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Completely summarize an article with correct English grammar and accurate spelling, but must also contain sufficient critical analysis to earn full points. Critical analysis (do NOT forget CRITICAL ANALYSIS) of a research paper requires critical or original thinking, which is expressed as a combination of the following: a brief synopsis of the experiments performed, description of deficiencies in the experimental design, comparison of experimental approach with other work, statement of the strengths of the paper, re-interpretation of results, suggestions for future experimentation based on results, and a critical review of authors’ conclusions. 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Please cite this article as doi: 10.1111/omi.12145 This article is protected by copyright. All rights reserved. Received Date : 22-Jul-2015 Revised Date : 12-Oct-2015 Accepted Date : 26-Oct-2015 Article type : Original Article Escape from the competence state in Streptococcus mutans is governed by the bacterial population density D. Dufour, C. Villemin, J.A. Perry* and C.M. Lévesque Dental Research Institute, Faculty of Dentistry, University of Toronto, Toronto, ON, Canada Correspondence: Céline M. Lévesque, Dental Research Institute, Faculty of Dentistry, University of Toronto, 124 Edward St., Room 454, Toronto, ON M5G 1G6, Canada. Tel.: +1 416 979 4917, ext. 4313; fax: +1 416 979 4936; E-mail: [email protected] * Present address: M. G. DeGroote Institute for Infectious Disease Research, Department of Biochemistry and Biomedical Sciences, DeGroote School of Medicine, McMaster University, Hamilton, ON L8S 4K1, Canada Running title: S. mutans competence shut-off Keywords: DNA transformation; cell density; quorum-sensing; dental caries; Streptococcus mutans Accepted Article This article is protected by copyright. All rights reserved. SUMMARY Horizontal gene transfer through natural DNA transformation is an important evolutionary mechanism among bacteria. Transformation requires that the bacteria are physiologically competent to take and incorporate free DNA directly from the environment. Although natural genetic transformation is a remarkable feature of many naturally competent bacteria, the process is energetically expensive for the cells. Consequently, a tight control of the competence state is necessary. The objective of the present work was to help decipher the molecular mechanisms regulating the escape from the competence state in Streptococcus mutans, the principal etiological agent responsible for tooth decay in humans. Our results showed that the cessation of competence in S. mutans was abrupt, and did not involve the accumulation of a competence inhibitor nor the depletion of a competence activator in the extracellular environment. The competence state was repressed at high cell population density via concomitant repression of sigX gene encoding the master regulator of the competence regulon. Co-culture experiments performed with oral and non-oral bacteria showed that S. mutans assesses its own population density and also the microbial density of its surroundings to regulate its competence escape. Interestingly, neither the intra- and extra-species quorum-sensing systems nor the other 13 twocomponent regulatory systems identified in S. mutans were involved in the cell-densitydependent escape of the competence state. Altogether, our results suggest a complex mechanism regulating the competence shut-off involving cell-density-dependent repression of sigX through an as yet undefined system, and possibly SigX protein stability. Accepted Article This article is protected by copyright. All rights reserved. INTRODUCTION Natural DNA transformation is a mechanism of horizontal gene transfer found in bacteria, involving the active internalization and incorporation of exogenous naked DNA molecules from the environment into the genome of a bacterial cell (Chen & Dubnau, 2004). To occur, natural DNA transformation requires a specific physiological state called ‘competence’. Until now natural DNA transformation has been reported through in vitro experiments in a total of 82 bacterial species belonging to diverse phyla of both Gram-positive and Gram-negative bacteria (Johnston et al., 2014). Among the naturally competent Gram-positive bacteria, streptococcal species have been the most investigated. Initially discovered in Streptococcus pneumoniae in 1928 by Griffith (Griffith, 1928), natural DNA transformation have been notably reported in almost all streptococcal species found in the oral cavity and upper respiratory tract of humans (Johnsborg et al., 2007; Johnston et al., 2014). Although most oral streptococci are considered commensal organisms, some of them can change a beneficial relationship into a pathogenic relationship for reasons that are not always completely elucidated. Oral streptococci are associated with oral infections (dental caries or tooth decay, refractory periodontitis, abscesses). Some are known to cause infective endocarditis when disseminated through the blood stream (Russell, 2006). Because naturally competent oral streptococci are able to sample the DNA pool of an entire community, they have the ability to acquire fitness-enhancing genes allowing them to adapt and survive in their natural habitat, including resistance to host defence mechanisms and antibiotic therapy. Recently, a study investigating the genomic history of 44 streptococcal species, including 14 human oral species, through gene loss, gene gain and genome expansion analysis, has underlined a very dynamic Accepted Article This article is protected by copyright. All rights reserved. pattern of genome evolution (Richards et al., 2014). It appeared that, for a large proportion, the pan-genome of streptococci has gone through massive lateral gene transfer. This finding is quite obvious and expected for oral streptococci, knowing their natural ecosystem and their ability for natural genetic transformation. Although beneficial for the bacteria, DNA transformation is an energetically expensive process requiring a group of interacting proteins involved in the uptake, translocation through the membranes, protection, and homologous DNA recombination (Dubnau, 1999; Chen and Dubnau, 2004). Hence, the development of genetic competence must be tightly regulated to ensure that it is not undertaken in a less than ideal environment. Over the past decade, major advances have occurred in the understanding of the modulation of genetic competence in S. mutans and S. pneumoniae by the CSP-ComDE quorum-sensing system composed of the CSP pheromone and the ComDE two-component system (TCS) (Senadheera et al., 2005b; Ahn et al., 2006; Martin et al., 2006; Oggioni & Morrison, 2008; Claverys et al., 2009). Yet, our understanding of the molecular mechanisms underlying the shut-off of competence in streptococci is still in its infancy. Two mechanisms of competence escape were proposed based on the quorum-sensing system activating SigX, the master regulator of the competence regulon, required for the expression of late competence genes involved in DNA processing, uptake, and recombination (Lee & Morrison, 1999; Aspiras et al., 2004). In S. pneumoniae, the shut-off of the competence state has been mainly attributed to the binding of the late competence protein DprA to the response regulator ComE to abolish transcription from ComE-activated promoters, leading to the arrest of SigX production (Mirouze et al., 2013). Accepted Article This article is protected by copyright. All rights reserved. In S. mutans, a second type of quorum-sensing system has been recently described. This new system is called ComRS and is composed of the small XIP peptide and its transcriptional regulator ComR (Mashburn-Warren et al., 2010). In S. mutans, both quorum-sensing systems (CSP-ComDE and ComRS) regulate genetic competence, and the ComRS system is necessary for the induction of SigX, which is essential for the development of competence (Federle & Morrison, 2012). SigX activity is highly sensitive to the growth medium used to cultivate the cells. CSP pheromone activates SigX in a nutrient-rich medium, while XIP stimulates SigX only in a chemically-defined medium devoid of exogenous peptides (Son et al., 2012; Reck et al., 2015). But still today the interconnection between the CSP and XIP signalling pathways controlling the regulation of competence is not completely understood. Work done by Li’s group showed that the shut-off of the competence state in S. mutans was associated with the proteolytic degradation of SigX by the MecA adaptor (Tian et al., 2013; Dong et al., 2014). They showed that MecA mediates the formation of a tertiary SigX–MecA–Clp complex that sequesters SigX, accelerating the escape of the cells from competence. Interestingly, MecA does not act as an anti-SigX factor when S. mutans is cultivated in a peptide-free medium that is also permissive for competence. The environmental cues triggering the shut-off of competence through the proteolysis of SigX still remains to be elucidated. The aim of this work was to investigate the environmental conditions governing the escape of the competence state of S. mutans cultivated in a nutrient-rich medium. Our results suggest that the exit from the competence state is abrupt and is intimately correlated to the whole bacterial population density. We also provided the evidence that S. mutans uses multiple sensing systems Accepted Article This article is protected by copyright. All rights reserved. sharing regulatory components to induce and shut down the expression of the competence regulon. METHODS Bacterial strains and growth conditions A summary of bacterial strains is provided in Table 1. All bacterial strains were grown in ToddHewitt medium supplemented with 0.3 % yeast extract (THYE) at pH 7.5. Streptococcal strains were incubated statically at 37ºC in air with 5% CO2, while Escherichia coli was cultivated aerobically at 37ºC. Nonpolar insertion-deletion mutants were constructed in S. mutans UA159 wild type strain by PCR ligation mutagenesis (Lau et al., 2002). To construct the ciaR overexpressing strain, the full-length coding region of ciaR was PCR amplified using S. mutans UA159 genomic DNA as a template and cloned under the control of the constitutive promoter P23 in the shuttle plasmid pIB166 (Biswas et al., 2008). For growth of S. mutans, antibiotics were added when necessary at the following final concentrations: chloramphenicol, 10 µg ml-1 ; erythromycin, 10 µg ml–1; kanamycin, 300 µg ml–1; spectinomycin, 1 mg ml–1. Cell growth was monitored by determining the optical density at 600 nm. Cell viability was assessed by counting colony forming unit (CFU) on replica agar plates. Natural DNA transformation assays Standard assay Overnight cultures of S. mutans were diluted (1:20) into fresh THYE broth (unbuffered) and incubated statically at 37°C. For the experiments using a pH-buffered THYE medium, overnight cultures were diluted (1:20) into fresh THYE medium buffered at pH 7.5 with 40 mM Accepted Article This article is protected by copyright. All rights reserved. phosphate/citrate buffer (Welin-Neilands & Svensäter, 2007). Samples (0.5 ml aliquots) were withdrawn at the indicated times and S. mutans ∆rgp genomic DNA (0.2 – 200 μg ml–1) containing the spectinomycin resistance marker inserted into the rgp locus of the UA159 strain, as inactivation of this locus has no effect on transformation efficiency (Perry et al., 2009), was added to the aliquots. DNase I (20 U ml–1) was added after 1 h, and the mixture was incubated for another 1.5 h to allow for DNA integration and phenotypic expression of the resistance marker. Cells were serially diluted and spot plated on THYE (non-selective) and THYEspectinomycin (selective) agar plates for CFU determination. The transformation efficiency (TE) was calculated as the percentage of spectinomycin-resistant transformants divided by the total number of recipient cells. All assays were performed in triplicate from three independent experiments. Cell-free supernatant assay Overnight cultures of S. mutans UA159 wild type strain were diluted (1:20) into fresh THYE broth and incubated statically at 37°C until an optical density at 600 nm of 0.1 was reached. Samples (0.5 ml aliquots) were withdrawn and 50 µl of cell-free supernatant of UA159 wild type cultures harvested at lag phase (optical density at 600 nm of ~0.1), mid-log phase (optical density at 600 nm of ~0.8), or stationary phase (optical density at 600 nm of ~1.6) was added. The mixtures were then incubated at 37°C for 2.5 h in the presence of 20 µg ml–1 of ∆rgp genomic DNA. Cells were serially diluted and spot plated on THYE and THYE-spectinomycin agar plates for CFU determination. TE was calculated as described above. Accepted Article This article is protected by copyright. All rights reserved. High-cell-density assay Overnight cultures of S. mutans UA159 wild type strain and its mutants were diluted (1:20) into fresh THYE broth and incubated statically at 37°C until an optical density at 600 nm of 0.1 was reached. Samples of 0.5 ml (1×), 2.5 ml (5×), 5 ml (10×), 7.5 ml (15×), and 10 ml (20×) were withdrawn and centrifuged at 4000 g for 10 min at 4°C. Bacterial cell pellets were resuspended in 0.5 ml of their own supernatant unless otherwise indicated. Ten micrograms of S. mutans ∆rgp genomic DNA was then added and the cultures were incubated at 37°C for 2.5 h. Cells were serially diluted and spot plated on THYE and THYE-spectinomycin agar plates for CFU determination. TE was calculated as described above. Mixed cultures assay Overnight cultures of S. mutans UA159Kan, Streptococcus salivarius ATCC 25975, and E. coli DH10B were diluted (1:20) into fresh THYE broth and incubated at 37°C until an optical density at 600 nm of 0.1 was reached. Cells were harvested by centrifugation and co-cultures were prepared by resuspending mixed cells in a final volume of 0.5 ml of fresh THYE broth. Cocultures were composed of S. mutans and S. salivarius cells or S. mutans and E. coli cells in ratios of 1:1, 1:5, 1:10, 1:15, and 1:20. The co-cultures were then incubated at 37°C for 2.5 h in the presence of 20 µg ml–1 of S. mutans ∆rgp genomic DNA. Cells were serially diluted and spot plated on THYE-kanamycin (for selection of S. mutans UA159Kan cells) and THYEkanamycin/spectinomycin (for selection of S. mutans UA159Kan spectinomycin-resistant transformants) agar plates for CFU determination. TE was calculated as the percentage of kanamycin/spectinyomycin-resistant transformants divided by the total number of S. mutans Accepted Article This article is protected by copyright. All rights reserved. UA159Kan recipient cells. All assays were performed in triplicate from three independent experiments. Gene expression analysis Transcriptional analysis of sigX gene during the growth of S. mutans UA159 wild type strain in THYE broth was conducted by quantitative real-time PCR (QPCR). Wild type 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 a first-strand cDNA synthesis kit (MBI Fermentas). QPCR was carried out using the SsoFast EvaGreen Supermix (Bio-Rad) and a CFX96 real-time PCR detection system (BioRad). The 16S rRNA gene was used as an internal reference. QPCR assays were performed in triplicate with RNA isolated from three independent experiments. Statistical significance was determined by using a Student t test and a P value of <0.01. RESULTS S. mutans abruptly exits from the competence state past the mid-logarithmic phase of growth In streptococcal species such as S. mutans, transformation occurs naturally. At variable times during growth, these naturally transformable bacteria become transiently competent to take up free DNA from the environment. In S. mutans, natural transformation occurs at low cell density in early logarithmic phase. To get a better understanding of the kinetics of competence development in S. mutans cultivated in nutrient-rich medium, we first performed a time-course experiment in which donor genomic DNA was provided at different phases of cell growth. As Accepted Article This article is protected by copyright. All rights reserved. shown in Figure 1, no significant variation of the transformation efficiency was observed during the lag phase until the early log phase. In fact, the transformation efficiency of S. mutans WT strain cultivated in rich medium was ~ 0.001% until the early log phase of growth. When cells reached mid-log phase (optical density at 600 nm of ~0.8), a slight decrease in transformation efficiency was observed. A significant decrease of transformation efficiency of at least 1 log– fold was observed immediately after mid-log phase (optical density at 600 nm of ~1.1). At an optical density at 600 nm of ~1.3 and higher, no transformants could be detected even after plating all of the transformation mixture on selective agar plates (Figure 1). S. mutans is well known for its capacity to produce acids from dietary sugars. A recent study reported that environmental pH can have a strong effect on the XIP-induced competence state in S. mutans cultivated in a chemically-defined medium (Guo et al., 2014). Results presented at Figure 1 showed a decrease in culture pH, with a pH value of ≤5.5 past mid-log phase, when cells were not transformable. To eliminate the possibility that an acidic pH was primarily responsible for the cessation of competence, additional transformation experiments were performed. Firstly, cells were harvested at different times, washed with phosphate-buffered saline, and resuspended into fresh unbuffered THYE broth at pH 7.5. In the second set of experiments, overnight cultures were diluted into a pH-buffered THYE broth to maintain the pH of the medium close to 7.5 throughout the duration of the experiments. Transformation assays were then performed as described previously (see Figure 1 legend for details). Interestingly, similar competence profiles were obtained for all sets of experiments with a sharp decrease of transformation efficiency observed past mid-log phase and no transformants detected at stationary phase (data not shown). These results suggest that most probably the environmental Accepted Article This article is protected by copyright. All rights reserved. pH does not influence the development of natural competence when S. mutans is cultivated in a nutrient-rich medium. In order to investigate if the decline in transformation efficiency past the mid-log phase was due to an effect of the DNA concentration, standard transformation assays were performed using increasing concentrations of donor genomic DNA. Our results showed a dose-dependent response when lag phase and mid-log phase cells were used (Figure 2). However, a saturation level was reached at over 20 µg ml–1 of donor DNA. Stationary phase cells produced no transformants even at the highest concentration of donor DNA tested suggesting that the amount of extracellular DNA available is not a limiting factor. Altogether, our results showed that S. mutans is not naturally competent for transformation throughout its growth cycle, but competence is observed during a short period of time. The fact that the amount of homologous donor DNA is not a limiting factor also suggests a fine-tuning of the competence window in S. mutans. To rule out the possibility that the nutritional potential of the medium could affect the ability of S. mutans cells to be transformed, standard DNA transformation experiments were performed using THYE broth diluted 5-fold. Our results showed that a dilution of the medium did not affect the ability of the cells to be transformed as cells cultivated in a 5-fold diluted THYE medium were transformed at frequencies similar to those obtained with cells cultivated in full-strength THYE broth (data not shown). Accepted Article This article is protected by copyright. All rights reserved. Neither the accumulation of an inhibitor nor the depletion of an activator is responsible for the competence escape Cessation of competence in S. mutans is abrupt and occurs after the mid-logarithmic phase of growth (Figure 1). These results prompted us to investigate if the accumulation of a competence inhibitor or the depletion of a competence activator in the extracellular environment was responsible for the competence shut off. Cell-free supernatants (10% (vol/vol)) harvested at midlog and stationary growth phases were first evaluated for inhibitory effect on transformation of S. mutans lag phase cells. No significant decrease in the transformation efficiency was observed (Table 2). On the other hand, if a competence activator was present during the lag phase until the mid-log phase, it would be possible to restore the competence phenotype of stationary phase cells by treating these cells with cell-free supernatant obtained from lag phase cultures. No significant increase in transformation efficiency was observed for the mid-log phase cells, and the stationary phase cells were still not transformable (Table 2). Since the addition of only 10% (vol/vol) of cell-free supernatant could be under-representative of the real concentration of a putative activator or inhibitor present in the extracellular medium, we repeated the experiments described above using lyophilised cell-free supernatants in order to obtain higher concentrations. Lag phase and mid-log phase cells were transformed at frequencies similar to those obtained with 10% (vol/vol) cell-free-supernatant, while stationary phase cells were still not transformable (data not shown). Altogether, these results suggest that the cessation of natural competence in S. mutans during growth in a nutrient-rich medium does not involve the accumulation of a competence inhibitor nor the depletion of a competence activator. Accepted Article This article is protected by copyright. All rights reserved. Competence escape is governed by bacterial cell density We next hypothesized that the escape from the competence state might be related to the increase in S. mutans cell population density. To test this hypothesis, transformable cells harvested during the lag phase were concentrated by centrifugation to obtain different densities. The cell pellets were resuspended in their own supernatant (Figure 3A) or fresh THYE broth (Figure 3B), and used in our high-cell-density transformation assay (see Materials and Methods for details). As shown in Figure 3A, the transformation efficiency decreased with increasing cell density. Interestingly, when the transformable lag phase cells were concentrated 20×, equivalent to the cell density observed at the onset of the stationary phase (stationary phase cells are not transformable; Figure 2), no transformants could be detected. In order to rule out the possibility that metabolic end-products could interfere with the competence phenotype, we repeated the same experiments but using lag phase cells resuspended into fresh THYE medium. The same profile was then obtained (Figure 3B). To test if S. mutans cells were still able to grow at higher cell densities, the total number of cells was determined by measurement of CFU on non-selective agar plates. Our results showed that the numbers of CFU were similar for each condition tested confirming that S. mutans cells were still dividing even at the highest cell density tested. Altogether, these results clearly showed a cell density-dependent decrease in transformation efficiency in S. mutans when cultivated in nutrient-rich medium. Accepted Article This article is protected by copyright. All rights reserved. The SigX factor essential for the activation of competence genes is repressed at high cell density The alternative SigX factor is required for the transcriptional control of the competence regulon (Aspiras et al., 2004). In fact, inactivation of sigX gene completely abolished competency in S. mutans. The transcriptional profile of sigX during normal growth of S. mutans under nutrientrich conditions was then determined concomitantly with the ability of the cells to become competent. S. mutans wild type cells were cultivated in THYE medium and harvested at the indicated times. Samples were split in half, where the first half was used in our standard transformation assay and the second half was processed for QPCR analysis. As expected, the expression of sigX gene was up-regulated during the early log and logarithmic growth phases where the cells are the most transformable (Table 3). In contrast, a sharp decrease in sigX expression was observed past mid-log phase until the onset of the stationary phase of growth. During the stationary phase, no transformants could be detected and sigX expression was strongly repressed. We also quantified sigX expression using transformable lag phase cells concentrated 10× and 20× by centrifugation to mimic high density cell cultures of optical density at 600 nm of ∼1.0 and ∼2.0, respectively. Twenty micrograms per ml of S. mutans ∆rgp genomic DNA was then added and the cultures were incubated at 37°C for 2.5 h before quantification of sigX expression. QPCR results showed that sigX gene was repressed by ~3–fold (–2.85 ± 0.42) and ~25–fold (– 25.42 ± 3.88) when cells were concentrated 10× and 20×, respectively, versus unconcentrated samples. Together, these results indicate that competence escape in S. mutans at high cell density correlates with the disappearance of SigX factor. Accepted Article This article is protected by copyright. All rights reserved. S. mutans senses the whole cell population density to shut down its competence state Our results showed that the exit of competence is dictated by the cell population density. Since S. mutans resides normally in a diverse, multi-species biofilm (Aas et al., 2005), we next investigated if S. mutans only senses its own population density or can also assess the whole microbial density to regulate competence escape. We performed transformation experiments using mixed cultures of S. mutans and Streptococcus salivarius, an early colonizer of the oral cavity (Cole et al., 2013), both harvested during the lag phase of growth. The mixtures with various ratios (1:1, 1:5, 1:10, 1:15, 1:20) of the two bacterial species were prepared by mixing a fixed amount of transformable S. mutans lag phase cells with increasing concentrations of S. salivarius lag phase cells. Controls consisted of transformable S. mutans lag phase cells concentrated by centrifugation and resuspended into fresh THYE broth to obtain different cell densities. As expected, a cell density-dependent decrease of transformation efficiency was observed for the S. mutans monoculture controls (Table 4). A similar evolution of the transformation efficiency was observed using mixed cultures of S. mutans and S. salivarius. Interestingly, competence shut-off occurred earlier in the mixed cultures, with no transformants obtained at a ratio of 1:15 (Table 4). In order to test if S. mutans responded differently in the presence of a non oral bacterial species, we repeated the transformation experiments using a mixed culture of S. mutans and E. coli. In this case, a significant effect on S. mutans transformation efficiency was observed at a ratio of 1:10, with no transformants detected even after plating all of the transformation mixture (Table 4). Growth kinetics analysis showed that co-cultivation of S. mutans and S. salivarius or S. mutans and E. coli did not affect growth of S. mutans under the conditions tested. Altogether, these results suggest that at low cell density the competence state of S. mutans is induced. Once a threshold level of cells (self and foreign Accepted Article This article is protected by copyright. All rights reserved. recognition) is reached, S. mutans shuts down its competence state; yet S. mutans exits from the competence state at a much lower cell density in the presence of non closely-related species. The CSP-ComDE quorum-sensing system does not participate in the exit of competence at high cell density Bacteria use quorum-sensing systems to assess their number and species composition in their environment (Waters & Bassler, 2005). In S. mutans, the CSP-ComDE quorum-sensing system controls a variety of cellular processes including the development of genetic competence (Li et al., 2001). We hypothesized that the CSP-ComDE system could be responsible for the cell density-dependent decrease in transformation efficiency. We first tested the direct impact of the ComDE TCS using cells of S. mutans ∆comD and ∆comE mutants, which are unable to sense and respond to the CSP pheromone, respectively. Transformation assays were performed using monocultures of S. mutans ∆comD, ∆comE, and ∆comDE mutants harvested during lag phase and concentrated by centrifugation to obtain different cell densities. Unexpectedly, the transformation efficiency decreased with increasing cell density for the single and double mutants (Table 5). In fact, mutant cells were transformed at frequencies similar to those obtained using wild type cells, suggesting that the CSP-ComDE quorum-sensing system was not implicated in the cell density-dependent decrease in transformation efficiency. Moreover, addition of increasing concentrations of exogenous synthetic CSP (up to 0.2 µM) during our standard natural DNA transformation assays led to a progressive increase in transformation efficiency, and a plateau was reached at concentrations higher that 0.2 µM CSP (data not shown). Mutant strains deficient in nlmTE and sepM genes, encoding the ABC transporter (Petersen & Scheie, 2000) and the SepM protease (Hossain & Biswas, 2012) for the export and Accepted Article This article is protected by copyright. All rights reserved. the final maturation of the CSP pheromone, respectively, were also tested for their ability to be transformed at increasing cell densities. Both mutants presented a similar transformation profile to that obtained for the wild type strain (Table 5). Altogether, these results suggest that the cell density-dependent decrease in transformation efficiency is not mediated by the CSP-ComDE quorum-sensing system, and that the CSP molecule is not solely responsible for regulating competence according to cell density in nutrient-rich medium. None of the known other two-component systems of S. mutans are involved in the exit of the competence state at high cell density S. mutans UA159 reference strain possesses thirteen other TCSs (Ajdic et al., 2002; Lévesque et al., 2007; Biswas et al., 2008) and one orphan response regulator (Song et al., 2012). We hypothesized that if a regulatory system was involved in competence escape, the cell densitydependent decrease would be abolished in the knockout strain. For each TCS, the histidine kinase and response regulator genes were individually inactivated (except for the VicR response regulator as a null mutation was found lethal; Senadheera et al., 2005a) and the effect of gene disruption was tested on the cell’s ability to be transformed at different cell densities. All mutants showed a cell density-dependent decrease in transformation efficiency similar to the wild type strain (Table 5). Interestingly, the response regulator CiaR, but not its cognate CiaH histidine kinase, was found essential for the development of natural competence under our conditions tested. To investigate the possibility that CiaR could play an important role in the competence shut-off, we first determined the transcriptional profile of ciaR gene during growth of S. mutans in THYE broth concomitantly with the ability of the cells to become naturally competent. Three time-points were investigated: i) log-phase, when cells are naturally competent; ii) past mid-log Accepted Article This article is protected by copyright. All rights reserved. phase, when transformation efficiency decreases; iii) at the onset of the stationary growth phase, when no transformants can be obtained. Our results showed that ciaR gene was expressed under all conditions tested even when the cells were not transformable. We also cloned ciaR gene under the control of a constitutive promoter and tested the evolution of natural competence of S. mutans(pIB166-ciaR) overexpressing CiaR vs. S. mutans(pIB166) carrying the empty plasmid (control strain). The natural transformation experiments were performed as described in the legend of Figure 1. A similar evolution of the transformation efficiency was observed between the overexpressing strain and its control (data not shown). Although CiaR response regulator plays an important role in the development of natural competence in S. mutans cultivated in THYE broth, these experiments suggest that CiaR regulator is not involved in the escape from the competence state under the conditions tested. We also tested the high-cell-density-responsive regulatory system HdrRM, a two-gene regulatory system involved in competence and bacteriocin production in S. mutans (Merritt et al., 2007). Similarly, individual mutants (∆hdrM, ∆hdrR) and a double mutant (∆hdrRM) were constructed and transformation tested at different cell densities. Our results showed a cell density-dependent decrease in transformation efficiency (Table 5). Finally, since S. mutans senses the whole bacterial population density to shut off its competence state at high cell density (Table 4), we inactivated the luxS gene responsible for the synthesis of the autoinducer 2 (AI-2), a universal or species-non-specific signal in bacteria (Straight & Kolter, 2009). S. mutans ∆luxS mutant was then tested for its ability to be transformed when co-cultured with increasing concentrations of E. coli cells. As shown in Table 5, LuxS does not participate in the cell density-dependent competence escape in S. mutans. Accepted Article This article is protected by copyright. All rights reserved. DISCUSSION The oral cavity contains a richly diverse community of resident bacteria composed of several hundred species (Aas et al., 2005). Given the enormous numbers of bacterial species and the large size of accessible exogenous transforming DNA, naturally competent bacteria can acquire new genetic information. In fact, the acquisition of potential useful genetic information, such as novel metabolic functions, virulence traits or antibiotic resistance can have a major impact on human health as pathogen strains more virulent than their ancestors are likely to emerge. S. mutans is a well-characterized resident of the complex multispecies biofilm formed on tooth enamel. This bacterium infects more than half of the world’s human population. S. mutans is widely recognized as a key etiological agent of dental caries, one of the most prevalent chronic infectious diseases worldwide (Loesche, 1986; Burne et al., 2012). Although the factors triggering the induction of genetic competence in S. mutans are well known, the mechanisms involved in the competence escape remain poorly understood. In this work, we showed that when cultivated in a nutritional rich medium, S. mutans regulates the development of genetic competence in a cell-density-dependent manner. At low cell population density, cells entry into the competence state. Once a threshold level of high cell density is reached, S. mutans cells exit from the competence state abruptly. Recent studies have proposed that the escape from the competence state in S. mutans cultivated in a complex medium involved a post-translational control of the SigX factor, through the recognition and the targeting of SigX by MecA adaptor for the degradation by the host protease Clp (Tian et al., 2013; Dong et al., 2014). Our results suggest that the exit from competence occurs first at a transcriptional level with the repression of sigX expression as cell population density increases. Based on these results, we propose the following model. At low cell population density, S. mutans activates the expression of sigX gene. Accepted Article This article is protected by copyright. All rights reserved. SigX factor accumulates to sufficient levels outcompeting the MecA adaptor and Clp proteins that are expressed at a constant level during cell growth. Therefore, free SigX can associate with the core RNA polymerase to activate transcription of the competence regulon. When a high cell population density is reached, S. mutans repressed the transcription of sigX. The free SigX population is retained at very low levels, and free SigX molecules are thus sequestered into SigX–MecA–Clp complexes for degradation, leading to competence shut down. Transformation experiments performed using co-cultures of S. mutans with S. salivarius, a commensal and pioneer colonizer of the oral cavity, clearly showed that S. mutans assesses its own population numbers and also the population density of other species of bacteria in the vicinity in exiting the competence sate. The same results were obtained using an non-oral bacterial species (E. coli). Interestingly, competence shut-off occurred earlier in the mixed cultures with E. coli than with S. salivarius, suggesting that S. mutans transformation works best with closely-related species. Intriguingly, S. mutans does not use its main intraspecies (CSPComDE) or interspecies (AI-2) quorum-sensing systems to sense the bacterial population density. We first speculated that another quorum-sensing system that generates an as yet undefined, diffusible signal molecule(s) to act in gauging intra- and interspecies population density could exist. This hypothesis was finally rejected since neither the accumulation of an inhibitor nor the depletion of an activator was found responsible for the competence escape under the conditions tested. In addition to the ComDE TCS, S. mutans UA159 wild type reference strain possesses multiples TCSs. These regulatory systems involve the transfer of phosphate between histidine and aspartate amino acid residues on membrane-bound histidine kinase sensors and cognate cytoplasmic response regulators, respectively. When activated, the response regulators act as transcription factors to repress and/or activate gene expression (Stock Accepted Article This article is protected by copyright. All rights reserved. et al., 2000). None of these TCSs were able to participate in the exit of competence at high cell density under the conditions tested. Merritt and co-workers characterized in S. mutans UA140 strain a two-gene operon called hdrRM, encoding a transcriptional regulator HdrR and a membrane protein HdrM (Merritt et al., 2007). The hdrRM operon was found to be induced >10- fold by conditions of high cell density and the HdrM membrane protein was identified as a negative regulator of the competence state as mutation of hdrM greatly increased the natural transformation efficiency. Unexpectedly, our high cell-density transformation assay failed to show any impact of the HdrRM system in the cell density-dependent shut off of the competence state in S. mutans UA159 strain. Moreover, mutation of the hdrM locus in UA159 strain did not induce an increase in transformation efficiency. This result suggests a different function of the hdrRM locus between the two S. mutans strains. Bacteria use quorum-sensing for the control of a wide variety of functions (Waters & Bassler, 2005). A sufficient body of experimental work using multicellular communities suggest that bacterial cells can also use another strategy, called contact-dependent signalling, to modulate gene expression. Contact-dependent signalling is used by bacteria found in close proximity such as within biofilms, to regulate bacterial growth of both sibling and competitors (Blango & Mulvey, 2009). Hence, physical interactions and subsequent modulation of gene expression have been particularly observed in oral microbial communities. For example, the periodontal pathogen Porphyromonas gingivalis showed an increased adhesive capacity to various substrates through the induction of genes encoding proteins with hemagglutinin adhesion domains when cultivated in close contact with Treponema denticola also a member of the red complex associated with severe forms of periodontal disease (Meuric et al., 2013). In E. coli, a form of contact-dependent communication, called contact-dependent inhibition, is used by some cells to inhibit the growth Accepted Article This article is protected by copyright. All rights reserved. of target E. coli cells competing for limited environmental resources (Aoki et al., 2010). Our cocultures results suggest that a similar cell contact-dependent phenomenon may occur in the regulation of genetic competence in S. mutans. We are currently investigating this interesting possibility. ACKNOWLEDGEMENTS We thank Indranil Biswas for the gift of pIB107 and pIB166 plasmids. This work was supported by a Canadian Institutes of Health Research (CIHR) grant MOP-93555 to C.M.L and Natural Sciences and Engineering Research Council of Canada (NSERC) grant RGPIN 355968 to C.M.L. C.M.L. is a recipient of a Canada Research Chair. REFERENCES Aas, J.A., Paster, B.J., Stokes, L.N., Olsen, I. and Dewhirst, F.E. (2005) Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 43: 5721–5732. Ahn, S., Wen, Z.T., and Burne, R.A. (2006) Multilevel control of competence development and stress tolerance in Streptococcus mutans UA159. Infect Immun 74: 1631–1642. Ajdic, D., McShan, W.M., McLaughlin, R.E. et al. (2002) Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc Natl Acad Sci U S A 99: 14434–14439. Aoki, S.K., Diner, E.J., de Roodenbeke, C.T. et al. 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Table 1 Bacterial strains used in this study Strains Characteristics References Escherichia coli DH10B Wild type Lab stock Streptococcus salivarius ATCC 25975 Wild type Lab stock Streptococcus mutans UA159 Wild type Lab stock UA159(pIB166) S. mutans UA159 harboring pIB166 plasmid; Cmlr Lab stock UA159(pIB166-ciaR) S. mutans UA159 harboring pIB166-ciaR construct; Cmlr This work UA159Kan S. mutans UA159 harboring a chromosomal kanamycin resistance marker inserted at the SMU.1405 locus; Kanr Biswas & Biswas (2006) Δrgp Nonpolar SMU.825–830 mutant derived from S. mutans UA159; Spcr Perry et al. (2009) ΔcomD Nonpolar SMU.1916 mutant derived from S. mutans UA159; Emr Perry et al. (2009) ΔcomE Nonpolar SMU.1917 mutant derived from S. mutans UA159; Emr Perry et al. (2009) ΔcomDE Nonpolar SMU.1916–1917 mutant derived from S. mutans UA159; Emr Perry et al. (2009) ΔsepM Nonpolar SMU.518 mutant derived from S. mutans UA159; Emr This work ΔcomAB Nonpolar SMU.286–287 mutant derived from S. mutans UA159; Emr Perry et al. (2009) ΔluxS Nonpolar SMU.474 mutant derived from S. mutans UA159; Emr Sztajer et al. (2008) ΔhdrR Nonpolar SMU.1854 mutant derived from S. mutans UA159; Emr This work ΔhdrM Nonpolar SMU.1855 mutant derived from S. mutans UA159; Emr This work ΔhdrRM Nonpolar SMU.1854–1855 mutant derived from S. mutans UA159; Emr This work ΔvicK Nonpolar SMU.1516 mutant derived from S. mutans UA159; Emr Lévesque et al. (2007) ΔciaH Nonpolar SMU.1128 mutant derived from S. mutans UA159; Emr Lévesque et al. (2007) ΔciaR Nonpolar SMU.1129 mutant derived from S. mutans UA159; Emr This work ΔcovS Nonpolar SMU.1145 mutant derived from S. mutans UA159; Emr Lévesque et al. (2007) ΔcovR Nonpolar SMU.1146 mutant derived from S. mutans UA159; Emr This work Accepted Article This article is protected by copyright. All rights reserved. ΔkinF Nonpolar SMU.928 mutant derived from S. mutans UA159; Emr Lévesque et al. (2007) ΔllrF Nonpolar SMU.927 mutant derived from S. mutans UA159; Emr This work ΔscnK Nonpolar SMU.1814 mutant derived from S. mutans UA159; Emr Lévesque et al. (2007) ΔscnR Nonpolar SMU.1815 mutant derived from S. mutans UA159; Emr This work ΔspaK Nonpolar SMU.660 mutant derived from S. mutans UA159; Emr Lévesque et al. (2007) ΔspaR Nonpolar SMU.659 mutant derived from S. mutans UA159; Emr This work ΔphoR Nonpolar SMU.1037 mutant derived from S. mutans UA159; Emr Lévesque et al. (2007) ΔycbL Nonpolar SMU.1038 mutant derived from S. mutans UA159; Emr This work ΔkinG Nonpolar SMU.1009 mutant derived from S. mutans UA159; Emr Lévesque et al. (2007) ΔllrG Nonpolar SMU.1008 mutant derived from S. mutans UA159; Emr This work ΔlevS Nonpolar SMU.1965 mutant derived from S. mutans UA159; Emr Lévesque et al. (2007) ΔlevR Nonpolar SMU.1964 mutant derived from S. mutans UA159; Emr This work ΔlytS Nonpolar SMU.577 mutant derived from S. mutans UA159; Emr Lévesque et al. (2007) ΔlytT Nonpolar SMU.576 mutant derived from S. mutans UA159; Emr This work ΔliaS Nonpolar SMU.486 mutant derived from S. mutans UA159; Emr Lévesque et al. (2007) ΔliaR Nonpolar SMU.487 mutant derived from S. mutans UA159; Emr Suntharalingam et al. (2009) Δhk12 Nonpolar SMU.1548 mutant derived from S. mutans UA159; Emr Lévesque et al. (2007) Δrr12 Nonpolar SMU.1547 mutant derived from S. mutans UA159; Emr This work Δ45 Nonpolar SMU.45 mutant derived from S. mutans UA159; Emr This work Δ46 Nonpolar SMU.46 mutant derived from S. mutans UA159; Emr This work ΔgcrR Nonpolar SMU.1924 mutant derived from S. mutans UA159; Emr This work Cmlr , chloramphenicol resistance; Emr , erythromycin resistance; Kanr , kanamycin resistance; Spcr , spectinomycin resistance. Accepted Article This article is protected by copyright. All rights reserved. Table 2 Transformation experiments using cell-free supernatants Transforming cells1 Cell-free supernatant2 TE ± SD (%)3 Lag phase cells None (control) 3.9 ± 0.2 (× 10–4) Lag phase cells Lag phase 3.1 ± 0.7 (× 10–4) Lag phase cells Mid-log 2.3 ± 1.0 (× 10–4) Lag phase cells Stationary phase 4.5 ± 2.2 (× 10–4) Mid-log phase cells None (control) 8.4 ± 6.3 (× 10–4) Mid-log phase cells Lag phase 13.3 ± 5.3 (× 10–4) Stationary phase cells None (control) 0 Stationary phase cells Lag phase 0 1 S. mutans UA159 wild type cells cultivated in THYE broth were harvested during lag phase, mid-log phase, and stationary phase, and used for the cell-free supernatant transformation assays (see Material and methods for details). 2 Cell-free supernatant of S. mutans UA159 wild type cultures cultivated in THYE broth and collected at lag phase, mid-log phase, and stationary phase (see Material and methods for details). 3 No statistical differences were observed versus control. Table 3 Evolution of sigX expression and natural competence during S. mutans growth under nutrient-rich conditions Time (h) 0.5 1.5 2.5 3.5 4.5 Growth phase Lag Early log Log Mid-log Stationary Optical density at 600 nm ~0.1 ~0.2 ~0.4 ~0.8 ~1.6 Transformation efficiency1 9.2 ± 1.6 (×10–4) 17.5 ± 1.6 (×10–4) 9.8 ± 2.3 (×10–4) 3.9 ± 1.1 (×10–4) 0 sigX expression2 +1.0 +2.91 ± 0.35 +3.91 ± 0.76 –1.20 ± 0.30 –8.24 ± 0.50 Accepted Article This article is protected by copyright. All rights reserved. 1 The transformation efficiency was calculated as the percentage of spectinomycin-resistant transformants divided by the total number of recipient cells. The data are the averages and standard deviations of results from three independent experiments. 2 Expression of sigX was set at 1 for t = 0.5 h. Gene expression is presented as the average fold change ± standard deviation compared with the expression at t = 0.5 h. Table 4 Transformation of S. mutans in mixed cultures S. mutans transformation efficiency1 Ratio2 OD600 3 S. mutans4 S. mutans/S. salivarius S. mutans/E. coli 1:1 0.1 1.04 ± 0.13 (10–3) 1.92 ± 0.46 (10–3) 0.56 ± 0.21 (10–3) 1:5 0.5 2.23 ± 0.46 (10–4) 2.50 ± 0.51 (10–4) 1.05 ± 0.31 (10–4) 1:10 1.0 1.31 ± 0.51 (10–5) 10.20 ± 5.85 (10–5) 0 1:15 1.5 7.09 ± 0.89 (10–6) 0 0 1:20 2.0 0 0 0 1 S. mutans UA159Kan cells cultivated in THYE broth were harvested during lag phase and used for the mixed cultures transformation assays (see Material and methods for details). The transformation efficiency was calculated as the percentage of kanamycin/spectinomycin-resistant transformants divided by the total number of recipient cells. The data are the averages and standard deviations of results from three independent experiments. 2 Ratio of bacterial cells. 3 Optical density at 600 nm (OD600) of the mono or mixed cultures. 4 Controls consisted of transformable S. mutans UA159Kan lag phase cells concentrated by centrifugation and resuspended into fresh THYE broth to obtain different cell densities. Accepted Article This article is protected by copyright. All rights reserved. Table 5 Transformation efficiency of S. mutans strains at different cell densities Transformation efficiency (×10–4) 1 Strain Deletion2 1× 5× 10× UA159 Wild type strain, no deletion 11.2 ± 1.5 2.9 ± 0.06 0.2 ± 0.2 ΔcomD ComD HK membrane receptor 5.1 ± 0.5 0.4 ± 0.4 0.2 ± 0.2 ΔcomE ComE RR 12.7 ± 1.5 0.2 ± 0.07 0.1 ± 0.02 ΔcomDE ComD HK and ComE RR 8.4 ± 1.7 0.4 ± 0.1 0.1 ± 0.01 ΔsepM SepM protease 6.5 ± 0.8 0.8 ± 0.1 0.2 ± 0.06 ΔcomAB ComAB membrane transporter 7.3 ± 1.3 1.1 ± 0.3 0.2 ± 0.06 ΔvicK VicK HK 2.7 ± 0.2 0 0 ΔciaH CiaH HK 36.2 ± 31.0 8.2 ± 3.8 0.3 ± 0.1 ΔciaR CiaR RR 0 0 0 ΔcovS CovS HK 5.5 ± 3.7 0.5 ± 0.2 0.08 ± 0.01 ΔcovR CovR RR 4.5 ± 0.5 0.2 ± 0.06 0.1 ± 0.03 ΔkinF KinF HK 33.1 ± 39.8 0.7 ± 0.09 0.1 ± 0.02 ΔllrF LlrF RR 3.0 ± 1.5 0.3 ± 0.2 0.06 ± 0.04 ΔscnK ScnK HK 5.5 ± 0.6 0.3 ± 0.1 0.1 ± 0.05 ΔscnR ScnR RR 4.0 ± 0.3 0.4 ± 0.1 0.2 ± 0.03 ΔspaK SpaK HK 10.3 ± 5.0 0.5 ± 0.4 0.1 ± 0.01 ΔspaR SpaR RR 2.1 ± 0.9 0.1 ± 0.01 0.08 ± 0.4 ΔphoR PhoR HK 5.4 ± 0.4 0.2 ± 0.06 0.06 ± 0.04 ΔycbL YcbL RR 1.9 ± 0.08 0.2 ± 0.1 0.05 ± 0.02 ΔkinG KinG HK 6.5 ± 0.8 0.2 ± 0.1 0.02 ± 0.01 ΔllrG LlrG RR 1.6 ± 0.6 0.2 ± 0.05 0.04 ± 0.01 ΔlevS LevS HK 2.2 ± 0.6 0.1 ± 0.01 0.05 ± 0.02 ΔlevR LevR RR 1.9 ± 0.4 0.3 ± 0.3 0.1 ± 0.01 ΔlytS LytS HK 5.4 ± 3.5 0.6 ± 0.07 0.05 ± 0.007 ΔlytT LytR RR 2.1 ± 0.05 0.1 ± 0.01 0.08 ± 0.02 ΔliaS LiaS HK 0.4 ± 0.05 0.1 ± 0.04 0.04 ± 0.003 ΔliaR LiaR RR 26.4 ± 6.5 1.0 ± 0.5 0.4 ± 0.1 Δhk12 HK12 7.1 ± 0.3 0.3 ± 0.1 0.2 ± 0.1 Δrr12 RR12 5.0 ± 0.1 0.2 ± 0.01 0.1 ± 0.01 Accepted Article This article is protected by copyright. All rights reserved. Δ45 HK14 3.2 ± 0.5 0.4 ± 0.1 0.1 ± 0.08 Δ46 RR14 2.3 ± 0.2 0.3 ± 0.05 0.1 ± 0.05 ΔgcrR GcrR orphan RR 2.8 ± 0.3 0.2 ± 0.04 0.08 ± 0.02 ΔhdrM HdrM membrane protein 7.3 ± 0.9 2.7 ± 0.04 0.3 ± 0.05 ΔhdrR HdrR transcriptional regulator 6.1 ± 0.5 0.7 ± 0.3 0.1 ± 0.1 ΔhdrRM HdrR and HdrM 5.9 ± 1.0 2.5 ± 0.6 0.6 ± 0.1 ΔluxS3 LuxS enzyme 20.1 ± 1.0 0.5 ± 0.1 0.3 ± 0.2 1 S. mutans cells harvested during lag phase were either not concentrated (1×, control) or concentrated 5× and 10× by centrifugation. The transformation efficiency was calculated as the percentage of antibiotic-resistant transformants divided by the total number of recipient cells. The data are the averages and standard deviations of results from three independent experiments. 2 HK, histidine kinase membrane-bound sensor; RR, cytoplasmic response regulator. 3 S. mutans cells harvested during lag phase were mixed with cultures of E. coli concentrated by centrifugation. The transformation efficiency was calculated and expressed as described above. FIGURE LEGENDS Figure 1 Evolution of natural transformation of S. mutans cultivated in nutrient-rich medium. Overnight cultures of S. mutans UA159 wild type strain were diluted in fresh THYE broth and incubated statically at 37°C. At the indicated times, cell samples were withdrawn and exposed for 1 h at 37°C to 20 µg ml–1 of ∆rgp genomic DNA. Samples were then treated with DNase and the mixture was incubated for another 1.5 h at 37°C before differential plating. The transformation efficiency (TE, triangle) was expressed as the percentage of spectinomycinresistant transformants divided by the total number of recipient cells. The data are the averages and standard deviations of results from three independent cultures. Bacterial growth was monitored spectrophotometrically (optical density at 600 nm (OD600), square). A representative Accepted Article This article is protected by copyright. All rights reserved. growth curve at 37°C is shown. Transformation efficiencies and terminal pH readings taken at the indicated times are shown below the graph. Figure 2 Effect of donor DNA concentration on transformation efficiency of S. mutans. Standard transformation assays were performed using S. mutans UA159 wild type cells harvested at lag phase (optical density at 600 nm of ~0.1), mid-log phase (optical density at 600 nm of ~0.8), or stationary phase (optical density at 600 nm of ~1.6), and in the presence of 0.2 µg ml–1 (white bars), 2 µg ml–1 (dashed bars), 20 µg ml–1 (grey bars), or 200 µg ml–1 (black bars) of donor genomic DNA carrying a spectinomycin resistance gene. The transformation efficiency (TE) was expressed as the percentage of spectinomycin-resistant transformants divided by the total number of recipient cells. The data are the averages and standard deviations of results from three independent cultures. Figure 3 Impact of the cell population density on S. mutans transformation efficiency. S. mutans UA159 wild type cells harvested during lag phase were either not concentrated (1×, control) or concentrated 5×, 10×, 15× and 20× by centrifugation, and resuspended in their own supernatant (A) or in fresh THYE medium (B). The transformation efficiency (TE) was expressed as the percentage of spectinomycin-resistant transformants divided by the total number of recipient cells. The data are the averages and standard deviations of results from three independent cultures. Accepted Article This article is protected by copyright. All rights reserved. Accepted Article This article is protected by copyright. All rights reserved.