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ABSTRACTArginine catabolism by the bacterial arginine deiminase system (ADS) has anticariogenic properties through the production of ammonia, which modulates the pH of the oral environment. Given the potential protective capacity of the ADS pathway, the exploitation of ADS-competent oral microbes through pre- or probiotic applications is a promising therapeutic target to prevent tooth decay. To date, most investigations of the ADS in the oral cavity and its relation to caries have focused on indirect measures of activity or on specific bacterial groups, yet the pervasiveness and rate of expression of the ADS operon in diverse mixed microbial communities in oral health and disease remain an open question. Here, we use a multivariate approach, combining ultra-deep metatranscriptomic sequencing with paired metataxonomic and in vitro citrulline quantification to characterize the microbial community and ADS operon expression in healthy and late-stage cavitated teeth. While ADS activity is higher in healthy teeth, we identify multiple bacterial lineages with upregulated ADS activity on cavitated teeth that are distinct from those found on healthy teeth using both reference-based mapping and de novo assembly methods. Our dual metataxonomic and metatranscriptomic approach demonstrates the importance of species abundance for gene expression data interpretation and that patterns of differential expression can be skewed by low-abundance groups. Finally, we identify several potential candidate probiotic bacterial lineages within species that may be useful therapeutic targets for the prevention of tooth decay and propose that the development of a strain-specific, mixed-microbial probiotic may be a beneficial approach given the heterogeneity of taxa identified here across health groups.IMPORTANCETooth decay is the most common preventable chronic disease, affecting more than two billion people globally. The development of caries on teeth is primarily a consequence of acid production by cariogenic bacteria that inhabit the plaque microbiome. Other bacterial strains in the oral cavity may suppress or prevent tooth decay by producing ammonia as a byproduct of the arginine deiminase metabolic pathway, increasing the pH of the plaque biofilm. While the benefits of arginine metabolism on oral health have been extensively documented in specific bacterial groups, the prevalence and consistency of arginine deiminase system (ADS) activity among oral bacteria in a community context remain an open question. In the current study, we use a multi-omics approach to document the pervasiveness of the expression of the ADS operon in both health and disease to better understand the conditions in which ADS activity may prevent tooth decay.

ABSTRACT Streptococcus mutans, the primary etiologic agent of human dental caries, and a variety of oral Streptococcus and Actinomyces spp. synthesize high molecular mass homopolymers of fructose (fructans) with predominantly β2,1- (inulins) or β2,6-linkages (levans). The ability of S. mutans to degrade fructans contributes to the severity of dental caries. The extracellular product of fruA of S. mutans is an exo- β-d-fructofuranosidase that releases fructose from levan and inulin. Located 70 bp downstream of fruA, fruB encodes a member of the glycoside hydrolase family 32, but the function of FruB has not been established. Growth assays performed using wild-type UA159 and fruB-deficient derivatives, with fructans as the sole carbohydrate source, showed a significant reduction in the growth rate of a fruB mutant on levan, but not on inulin. A purified, recombinant FruB protein degraded levan to release mainly fructooligosaccharides. Driven by the fruA promoter and a secondary promoter located in the 3′ region of the fruA sequence, the fruB gene is inducible by fructose and especially by levan, but a stable stem-loop structure in the intergenic region likely modulates transcriptional read-through from fruA. Transcriptomic analysis of UA159 and a fruB mutant grown on 0.2% levan revealed differential expression of genes encoding ABC transporters, transcriptional regulators and genes involved in growth and stress tolerance. The ability of FruB to enhance levan metabolism and the high degree of conservation of FruB across S. mutans isolates imply a significant contribution of FruB to the fitness and virulence of this pathogen in human dental biofilms. IMPORTANCE Carbohydrate metabolism and acid production are essential for the development of dental caries. As a by-product of sucrose metabolism, formation, and degradation of fructans enhances the severity of caries by S. mutans in animal models. This study highlights a significant breakthrough in identifying FruB in S. mutans as an endolevanase that contributes to efficient utilization of levan, a specific type of fructan produced by certain commensals but not S. mutans. Transcriptomic analysis revealed that FruB-dependent levan metabolism impacted global gene regulation, including a large number of novel genes. Considering the preference for levan by both FruA and FruB, the conservation of fruAB in S. mutans might represent a competitive advantage in access to the energy storage produced by dental microbiome. This is the first report demonstrating the presence of an endolevanase in S. mutans, therefore should be of broad interest to the fields of dental caries and complex carbohydrate metabolism.

A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

ABSTRACT Health-associated oral Streptococcus species are promising probiotic candidates to protect against dental caries. Ammonia production through the arginine deiminase system (ADS), which can increase the pH of oral biofilms, and direct antagonism of caries-associated bacterial species are desirable properties for oral probiotic strains. ADS and antagonistic activities can vary dramatically among individuals, but the genetic basis for these differences is unknown. We sequenced whole genomes of a diverse set of clinical oral Streptococcus isolates and examined the genetic basis of variability in ADS and antagonistic activities. A total of 113 isolates were included and represented 10 species: Streptococcus australis, A12-like, S. cristatus, S. gordonii, S. intermedius, S. mitis, S. oralis including S. oralis subsp. dentisani, S. parasanguinis, S. salivarius, and S. sanguinis. Mean ADS activity and antagonism on Streptococcus mutans UA159 were measured for each isolate, and each isolate was whole genome shotgun sequenced on an Illumina MiSeq. Phylogenies were built of genes known to be involved in ADS activity and antagonism. Several approaches to correlate the pan-genome with phenotypes were performed. Phylogenies of genes previously identified in ADS activity and antagonism grouped isolates by species, but not by phenotype. A genome-wide association study (GWAS) identified additional genes potentially involved in ADS activity or antagonism across all the isolates we sequenced as well as within several species. Phenotypic heterogeneity in oral streptococci is not necessarily reflected by genotype and is not species specific. Probiotic strains must be carefully selected based on characterization of each strain and not based on inclusion within a certain species. IMPORTANCE Representative type strains are commonly used to characterize bacterial species, yet species are phenotypically and genotypically heterogeneous. Conclusions about strain physiology and activity based on a single strain therefore may be inappropriate and misleading. When selecting strains for probiotic use, the assumption that all strains within a species share the same desired probiotic characteristics may result in selection of a strain that lacks the desired traits, and therefore makes a minimally effective or ineffective probiotic. Health-associated oral streptococci are promising candidates for anticaries probiotics, but strains need to be carefully selected based on observed phenotypes. We characterized the genotypes and anticaries phenotypes of strains from 10 species of oral streptococci and demonstrate poor correlation between genotype and phenotype across all species.

ABSTRACT Health-associated oral Streptococcus species are promising probiotic candidates to protect against dental caries. Ammonia production through the arginine deiminase system (ADS), which can increase the pH of oral biofilms, and direct antagonism of caries-associated bacterial species are desirable properties for oral probiotic strains. ADS and antagonistic activities can vary dramatically among individuals, but the genetic basis for these differences is unknown. We sequenced whole genomes of a diverse set of clinical oral Streptococcus isolates and examined the genetic basis of variability in ADS and antagonistic activities. A total of 113 isolates were included and represented 10 species: Streptococcus australis, A12-like, S. cristatus, S. gordonii, S. intermedius, S. mitis, S. oralis including S. oralis subsp. dentisani, S. parasanguinis, S. salivarius, and S. sanguinis. Mean ADS activity and antagonism on Streptococcus mutans UA159 were measured for each isolate, and each isolate was whole genome shotgun sequenced on an Illumina MiSeq. Phylogenies were built of genes known to be involved in ADS activity and antagonism. Several approaches to correlate the pan-genome with phenotypes were performed. Phylogenies of genes previously identified in ADS activity and antagonism grouped isolates by species, but not by phenotype. A genome-wide association study (GWAS) identified additional genes potentially involved in ADS activity or antagonism across all the isolates we sequenced as well as within several species. Phenotypic heterogeneity in oral streptococci is not necessarily reflected by genotype and is not species specific. Probiotic strains must be carefully selected based on characterization of each strain and not based on inclusion within a certain species. IMPORTANCE Representative type strains are commonly used to characterize bacterial species, yet species are phenotypically and genotypically heterogeneous. Conclusions about strain physiology and activity based on a single strain therefore may be inappropriate and misleading. When selecting strains for probiotic use, the assumption that all strains within a species share the same desired probiotic characteristics may result in selection of a strain that lacks the desired traits, and therefore makes a minimally effective or ineffective probiotic. Health-associated oral streptococci are promising candidates for anticaries probiotics, but strains need to be carefully selected based on observed phenotypes. We characterized the genotypes and anticaries phenotypes of strains from 10 species of oral streptococci and demonstrate poor correlation between genotype and phenotype across all species.

Interspecific competition in bacteria governs colony growth dynamics and pattern formation. Here, we demonstrate an interesting phenomenon of interspecific competition between Bacillus cereus MSM-S1 and Pseudomonas sp. MSM-M1, where secretion of an inhibitor by Pseudomonas sp. is used as a strategy for survival. Although B. cereus grows faster than Pseudomonas sp., in the presence of Pseudomonas sp. the population of B. cereus reduces significantly, whereas Pseudomonas sp. do not show any marked alteration in their population growth. Appearance of a zone of inhibition between growing colonies of two species on nutrient agar prevents the expanding front of the MSM-S1 colony from accessing and depleting nutrients in the region occupied by MSM-M1, thereby aiding the survival of the slower growing MSM-M1 colonies. To support our experimental results, we present simulations, based on a chemotactic model of colony growth dynamics. We demonstrate that the chemical(s) secreted by Pseudomonas sp. is responsible for the observed inhibition of growth and spatial pattern of the B. cereus MSM-S1 colony. Our experimental results are in excellent agreement with the numerical results and confirm that secreted inhibitors enable Pseudomonas sp. to survive and coexist in the presence of faster growing B. cereus, in a common niche.