATST-----SNVAEIYQFP----AEAIAES--HDVPLVGRIAAGVPITAEQHVDDVMRLP-ERLTGSGTLFML 865 ** 866 867 >ECOLI_LexA RVSGMSMKDIGIMDGDLLAVHKTQDVRNGQVVVARI----DDE-VTVKRLKKQGNKVELLPEN--SEFKPIVVDLRQQSFTIEGLAVGVIRNGDWL---- 868 >LLACT_HdiR RVKGESMEPD-FPNFSIATFLHTGFGRSGDVYAIAEGDLGEERLYIKQVFEEEDGNFRIHSLNPDPQYKDFYLG-QEDNFRIIGPVVDNFEEIEESQIID 869 >SMUTA_2027 WIFGDSMEPI-FLNGEVALIKQTGFDYDGAIYAIDW----DGQTYIKKVYREETG-LRLVSLN--KKYADKFAP-YDENPRIIGLIVGNFIPLEG----- 870 >STAPH_REPR KVNGDSMEPM-FKDGEIIFVEKTHNIKNGQIGIFII----EEEAYVKKVFVEDDR-LTLVSLN--KDYDDLHFY-RNESVRLIGKVIL------------ 871 >SUBER_HDIR WVFGDSMEPT-YLNGEVVLIKQEGFDYDGAIYAVEW----DGQTYIKKVYREEDG-LRLVSLN--KKYSDKFAP-FDENPRIIGKIIANFMPLEV----- 872 >RSPHA_LexA EVKGDSMIAAGINDGDIVVIREQQTADNGDIVVALVA---DHE-ATLKRYRRRGGMIALEPAN--DSYETQVYP--EQMVKVQGRLVGLIRSY------- 873 >BLONG_LexA EVHGDSMVDAAICDGDYVVVREQNSAVNGDIVAALL----DDE-ATVKTFRKENGHVWLMPHN--PAYSPIDGT----HATIMGKVVTVLRKL------- 874 * * 875 876

877

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FIG. 3 878

A) 879

880

B) 881

882

883

884

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FIG. 4 885

886

887

888

889

6.77

0.74

2.36

0.85

2.29

1.38

2.42

2.76

0

1

2

3

4

5

6

7

8

9

10

UA159 ΔlexA

log

-fo

ld c

ha

ng

e in

cel

l su

rviv

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(no

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CS

P p

re-s

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s)

Ofloxacin

Oxacillin

Rifampicin

Vancomycin

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FIG. 5 890

891

892

893

894

0.0001

0.001

0.01

0.1

1

UA159 ΔlexA

TE

(%

)

No CSP

CSP

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FIG. 6 895

896

897

3.03 2.91

1.00 0.90

0.84

0

1

2

3

4

5

UA159 Δ63 Δ984 ΔscrA Δcsn2

log

-fo

ld c

ha

ng

e ce

ll s

urv

ival

(no

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P p

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