Oral Microbial Communities: Genomic Inquiry and Interspecies Communication

Multispecies communities, not a collection of single-species colonies, compose human oral biofilms. The human oral microbiome is the most intensely studied and, perhaps, the best-characterized ecosystem in humans, but the organization of these species into multispecies communities at any point in time remains poorly understood. Genomic inquiry of community members will enable a fuller vision of each member’s metabolic contribution to the community. A central contributor to colonization of oral bacteria in biofilms is the universal ability of these species to attach to a surface: to teeth or to epithelial cell surfaces or to the bacterial surface of an already attached cell. Cells suspended in saliva are unable to multiply within a typical between-swallows interval due to the rapid salivary flow. Thus, adherence is essential before multispecies communities can develop and colonize the surface. Following coadherence and coaggregation, attached cells grow, as evidenced by incorporation of radiolabeled nucleosides and by cell division planes observed in the biofilms of intraoral mounted pieces of enamel or glass. Evidence-based two-species and three-species biofilms contribute in understanding species succession leading to a crossroad between health and disease in dental plaque biofilms. Virulence mechanisms exhibited by red complex periodontal pathogens are discussed in an inspired review of the interplay between pathogenic and commensal bacteria in disrupting host homeostasis. It is recognized that multispecies communities respond differently than pure cultures to antimicrobials.

This chapter summarizes the recent advancement of our knowledge of the healthy oral microbiome, and address the potential role of the oral microbiome in systemic diseases, including cardiovascular disease and pneumonia. An understanding of the composition of the oral microbial community with respect to oral health is essential for diagnosis, prevention, and treatment of oral diseases. The chapter describes oral microbiome associated with periodontal disease, microbiome associated with endodontic infections, apical periodontitis and tooth decay, and oral microbiome associated with oral cancer. Diseases caused by the oral microbiome are not limited to oral infections. Recognition that disease conditions associated with the oral microbiome contribute to systemic infections may require clinicians to consider alternative preventative and therapeutic approaches. The current data on two of the most-studied systemic conditions, cardiovascular and respiratory diseases are reviewed in the chapter. A community-and microbial ecology-based pathogenic concept that forms the basis for understanding relationships between the oral microbiome and the host, as well as developing novel strategies for therapeutics and disease prevention is discussed in the chapter. A better understanding of the oral microbiome structure and function and the dynamics between the commensals and pathogens is needed to selectively modulate composition of the microbiome and prevent oral disease.

This chapter talks about the closest relatives of Streptococcus mitis that are the commensals S. oralis, S. infantis, and, in particular, the important pathogen Streptococcus pneumoniae and the still relatively unknown Streptococcus pseudopneumoniae. The genetic diversity among S. mitis strains may have important consequences in the oral cavity. Draft genomes display an artificially high total number of genes and number of paralogs due to sequencing errors and gaps leading to gene fragmentation, as well as low-quality redundant sequences at contig ends. The verification results in the conclusion that this observation is not due to the fact that (i) all but one of the non-S. pneumoniae genomes studied are draft genomes, and (ii) gene prediction standards differ among sequencing centers. First, the closed S. mitis B6 genome displays a higher coding percentage. Second, the coding density was measured in three unpublished draft S. pneumoniae genomes obtained from three different sequencing centers, and the average coding percentage was 84.9%. The previous observation that virtually every independent isolate of S. mitis represents a distinct species according to traditional taxonomic principles is supported by our multigenome analysis. The significant sharing of core genes between S. mitis and S. pneumoniae reinforces the conclusion that S. pneumoniae is one lineage of the S. mitis complex.

This chapter focuses on the contribution of the genome sequence of Streptococcus sanguinis to enhance one's understanding of its interactions in the oral cavity. It was determined that streptococci resembling S. sanguinis constituted about half of the streptococcal component of dental plaque. This study likely identified as S. sanguinis some strains that would currently be classified as Streptococcus oralis or Streptococcus gordonii. This chapter points out that studies with related species may provide a framework for future hypothesis-driven investigation of S. sanguinis cell wall-anchored (Cwa) proteins associated with oral adhesion and aggregation. It was later determined that competence is upregulated in S. sanguinis in the presence of oxygen, as is also true for S. pneumoniae and S. mutans. The chapter concludes with the hope that the availability of the genome sequence stimulates new research related to the role of S. sanguinis within the oral community.

Among Actinomyces spp., Actinomyces oris is the most abundant species in the human oral cavity. This chapter provides an account of the current knowledge of a key adhesive principle, the fimbriae of A. oris, which are considered to be the main players for the cell-cell and cell-substratum interactions involving the early colonizers of dental plaque. The genome sequence of A. oris has led to the identification and characterization of various fimbrial components and the specific enzymes, called sortases, that are responsible for the ordered assembly of fimbrial subunits into covalently cross-linked polymers and subsequent incorporation into the cell wall. The genome sequence also revealed the presence of an unusually large number of putative cell wall-anchored proteins, some of which must serve as additional adhesive principles facilitating the adherence of bridge organisms and late colonizers. Importantly, not only is the S. aureus srtA mutant defective in cell wall anchoring of LPXTG-containing surface proteins but also it is attenuated in animal models of infection. The Actinomyces fimbrial system is a versatile adhesive principle for promoting bacterial coaggregation and host tissue adherence that leads to the development of one of the most complex biofilms, the dental plaque.

The recent availability of genome sequences for Veillonella species provides an exceptional opportunity to probe their biology and role in the microbial ecology of the mouth. The recent genome-scale stoichiometric model of Porphyromonas gingivalis metabolic networks is an exceptional example of such an approach and illustrates the potential of advancements in genome sequencing and bioinformatic tools in studies of bacterial metabolism and ecology. Fifteen complete genomes, three metagenomes of TM7, and 38 genome surveys of oral isolates are available at the human oral microbiome database (HOMD). Seven Veillonella genomes are currently available as genome surveys at the HOMD. Almost 10% of the total cultivable biota from the tongue consists of Veillonellae. Veillonellae lack hexokinase activity but have other glycolytic enzymes. Veillonellae are also unable to incorporate radiolabeled glucose into bacterial cell compounds and seem to lack a glucose phosphotransferase system. The observations on the Veillonella physiology can now be confirmed and visualized in the carbohydrate metabolism glycolysis-gluconeogenesis pathway constructed with Veillonella parvula ATCC 10790 genome data. This chapter presents the genomic evidence for the presence of both nitrate reduction enzymes and membrane-bound proton-translocating ATPases. Interactions between Streptococcus spp. and Veillonella spp. naturally occur in vivo and emphasize the natural relationship of species in the development of biofilms. The availability of complete Veillonella genome sequences provides a unique opportunity for developing genetic systems for Veillonellae.

Fusobacterium nucleatum is an oral anaerobe and the most common gram-negative isolate from both healthy and diseased oral sites. Association of F. nucleatum with gingivitis and with its progression to periodontal disease has been based on the fact that numbers of F. nucleatum organisms greatly increase in samples taken from diseased sites compared to those from healthy ones. The author feels that the sharing of both gram-positive and gram-negative properties might facilitate communication of fusobacteria with both gram-positive early colonizers and gram-negative late colonizers, an essential stage in the development of the periopathogenic dental biofilm. The species complexity of oral biofilms is probably the greatest hurdle in studying the fusobacterial role in oral health and disease. The author proposes that fusobacterial virulence relies on the presence of its adhesins and not on classical toxins and proteases. Microarrays can be designed to interact uniquely with F. nucleatum gene transcripts. These microarrays can be reacted with total RNAs extracted from dental biofilms sampled from healthy and diseased periodontal sites of increasing disease severity. In vitro studies have suggested that fusobacterial bridging coaggregation interactions are important for the shift in the composition of the microbial community associated with transition from health to disease. Ongoing introduction of tools for genetic manipulation of fusobacteria will enable the investigation of fusobacterial virulence-associated molecular elements. Integration species-specific and general molecular tools is expected to enable the study and the understanding of F. nucleatum's role in bridging from oral health to disease.

Aggregatibacter actinomycetemcomitans is a small, gram-negative, facultatively anaerobic coccobacillus that belongs in the family Pasteurellaceae. A localized form of the disease, localized aggressive periodontitis, was originally found to be associated with the presence of A. actinomycetemcomitans in periodontal pockets. Genetic analyses show that the JP2 clone constitutes a subpopulation of restriction fragment length polymorphism (RFLP) group II. All isolates of the JP2 clone are serotype b, are identical in multilocus enzyme electrophoresis, and have the same DNA fingerprint using the restriction enzyme MspI, but show variation in ribotype. The RFLP group II strains, including strain JP2, have elevated expression of cytolethal distending toxin (CDT) measured as cytotoxicity for Chinese hamster ovary cells. Genomic islands that carry the leukotoxin gene cluster, the lipooligosaccharide biosynthesis gene cluster, the tight adherence gene cluster, and the CDT gene cluster are common to HK1651, D11S-1, and D7S-1 strains, whereas the other islands are strain specific. Members of the author's group used multilocus sequence analysis to study microevolution of the JP2 clone. Based on the assumptions that the variations were caused by single-site mutations and that each mutation occurred only once, it was possible to deduce an evolutionary model for A. actinomycetemcomitans serotype b, including the origin of the JP2 clone.

Studies have shown that Porphyromonas gingivalis surface structures play an important role in the life cycle of the oral anaerobe. In particular, cysteine proteases, fimbriae, membrane vesicles (MVs), and surface polysaccharides have been of primary interest. P. gingivalis is a gram-negative obligate anaerobe that persists in the subgingival crevice of the oral cavity. The chapter provides a review of molecular genetic studies on cysteine proteases, fimbriae, MVs, and surface polysaccharides. It discusses the use of genome sequence information in deciphering the role of surface structures in biofilm development and pathogenicity. The high proteolytic activity of P. gingivalis and its role in nutrient acquisition and virulence are discussed. Surface polysaccharides are key virulence factors of P. gingivalis, as well as of many other gram-negative pathogens. The switch from being a benign member of the oral biofilm to being a proliferating virulent pathogen is central to the pathogenicity of this anaerobe, and changes in expression of surface structures play an important role in this switch. Expression of these surface structures modulates cell-cell and cell-host interactions and thus biofilm development; hence, MVs, gingipains, fimbriae, and surface polysaccharides play a central role in both virulence and biofilm growth.

This chapter predicts and highlights the genome functions of Tannerella forsythia related to bacterial community development. The major intent is to identify putative genes that are likely to be important in T. forsythia interactions with the members of the bacterial community. The chapter focuses on a predicted function derived from the recently completed T. forsythia genome. It was demonstrated that T. forsythia growth required an exogenous source of N-acetylmuramic acid (MurNAc). Investigation of the genome functions that assist T. forsythia in scavenging MurNAc from coinhabiting species could lead to the identification of novel mechanisms for MurNAc uptake in bacteria, as well as to the design of novel strategies for blocking MurNAc uptake by T. forsythia and controlling periodontitis. The chapter talks about surface components, surface layer (S-layer) glycoproteins, and LRR proteins. Only a few virulence factors have been identified in T. forsythia. The metabolic conditions of the host could influence T. forsythia growth and virulence gene expression. Recent studies have shown that expression of the T. forsythia virulence protein BspA and its homologues is affected in response to environmental cues. T. forsythia possesses several conjugative transposon (CTn) elements belonging to the Bacteroides CTnDOT family. In the near future, computational approaches supported by direct laboratory experimentation will be necessary to decipher some of the complex bacterial interactions.

This chapter reviews current progress in applying Treponema denticola genome information to the research areas and suggests some topics that have yet to be addressed adequately by the research community. A group of seven genes was identified by homology with TP0155, a fibronectin-binding protein of T. pallidum. While five of these proteins showed at least some fibronectin-binding activity in recombinant form, only one was clearly demonstrated to be surface localized. This study provides a rational basis for further examination of the relative contribution of these proteins to T. denticola interactions with fibronectin. The T. pallidum repeat (tpr) genes, which encode paralogous proteins with sequence similarity to major surface protein (Msp) of T. denticola, were expressed at relatively low levels. This study, as well as numerous similar studies of other pathogens, demonstrates the potential utility of in vivo microarray analysis of bacterial gene expression. The current paucity of microarray-based T. denticola global expression analysis is likely the result of several factors. As in many bacteria, the T. denticola genome contains genes that appear to be more closely related to those of eukaryotes or archaea than to those of other bacteria. Several groups are investigating various aspects of T. denticola molecular physiology, especially as it relates to microbial community ecology and periodontal pathogenesi. In addition to the continued need to characterize putative virulence determinants very little is known about several important biological components of these cells, including nutrient uptake and processing, secretion and efflux systems, and various lipids and lipid-containing molecules.

Catheters and other prostheses provide novel surfaces for colonization by Candida albicans and other microorganisms and the formation of biofilms. The polyene and azole antifungal compounds, such as amphotericin B and fluconazole, are principal agents used to treat subjects with various Candida infections. It is well established that microorganisms colonizing surfaces grow as biofilm communities. Bcr1 regulates expression of the glycosylphosphatidylinositol (GPI)-anchored adhesins Als1, Als3, and Hwp1, which contribute to biofilm formation. These proteins promote fungal-cell-to-fungal-cell interactions in biofilm formation, with Hwp1 binding to Als1 and Als3. These developments in understanding the mechanisms of biofilm formation by C. albicans have evolved directly through applications of genomic sequence information. The availability of the C. albicans genome sequence has facilitated genome-wide bioinformatics analysis to predict every protein that may be modified by the addition of a GPI anchor. Most colonizing microorganisms on mucosal surfaces have to survive in mixed microbial communities. There are three steps in the initiation of disease by C. albicans: (i) establishment within a mixed-species community, (ii) outgrowth of C. albicans, and (iii) infection of tissues. It is hypothesized that the ability of oral streptococci and C.albicans to engage in physical and chemical communication promotes the colonization of C.albicans in the oral cavity and the development of mixed-species communities of bacteria and fungi. C.albicans is a highly successful colonizer of humans and may exist in a carriage state at mucosal surfaces in the gastrointestinal (GI) and genitourinary tracts.

This chapter addresses how genomic sciences are revealing why Streptococcus mutans is such an effective caries pathogen in humans. Recognizing the importance of central metabolism and acid production in pathogenesis by S. mutans, many laboratories began functional studies in the post-genomic era with a focus on gene regulation, stress tolerance, and biofilm formation. The AtlA protein of S. mutans does not share substantial primary sequence homology with many other proteins in the databases, so AtlA may be a good target for novel therapeutics. A variety of other examples can be cited of how functional genomics revealed new properties of S. mutans. The translation of environmental stimuli into changes in gene expression in bacteria frequently involves two-component signal transduction systems (TCSs) consisting of a membrane-bound sensor histidine kinase (HK) and a cytoplasmic response regulator (RR). A common theme that emerged from these studies is that S. mutans has streamlined its genome by using pathways to cope with environmental insults to regulate a variety of virulence attributes. The expression of fruA is inducible by its substrates and is sensitive to carbon catabolite repression (CCR)-inducing reagents, including glucose, fructose, and mannose. Galactose gives the least repression of CCR-sensitive genes in S. mutans. In a study, only one strain of S. mutans, UA159, was tested, and although it was able to reach the intracellular compartment in very low numbers, the fate of UA159 in the host cytoplasm, as well as the consequences of invasion for the host cell, was not investigated.

Gingival epithelial cells, the initial lining of gingival mucosa functioning as an important part of the innate immune system, are among the first host cells colonized by Porphyromonas gingivalis. This chapter presents a study which showed that the gingival epithelial cells display a high level of death upon treatment with ATPe and that treatment with purified recombinant Ndk inhibits ATP-mediated host cell plasma membrane permeabilization in a dose-dependent manner. Cytochalasin D, a fungal toxin that disrupts the microfilaments and inhibits actin polymerization, caused significant inhibition of the level of intercellular spread, confirming that the stimulation of actin polymerization is an underlying factor for dissemination of P. gingivalis within the host epithelium. In addition to constituting a physical barrier to the ingress of organisms, epithelial cells can sense and respond to the presence of bacteria following stimulation of pattern recognition receptors (PRRs), resulting in secretion of inflammatory cytokines and differentiation of the epithelial cells. The current challenges include accurate definition of species and identification of the species present at subthresholds. The initiation of inflammation and succession of disease might require a large number of potentially pathogenic populations acting in concert. The current innovative approaches for determining biphasic interaction between host and microbe, particularly identification of mutualistic and/or pathogenic genes and proteins, include suppression subtractive hybridization, comparative genomic analyses by DNA microarrays, and various proteomics technologies.

Fusobacterium nucleatum is a gram-negative filamentous anaerobe ubiquitous to the oral cavity. While the focus of this chapter is on F. nucleatum interaction with host cells, the chapter also explains the role of coaggregation in oral microbial community interaction with the host. F. nucleatum is an opportunistic pathogen implicated in various forms of periodontal disease. During periodontal infection, the cell mass of F. nucleatum can increase as much as 10,000-fold, making it one of the most abundant anaerobic species in the diseased sites. In particular, F. nucleatum is one of the leading organisms identified in intrauterine infections causing adverse pregnancy outcomes, including spontaneous miscarriage, preterm birth, and stillbirth. One common feature shared by bacterial pathogens is their ability to adhere to and invade host cells. F. nucleatum modulates an array of host responses upon attachment to and invasion of host cells. A protein complex, FIP, composed of two subunits of 44 and 48 kDa, mediates T-cell suppression. Sequence analysis of TM7a shows that while the majority of the genes are only distantly related to genes found in other organisms, a minority share high sequence similarity with genes found in members of the classes Bacilli, Clostridia, and Fusobacteria, which may result from horizontal gene transfer in the oral cavity. FadA is a unique adhesin in that both the secreted mFadA and the intact pre-FadA are required for function.

The first studies focusing on the competence-stimulating peptide (CSP) system were prompted by investigations to understand competence development for natural transformation. CSP signals and regulatory pathways are found in most oral streptococci. The molecular mechanisms involved in CSP signaling have been described in more detail for Streptococcus pneumoniae than for other streptococci. The CSP autoinducing system is remarkably closely related to well-characterized bacteriocin autoinducing systems. Genetic competence and bacteriocin production appear to be closely linked in streptococci. It is possible, that the mechanisms involved in competence for genetic transformation are somehow uncoupled with the stress response to spectinomycin. The absence of one of the two components of the histidine kinase/response regulator ComDE impairs the increased biofilm formed in the presence of CSP, supporting the specificity of the response. The possible contribution of lysis and DNA binding to the CSP effect on biofilm formation is supported by the findings that mutants of Staphylococcus mutans deficient in the DNA binding and uptake machinery form less biofilm and that degradation of extracellular DNA reduces biofilm formation in streptococci. In the multicellular communities found in the oral cavity, natural interference with autoinducing signaling systems such as the CSP signaling pathway might be a natural scenario, depending on how the bacteria are structurally distributed in biofilms. Present efforts to sequence microbes commonly colonizing humans will certainly provide important tools to deepen our understanding of the interactions between microorganisms in complex communities, and the human responses to these communities.

This chapter explores the mechanism of interspecies cell-cell communication in the oral biofilm, with emphasis on the cariogenic organism Streptococcus mutans. Bioluminescence in the marine organism Vibrio harveyi was one of the first examples of quorum sensing behavior described to occur in nature. The gene required for autoinducer 2 (AI-2) production encodes the enzyme LuxS, which functions in the S-adenosylmethionine (SAM) utilization pathway. Biofilms are complex communities where close interactions between heterogeneous species are common. Alterations in AI-2 signaling might affect biofilm formation in several ways, and changes in the cells themselves during the physiologically distinct biofilm mode of growth might reciprocally affect AI-2 signaling. Exopolysaccharide (EPS) production occurs after attachment and is involved in the latter stages of biofilm maturation. The genetic basis for altered biofilm structure in S. mutans luxS mutants has been attributed to an overexpression of the general stress response genes encoding GroEL and DnaK or to increased glucosyltransferase expression. The majority of genes found to respond to the AI-2 signal were genes involved in protein synthesis and genes for hypothetical proteins. A convincing demonstration of S. mutans complementing a luxS deletion in another species would be helpful in establishing the true nature of this signal. Interference with AI-2-mediated signaling occurs between competing microorganisms that share the same niche.

Caries and periodontal disease are biofilm-mediated diseases that remain widespread and serious health problems. The dental biofilm is a complex microbial community that can comprise up to 700 bacterial species. Recent studies have made substantial progress in defining these signaling circuits and the impact that bacterial communication has on the formation and persistence of the dental biofilm. Several distinct classes of autoinducers have been identified, including numerous structurally distinct acylhomoserine lactones (AHLs), oligopeptides, quinolones, and derivatives of tetrahydrofuran. LuxS is an enzyme in the activated methyl cycle which functions in the turnover of S-adenosylhomocysteine (SAH) and generation of S-adenosylmethionine. To determine if luxS influenced virulence properties, the expression of several genes that contribute to Aggregatibacter actinomycetemcomitans virulence was analyzed after inactivation of luxS. Real-time PCR revealed that the expression of numerous genes encoding proteins involved in iron acquisition and storage was significantly altered in the luxS mutant. Biofilm growth was restored to wild-type levels either by transforming the mutant strain with a functional copy of luxS or by the addition of partially purified AI-2 to the growth medium. To determine if AI-2 represents a true quorum-sensing signal, Rezzonico and Duffy compiled and searched a microbial genome database for known AI-2 receptors, i.e., LuxQP and LsrB, and then compared the distribution of these proteins with published studies that examined quorum-sensing phenotypes in the organisms. The widespread distribution of LuxS in both gram-negative and gram-positive bacteria has led to the suggestion that AI-2 represents a universal quorum-sensing system that mediates interspecies communication.

Microbial interactions involve sensing and responding to chemical signals or cues that are released from cells. In the early stages of plaque accumulation on freshly cleaned teeth, most interactions occur between the initial colonizers such as Streptococcus spp., Actinomyces spp., and Veillonella spp. However, mature dental plaque biofilms may contain up to 200 different phylotypes of bacteria, resulting potentially in almost 20,000 different pairwise interactions. This chapter focuses on just one of these interactions: that between two initial colonizers of oral biofilms, Streptococcus gordonii and Actinomyces oris. Interbacterial binding between oral bacteria has been investigated extensively in vitro using coaggregation assays. In these experiments, pure cultures of genetically distinct bacteria are mixed in test tubes and interactions are scored based on the extent of clumping (coaggregation). Coaggregation is used to model biofilm communities containing S. gordonii and A. oris and investigate gene regulation in mixed-species cultures. A powerful application of postgenomic technologies is the analysis of gene expression using DNA microarrays. The first genome-level analysis of S. gordonii-A. oris interactions employed microarrays to identify genes in S. gordonii that were regulated in response to coaggregation with A. oris. This study demonstrated that S. gordonii specifically activates a set of genes in response to cell-cell contact (coaggregation) with A. oris. In theory, microarrays and other genome-based expression technologies can be used to investigate cell-cell contact-induced gene regulation in any bacterial species for which the genome sequence is known.

The microbial composition of the human oral cavity has been well characterized using genomic and metagenomic techniques that have enabled rapid identification of microbial species. These studies indicate the presence of organisms in an environment, yet they do not provide information regarding the metabolic events that contribute to the oral pathology. By performing pure culture studies with secondary metabolites that A. actinomycetemcomitans encounters, novel responses to lactate and H2O2 were discovered. The future work will reveal additional polymicrobial interactions that enhance bacterial resistance to innate immunity. Riboswitches often control genes involved in biosynthesis and transport of metabolites, and they allow cells to rapidly change gene expression in response to intracellular metabolite levels. Additionally, several noncoding regulatory RNAs (ncRNAs) were upregulated during biofilm growth, indicating potential roles in biofilm development and bacterial group behavior. Overall, this study highlights the importance of ncRNAs in A. actinomycetemcomitans, particularly their potential roles in metabolism and group behavior.

The composition of oral biofilm communities, and their pathogenic potential, may depend on interspecies binding and communication interactions. Multivalent coadhesive interactions characterize the binding of Porphyromonas gingivalis to oral streptococci such as Streptococcus gordonii and drive subsequent development of heterotypic S. gordonii-P. gingivalis communities. Gene regulation by two-component systems (TCSs) is a common mechanism used by bacteria to modulate cell behavior in response to environmental changes. Following the initial binding interaction between P. gingivalis and S. gordonii, adherent P. gingivalis cells undergo a programmatic phenotypic shift in order to prepare for community living. P. gingivalis produces autoinducer 2 (AI-2) and responds both to homologous signal and to AI-2 from other organisms such as S. gordonii and Actinobacillus actinomycetemcomitans. The expression of GTF, Rgg, and exo-β;-D-fructosidase (fructanase) is downregulated in the absence of LuxS, whereas expression of tagatose 1,6-diphosphate aldolase is elevated. Contact with S. cristatus propagates a signal in P. gingivalis that causes downregulation of fimA expression. Signaling is mediated by arginine deiminase (ArcA) on the surface of Streptococcus cristatus. Biofilms tend to be polymicrobial in nature, and the subgingival plaque biofilm is an exemplar of this situation, with as many as 200 species present in any one individual.

Interactions between oral streptococci have developed because of ecological pressures within the oral environment. The high abundance of oral streptococci makes it very likely that they have evolved close relationships manifested in diverse interspecies interactions. Interspecies interactions in the oral biofilm can be defined by the purpose and nature of the interactions. Although there are many potential interactions between oral streptococci and other members of the oral biofilm community as well as the host, this chapter specifically focuses on streptococcus-streptococcus interactions. By promoting genetic exchange, the interspecies interaction, although not beneficial for the lysed bacterial species, is important in the context of evolution. A counteroffensive strategy was recently identified for Streptococcus oligofermentans, another oral streptococcal species and competitor of S. mutans. Surprisingly, S. oligofermentans is able to utilize the lactic acid produced by cariogenic species, such as S. mutans, to generate hydrogen peroxide, leading to growth inhibition of the lactic acid producer. Expanding research into multispecies models is desirable and would most likely reveal interesting synergistic behavior of oral streptococci. The close relationship of the oral streptococci enables the reconstruction of biochemical networks from already-existing metabolic functions identified in other streptococci. The spatial-temporal developmental patterns of oral streptococci in the biofilm, including the molecular mechanisms, are characterized. The oral biofilm is easily accessible, and the variety of genetic manipulation options for oral streptococci allows for experimental verification of in silico predictions.

This chapter focuses on the role of the candidate interspecies signaling molecule autoinducer-2 (AI-2), which is produced by the activity of the enzyme LuxS. It discusses its contribution to the development of dental plaque. A number of studies have been conducted on the impact of AI-2 and LuxS on dental plaque development. While investigations of the role(s) of AI-2 are still in their infancy, it is clear that the activity of LuxS and AI-2 moderates certain phenotypic characteristics of oral commensal and oral pathogenic bacteria. Particular notice in this chapter is given to LuxS-and AI-2-mediated interactions between the commensals Streptococcus oralis 34 and Actinomyces oris T14V, which are two initial colonizers of dental plaque. LsrB-like ribose transport periplasmic binding protein, in concert with LsrB, enhances biofilm formation through the recognition and uptake of AI-2. Of relevance to the oral streptococci and this chapter, a cursory examination of streptococcal genomes indicates that various Streptococcus spp. express RbsB-like protein. Thus, membrane-bound receptors and/or transport proteins may facilitate accumulation of AI-2 and intracellular signaling in streptococci. This chapter discusses studies of S. oralis 34 and A. oris T14V and their implication in future studies of AI-2-mediated communication. An interesting aspect of Sorbarod studies was that the data suggested that colocalization of S. oralis 34 and A. oris T14V facilitates the uptake and/or sequestering of AI-2 by one or both species.

This chapter integrates unpublished data with the published reports on the synergism between veillonellae and streptococci. It speculates on how the genome sequence of veillonellae will help one understand its biology and potential role in the pathogenicity of the dental biofilm community. It was discovered that hydrogen peroxide (H2O2) resistance in Streptococcus mutans was increased 100 to 1,000-fold over monospecies culture in cocultures with Veillonella sp. strain PK1910. This chapter focuses on some important findings from the Veillonella sp. strain PK1910 genome. In the 1964 description of Veillonellasp. by Rogosa, the species V. alcalescens was distinguished from V. parvula by the ability to decompose H2O2. In the 1982 revision of the species, the former subspecies in V. alcalescens were classified as three species: V. ratti, V. criceti, and V. dispar. Veillonella species are among the most prevalent early colonizers of oral biofilm. In-depth investigations on the mechanism of interactions of veillonellae with other oral microbial species will contribute significantly to one's overall understanding of the ecology of oral biofilms in the human host. Different from a metagenomics approach, metatranscriptomics focuses on community member functions at a particular time under a particular condition.

To control multispecies community function within dental plaque, one must first understand the spatiotemporal organization of the species within the community. Oral multispecies communities are not randomly organized. This chapter illustrates the application of metatranscriptomics to in vitro biofilm communities constructed with defined species. It describes the application of a sensitive procedure called catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) that measures mRNA at the single-cell level in vivo. A strategy to use the species to investigate metatranscriptomics of their communities, is presented in the chapter. Metatranscriptomic analysis of this community could document some gene expression changes accompanying this burst of growth. In summary, it appears that each of the three initial colonizers favors interaction with specific partners: Streptococcus oralis-Veillonella sp., Streptococcus gordonii-P. gingivalis, and Actinomyces oris-Fusobacterium nucleatum. With the availability of numerous genome-sequenced strains from the Human Oral Microbiome initiative, great progress can be achieved through metatranscriptomic analyses of two-, three-, and four-species biofilms growing on saliva as the sole source of nutrition. By using genome-sequenced strains, assigning gene functions to cDNA sequences will be much easier, and rapid progress can be made in understanding the relationship of community membership, spatiotemporal organization, and multispecies community growth.