As a major etiological agent of human dental caries, Streptococcus mutans resides primarily in biofilms that form on the tooth surfaces, also known as dental plaque. In addition to caries, S. mutans is responsible for cases of infective endocarditis with a subset of strains being indirectly implicated with the onset of additional extraoral pathologies. During the past 4 decades, functional studies of S. mutans have focused on understanding the molecular mechanisms the organism employs to form robust biofilms on tooth surfaces, to rapidly metabolize a wide variety of carbohydrates obtained from the host diet, and to survive numerous (and frequent) environmental challenges encountered in oral biofilms. In these areas of research, S. mutans has served as a model organism for ground-breaking new discoveries that have, at times, challenged long-standing dogmas based on bacterial paradigms such as Escherichia coli and Bacillus subtilis . In addition to sections dedicated to carbohydrate metabolism, biofilm formation, and stress responses, this article discusses newer developments in S. mutans biology research, namely, how S. mutans interspecies and cross-kingdom interactions dictate the development and pathogenic potential of oral biofilms and how next-generation sequencing technologies have led to a much better understanding of the physiology and diversity of S. mutans as a species.

f Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5-and 24-h biofilms was increased by >3-and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA.

Summary Bacteria belonging to the genus Streptococcus are the first inhabitants of the oral cavity which can be acquired right after birth and thus play an important role in the assembly of the oral microbiota. In this chapter, we will discuss the different oral environments inhabited by streptococci and the species that occupy each niche. Special attention is given to the taxonomy of Streptococcus as this genus is now divided into 8 distinct groups where oral species are found in 6 of them. Oral streptococci produce an arsenal of adhesive molecules that allow them to efficiently colonize different tissues in the mouth. Also, they have a remarkable ability to metabolize carbohydrates via fermentation thereby generating acids as byproducts. Excessive acidification of the oral environment by aciduric species such as Streptococcus mutans is directly associated with the development of dental caries. However, less acid-tolerant species such as Streptococcus salivarius and Streptococcus gordonii produce large amounts of alkali displaying and important role in the acid-base physiology of the oral cavity. Another important characteristic of certain oral streptococci is their ability to generate hydrogen peroxide that can inhibit the growth of S. mutans. Thus, oral streptococci can also be beneficial to the host by producing molecules that are inhibitory to pathogenic species. Lastly, commensal and pathogenic streptococci residing in the oral cavity can eventually gain access to the bloodstream and cause systemic infections such as infective endocarditis.

LuxS-mediated quorum sensing has recently been shown to regulate important physiologic functions and virulence in a variety of bacteria. In this study, the role of luxS of Streptococcus mutans in the regulation of traits crucial to pathogenesis was investigated. Reporter gene fusions showed that inactivation of luxS resulted in a down-regulation of fructanase, a demonstrated virulence determinant, by more than 50%. The LuxS-deficient strain (TW26) showed increased sensitivity to acid killing but could still undergo acid adaptation. Northern hybridization revealed that the expression of RecA, SmnA (AP endonuclease), and Nth (endonuclease) were down-regulated in TW26, especially in early-exponential-phase cells. Other down-regulated genes included ffh (a signal recognition particle subunit) and brpA (biofilm regulatory protein A). Interestingly, the luxS mutant showed an increase in survival rate in the presence of hydrogen peroxide (58.8 mM). The luxS mutant formed less biofilm on hydroxylapatite disks, especially when grown in biofilm medium with sucrose, and the mutant biofilms appeared loose and hive-like, whereas the biofilms of the wild type were smooth and confluent. The mutant phenotypes were complemented by exposure to supernatants from wild-type cultures. Two loci, smu486 and smu487, were identified and predicted to encode a histidine kinase and a response regulator. The phenotypes of the smu486 smu487 mutant were, in almost all cases, similar to those of the luxS mutant, although our results suggest that this is not due to AI-2 signal transduction via Smu486 and Smu487. This study demonstrates that luxS-dependent signaling plays critical roles in modulating key virulence properties of S. mutans.Streptococcus mutans is recognized as the principal etiological agent of dental caries, one of the most prevalent human infectious diseases. The abilities to adhere to and to form biofilms on the tooth surface, to metabolize carbohydrates, and to survive low pH and other environmental insults are believed to be critical to the cariogenicity of this human pathogen (4). Known for its high degree of acid tolerance (aciduricity) and its high capacity to produce acids (acidogenicity), S. mutans has evolved strategies to survive, grow, and carry out glycolysis at extracellular pH values well below those at which demineralization of tooth enamel begins to take place. As an obligate biofilm-forming bacterium (4), S. mutans lives almost exclusively on the tooth surface at high cell density in a high-diversity ecosystem better known as dental plaque. The structure and composition of the plaque are known to be strongly influenced by such factors as the source and availability of nutrients, the pH in the oral cavity, and the ability of its constituents to adapt to fluctuations in environmental conditions (4, 38).In a process termed quorum sensing (QS), which was first discovered in the marine bacterium Vibrio fischeri, bacterial populations coordinate gene expression and, thus, the behavior of the community. It is now appare...

Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxS Sm ) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.Streptococcus mutans, an "obligate" biofilm-forming bacterium (5) and the primary etiological agent of human dental caries, has developed a variety of mechanisms to colonize the tooth surfaces and, under certain conditions, to become a numerically significant species in cariogenic biofilms (4). Moreover, this bacterium possesses the ability to scavenge multiple fermentable sugars at micromolar concentrations and to grow and carry out glycolysis at the low plaque pH values attained in the oral cavity. The production of acids by S. mutans causes dissolution of minerals in tooth enamel and formation of dental caries.Despite the complexity of molecular mechanisms implicated in the process, biofilm formation is generally considered to be a two-step sequential process requiring attachment of the bacterial cells to a surface (early adherence), followed by growthdependent accumulation of bacteria in multilayered cell clusters, involving intra-and intergeneric adhesion events. Considerable effort has been devoted to identifying factors of S. mutans and other oral streptococci that participate in biofilm initiation and development. Surface-associated proteins, such as SpaP and Fap1, have been found to function as high-affinity adhesins and play an important role in the initiation of biofilm formation by oral streptococci (2, 14). Extracellular glucans, especially the insoluble glucans synthesized from sucrose by the glucosyltransferases, are of central importance in adhesive interactions by mutans streptococci and are key factors in the accumulation of mutans streptococci on smooth surfaces (22). Reduction in the synthesis of water-insoluble glucan throug...

CcpA globally regulates transcription in response to carbohydrate availability in many gram-positive bacteria, but its role in Streptococcus mutans remains enigmatic. Using the fructan hydrolase (fruA) gene of S. mutans as a model, we demonstrated that CcpA plays a direct role in carbon catabolite repression (CCR). Subsequently, the expression of 170 genes was shown to be differently expressed (>2-fold) in glucose-grown wild-type (UA159) and CcpA-deficient (TW1) strains (P < 0.001). However, there were differences in expression of only 96 genes between UA159 and TW1 when cells were cultivated with the poorly repressing substrate galactose. Interestingly, 90 genes were expressed differently in wild-type S. mutans when glucose-and galactosegrown cells were compared, but the expression of 515 genes was altered in the CcpA-deficient strain in a similar comparison. Overall, our results supported the hypothesis that CcpA has a major role in CCR and regulation of gene expression but revealed that in S. mutans there is a substantial CcpA-independent network that regulates gene expression in response to the carbohydrate source. Based on the genetic studies, biochemical and physiological experiments demonstrated that loss of CcpA impacts the ability of S. mutans to transport and grow on selected sugars. Also, the CcpA-deficient strain displayed an enhanced capacity to produce acid from intracellular stores of polysaccharides, could grow faster at pH 5.5, and could acidify the environment more rapidly and to a greater extent than the parental strain. Thus, CcpA directly modulates the pathogenic potential of S. mutans through global control of gene expression.

Genetic competence appears to be important in establishment of biofilms and tolerance of environmental insults. We report here that the development of competence is controlled at multiple levels in a complex network that includes two signal-transducing two-component systems (TCS). Using Streptococcus mutans strain UA159, we demonstrate that the histidine kinase CiaH, but not the response regulator CiaR, causes a dramatic decrease in biofilm formation and in transformation efficiency. Inactivation of comE or comD had no effect on stress tolerance, but transformability of the mutants was poor and was not restored by addition of competencestimulating peptide (CSP). Horse serum (HS) or bovine serum albumin (BSA) had no impact on transformability of any strains. Interestingly, though, the presence of HS or BSA in combination with CSP was required for efficient induction of comD, comX, and comYA, and induction was dependent on ComDE and CiaH, but not CiaR. Inactivation of comC, encoding CSP, had no impact on transformation, and CiaH was shown to be required for optimal comC expression. This study reveals that S. mutans integrates multiple environmental signals through CiaHR and ComDE to coordinate induction of com genes and that CiaH can exert its influence through CiaR and as-yet-unidentified regulators. The results highlight critical differences in the role and regulation of CiaRH and com genes in different S. mutans isolates and between S. mutans and Streptococcus pneumoniae, indicating that substantial divergence in the role and regulation of TCS and competence genes has occurred in streptococci.Streptococcus mutans, one of the principle causative agents of dental caries and a leading cause of infective endocarditis, has developed a variety of mechanisms to colonize tooth surfaces and to tolerate the stresses commonly experienced in the oral cavity. The abilities to form tenacious biofilms and to tolerate environmental insults are considered major virulence attributes of this organism (5,7,27,28). Rapid and substantial variation in metabolizable energy sources and external pH are common in the human mouth and have a profound impact on oral biofilm ecology. Nutrient limitation and acidic conditions induce many phenotypic changes that can modulate virulence in ways that enhance the capacity of S. mutans to form and persist in biofilms (7,17,36,61,62). A number of recent reports have disclosed an intimate linkage between stress tolerance and efficient biofilm formation (32,57,64,65). For example, mutations that affect cellular levels of the (p)ppGpp synthase RelA (33), ClpP protease (34), DnaK (24,25,29,35), and the serine protease and chaperone HtrA (1, 11) in S. mutans concomitantly influence stress resistance and the ability of the cells to form biofilms. In addition, stress tolerance and biofilm formation are also dependent on quorum-sensing systems in S. mutans, including the LuxS-dependent autoinducer 2 (47, 64, 66) and the competence regulon, which governs genetic transformation (37,39).Genetic transformation ...

Streptococcus mutans, the primary etiological agent of human dental caries, has developed multiple mechanisms to colonize and form biofilms on the tooth surface. The brpA gene codes for a predicted surfaceassociated protein with apparent roles in biofilm formation, autolysis, and cell division. In this study, we used two models to further characterize the biofilm-forming characteristics of a BrpA-deficient mutant, strain TW14. Compared to those of the parent strain, UA159, TW14 formed long chains and sparse microcolonies on hydroxylapatite disks but failed to accumulate and form three-dimensional biofilms when grown on glucose as the carbohydrate source. The biofilm formation defect was also readily apparent by confocal laser scanning microscopy when flow cells were used to grow biofilms. When subjected to acid killing at pH 2.8 for 45 min, the survival rate of strain TW14 was more than 1 log lower than that of the wild-type strain. TW14 was at least 3 logs more susceptible to killing by 0.2% hydrogen peroxide than was UA159. The expression of more than 200 genes was found by microarray analysis to be altered in cells lacking BrpA (P < 0.01). These results suggest that the loss of BrpA can dramatically influence the transcriptome and significantly affects the regulation of acid and oxidative stress tolerance and biofilm formation in S. mutans, which are key virulence attributes of the organism.Streptococcus mutans, the primary etiological agent of human dental caries, exists almost exclusively in biofilms on tooth surfaces. The pathogenic potential of S. mutans is attributable to potent biofilm-forming abilities, a high capacity to produce acids, a high degree of acid tolerance, and the possession of high-affinity systems for the assimilation of many carbohydrate sources (9).Biofilms are surface-attached, structurally and compositionally complex bacterial communities (17,(31)(32)(33). Accumulating data suggest that bacterial cells in biofilms interact with and coordinate the expression of a wide range of genes in response to evolving environmental conditions, including pH, oxygen, carbon source and nutrient availability, cell density, and the presence of a solid surface. A series of highly coordinated physiological and biochemical functions is required for the bacteria to form mature biofilms in response to environmental cues (17,(31)(32)(33). The development of highly structured biofilms, however, gives the adherent populations the flexibility to cope with fluctuating environments and the selective advantages that surface association offers (17).Recent studies have revealed that several genetic regulatory networks in S. mutans are required for bacterial adherence, biofilm accumulation, and growth under the conditions encountered during biofilm formation. The cell-density-dependent Com system, which is known to regulate genetic competence in S. mutans and other naturally competent streptococci, plays a significant role in biofilm formation and acid tolerance (20, 21). The LuxS enzyme, which is responsible for th...