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.

Streptococcus mutans is widely recognized as one of the key etiological agents of human dental caries. Despite its role in this important disease, our present knowledge of gene content variability across the species and its relationship to adaptation is minimal. Estimates of its demographic history are not available. In this study, we generated genome sequences of 57 S. mutans isolates, as well as representative strains of the most closely related species to S. mutans (S. ratti, S. macaccae, and S. criceti), to identify the overall structure and potential adaptive features of the dispensable and core components of the genome. We also performed population genetic analyses on the core genome of the species aimed at understanding the demographic history, and impact of selection shaping its genetic variation. The maximum gene content divergence among strains was approximately 23%, with the majority of strains diverging by 5-15%. The core genome consisted of 1,490 genes and the pan-genome approximately 3,296. Maximum likelihood analysis of the synonymous site frequency spectrum (SFS) suggested that the S. mutans population started expanding exponentially approximately 10,000 years ago (95% confidence interval [CI]: 3,268-14,344 years ago), coincidental with the onset of human agriculture. Analysis of the replacement SFS indicated that a majority of these substitutions are under strong negative selection, and the remainder evolved neutrally. A set of 14 genes was identified as being under positive selection, most of which were involved in either sugar metabolism or acid tolerance. Analysis of the core genome suggested that among 73 genes present in all isolates of S. mutans but absent in other species of the mutans taxonomic group, the majority can be associated with metabolic processes that could have contributed to the successful adaptation of S. mutans to its new niche, the human mouth, and with the dietary changes that accompanied the origin of agriculture.

The oral microbiota associated with the initiation and progression of dental caries has yet to be fully characterized. The Human Oral Microbe Identification Using Next-Generation Sequencing (HOMINGS) approach was used to analyze the microbiomes of site-specific supragingival dental plaques from children with different caries status. Fifty-five children (2 to 7 years of age) were assessed at baseline and at 12 months and grouped as caries free (CF), caries active with enamel lesions (CAE), and caries active with dentin carious lesions (CA). Plaque samples from cariesfree tooth surfaces (PF) and from enamel carious lesions (PE) and dentin carious lesions (PD) were collected. 16S community profiles were obtained by HOMINGS, and 408 bacterial species and 84 genus probes were assigned. Plaque bacterial communities showed temporal stability, as there was no significant difference in beta diversity values between the baseline and 12-month samples. Irrespective of collection time points, the microbiomes of healthy tooth surfaces differed substantially from those found during caries activity. All pairwise comparisons of beta diversity values between groups were significantly different (P Ͻ 0.05), except for comparisons between the CA-PF, CAE-PE, and CA-PE groups. Streptococcus genus probe 4 and Neisseria genus probe 2 were the most frequently detected taxa across the plaque groups, followed by Streptococcus sanguinis, which was highly abundant in CF-PF. Well-known acidogenic/aciduric species such as Streptococcus mutans, Scardovia wiggsiae, Parascardovia denticolens, and Lactobacillus salivarius were found almost exclusively in CA-PD. The microbiomes of supragingival dental plaque differ substantially among tooth surfaces and children of different caries activities. In support of the ecological nature of caries etiology, a steady transition in community species composition was observed with disease progression.

In addition to causing severe invasive infections in humans, Streptococcus agalactiae, or group B Streptococcus (GBS), is also a major cause of bovine mastitis. Here we provide the first genome sequence for S. agalactiae isolated from a cow diagnosed with clinical mastitis (strain FSL S3-026). Comparison to eight S. agalactiae genomes obtained from human disease isolates revealed 183 genes specific to the bovine strain. Subsequent polymerase chain reaction (PCR) screening for the presence/absence of a subset of these loci in additional bovine and human strains revealed strong differentiation between the two groups (Fisher exact test: p < 0.0001). The majority of the bovine strain-specific genes (~85%) clustered tightly into eight genomic islands, suggesting these genes were acquired through lateral gene transfer (LGT). This bovine GBS also contained an unusually high proportion of insertion sequences (4.3% of the total genome), suggesting frequent genomic rearrangement. Comparison to other mastitis-causing species of bacteria provided strong evidence for two cases of interspecies LGT within the shared bovine environment: bovine S. agalactiae with Streptococcus uberis (nisin U operon) and Streptococcus dysgalactiae subsp. dysgalactiae (lactose operon). We also found evidence for LGT, involving the salivaricin operon, between the bovine S. agalactiae strain and either Streptococcus pyogenes or Streptococcus salivarius. Our findings provide insight intomechanismsfacilitatingenvironmentaladaptationandacquisitionofpotential virulence factors, while highlighting both the key role LGT has played in the recent evolution of the bovine S. agalactiae strain, and the importance of LGT among pathogens within a shared environment.

The genus Streptococcus comprises important pathogens that have a severe impact on human health and are responsible for substantial economic losses to agriculture. Here, we utilize 46 Streptococcus genome sequences (44 species), including eight species sequenced here, to provide the first genomic level insight into the evolutionary history and genetic basis underlying the functional diversity of all major groups of this genus. Gene gain/loss analysis revealed a dynamic pattern of genome evolution characterized by an initial period of gene gain followed by a period of loss, as the major groups within the genus diversified. This was followed by a period of genome expansion associated with the origins of the present extant species. The pattern is concordant with an emerging view that genomes evolve through a dynamic process of expansion and streamlining. A large proportion of the pan-genome has experienced lateral gene transfer (LGT) with causative factors, such as relatedness and shared environment, operating over different evolutionary scales. Multiple gene ontology terms were significantly enriched for each group, and mapping terms onto the phylogeny showed that those corresponding to genes born on branches leading to the major groups represented approximately one-fifth of those enriched. Furthermore, despite the extensive LGT, several biochemical characteristics have been retained since group formation, suggesting genomic cohesiveness through time, and that these characteristics may be fundamental to each group. For example, proteolysis: mitis group; urea metabolism: salivarius group; carbohydrate metabolism: pyogenic group; and transcription regulation: bovis group.

The ability of certain oral biofilm bacteria to moderate pH through arginine metabolism by the arginine deiminase system (ADS) is a deterrent to the development of dental caries. Here, we characterize a novel Streptococcus strain, designated strain A12, isolated from supragingival dental plaque of a caries-free individual. A12 not only expressed the ADS pathway at high levels under a variety of conditions but also effectively inhibited growth and two intercellular signaling pathways of the dental caries pathogen Streptococcus mutans. A12 produced copious amounts of H 2 O 2 via the pyruvate oxidase enzyme that were sufficient to arrest the growth of S. mutans. A12 also produced a protease similar to challisin (Sgc) of Streptococcus gordonii that was able to block the competence-stimulating peptide (CSP)-ComDE signaling system, which is essential for bacteriocin production by S. mutans. Wild-type A12, but not an sgc mutant derivative, could protect the sensitive indicator strain Streptococcus sanguinis SK150 from killing by the bacteriocins of S. mutans. A12, but not S. gordonii, could also block the XIP (comX-inducing peptide) signaling pathway, which is the proximal regulator of genetic competence in S. mutans, but Sgc was not required for this activity. The complete genome sequence of A12 was determined, and phylogenomic analyses compared A12 to streptococcal reference genomes. A12 was most similar to Streptococcus australis and Streptococcus parasanguinis but sufficiently different that it may represent a new species. A12-like organisms may play crucial roles in the promotion of stable, health-associated oral biofilm communities by moderating plaque pH and interfering with the growth and virulence of caries pathogens. Dental caries remains an enormous health problem worldwide (1-4). The current view of the initiation and progression of dental caries is one of a dynamic process in which the normal balance between the complex environmental factors that control demineralization and remineralization of the tooth is disrupted in a way that favors the loss of tooth mineral. Demineralization is driven primarily by microbial fermentation of carbohydrates to organic acids, which can lower the pH of oral biofilms to the extent that there is significant dissolution of tooth mineral. Alkalization of oral biofilms occurs during fasting periods and is attributable to salivary clearance of acids, buffering of oral biofilms by host-derived and microbially derived components, and the production of basic compounds by microbial metabolism of amino acids, polyamines, urea, and other compounds (5-10). Development or worsening of a carious lesion occurs when acid-mediated enamel demineralization predominates and is associated with changes in the composition and biochemical activities of the associated microflora. Evidence is now accumulating from a variety of sources that supports that alkali generation, particularly in the form of ammonia, plays a major role in pH homeostasis in oral biofilms and inhibits the initiation and progre...

High coverage, whole genome shotgun (WGS) sequencing of 57 geographically- and genetically-diverse isolates of Streptococcus mutans from individuals of known dental caries status was recently completed. Of the 57 sequenced strains, fifteen isolates, were selected based primarily on differences in gene content and phenotypic characteristics known to affect virulence and compared with the reference strain UA159. A high degree of variability in these properties was observed between strains, with a broad spectrum of sensitivities to low pH, oxidative stress (air and paraquat) and exposure to competence stimulating peptide (CSP). Significant differences in autolytic behavior and in biofilm development in glucose or sucrose were also observed. Natural genetic competence varied among isolates, and this was correlated to the presence or absence of competence genes, comCDE and comX, and to bacteriocins. In general strains that lacked the ability to become competent possessed fewer genes for bacteriocins and immunity proteins or contained polymorphic variants of these genes. WGS sequence analysis of the pan-genome revealed, for the first time, components of a Type VII secretion system in several S. mutans strains, as well as two putative ORFs that encode possible collagen binding proteins located upstream of the cnm gene, which is associated with host cell invasiveness. The virulence of these particular strains was assessed in a wax-worm model. This is the first study to combine a comprehensive analysis of key virulence-related phenotypes with extensive genomic analysis of a pathogen that evolved closely with humans. Our analysis highlights the phenotypic diversity of S. mutans isolates and indicates that the species has evolved a variety of adaptive strategies to persist in the human oral cavity and, when conditions are favorable, to initiate disease.

The white shark (Carcharodon carcharias; Chondrichthyes, Elasmobranchii) is one of the most publicly recognized marine animals. Here we report the genome sequence of the white shark and comparative evolutionary genomic analyses to the chondrichthyans, whale shark (Elasmobranchii) and elephant shark (Holocephali), as well as various vertebrates. The 4.63-Gbp white shark genome contains 24,520 predicted genes, and has a repeat content of 58.5%. We provide evidence for a history of positive selection and gene-content enrichments regarding important genome stability-related genes and functional categories, particularly so for the two elasmobranchs. We hypothesize that the molecular adaptive emphasis on genome stability in white and whale sharks may reflect the combined selective pressure of large genome sizes, high repeat content, high long-interspersed element retrotransposon representation, large body size, and long lifespans, represented across these two species. Molecular adaptation for wound healing was also evident, with positive selection in key genes involved in the wound-healing process, as well as Gene Ontology enrichments in fundamental wound-healing pathways. Sharks, particularly apex predators such as the white shark, are believed to have an acute sense of smell. However, we found very few olfactory receptor genes, very few trace amine-associated receptors, and extremely low numbers of G protein-coupled receptors. We did however, identify 13 copies of vomeronasal type 2 (V2R) genes in white shark and 10 in whale shark; this, combined with the over 30 V2Rs reported previously for elephant shark, suggests this gene family may underlie the keen odorant reception of chondrichthyans.