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Conserved pheromone production, response and degradation by Streptococcus mutans

Streptococcus mutans, a bacterium with high cariogenic potential, coordinates competence for natural transformation and bacteriocin production via the XIP and CSP pheromones. CSP is effective in inducing bacteriocin responses, but not competence in chemically defined media (CDM). This is in contrast to XIP, which is a strong inducer of competence in CDM, but can also stimulate bacteriocin genes as a late response. Inter-connections between the pathways activated by the two pheromones have been characterized in certain detail in S. mutans UA159, but it is mostly unknown whether such findings are representative for the species. In this study, we used bioassays based on luciferase reporters for the bacteriocin gene cipB and the alternative sigma factor sigX to investigate various S. mutans isolates for production and response to CSP and XIP pheromones in CDM. Similar to S. mutans UA159, endogenous CSP was undetectable in the culture supernatants of all tested strains. During optimization of the bioassay using the cipB reporter, we discovered that the acivity of exogenous CSP used as a standard was reduced over time during S. mutans growth. Using a FRET-CSP reporter peptide, we found that S. mutans UA159 was indeed able to degrade CSP, and that such proteolytic activity was not significantly different in isogenic mutants with deletion of the protease gene htrA, or the competence genes sigX, oppD, and comR. CSP proteolysis was also detected in all the wild type strains, indicating that such activity is conserved in S. mutans. For the XIP pheromone, endogenous production was observed in the supernatants of all 34 tested strains at peak concentrations in culture supernatants that varied between 200 nM and 26000 nM. Transformation in the presence of exogenous XIP was detected in all, but one, of the isolates. The efficiency of transformation varied, however, among the different strains, and for those with the highest transformation rates, endogenous XIP peak concentrations in the supernatants were above 2000 nM XIP. We conclude that XIP production and inducing effect on transformation, as well as proteolytic activity leading to the inactivation of CSP are conserved functions among different S. mutans isolates. Understanding the functionality and conservation of pheromone systems in S. mutans may lead to novel strategies to prevent or treat unbalances in oral microbiomes that may favour diseases.


INTRODUCTION
Natural genetic transformation is widely distributed in bacteria. In streptococci and in bacillus it occurs during a genetically programmed differentiated state called competence. During this state the bacteria become capable to take up DNA from the environment and incorporate it into their genomes. The capacity for natural transformation has been reported for more than 80 bacterial species (1,2). Among streptococci, competence for natural transformation includes most species of the mitis, salivarius, bovis, anginosus and mutans groups (1,(3)(4)(5). The core of the machinery necessary for streptococcal transformation relies on the transcription of the conserved alternative sigma factor SigX, also known as ComX. SigX orchestrates a core response in streptococcal species characterized by the induction of 27 to 30 genes (6). The functions of the core genes are predominantly related to transformation, most of them coding for competence effector proteins for DNA binding, uptake and recombination (6)(7)(8).
Streptococcus mutans is a member of the mutans group and part of the human oral microbiota. Environmental factors disturbing the ecological balance in the oral cavity, such as increased sugar intake, may favor S. mutans growth. The S. mutans acidogenic and aciduric properties may then contribute to tooth demineralization and dental caries (9). Natural transformation was first reported in S. mutans in 1981 (10). This and later studies showed that transformation in S. mutans, at least in the laboratory setting, is restricted to a limited range of strains, and depends on environmental conditions not yet fully understood (10). Studies investigating the S. mutans pan-genome have recently revealed the ubiquity of sigX and competence effector genes in S. mutans (11)(12)(13), indicating that competence may be a conserved feature in S. mutans. Moreover, the extensive horizontal gene transfer observed in the genomes of S. mutans clinical isolates (11), indicates that transformation occurs in their natural habitat, and may be a widespread feature in the species.
In S. mutans the competent state is triggered by two linear peptides, the CSP (competence stimulating peptide) (7) and the XIP (sigX-inducing peptide) ( Figure 1) (8). CSP was the first described pheromone and, until recently, the only one known to activate the competence system for genetic transformation in S. mutans. The comC gene, which encodes CSP, is downstream the comDE operon, encoding the ComD histidine kinase and the ComE response regulator. At least in the synthetic form, CSP is thought to bind to the ComD histidine kinase of the ComDE twocomponent system, leading to phosphorylation of the ComE response regulator (14).
Phosphorylated ComE directly up-regulates the transcription of clusters of bacteriocin-related genes (6,8,15,16). These include among others the SMU_1914 gene (also known as nlmC and cipB), encoding mutacin V, and the putative immunity protein SMU_1913, found in a comE downstream region. Mutacin V has been proposed to link the early CSP bacteriocin-inducing response to the competence response (17)(18)(19)(20)(21). Notably, the encoding gene SMU_1914 is absent in a range of S. mutans strains (13), and other genetic elements, perhaps more conserved, may link the two processes. Essential for competence development is the activation of the XIP pheromone encoding gene, comS, followed by the upregulation of the alternative sigma factor SigX, the master core regulator of competence. In rich media, ComS seems to be processed to XIP inside the cells. Intracellular XIP and/or ComS then binds to ComR to activate the competence pathway (22). The predicted CSP is in general conserved among S. mutans strains (7,16), and there are indications that S. mutans may form a single CSP pherotype (16,23). At least in complex medium, the CSP precursor peptide is thought to be processed during and after export, resulting in the active CSP (16,23). The XIP pheromone system is found in several streptococci (3,5,8,24,25). In S. mutans UA159 the XIP pheromone encoded by comS has been identified in culture supernatants grown in chemically defined medium (CDM) lacking peptides (8,26). XIP is produced as a pro-peptide (ComS) that is possibly exported and processed into the active XIP (N-GLDWWSL) (4,8,26).
The presence of an exporter has, however, never been demonstrated, and recent studies inidicate that release of XIP or ComS by autolysis is sufficient to promote intercellular communication in CDM (27). Under such conditions, response to the XIP depends on the Opp oligopeptide permease system, indicating that the peptide is internalized (8). Once inside the cells XIP is thought to bind to the Rgg-like regulator ComR to activate transcription of sigX and comS. In contrast with XIP, CSP shows no activity or low potency in triggering competence in CDM (28)(29)(30), but can still induce the expression of bacteriocin-related genes of the ComE regulon (28). When it comes to transformation, the use of synthetic CSP has led to higher levels of S. mutans transformation (31,32), but has not extended the range of strains transformed in the absence of the synthetic pheromone. As for the synthetic XIP, evidence for competence induction and endogenous pheromone production has so far been restricted to the reference strain UA159, with few exceptions (12).
In this study we investigated the functional conservation of the S. mutans CSP and XIP pheromone signaling systems in CDM. Extracellular CSP activity was not detected in any of the tested strains. Exposure to synthetic CSP revealed that S. mutans can indeed degrade CSP, a behavior that was conserved in the examined strains, and that at least in strain UA159 was not abolished by deletion of sigX, comR, oppD, or the htrA protease gene. For the XIP pheromone, endogenous production was found in all tested strains, and transformation was found in all, but one of the isolates grown in CDM. The results thus indicate the presence of a single S. mutans XIP pherogroup, and suggest that S. mutans has a conserved ability to suppress CSP activity in CDM.

Bacterial strains and media
The Streptococcus mutans strains used in this study and their relevant characteristics are listed in Table 1. The strains used in the study were selected from the collection of strains in our laboratory, representing strains known to be transformed (UA159, V403, OMZ175, LML-2, LML-4, GS5, NG8, BM71, LT11) and others in which genetic competence has not yet been characterized. Todd-Hewitt broth (THB; Becton Dickinson) was used to grow all the strains used for DNA extraction and DNA sequencing. Chemically Defined Medium (CDM) (8) was used to perform the genetic transformation assays and bioassay to measure the activity of exogenously added CSP in the supernatants of S. mutans, and also to verify and quantify the concentration of extracellular native XIP in culture supernatants (4,26). The antibiotics erythromycin, kanamycin and spectinomycin used for mutant construction were used at final concentrations of 10 µg mL -1 , 500 µg mL -1 and 500 µg mL -1 , respectively. THB agar and THB agar supplemented with erythromycin were used to enumerate the total and transformed number of S. mutans.

Construction of mutants.
The deletion mutants were constructed using the PCR-ligation mutagenesis strategy (33). Sequence information was obtained from the S. mutans UA159 genome.
Mutants with deletion of comX and htrA were constructed. AscI or FseI sites were incorporated into the 5`-ends of the oligonucleotide primers and both ends of the resistance cassette. The The resultant amplicons were digested with AscI or FseI whereas the kanamycin resistance cassette was digested with both enzymes. The upstream and downstream amplicons of target genes were ligated to the kanamycin cassette using T4 DNA ligase. The two ligated products were mixed and PCR amplified with distal primers. The resultant amplicons were used to transform S. mutans.
Gene deletion was confirmed by PCR amplification and gel electrophoresis.

DNA sequence analysis
The chromosomal DNA of S. mutans strains were isolated as previously described (34). The mutans UA159 sequence as a reference.

Bioassay of CSP and XIP activity in culture supernatants
The CSP bioassay was performed to measure the production of native CSP and activity of exogenously added CSP in the supernatants of S. mutans during growth, and the XIP bioassay was to verify and quantify the concentration of extracellular native XIP in culture supernatants. A PcipBluc reporter (SM059) was used in the CSP bioassay as previously described (26), and a PsigX-luc reporter in a comS deletion background (SM091) was used to perform the XIP bioassay as described by Desai et al. (4), with slight modifications (26).
To estimate CSP production and response, the supernatants were collected by 10x dilution of precultures at OD600 of 0,6 in CDM containing 10% Bovine serum albumin (BSA). When added, 250nM CSP concentration was used. Supernatants were collected for 6 hours during growth. For

Genetic transformation
Overnight cultures of all S. mutans strains were diluted 1:10 in fresh CDM and incubated in 5% CO2 at 37°C. The OD600 was followed until the cultures reached an absorbance value of 0.6, then 10% of glycerol was added and they were stored at -80°C. To perform the assay the stored cultures were diluted in fresh CDM 1:10 to an OD600 of 0.05 and incubated in air for 2h at 37°C.
The OD was measured and aliquots of 150 µL of the bacterial suspensions were transferred to 1.5 mL microcentrifuge tubes. The plasmid pVA838 (Table) was added to all tubes at a final concentration of 1 µg mL -1 and XIP was added in the experimental group at a final concentration of 1,000 nM. The samples were then incubated in air for additional 4 h at 37°C, at which time the OD was measured again and the bacterial suspensions were serially diluted (up to 1 to 10 6 ) in PBS.
The suspensions were plated in duplicate on THB agar and THB agar supplemented with erythromycin 10 µg mL -1 . The plates were incubated in 5% CO2 for 48 h at 37°C before counting the colony-forming units (CFU). The number of genetically transformed cells was divided per total CFU to obtain the values for transformation frequency. Three independent experiments were performed to evaluate transformation in CDM.
In order to evaluate genetic transformation in the non-responsive strains, an independent experiment extending the incubation time to 10 h before plating was performed. In addition, at 6 h an extra load of pVA838 was added, which increased the final concentration to 2 µg mL -1 . For the remaining non-responsive strains, transformation was attempted using the 6.3kb aRJ02 chromosomal PCR amplicon with a kanamycin marker, which is expected to have a higher sensitivity for detection of transformability (Table) (35,36).

Bioassay for CSP detection in CDM.
In this study we used a PcipB-luc reporter (Pluc1914) for detection of extracellular CSP activity, as previously described (26), except that high sensitivity was only achieved in the presence of BSA (Figure 2A). BSA was chosen because it is known to enhance competence of S. mutans in rich media (37), and in S. pneumoniae, BSA prevents CSP proteolysis (38).
The potency of CSP in inducing the activiy of the cipB promoter was increased by approximately 33 fold in CDM supplemented with BSA compared with CDM alone (Fig. 2A).
Failure of CSP to induce sigX in CDM was independent of carbohydrate source. Since the induction of cipB was significangly enhanced in the presence of BSA, we decided to further examine the intriguingly fact that CSP fails to stimulate sigX in CDM (28)(29)(30). Our hypothesis was that increased stimulation of the early response by CSP, as observed with BSA supplementation, would enable the activation of sigX, which is a late response in complex media. We used a similar bioassay as described above, but using the promoter of sigX linked to the luciferase gene instead.
The results showed that CSP concentrations up to 1000 nM failed to induce the PsigX-luc reporter in CDM supplemented with BSA and glucose ( Figure 2B).
Since the carbohydrate source, at least in complex media, has a large influence on the induction of sigX expression by CSP (39), we also investigated sigX expression in CDM supplemented with fructose and galactose ( Figures 2C and 2D). CSP still failed in demonstrating sigX induction. Thus, although BSA enables prompt early response to exogenous CSP in CDM, the CSP well-known inducing effect on sigX expression in complex media could not be rescued by optimal activation of the early response to CSP.

Endogenous CSP activity was not detected in the supernatants of S. mutans. Lack of endogenous CSP activity in the supernatants of S. mutans UA159 grown in CDM has been
previously reported by our group (26). It is, however, unknown whether this is a conserved feature in the species. We investigated 6 other strains of S. mutans, and the comC deletion mutant SM004, for the CSP activity in their supernatants, by growing them in CDM in the presence of BSA ( Figure   2E). No cipB-inducing activity was detected in culture supernatants collected at early-, mid-, and late-exponential phases of growth, thus indicating that the lack of extracellular CSP activity under such growth conditions may represent a conserved feature in S. mutans.

Suppression of CSP activity during growth in CDM.
Since (1) activation of the CSP late response resulting in induction of sigX was abolished in CDM, but not the early response leading to increased expression of cipB, and since BSA enhanced the early response, we decided to investigate whether CSP may be degraded over time by S. mutans. Supernatants of S. mutans cultures exposed to 250 nM synthetic CSP were collected at different time points during growth in CDM. CSP activity in the supernatants of S. mutans UA159 was measured by using the cipB luciferase reporter described above. Within the first 3 hours, CSP had almost completely disappeared from the culture supernatants ( Figure 3A).
Due to the recognizable role of CSP in the induction of competence, it was crucial to determine if the loss of CSP activity was dependent on the development of competence. For this, we investigated the CSP activity in the culture supernatants of UA159 deletion mutants for sigX, oppD, and comR (Table). In all of them, a dramatic reduction in activity of exogenously added CSP was observed ( Figure 3B).
To determine whether such an inhibitory effect on CSP activity could be due to proteolytic cleavage, we supplemented the CDM medium with a FRET-18CSP reporter peptide ( Figure 3C).
We found that all three isogenic mutants (sigX, oppD, and comR) grown under such conditions promoted increase in fluorescence activity, as measured after 240 min incubation at 37C ( Figure   3C), thus suggesting that the ability of S. mutans to proteolyticially inactivate CSP does not require expression of key elements of the competence regulon. We also examined the effect of inactivating the htrA gene coding for the S. mutans HtrA protease, which in S. pneumoniae inactivates the CSP and cleaves misfolded proteins. No reduction in CSP degradation was observed for the htrA mutant ( Figure 3C). CSP activity was indeed slightly higher for the culture supernatants of the htrA mutant at mid-and late-exponential phases of growth ( Figure 3D).
Finally, we tested whether suppression of CSP activity is extended to other S. mutans strains. In all the strains tested, reduction in CSP activity was observed, though at different levels ( Figure 3E). When grown in the presence of the FRET-18CSP reporter peptide, an increase in fluorescence was observed for all strains. We conclude that S. mutans suppresses CSP activity during growth in CDM, and that the mechanisms involved are active in the absence of htrA, sigX, comR or oppD, indicating the involvement of competence-regulated independent factors.
Proteolytic activity leading to CSP inactivation seems to be conserved in S. mutans. at the mid-exponential growth phase, and reaching maximal values at early stationary phase (data not shown). We conclude that the growth conditions that support extracellular XIP pheromone accumulation by UA159 may also sustain XIP production by a majority of the strains.

XIP activity is detected in
Thirty-three out of thirty-four strains were naturally transformable. Natural transformation without the addition of synthetic pheromone was detected in 23 of the 34 strains (data not shown).
Because optimal levels of XIP may not be present at the time of competence under in vitro conditions, it was of interest to learn whether addition of synthetic XIP would result in higher transformation levels. Our results showed that synthetic XIP extended the range of transformed strains. Three of the strains with detectable XIP levels in the supernatants, including OMZ175, UAB90, and KM1 were transformed only upon addition of synthetic XIP. In the strains that were not transformed with the initial protocol, we increased the time during which they grew in the presence of synthetic XIP and donor DNA to 10 h, but even then no transformants were obtained. (40). We next used a more sensitive protocol based on the use of PCR large fragments as donor DNA (35,36). Only strain At10 was not transformed, thus indicating that a majority of S. mutans are amenable to transformation. Overall, strains that exhibited lower transformation frequencies showed lower levels of XIP activity in their culture supernatants ( Figure 4B).
The comS gene was conserved in the 34 S. mutans strains. The S. mutans comS gene encoding the XIP precursor is essential for competence development. Given the variability in transformation efficiency observed above, and that some strains were not amenable to transformation, we investigated whether the comS gene and its putative promoter sequences were conserved in the S.
mutans strains included in the study. The results showed that comS was identical in all the 34 strains ( Figure 4C). Highly conserved sequences were also found in the comS putative promoter. In this region the only difference in relation to UA159 was the presence or absence of an additional adenine between the -10 element and the comS translation initiation site. The additional adenine was present in 22 out of the 34 strains evaluated. In all the strains, comS was located 57-58 nt downstream of the tRNA transcriptional terminator of comR (SMU_61) and the putative -10 element was 32-33 nt upstream of the comS translation initiation site. While this study was being performed, new genomic sequences for more 78 strains of S. mutans were made available on public databases (41). In order to compare our results with the new genomic sequences we ran BLAST using the sequence shown in

DISCUSSION
In complex media, the CSP pheromone triggers the expression of bacteriocin-related genes, followed by a late response characterized by increased expression of the XIP-encoding gene comS, and competence development (43,44). The mechanisms leading to activation of comS expression and competence remain unknown. It is also still unknown the reason why CSP fails in stimulating competence in peptide-free defined medium. It has been suggested that S. mutans may perhaps produce a protease that, similar to the HtrA protease in S. pneumoniae, could inactivate the CSP (29). This possible explanation, however, did not seem to match with the later finding that CSP is actually active in CDM, in that it can stimulate cipB and all other genes regulated by ComED (28).
Our results show, however, that S. mutans can indeed inactivate CSP by a mechanism that most probably involve proteolysis, given the results obtained with the FRET-18CSP reporter peptide.
We confirmed previous results that CSP induces cipB (28), and found that a CSP concentration as low as 1 nM in the presence of BSA was sufficient to activate the early bacteriocin response. This is in oppose to the CSP concentration required to activate sigX, which can be observed with values that exceed the maximum concentration used in this study (1000 nM) (data not shown). Inactivation of the S. mutans CSP by proteases produced by other streptococcal specieshave been known for more than ten years (45). However, this is the first indication that S. mutans can also inhibit the activity of its own CSP, most probably via proteolytic activity. Unlike for S. pneumoniae (38), however, the HtrA protease in S. mutans was not found to be required for CSP inactivation.
In S. pneumoniae, luciferase and gfp reporter strains for CSP activity have been successfully used to identify S. pneumoniae CSP pheromones in culture supernatants (46). Also in the seminal study that identified the S. pneumoniae competence factor, the CSP was isolated and purified from the supernatant (47). Also for S. mutans UA159 grown in complex medium, CSP has been identified by mass spectrometry in the supernatant fraction (23). Based on these findings, we hypothesized that if S. mutans produces endogenous CSP, we would probably be able to detect it in its culture supernatant. The protocol we developed in this study using a cipB luciferase reporter suspended in CDM supplemented with BSA enabled the identification of concentrations as low as 1 nM. Despite this high sensitivity, we could not detect any CSP inducing activity in the supernatants of the different strains tested, indicating therefore that endogenous CSP was either absent, present at very low levels or not present in the supernatant fraction.
In contrast to CSP, endogenous XIP activity was present in the supernatants of most strains.
Moreover, activation of the XIP system in CDM enabled transformation of all but one of the 34 strains tested. The comS gene encoding the pre-processed form of XIP was indeed found in all the strains, which is in line with the results of a recent study reporting comS as part of the S. mutans core genome (11). In general, higher levels of XIP detection in the supernatants correlated with increased transformability. Of note, some strains that produced high concentrations of endogenous XIP, needed to be stimulated by synthetic XIP to transform. This was, however, not surprising, given the fact that maximum concentrations were in most cases observed only close to the stationary phase, which is a period during which competence under laboratory conditions is already shut-off. For some of the strains that produced low levels of XIP, transformation was only detected by using a large PCR amplicon as DNA donor, which is known to result in increased recovery of transformants (35,48).    ComE is also thought to induce comC, which product is pre-processed and exported by an ABCexporter, and cleaved to its active form outside the cells (18-CSP pheromone). In CDM, CSP induces the ComED pathway, but it is unknwon whether the CSP pheromone system is endogenously activated. It is also unknown why in complex media the CSP pheromone induces competence, but in CDM, only the bacteriocin pathway is stimulated. To interrogate the conservation of the ComRS and ComED signaling pathways in other members of the species, sigX and cipB luciferase reporter bioassays were employed.