Justin Merritt

Competition and Coexistence between Streptococcus mutans and Streptococcus sanguinis in the Dental Biofilm

The human mucosal surface is colonized by the indigenous microflora, which normally maintains an ecological balance among different species. Certain environmental or biological factors, however, may trigger disruption of this balance, leading to microbial diseases. In this study, we used two oral bacterial species, Streptococcus mutans and Streptococcus sanguinis (formerly S. sanguis), as a model to probe the possible mechanisms of competition/coexistence between different species which occupy the same ecological niche. We show that the two species engage in a multitude of antagonistic interactions temporally and spatially; occupation of a niche by one species precludes colonization by the other, while simultaneous colonization by both species results in coexistence. Environmental conditions, such as cell density, nutritional availability, and pH, play important roles in determining the outcome of these interactions. Genetic and biochemical analyses reveal that these interspecies interactions are possibly mediated through a well-regulated production of chemicals, such as bacteriocins (produced by S. mutans) and hydrogen peroxide (produced by S. sanguinis). Consistent with the phenotypic characteristics, production of bacteriocins and H2O2 are regulated by environmental conditions, as well as by juxtaposition of the two species. These sophisticated interspecies interactions could play an essential part in balancing competition/coexistence within multispecies microbial communities.

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.

Inactivation of the ciaH gene in Streptococcus mutans diminishes mutacin production and competence development, alters sucrose-dependent biofilm formation, and reduces stress tolerance

ABSTRACT Many clinical isolates of Streptococcus mutans produce peptide antibiotics called mutacins. Mutacin production may play an important role in the ecology of S. mutans in dental plaque. In this study, inactivation of a histidine kinase gene, ciaH, abolished mutacin production. Surprisingly, the same mutation also diminished competence development, stress tolerance, and sucrose-dependent biofilm formation.

Streptococcus oligofermentans inhibits Streptococcus mutans through conversion of lactic acid into inhibitory H2O2: a possible counteroffensive strategy for interspecies competition.

The oral microbial flora contains over 500 different microbial species that often interact as a means to compete for limited space and nutritional resources. Streptococcus mutans, a major caries‐causing pathogen, is a species which tends to interact competitively with other species in the oral cavity, largely due to its ability to generate copious quantities of the toxic metabolite, lactic acid. However, during a recent clinical study, we discovered a novel oral streptococcal species, Streptococcus oligofermentans, whose abundance appeared to be inversely correlated with that of S.u2003mutans within dental plaque samples and thus suggested a possible antagonistic relationship with S.u2003mutans. In this study, we used a defined in vitro interspecies interaction assay to confirm that S.u2003oligofermentans was indeed able to inhibit the growth of S.u2003mutans. Interestingly, this inhibitory effect was relatively specific to S.u2003mutans and was actually enhanced by the presence of lactic acid. Biochemical analyses revealed that S.u2003oligofermentans inhibited the growth of S.u2003mutans via the production of hydrogen peroxide with lactic acid as the substrate. Further genetic and molecular analysis led to the discovery of the lactate oxidase (lox) gene of S.u2003oligofermentans as responsible for this biological activity. Consequently, the lox mutant of S.u2003oligofermentans also showed dramatically reduced inhibitory effects against S.u2003mutans and also exhibited greatly impaired growth in the presence of the lactate produced by S.u2003mutans. These data indicate that S.u2003oligofermentans possesses the capacity to convert its competitors main ‘weapon’ (lactic acid) into an inhibitory chemical (H2O2) in order to gain a competitive growth advantage. This fascinating ability may be an example of a counteroffensive strategy used during chemical warfare within the oral microbial community.

LuxS controls bacteriocin production in Streptococcus mutans through a novel regulatory component

The oral pathogen Streptococcus mutans employs a variety of mechanisms to maintain a competitive advantage over many other oral bacteria which occupy the same ecological niche. Production of the bacteriocin, mutacin I, is one such mechanism. However, little is known about the regulatory mechanisms associated with mutacin I production. Previous work has demonstrated that the production of mutacin I greatly increased with cell density. In this study, we found that high cell density also triggered high level mutacin I gene transcription. However, this response was abolished upon deletion of luxS. Further analysis using real‐time reverse transcription polymerase chain reaction (RT‐PCR) demonstrated that in the luxS mutant transcription of both the mutacin I structural gene mutA and the mutacin I transcriptional activator mutR was impaired. Through microarray analysis, a putative transcription repressor annotated as Smu1274 in the Los Alamos National Laboratory Oral Pathogens Sequence Database was identified, which was strongly induced in the luxS mutant. Characterization of Smu1274, which we referred to as irvA, suggested that it may act as an inducible repressor to suppress mutacin I gene expression. A luxS and irvA double mutant regained the ability to produce mutacin I; whereas a constitutive irvA‐producing strain was impaired in mutacin I production. These findings reveal a novel regulatory pathway for mutacin I gene expression, which may provide clues to the regulatory mechanisms of other cellular functions regulated by luxS in S. mutans.

Quantitative analyses of Streptococcus mutans biofilms with quartz crystal microbalance, microjet impingement and confocal microscopy

Microbial biofilm formation can be influenced by many physiological and genetic factors. The conventional microtiter plate assay provides useful but limited information about biofilm formation. With the fast expansion of the biofilm research field, there are urgent needs for more informative techniques to quantify the major parameters of a biofilm, such as adhesive strength and total biomass. It would be even more ideal if these measurements could be conducted in a real-time, non-invasive manner. In this study, we used quartz crystal microbalance (QCM) and microjet impingement (MJI) to measure total biomass and adhesive strength, respectively, of S. mutans biofilms formed under different sucrose concentrations. In conjunction with confocal laser scanning microscopy (CLSM) and the COMSTAT software, we show that sucrose concentration affects the biofilm strength, total biomass, and architecture in both qualitative and quantitative manners. Our data correlate well with previous observations about the effect of sucrose on the adherence of S. mutans to the tooth surface, and demonstrate that QCM is a useful tool for studying the kinetics of biofilm formation in real time and that MJI is a sensitive, easy-to-use device to measure the adhesive strength of a biofilm.