A quorum-sensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation

In a previous study, a quorum-sensing signaling system essential for genetic competence in Streptococcus mutans was identified, characterized, and found to function optimally in biofilms (Li et al., J. Bacteriol. 183:897-908, 2001). Here, we demonstrate that this system also plays a role in the ability of S. mutans to initiate biofilm formation. To test this hypothesis, S. mutans wild-type strain NG8 and its knockout mutants defective in comC, comD, comE, and comX, as well as a comCDE deletion mutant, were assayed for their ability to initiate biofilm formation. The spatial distribution and architecture of the biofilms were examined by scanning electron microscopy and confocal scanning laser microscopy. The results showed that inactivation of any of the individual genes under study resulted in the formation of an abnormal biofilm. The comC mutant, unable to produce or secrete a competence-stimulating peptide (CSP), formed biofilms with altered architecture, whereas the comD and comE mutants, which were defective in sensing and responding to the CSP, formed biofilms with reduced biomass. Exogenous addition of the CSP and complementation with a plasmid containing the wild-type comC gene into the cultures restored the wild-type biofilm architecture of comC mutants but showed no effect on the comD, comE, or comX mutant biofilms. The fact that biofilms formed by comC mutants differed from the comD, comE, and comX mutant biofilms suggested that multiple signal transduction pathways were affected by CSP. Addition of synthetic CSP into the culture medium or introduction of the wild-type comC gene on a shuttle vector into the comCDE deletion mutant partially restored the wild-type biofilm architecture and further supported this idea. We conclude that the quorum-sensing signaling system essential for genetic competence in S. mutans is important for the formation of biofilms by this gram-positive organism.     

Biofilm mode of growth of Streptococcus intermedius favored by a competence-stimulating signaling peptide

Gram-positive and gram-negative bacteria use quorum sensing to coordinate population behavior. In several streptococci, quorum sensing mediated by competence-stimulating peptides (CSP) is associated with development of competence for transformation. We show here that a synthetic CSP favored the biofilm mode of growth of Streptococcus intermedius without affecting the rate of culture growth.     

The Streptococcus sanguinis competence regulon is not required for infective endocarditis virulence in a rabbit model

Streptococcus sanguinis is an important component of dental plaque and a leading cause of infective endocarditis. Genetic competence in S. sanguinis requires a quorum sensing system encoded by the early comCDE genes, as well as late genes controlled by the alternative sigma factor, ComX. Previous studies of Streptococcus pneumoniae and Streptococcus mutans have identified functions for the >100-gene com regulon in addition to DNA uptake, including virulence. We investigated this possibility in S. sanguinis. Strains deleted for the comCDE or comX master regulatory genes were created. Using a rabbit endocarditis model in conjunction with a variety of virulence assays, we determined that both mutants possessed infectivity equivalent to that of a virulent control strain, and that measures of disease were similar in rabbits infected with each strain. These results suggest that the com regulon is not required for S. sanguinis infective endocarditis virulence in this model. We propose that the different roles of the S. sanguinis, S. pneumoniae, and S. mutans com regulons in virulence can be understood in relation to the pathogenic mechanisms employed by each species.     

Natural genetic transformation of Streptococcus mutans growing in biofilms

Streptococcus mutans is a bacterium that has evolved to be dependent upon a biofilm "lifestyle" for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in the development of genetic competence in S. mutans and to assay the natural genetic transformability of biofilm-grown cells. Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci. The genetic locus of this system comprises three genes, comC, comD, and comE, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulator, respectively. We deduced the sequence of comC and its active pheromone product and chemically synthesized the corresponding 21-amino-acid competence-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilitated increased transformation frequencies, with typically 1% of the total cell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesis or allelic replacement followed by assays of transformation efficiency. We also demonstrated that biofilm-grown S. mutans cells were transformed at a rate 10- to 600-fold higher than planktonic S. mutans cells. Donor DNA included a suicide plasmid, S. mutans chromosomal DNA harboring a heterologous erythromycin resistance gene, and a replicative plasmid. The cells were optimally transformed during the formation of 8- to 16-h-old biofilms primarily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibiotic resistance determinant. This work demonstrated that a peptide pheromone system controls genetic competence in S. mutans and that the system functions optimally when the cells are living in actively growing biofilms.     

Peptide pheromone induced cell death of Streptococcus mutans

Streptococcus mutans is considered one of the main causative agents of human dental caries. Cell-cell communication through two-component signal transduction systems (TCSTS) plays an important role in the pathogenesis of S. mutans. One of the S. mutans TCSTS, ComDE, controls both competence development and biofilm formation. In this study, we showed that addition of exogenous competence-stimulating peptide (CSP) beyond the levels necessary for competence inhibited the growth of S. mutans in a ComDE-dependent manner. We also demonstrated that further increases of CSP stopped S. mutans cell division leading to cell death. Use of CSP as a possible therapeutic agent is discussed.     

Interactions between oral bacteria: inhibition of Streptococcus mutans bacteriocin production by Streptococcus gordonii

Streptococcus mutans has been recognized as an important etiological agent in human dental caries. Some strains of S. mutans also produce bacteriocins. In this study, we sought to demonstrate that bacteriocin production by S. mutans strains GS5 and BM71 was mediated by quorum sensing, which is dependent on a competence-stimulating peptide (CSP) signaling system encoded by the com genes. We also demonstrated that interactions with some other oral streptococci interfered with S. mutans bacteriocin production both in broth and in biofilms. The inhibition of S. mutans bacteriocin production by oral bacteria was stronger in biofilms than in broth. Using transposon Tn916 mutagenesis, we identified a gene (sgc; named for Streptococcus gordonii challisin) responsible for the inhibition of S. mutans bacteriocin production by S. gordonii Challis. Interruption of the sgc gene in S. gordonii Challis resulted in attenuated inhibition of S. mutans bacteriocin production. The supernatant fluids from the sgc mutant did not inactivate the exogenous S. mutans CSP as did those from the parent strain Challis. S. gordonii Challis did not inactivate bacteriocin produced by S. mutans GS5. Because S. mutans uses quorum sensing to regulate virulence, strategies designed to interfere with these signaling systems may have broad applicability for biological control of this caries-causing organism.     

Identification of ComW as a new component in the regulation of genetic transformation in Streptococcus pneumoniae

Regulation of competence for genetic transformation in Streptococcus pneumoniae depends on a quorum-sensing system, genes involved in DNA uptake and recombination and a link between these two gene sets. The alternative sigma factor ComX provides this link. ComE, the response regulator of the quorum-sensing system, is required for expression of ComX and other early genes. However, an unknown ComE-dependent regulator is also required for competence when comX is expressed under control of the raffinose-responsive promoter of the aga operon. The gene comW (SP0018) is required for a high level of competence and is regulated by the quorum-sensing system, but its function is unknown. To explore its role further, comW was cloned into the multicopy plasmid pMSP3535, under the control of a nisin-inducible promoter (P(N)), and transformed into pneumococcal strains containing a raffinose-inducible comX gene (P(R)::comX). Further introduction of a comE deletion blocked the endogenous CSP signal transduction pathway. In the resulting strain, competence was independent of CSP but depended on treatment with both nisin and raffinose, showing that coexpression of comW and comX complemented the comE deficiency. ComX protein accumulation and expression of a late competence gene in the above strain support the conclusion that ComW is a new positive factor involved in competence regulation.     

Structure-activity analysis of quorum-sensing signaling peptides from Streptococcus mutans

Streptococcus mutans secretes and utilizes a 21-amino-acid signaling peptide pheromone to initiate quorum sensing for genetic competence, biofilm formation, stress responses, and bacteriocin production. In this study, we designed and synthesized a series of truncated peptides and peptides with amino acid substitutions to investigate their structure-activity relationships based on the three-dimensional structures of S. mutans wild-type signaling peptide UA159sp and C-terminally truncated peptide TPC3 from mutant JH1005 defective in genetic competence. By analyzing these peptides, we demonstrated that the signaling peptide of S. mutans has at least two functional domains. The C-terminal structural motif consisting of a sequence of polar hydrophobic charged residues is crucial for activation of the signal transduction pathway, while the core alpha-helical structure extending from residue 5 to the end of the peptide is required for receptor binding. Peptides in which three or more residues were deleted from the C terminus did not induce genetic competence but competitively inhibited quorum sensing activated by UA159sp. Disruption of the amphipathic alpha-helix by replacing the Phe-7, Phe-11, or Phe-15 residue with a hydrophilic residue resulted in a significant reduction in or complete loss of the activity of the peptide. In contrast to the C-terminally truncated peptides, these peptides with amino acid substitutions did not compete with UA159sp to activate quorum sensing, suggesting that disruption of the hydrophobic face of the alpha-helical structure results in a peptide that is not able to bind to the receptor. This study is the first study to recognize the importance of the signaling peptide C-terminal residues in streptococcal quorum sensing.     

Cell death in Streptococcus mutans biofilms: a link between CSP and extracellular DNA

Streptococcal competence-stimulating peptides (CSPs) were once thought to passively communicate population density in a process known classically as quorum sensing. However, recent evidence has shown that these peptides may also be inducible 'alarmones,' capable of conveying sophisticated messages in a population including the induction of altruistic cellular suicide under stressful conditions. We have previously characterized the alarmone response in Streptococcus mutans , a cariogenic resident of the oral flora, in which a novel bacteriocin-like peptide causes cell death in a subset of the population. Our objective in this work was to characterize the mechanism of immunity to cell death in S. mutans . Toward this goal, we have identified the conditions under which immunity is induced, and identified the regulatory system responsible for differential (and protective) expression of immunity. We also showed that CSP-induced death contributes to S. mutans biofilm formation through the release of chromosomal DNA into the extracellular matrix, providing a long sought-after mechanistic explanation for the role of CSP in S. mutans biofilm formation.     

Cell Density Modulates Acid Adaptation in Streptococcus mutans: Implications for Survival in Biofilms

Streptococcus mutans normally colonizes dental biofilms and is regularly exposed to continual cycles of acidic pH during ingestion of fermentable dietary carbohydrates. The ability of S. mutans to survive at low pH is an important virulence factor in the pathogenesis of dental caries. Despite a few studies of the acid adaptation mechanism of this organism, little work has focused on the acid tolerance of S. mutans growing in high-cell-density biofilms. It is unknown whether biofilm growth mode or high cell density affects acid adaptation by S. mutans. This study was initiated to examine the acid tolerance response (ATR) of S. mutans biofilm cells and to determine the effect of cell density on the induction of acid adaptation. S. mutans BM71 cells were first grown in broth cultures to examine acid adaptation associated with growth phase, cell density, carbon starvation, and induction by culture filtrates. The cells were also grown in a chemostat-based biofilm fermentor for biofilm formation. Adaptation of biofilm cells to low pH was established in the chemostat by the acid generated from excess glucose metabolism, followed by a pH 3.5 acid shock for 3 h. Both biofilm and planktonic cells were removed to assay percentages of survival. The results showed that S. mutans BM71 exhibited a log-phase ATR induced by low pH and a stationary-phase acid resistance induced by carbon starvation. Cell density was found to modulate acid adaptation in S. mutans log-phase cells, since pre-adapted cells at a higher cell density or from a dense biofilm displayed significantly higher resistance to the killing pH than the cells at a lower cell density. The log-phase ATR could also be induced by a neutralized culture filtrate collected from a low-pH culture, suggesting that the culture filtrate contained an extracellular induction component(s) involved in acid adaptation in S. mutans. Heat or proteinase treatment abolished the induction by the culture filtrate. The results also showed that mutants defective in the comC, -D, or -E genes, which encode a quorum sensing system essential for cell density-dependent induction of genetic competence, had a diminished log-phase ATR. Addition of synthetic competence stimulating peptide (CSP) to the comC mutant restored the ATR. This study demonstrated that cell density and biofilm growth mode modulated acid adaptation in S. mutans, suggesting that optimal development of acid adaptation in this organism involves both low pH induction and cell-cell communication.     

Natural genetic transformation in Streptococcus gordonii: comX imparts spontaneous competence on strain wicky.

Streptococcus gordonii Wicky becomes competent only after stimulation with conditioned medium from strain Challis as a source of competence factor (CF). A 3.2-kbp genomic fragment from Challis was found to impart spontaneous competence on Wicky by a complementation assay. Wicky clones containing the fragment secreted a heat-sensitive activity that induced competence in Wicky and in a comA insertion mutant of Challis. Activity was localized to a putative open reading frame, comX, with the potential to encode a 52-amino-acid peptide. comX had no similarity to known sequences, and a comX::ermAM insertion mutant of Challis transformed normally and secreted CF. These data suggest that a CF-independent pathway for competence induction exists in S. gordonii.     

Co-ordinated bacteriocin production and competence development: a possible mechanism for taking up DNA from neighbouring species

It is important to ensure DNA availability when bacterial cells develop competence. Previous studies in Streptococcus pneumoniae demonstrated that the competence-stimulating peptide (CSP) induced autolysin production and cell lysis of its own non-competent cells, suggesting a possible active mechanism to secure a homologous DNA pool for uptake and recombination. In this study, we found that in Streptococcus mutans CSP induced co-ordinated expression of competence and mutacin production genes. This mutacin (mutacin IV) is a non-lantibiotic bacteriocin which kills closely related Streptococcal species such as S. gordonii. In mixed cultures of S. mutans and S. gordonii harbouring a shuttle plasmid, plasmid DNA transfer from S. gordonii to S. mutans was observed in a CSP and mutacin IV-dependent manner. Further analysis demonstrated an increased DNA release from S. gordonii upon addition of the partially purified mutacin IV extract. On the basis of these findings, we propose that Streptococcus mutans, which resides in a multispecies oral biofilm, may utilize the competence-induced bacteriocin production to acquire transforming DNA from other species living in the same ecological niche. This hypothesis is also consistent with a well-known phenomenon that a large genomic diversity exists among different S. mutans strains. This diversity may have resulted from extensive horizontal gene transfer.     

Peptide alarmone signalling triggers an auto-active bacteriocin necessary for genetic competence

The induction of genetic competence is a strategy used by bacteria to increase their genetic repertoire under stressful environmental conditions. Recently, Streptococcus pneumoniae has been shown to co-ordinate the uptake of transforming DNA with fratricide via increased expression of the peptide pheromone responsible for competence induction. Here, we document that environmental stress-induced expression of the peptide pheromone competence-stimulating peptide (CSP) in the oral pathogen Streptococcus mutans . We showed that CSP is involved in the stress response and determined the CSP-induced regulon in S. mutans by microarray analysis. Contrary to pneumococcus, S. mutans responds to increased concentrations of CSP by cell lysis in only a fraction of the population. We have focused on the mechanism of cell lysis and have identified a novel bacteriocin as the 'death effector'. Most importantly, we showed that this bacteriocin causes cell death via a novel mechanism of action: intracellular action against self. We have also identified the cognate bacteriocin immunity protein, which resides in a separate unlinked genetic locus to allow its differential regulation. The role of the lytic response in S. mutans competence is also discussed. Together, these findings reveal a novel autolytic pathway in S. mutans which may be involved in the dissemination of fitness-enhancing genes in the oral biofilm.     

Competence-dependent endogenous DNA rearrangement and uptake of extracellular DNA give a natural variant of Streptococcus mutans without biofilm formation

The production of water-insoluble glucan (WIG) enables Streptococcus mutans to survive and persist in the oral niche. WIG is produced from sucrose by glucosyltransferase encoded tandemly by the highly homologous gtfB and gtfC genes. Conversely, a single hybrid gene from the endogenous recombination of gtfB and gtfC is easily generated using RecA, resulting in S. mutans UA159 WIG- (rate of ～1.0×10(-3)). The pneumococcus recA gene is regulated as a late competence gene. comX gene mutations did not lead to the appearance of WIG- cells. The biofilm collected from the flow cell had more WIG- cells than among the planktonic cells. Among the planktonic cells, WIG- cells appeared after 16 h and increased ～10-fold after 32 h of cultivation, suggesting an increase in planktonic WIG- cells after longer culture. The strain may be derived from the biofilm environment. In coculture with donor WIG+ and recipient WIG- cells, the recipient cells reverted to WIG+ and acquired an intact gtfBC region from the environment, indicating that the uptake of extracellular DNA resulted in the phenotypic change. Here we demonstrate that endogenous DNA rearrangement and uptake of extracellular DNA generate WIG- cells and that both are induced by the same signal transducer, the com system. Our findings may help in understanding how S. mutans can adapt to the oral environment and may explain the evolution of S. mutans.     

Establishing a genetic system for ecological studies of Streptococcus oligofermentans

Streptococcus oligofermentans is a newly characterized species belonging to the mitis group of oral streptococci. So far no correlation has been demonstrated between S. oligofermentans and dental caries. Furthermore, a reverse correlation has been observed between the number of S. oligofermentans and the number of Streptococcus mutans, a major cariogenic pathogen, in the oral cavity. These properties suggest that S. oligofermentans may have a potential to be used as a 'probiotics' for caries prevention. In this study, we aim to establish a genetic system in S. oligofermentans to further study the biology of this new species. Using homologous regions of the comCDE locus in other streptococci, the comC gene was isolated and sequenced. A synthetic competence-stimulating peptide (CSP) was synthesized and shown to be able to effectively induce competence in S. oligofermentans. This CSP-induced transformation system in S. oligofermentans was used to construct green fluorescent protein (gfp) and luciferase (luc) reporter systems, both of which are driven by the lactate dehydrogenase (ldh) promoter. These reporter systems were further shown to be highly expressed in planktonic and biofilm cells, suggesting that these reporter systems can be used in future ecological studies of S. oligofermentans.     

Effects of quorum sensing on cell viability in Streptococcus mutans biofilm formation

Streptococcus mutans (S. mutans) uses a quorum sensing (QS) signaling system, which is dependent on competence stimulating peptide (CSP), to regulate diverse physiological activities including bacteriocin production, genetic transformation, and biofilm formation. However, the mechanism of the QS system-induced biofilm formation remains unclear. Here, we demonstrated that the late-stage biofilm formation was increased by the addition of exogenous CSP in S. mutans. The numbers of dead cells in biofilms formed in presence of CSP was 64.5% higher than that without CSP after 12 h (p < 0.05) and 76.3% higher after 24 h (p < 0.05), the numbers of live cells in biofilms formed in presence of CSP were 89.3% higher than that without CSP after 24 h (p < 0.01). The expression of QS-associated genes was increased 3.4-5.3-fold by CSP in biofilms. Our results revealed that cell viability of S. mutans grown in biofilms is affected by the CSP-dependent QS system.