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Dental caries, the most common chronic infectious disease worldwide, has a complex etiology involving the interplay of microbial and host factors that are not completely understood. In this study, the oral microbiome and 38 host cytokines and chemokines were analyzed across 23 children with caries and 24 children with healthy dentition. De novo assembly of metagenomic sequencing obtained 527 metagenome-assembled genomes (MAGs), representing 150 bacterial species. Forty-two of these species had no genomes in public repositories, thereby representing novel taxa. These new genomes greatly expanded the known pangenomes of many oral clades, including the enigmatic Saccharibacteria clades G3 and G6, which had distinct functional repertoires compared to other oral Saccharibacteria. Saccharibacteria are understood to be obligate epibionts, which are dependent on host bacteria. These data suggest that the various Saccharibacteria clades may rely on their hosts for highly distinct metabolic requirements, which would have significant evolutionary and ecological implications. Across the study group, Rothia, Neisseria, and Haemophilus spp. were associated with good dental health, whereas Prevotella spp., Streptococcus mutans, and Human herpesvirus 4 (Epstein-Barr virus [EBV]) were more prevalent in children with caries. Finally, 10 of the host immunological markers were significantly elevated in the caries group, and co-occurrence analysis provided an atlas of potential relationships between microbes and host immunological molecules. Overall, this study illustrated the oral microbiome at an unprecedented resolution and contributed several leads for further study that will increase the understanding of caries pathogenesis and guide therapeutic development.
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Cárie Dentária , Metagenômica , Microbiota , Bactérias , Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Humanos , Microbiota/genéticaRESUMO
Streptococcus mutans is considered a primary etiologic agent of dental caries, which is the most common chronic infectious disease worldwide. S. mutans B04Sm5 was recently shown to produce reutericyclins and mutanocyclin through the muc biosynthetic gene cluster and to utilize reutericyclins to inhibit the growth of neighboring commensal streptococci. In this study, examination of S. mutans and muc phylogeny suggested evolution of an ancestral S. mutans muc into three lineages within one S. mutans clade and then horizontal transfer of muc to other S. mutans clades. The roles of the mucG and mucH transcriptional regulators and the mucI transporter were also examined. mucH was demonstrated to encode a transcriptional activator of muc. mucH deletion reduced production of mutanocyclin and reutericyclins and eliminated the impaired growth and inhibition of neighboring streptococci phenotypes, which are associated with reutericyclin production. ΔmucG had increased mutanocyclin and reutericyclin production, which impaired growth and increased the ability to inhibit neighboring streptococci. However, deletion of mucG also caused reduced expression of mucD, mucE, and mucI. Deletion of mucI reduced mutanocyclin and reutericylin production but enhanced growth, suggesting that mucI may not transport reutericyclin as its homolog does in Limosilactobacillus reuteri. Further research is needed to determine the roles of mucG and mucI and to identify any cofactors affecting the activity of the mucG and mucH regulators. Overall, this study provided pangenome and phylogenetic analyses that serve as a resource for S. mutans research and began elucidation of the regulation of reutericyclins and mutanocyclin production in S. mutans. IMPORTANCE S. mutans must be able to outcompete neighboring organisms in its ecological niche in order to cause dental caries. S. mutans B04Sm5 inhibited the growth of neighboring commensal streptococci through production of reutericyclins via the muc biosynthetic gene cluster. In this study, an S. mutans pangenome database and updated phylogenetic tree were generated that will serve as valuable resources for the S. mutans research community and that provide insights into the carriage and evolution of S. mutans muc. The MucG and MucH regulators, and the MucI transporter, were shown to modulate production of reutericyclins and mutanocyclin. These genes also affected the ability of S. mutans to inhibit neighboring commensals, suggesting that they may play a role in S. mutans virulence.
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Cárie Dentária , Streptococcus mutans , Biofilmes , Humanos , Filogenia , Streptococcus mutans/metabolismo , Ácido Tenuazônico/análogos & derivados , Ácido Tenuazônico/metabolismoRESUMO
It is well-understood that many bacteria have evolved to survive catastrophic events using a variety of mechanisms, which include expression of stress-response genes, quiescence, necrotrophy, and metabolic advantages obtained through mutation. However, the dynamics of individuals leveraging these abilities to gain a competitive advantage in an ecologically complex setting remain unstudied. In this study, we observed the saliva microbiome throughout the ecological perturbation of long-term starvation, allowing only the species best equipped to access and use the limited resources to survive. During the first several days, the community underwent a death phase that resulted in a â¼50-100-fold reduction in the number of viable cells. Interestingly, after this death phase, only three species, Klebsiella pneumoniae, Klebsiella oxytoca, and Providencia alcalifaciens, all members of the family Enterobacteriaceae, appeared to be transcriptionally active and recoverable. Klebsiella are significant human pathogens, frequently resistant to multiple antibiotics, and recently, ectopic colonization of the gut by oral Klebsiella was documented to induce dysbiosis and inflammation. MetaOmics analyses provided several leads for further investigation regarding the ecological success of the Enterobacteriaceae. The isolates accumulated single nucleotide polymorphisms in known growth advantage in stationary phase alleles and produced natural products closely resembling antimicrobial cyclic depsipeptides. The results presented in this study suggest that pathogenic Enterobacteriaceae persist much longer than their more benign neighbors in the salivary microbiome when faced with starvation. This is particularly significant, given that hospital surfaces contaminated with oral fluids, especially sinks and drains, are well-established sources of outbreaks of drug-resistant Enterobacteriaceae.
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Microbioma Gastrointestinal/fisiologia , Klebsiella/fisiologia , Viabilidade Microbiana , Boca/microbiologia , Providencia/fisiologia , Humanos , Saliva/microbiologiaRESUMO
Ecological networking and in vitro studies predict that anaerobic, mucus-degrading bacteria are keystone species in cystic fibrosis (CF) microbiomes. The metabolic byproducts from these bacteria facilitate the colonization and growth of CF pathogens like Pseudomonas aeruginosa. Here, a multi-omics study informed the control of putative anaerobic keystone species during a transition in antibiotic therapy of a CF patient. A quantitative metagenomics approach combining sequence data with epifluorescence microscopy showed that during periods of rapid lung function loss, the patient's lung microbiome was dominated by the anaerobic, mucus-degrading bacteria belonging to Streptococcus, Veillonella, and Prevotella genera. Untargeted metabolomics and community cultures identified high rates of fermentation in these sputa, with the accumulation of lactic acid, citric acid, and acetic acid. P. aeruginosa utilized these fermentation products for growth, as indicated by quantitative transcriptomics data. Transcription levels of P. aeruginosa genes for the utilization of fermentation products were proportional to the abundance of anaerobic bacteria. Clindamycin therapy targeting Gram-positive anaerobes rapidly suppressed anaerobic bacteria and the accumulation of fermentation products. Clindamycin also lowered the abundance and transcription of P. aeruginosa, even though this patient's strain was resistant to this antibiotic. The treatment stabilized the patient's lung function and improved respiratory health for two months, lengthening by a factor of four the between-hospitalization time for this patient. Killing anaerobes indirectly limited the growth of P. aeruginosa by disrupting the cross-feeding of fermentation products. This case study supports the hypothesis that facultative anaerobes operated as keystone species in this CF microbiome. Personalized multi-omics may become a viable approach for routine clinical diagnostics in the future, providing critical information to inform treatment decisions.
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Fibrose Cística/microbiologia , Metagenômica/métodos , Microbiota , Adulto , Antibacterianos/uso terapêutico , Fibrose Cística/complicações , Fibrose Cística/terapia , Genômica/métodos , Humanos , Pulmão/microbiologia , Masculino , Metabolômica/métodos , Microbiota/genética , Testes de Função Respiratória , Insuficiência Respiratória/genética , Insuficiência Respiratória/metabolismo , Insuficiência Respiratória/microbiologia , Insuficiência Respiratória/terapia , Escarro/microbiologiaRESUMO
While many studies have examined the bacterial taxa associated with dental caries, the most common chronic infectious disease globally, little is known about the caries-associated virome. In this study, the salivary viromes of 21 children with severe caries (>2 dentin lesions) and 23 children with healthy dentition were examined. 2,485 viral metagenome-assembled genomes (vMAGs) were identified, binned, and quantified from the metagenomic assemblies. These vMAGs were mostly phage, and represented 1,547 unique species-level vOTUs, 247 of which appear to be novel. The metagenomes were also queried for all 3,835 unique species-level vOTUs of DNA viruses with a human host on NCBI Virus, however all but Human betaherpesvirus 7 were at very low abundance in the saliva. The oral viromes of the children with caries exhibited significantly different beta diversity compared to the oral virome of the children with healthy dentition; several vOTUs predicted to infect Pauljensenia and Neisseria were strongly correlated with health, and two vOTUs predicted to infect Saccharibacteria and Prevotella histicola, respectively, were correlated with caries. Co-occurrence analysis indicated that phage typically co-occurred with both their predicted hosts and with bacteria that were themselves associated with the same disease status. Overall, this study provided the sequences of 53 complete or nearly complete novel oral phages and illustrated the significance of the oral virome in the context of dental caries, which has been largely overlooked. This work represents an important step towards the identification and study of phage therapy candidates which treat or prevent caries pathogenesis.
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The human oral microbiota is highly diverse and has a complex ecology, comprising bacteria, microeukaryotes, archaea and viruses. These communities have elaborate and highly structured biogeography that shapes metabolic exchange on a local scale and results from the diverse microenvironments present in the oral cavity. The oral microbiota also interfaces with the immune system of the human host and has an important role in not only the health of the oral cavity but also systemic health. In this Review, we highlight recent advances including novel insights into the biogeography of several oral niches at the species level, as well as the ecological role of candidate phyla radiation bacteria and non-bacterial members of the oral microbiome. In addition, we summarize the relationship between the oral microbiota and the pathology of oral diseases and systemic diseases. Together, these advances move the field towards a more holistic understanding of the oral microbiota and its role in health, which in turn opens the door to the study of novel preventive and therapeutic strategies.
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Microbiota , Vírus , Humanos , Boca/microbiologia , Bactérias/genética , ArchaeaRESUMO
While genome-wide transposon mutagenesis screens have identified numerous essential genes in the significant human pathogen Streptococcus pyogenes (group A Streptococcus or GAS), many of their functions remain elusive. This knowledge gap is attributed in part to the limited molecular toolbox for controlling GAS gene expression and the bacterium's poor genetic transformability. CRISPR interference (CRISPRi), using catalytically inactive GAS Cas9 (dCas9), is a powerful approach to specifically repress gene expression in both bacteria and eukaryotes, but ironically, it has never been harnessed for controlled gene expression in GAS. In this study, we present a highly transformable and fully virulent serotype M1T1 GAS strain and introduce a doxycycline-inducible CRISPRi system for efficient repression of bacterial gene expression. We demonstrate highly efficient, oligo-based single guide RNA cloning directly to GAS, enabling the construction of a gene knockdown strain in just 2 days, in contrast to the several weeks typically required. The system is shown to be titratable and functional both in vitro and in vivo using a murine model of GAS infection. Furthermore, we provide direct in vivo evidence that the expression of the conserved cell division gene ftsZ is essential for GAS virulence, highlighting its promise as a target for emerging FtsZ inhibitors. Finally, we introduce SpyBrowse (https://veeninglab.com/SpyBrowse), a comprehensive and user-friendly online resource for visually inspecting and exploring GAS genetic features. The tools and methodologies described in this work are poised to facilitate fundamental research in GAS, contribute to vaccine development, and aid in the discovery of antibiotic targets. IMPORTANCE: While group A Streptococcus (GAS) remains a predominant cause of bacterial infections worldwide, there are limited genetic tools available to study its basic cell biology. Here, we bridge this gap by creating a highly transformable, fully virulent M1T1 GAS strain. In addition, we established a tight and titratable doxycycline-inducible system and developed CRISPR interference (CRISPRi) for controlled gene expression in GAS. We show that CRISPRi is functional in vivo in a mouse infection model. Additionally, we present SpyBrowse, an intuitive and accessible genome browser (https://veeninglab.com/SpyBrowse). Overall, this work overcomes significant technical challenges of working with GAS and, together with SpyBrowse, represents a valuable resource for researchers in the GAS field.
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Sistemas CRISPR-Cas , Infecções Estreptocócicas , Streptococcus pyogenes , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Animais , Camundongos , Infecções Estreptocócicas/microbiologia , Virulência/genética , Regulação Bacteriana da Expressão Gênica , Modelos Animais de Doenças , Feminino , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
BACKGROUND: The human oral and nasal cavities can act as reservoirs for opportunistic pathogens capable of causing acute infection. These microbes asymptomatically colonize the human oral and nasal cavities which facilitates transmission within human populations via the environment, and they routinely possess clinically significant antibiotic resistance genes. Among these opportunistic pathogens, the Klebsiella genus stands out as a notable example, with its members frequently linked to nosocomial infections and multidrug resistance. As with many colonizing opportunistic pathogens, the essential transmission factors influencing the spread of Klebsiella species among both healthy and diseased individuals remain unclear. RESULTS: Here, we explored a possible explanation by investigating the ability of oral and nasal Klebsiella species to outcompete their native microbial community members under in vitro starvation conditions, which could be analogous to external hospital environments or the microenvironment of mechanical ventilators. When K. pneumoniae and K. aerogenes were present within a healthy human oral or nasal sample, the bacterial community composition shifted dramatically under starvation conditions and typically became enriched in Klebsiella species. Furthermore, introducing K. pneumoniae exogenously into a native microbial community lacking K. pneumoniae, even at low inoculum, led to repeated enrichment under starvation. Precise monitoring of K. pneumoniae within these communities undergoing starvation indicated rapid initial growth and prolonged viability compared to other members of the microbiome. K. pneumoniae strains isolated from healthy individuals' oral and nasal cavities also exhibited resistance to multiple classes of antibiotics and were genetically similar to clinical and gut isolates. In addition, we found that in the absence of Klebsiella species, other understudied opportunistic pathogens, such as Peptostreptococcus, increased in relative abundance under starvation conditions. CONCLUSIONS: Our findings establish an environmental and microbiome community circumstance that allows for the enrichment of Klebsiella species and other opportunistic pathogens. Klebsiella's enrichment may hinge on its ability to quickly outgrow other members of the microbiome. The ability to outcompete other commensal bacteria and to persist under harsh environmental conditions could be an important factor that contributes to enhanced transmission in both commensal and pathogenic contexts. Video Abstract.
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Farmacorresistência Bacteriana Múltipla , Klebsiella , Microbiota , Boca , Humanos , Farmacorresistência Bacteriana Múltipla/genética , Klebsiella/genética , Klebsiella/isolamento & purificação , Klebsiella/efeitos dos fármacos , Boca/microbiologia , Microbiota/efeitos dos fármacos , Microbiota/genética , Infecções por Klebsiella/microbiologia , Antibacterianos/farmacologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/efeitos dos fármacos , Inanição , Cavidade Nasal/microbiologia , Nariz/microbiologiaRESUMO
The oral microbiota has an enormous impact on human health, with oral dysbiosis now linked to many oral and systemic diseases. Recent advancements in sequencing, mass spectrometry, bioinformatics, computational biology, and machine learning are revolutionizing oral microbiome research, enabling analysis at an unprecedented scale and level of resolution using omics approaches. This review contains a comprehensive perspective of the current state-of-the-art tools available to perform genomics, metagenomics, phylogenomics, pangenomics, transcriptomics, proteomics, metabolomics, lipidomics, and multi-omics analysis on (all) microbiomes, and then provides examples of how the techniques have been applied to research of the oral microbiome, specifically. Key findings of these studies and remaining challenges for the field are highlighted. Although the methods discussed here are placed in the context of their contributions to oral microbiome research specifically, they are pertinent to the study of any microbiome, and the intended audience of this includes researchers would simply like to get an introduction to microbial omics and/or an update on the latest omics methods. Continued research of the oral microbiota using omics approaches is crucial and will lead to dramatic improvements in human health, longevity, and quality of life.
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Microbiota , Qualidade de Vida , Humanos , Biologia Computacional , Genômica , Metabolômica , Microbiota/genéticaRESUMO
The microorganisms living in the human oral cavity, collectively known as the oral microbiota, play a critical role in not only oral health, but systemic and overall health. The Baker Lab leverages emerging technologies in bioinformatics and molecular biology to answer fundamental questions regarding the ecology, physiology, and pathogenesis of the oral microbiota. We use a microbial 'omics approach, which has included pioneering the use of nanopore sequencing on saliva and oral bacterial RNA. The resulting work discovered novel bacterial species and biosynthetic pathways which impact the ecology of the oral microbiota and its relationship to human disease. This article will briefly define the oral microbiota. It will also summarize how bioinformatics and 'omics-based research have revolutionized oral health research. The article will then provide a broad summary of our past, present and future research and educational programs.
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The human oral and nasal cavities can act as reservoirs for opportunistic pathogens capable of causing acute infection. These microbes asymptomatically colonize the human oral and nasal cavities which facilitates transmission within human populations via the environment, and they routinely possess a clinically-significant antibiotic-resistance genes. Among these opportunistic pathogens, the Klebsiella genus stands out as a notable example, with its members frequently linked to nosocomial infections and multidrug resistance. As with many colonizing opportunistic pathogens, how Klebsiella transitions from an asymptomatic colonizer to a pathogen remains unclear. Here, we explored a possible explanation by investigating the ability of oral and nasal Klebsiella to outcompete their native microbial community members under in vitro starvation conditions, which could be analogous to external hospital environments. When Klebsiella was present within a healthy human oral or nasal sample, the bacterial community composition shifted dramatically under starvation conditions and typically became dominated by Klebsiella. Furthermore, introducing K. pneumoniae exogenously into a native microbial community lacking K. pneumoniae, even at low inoculum, led to repeated dominance under starvation. K.pneumoniae strains isolated from healthy individuals' oral and nasal cavities also exhibited resistance to multiple classes of antibiotics and were genetically similar to clinical and gut isolates. In addition, we found that in the absence of Klebsiella, other understudied opportunistic pathogens, such as Peptostreptococcus, dominate under starvation conditions. Our findings establish an environmental circumstance that allows for the outgrowth of Klebsiella and other opportunistic pathogens. The ability to outcompete other commensal bacteria and to persist under harsh environmental conditions may contribute to the colonization-to-infection transition of these opportunistic pathogens.
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Obtaining complete, high-quality reference genomes is essential to the study of any organism. Recent advances in nanopore sequencing, as well as genome assembly and analysis methods, have made it possible to obtain complete bacterial genomes from metagenomic (i.e., multispecies) samples, including those from the human microbiome. In this study, methods are presented to obtain complete bacterial genomes from human saliva using complementary Oxford Nanopore (ONT) and Illumina sequencing. Applied to 3 human saliva samples, these methods resulted in 11 complete bacterial genomes: 3 Saccharibacteria clade G6 (also known as Ca. Nanogingivalaceae HMT-870), 1 Saccharibacteria clade G1 HMT-348, 2 Rothia mucilaginosa, 2 Actinomyces graevenitzii, 1 Mogibacterium diversum, 1 Lachnospiraceae HMT-096, and 1 Lancefieldella parvula; and one circular chromosome of Ruminococcaceae HMT-075 (which likely has at least 2 chromosomes). The 4 Saccharibacteria genomes, as well as the Actinomyces graeventizii genomes, represented the first complete genomes from their respective bacterial taxa. Aside from the complete genomes, the assemblies contained 147 contigs of over 500,000 bp each and thousands of smaller contigs, together representing a myriad of additional draft genomes including many which are likely nearly complete. The complete genomes enabled highly accurate pangenome analysis, which identified unique and missing features of each genome compared to its closest relatives with complete genomes available in public repositories. These features provide clues as to the lifestyle and ecological role of these bacteria within the human oral microbiota, which will be particularly useful in designing future studies of the taxa that have never been isolated or cultivated. IMPORTANCE Obtaining complete and accurate genomes is crucial to the study of any organism. Previously, obtaining complete genomes of bacteria, including those of the human microbiome, frequently required isolation of the organism, as well as low-throughput, manual sequencing methods to resolve repeat regions. Advancements in long-read sequencing technologies, including Oxford Nanopore (ONT), have made it possible to obtain complete, closed bacterial genomes from metagenomic samples. This study reports methods to obtain complete genomes from the human oral microbiome using complementary ONT and Illumina sequencing of saliva samples. Eleven complete genomes were obtained from 3 human saliva samples, with genomes of Saccharibacteria HMT-870, Saccharibacteria HMT-348, and Actinomyces graeventzii being the first complete genomes from their respective taxa. Obtaining complete bacterial genomes in a high-throughput manner will help illuminate the metabolic and ecological roles of important members of the human microbiota, particularly those that have remained recalcitrant to isolation and cultivation.
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Microbiota , Sequenciamento por Nanoporos , Humanos , Análise de Sequência de DNA/métodos , Sequenciamento por Nanoporos/métodos , Saliva , Genoma Bacteriano/genética , Microbiota/genética , Bactérias/genéticaRESUMO
Saccharibacteria are abundant and diverse members of the human oral microbiome; however, they are poorly understood and appear to exhibit an epibiont/parasitic lifestyle dependent on host bacteria. Here, a complete metagenome-assembled genome (MAG) sequence of an organism from Saccharibacteria clade G1 human microbial taxon (HMT) 348 is reported, strain HMT-348_TM7c-JB.
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Streptococcus mutans promotes a tooth-damaging dysbiosis in the oral microbiota because it can form biofilms and survive acid stress better than most of its ecological competitors, which are typically health associated. Many of these commensals produce hydrogen peroxide; therefore, S. mutans must manage both oxidative stress and acid stress with coordinated and complex physiological responses. In this study, the proteome of S. mutans was examined during regulated growth in acid and oxidative stresses as well as in deletion mutants with impaired oxidative stress phenotypes, Δnox and ΔtreR. A total of 607 proteins exhibited significantly different abundances across the conditions tested, and correlation network analysis identified modules of coexpressed proteins that were responsive to the deletion of nox and/or treR as well as acid and oxidative stress. The data explained the reactive oxygen species (ROS)-sensitive and mutacin-deficient phenotypes exhibited by the ΔtreR strain. SMU.1069-1070, a poorly understood LytTR system, had an elevated abundance in the ΔtreR strain. S. mutans LytTR systems regulate mutacin production and competence, which may explain how TreR affects mutacin production. Furthermore, the protein cluster that produces mutanobactin, a lipopeptide important in ROS tolerance, displayed a reduced abundance in the ΔtreR strain. The role of Nox as a keystone in the oxidative stress response was also emphasized. Crucially, this data set provides oral health researchers with a proteome atlas that will enable a more complete understanding of the S. mutans stress responses that are required for pathogenesis, and will facilitate the development of new and improved therapeutic approaches for dental caries. IMPORTANCE Dental caries is the most common chronic infectious disease worldwide and disproportionately affects marginalized socioeconomic groups. Streptococcus mutans is considered a primary etiological agent of caries, with its pathogenicity being dependent on coordinated physiological stress responses that mitigate the damage caused by the oxidative and acid stress common within dental plaque. In this study, the proteome of S. mutans was examined during growth in acidic and oxidative stresses as well in nox and treR deletion mutants. A total of 607 proteins were differentially expressed across the strains/growth conditions, and modules of coexpressed proteins were identified, which enabled mapping the acid and oxidative stress responses across S. mutans metabolism. The presence of TreR was linked to mutacin production via LytTR system signaling and to oxidative stress via mutanobactin production. The data provided by this study will guide future research elucidating S. mutans pathogenesis and developing improved preventative and treatment modalities for dental caries.
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Proteínas de Bactérias , Cárie Dentária , Humanos , Proteínas de Bactérias/genética , Proteoma/metabolismo , Streptococcus mutans/genética , Espécies Reativas de Oxigênio/metabolismo , Estresse OxidativoRESUMO
Saccharibacteria (formerly TM7) have reduced genomes and a small cell size and appear to have a parasitic lifestyle dependent on a bacterial host. Although there are at least 6 major clades of Saccharibacteria inhabiting the human oral cavity, complete genomes of oral Saccharibacteria were previously limited to the G1 clade. In this study, nanopore sequencing was used to obtain three complete genome sequences from clade G6. Phylogenetic analysis suggested the presence of at least 3 to 5 distinct species within G6, with two discrete taxa represented by the 3 complete genomes. G6 Saccharibacteria were highly divergent from the more-well-studied clade G1 and had the smallest genomes and lowest GC content of all Saccharibacteria. Pangenome analysis showed that although 97% of shared pan-Saccharibacteria core genes and 89% of G1-specific core genes had putative functions, only 50% of the 244 G6-specific core genes had putative functions, highlighting the novelty of this group. Compared to G1, G6 harbored divergent metabolic pathways. G6 genomes lacked an F1Fo ATPase, the pentose phosphate pathway, and several genes involved in nucleotide metabolism, which were all core genes for G1. G6 genomes were also unique compared to that of G1 in that they encoded d-lactate dehydrogenase, adenylate cyclase, limited glycerolipid metabolism, a homolog to a lipoarabinomannan biosynthesis enzyme, and the means to degrade starch. These differences at key metabolic steps suggest a distinct lifestyle and ecological niche for clade G6, possibly with alternative hosts and/or host dependencies, which would have significant ecological, evolutionary, and likely pathogenic implications. IMPORTANCESaccharibacteria are ultrasmall parasitic bacteria that are common members of the oral microbiota and have been increasingly linked to disease and inflammation. However, the lifestyle and impact on human health of Saccharibacteria remain poorly understood, especially for the clades with no complete genomes (G2 to G6) or cultured isolates (G2 and G4 to G6). Obtaining complete genomes is of particular importance for Saccharibacteria, because they lack many of the "essential" core genes used for determining draft genome completeness, and few references exist outside clade G1. In this study, complete genomes of 3 G6 strains, representing two candidate species, were obtained and analyzed. The G6 genomes were highly divergent from that of G1 and enigmatic, with 50% of the G6 core genes having no putative functions. The significant difference in encoded functional pathways is suggestive of a distinct lifestyle and ecological niche, probably with alternative hosts and/or host dependencies, which would have major implications in ecology, evolution, and pathogenesis.
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Acetobacteraceae/classificação , Acetobacteraceae/genética , Genoma Bacteriano , Boca/microbiologia , Filogenia , Acetobacteraceae/metabolismo , Redes e Vias Metabólicas/genética , Microbiota , Análise de Sequência de DNA/métodosRESUMO
At least 6 highly diverse clades of Saccharibacteria inhabit the human oral cavity. However, all oral Saccharibacteria strains with currently available complete genome sequences or cultured isolates belong to clade G1, leaving clades G2 through G6 poorly understood. Here, a complete genome sequence of JB001, a clade G6 ("Candidatus Nanogingivalaceae") Saccharibacteria strain, is reported.
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Small molecules are a primary communication media of the microbial world, and play crucial, yet largely unidentified, roles in microbial ecology and disease pathogenesis. Many small molecules are produced by biosynthetic gene clusters, which can be predicted and analyzed computationally given a genome. A recent study examined the biosynthetic repertoire of the oral microbiome and cross-referenced this information against the disease status of the human host, providing leads for biosynthetic gene clusters, and their natural products, which may be key in the oral microbial ecology affecting dental caries and periodontitis. This chapter provides a step-by-step tutorial to bioinformatically to locate biosynthetic gene clusters within genomes, predict the type of natural products that are produced, and cross-reference the identified biosynthetic gene clusters to microbiomes associated with disease or health.
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Cárie Dentária , Microbiota , Produtos Biológicos , Suscetibilidade à Cárie Dentária , Humanos , Família Multigênica , Streptococcus mutansRESUMO
The human oral microbiome consists of diverse microbes actively communicating and interacting through a variety of biochemical mechanisms. Dental caries is a major public health issue caused by fermentable carbohydrate consumption that leads to dysbiosis of the oral microbiome. Streptococcus mutans is a known major contributor to caries pathogenesis, due to its exceptional ability to form biofilms in the presence of sucrose, as well as to its acidophilic lifestyle. S. mutans can also kill competing bacteria, which are typically health associated, through the production of bacteriocins and other small molecules. A subset of S. mutans strains encode the muc biosynthetic gene cluster (BGC), which was recently shown to produce the tetramic acids, mutanocyclin and reutericyclins A, B, and C. Reutericyclin A displayed strong antimicrobial activity and mutanocyclin appeared to be anti-inflammatory; however the effect of these compounds, and the carriage of muc by S. mutans, on the ecology of the oral microbiota is not known, and was examined here using a previously developed in vitro biofilm model derived from human saliva. While reutericyclin significantly inhibited in vitro biofilm formation and acid production at sub-nanomolar concentrations, mutanocyclin did not present any activity until the high micromolar range. 16S rRNA gene sequencing revealed that reutericyclin drastically altered the biofilm community composition, while mutanocyclin showed a more specific effect, reducing the relative abundance of cariogenic Limosilactobacillus fermentum. Mutanocyclin or reutericyclin produced by the S. mutans strains amended to the community did not appear to affect the community in the same way as the purified compounds, although the results were somewhat confounded by the differing growth rates of the S. mutans strains. Regardless of the strain added, the addition of S. mutans to the in vitro community significantly increased the abundance of S. mutans and Veillonella infantium, only. Overall, this study illustrates that reutericyclin A and mutanocyclin do impact the ecology of a complex in vitro oral biofilm; however, further research is needed to determine the extent to which the production of these compounds affects the virulence of S. mutans.
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Streptococcus mutans strain B04Sm5 was recently shown to inhibit the growth of neighboring commensal bacteria using reutericyclin, an acylated tetramic acid produced by the muc biosynthetic gene cluster. Here, a complete genome sequence of B04Sm5 is reported.
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Streptococcus mutans is a major etiologic agent of dental caries, which is the most common chronic infectious disease worldwide. S. mutans is particularly adept at causing caries due to its exceptional capacity to form biofilms and its ability to survive acidic conditions that arrest acid production and growth in many more benign members of the oral microbiota. Two mechanisms utilized by S. mutans to tolerate acid are: modulation of the membrane fatty acid content and utilization of the F1 F0 -ATPase to pump protons out of the cytosol. In this study, the role of the spxA2 transcriptional regulator in these two pathways, and overall cell envelope homeostasis, was examined. Loss of spxA2 resulted in an increase in the proportion of saturated fatty acids in the S. mutans membrane and altered transcription of several genes involved in the production of these membrane fatty acids, including fabT and fabM. Furthermore, activity of the F1 F0 -ATPase was increased in the ∆spxA2 strain. Transcription of spxA2 was elevated in the presence of a variety of membrane stressors, and highly dependent on the liaR component of the LiaFSR system, which is known to sense cell envelope stress in many Gram-positive bacteria. Finally, deletion of ∆spxA2 led to altered susceptibility of S. mutans to membrane stressors. Overall, the results of this study indicate that spxA2 serves a crucial role in transmitting the signal of cell wall/membrane damage from the LiaFSR sensor to downstream effectors in the SpxA2 regulon which restore and maintain membrane and cell wall homeostasis.