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Genomics and metabolomics are widely used to explore specialized metabolite diversity. The Paired Omics Data Platform is a community initiative to systematically document links between metabolome and (meta)genome data, aiding identification of natural product biosynthetic origins and metabolite structures.

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.

Next-generation sequencing studies of saliva and dental plaque from subjects in both healthy and diseased states have identified bacteria belonging to the Rothia genus as ubiquitous members of the oral microbiota. To gain a deeper understanding of molecular mechanisms underlying the chemical ecology of this unexplored group, we applied a genome mining approach that targets functionally important biosynthetic gene clusters (BGCs). All 45 genomes that were mined, representing Rothia mucilaginosa, Rothia dentocariosa, and Rothia aeria, harbored a catechol-siderophore-like BGC. To explore siderophore production further, we grew the previously characterized R. mucilaginosa ATCC 25296 in liquid cultures, amended with glycerol, which led to the identification of the archetype siderophore enterobactin by using tandem liquid chromatography-mass spectrometry (LC-MS/MS), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR) spectroscopy. Normally attributed to pathogenic gut bacteria, R. mucilaginosa is the first commensal oral bacterium found to produce enterobactin. Cocultivation studies including R. mucilaginosa or purified enterobactin revealed that enterobactin reduced growth of certain strains of cariogenic Streptococcus mutans and pathogenic strains of Staphylococcus aureus Commensal oral bacteria were either unaffected, reduced in growth, or induced to grow adjacent to enterobactin-producing R. mucilaginosa or the pure compound. Taken together with Rothia's known capacity to ferment a variety of carbohydrates and amino acids, our findings of enterobactin production add an additional level of explanation to R. mucilaginosa's prevalence in the oral cavity. Enterobactin is the strongest Fe(III) binding siderophore known, and its role in oral health requires further investigation.IMPORTANCE The communication language of the human oral microbiota is vastly underexplored. However, a few studies have shown that specialized small molecules encoded by BGCs have critical roles such as in colonization resistance against pathogens and quorum sensing. Here, by using a genome mining approach in combination with compound screening of growth cultures, we identified that the commensal oral community member R. mucilaginosa harbors a catecholate-siderophore BGC, which is responsible for the biosynthesis of enterobactin. The iron-scavenging role of enterobactin is known to have positive effects on the host's iron pool and negative effects on host immune function; however, its role in oral health remains unexplored. R. mucilaginosa was previously identified as an abundant community member in cystic fibrosis, where bacterial iron cycling plays a major role in virulence development. With respect to iron's broad biological importance, iron-chelating enterobactin may explain R. mucilaginosa's colonization success in both health and disease.

Streptococcus mutans is a common constituent of dental plaque and a major etiologic agent of dental caries (tooth decay). In this study, we elucidated the biosynthetic pathway encoded by muc, a hybrid polyketide synthase and nonribosomal peptide synthetase (PKS/NRPS) biosynthetic gene cluster (BGC), present in a number of globally distributed S. mutans strains. The natural products synthesized by muc included three N-acyl tetramic acid compounds (reutericyclin and two novel analogues) and an unacylated tetramic acid (mutanocyclin). Furthermore, the enzyme encoded by mucF was identified as a novel class of membrane-associated aminoacylases and was responsible for the deacylation of reutericyclin to mutanocyclin. A large number of hypothetical proteins across a broad diversity of bacteria were homologous to MucF, suggesting that this may represent a large family of unexplored acylases. Finally, S. mutans utilized the reutericyclin produced by muc to impair the growth of neighboring oral commensal bacteria. Since S. mutans must be able to out-compete these health-associated organisms to persist in the oral microbiota and cause disease, the competitive advantage conferred by muc suggests that this BGC is likely to be involved in S. mutans ecology and therefore dental plaque dysbiosis and the resulting caries pathogenesis.

Differential abundance analysis is controversial throughout microbiome research. Gold standard approaches require laborious measurements of total microbial load, or absolute number of microorganisms, to accurately determine taxonomic shifts. Therefore, most studies rely on relative abundance data. Here, we demonstrate common pitfalls in comparing relative abundance across samples and identify two solutions that reveal microbial changes without the need to estimate total microbial load. We define the notion of "reference frames", which provide deep intuition about the compositional nature of microbiome data. In an oral time series experiment, reference frames alleviate false positives and produce consistent results on both raw and cell-count normalized data. Furthermore, reference frames identify consistent, differentially abundant microbes previously undetected in two independent published datasets from subjects with atopic dermatitis. These methods allow reassessment of published relative abundance data to reveal reproducible microbial changes from standard sequencing output without the need for new assays.

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 ∼50-100-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.

[This corrects the article DOI: 10.1128/mSystems.00166-16.].

The ultra-small, obligate parasitic epibiont, TM7x, the first and only current member of the long-elusive Saccharibacteria (formerly the TM7 phylum) phylum to be cultivated, was isolated in co-culture with its bacterial host, Actinomyces odontolyticus subspecies actinosynbacter, XH001. Initial phenotypic characterization of the TM7x-associated XH001 co-culture revealed enhanced biofilm formation in the presence of TM7x compared to XH001 as monoculture. Genomic analysis and previously published transcriptomic profiling of XH001 also revealed the presence of a putative AI-2 quorum sensing (QS) operon, which was highly upregulated upon association of TM7x with XH001. This analysis revealed that the most highly induced gene in XH001 was an lsrB ortholog, which encodes a putative periplasmic binding protein for the auto inducer (AI)-2 QS signaling molecule. Further genomic analyses suggested the lsrB operon in XH001 is a putative hybrid AI-2/ribose transport operon as well as the existence of a luxS ortholog, which encodes the AI-2 synthase. In this study, the potential role of AI-2 QS in the epibiotic-parasitic relationship between XH001 and TM7x in the context of biofilm formation was investigated. A genetic system for XH001 was developed to generate lsrB and luxS gene deletion mutants in XH001. Phenotypic characterization demonstrated that deletion mutations in either lsrB or luxS did not affect XH001's growth dynamic, mono-species biofilm formation capability, nor its ability to associate with TM7x. TM7x association with XH001 induced lsrB gene expression in a luxS-dependent manner. Intriguingly, unlike wild type XH001, which displayed significantly increased biofilm formation upon establishing the epibiotic-parasitic relationship with TM7x, XH001ΔlsrB, and XH001ΔluxS mutants failed to achieve enhanced biofilm formation when associated with TM7x. In conclusion, we demonstrated a significant role for AI-2 QS in modulating dual-species biofilm formation when XH001 and TM7x establish their epibiotic-parasitic relationship.

The horseshoe effect is a phenomenon that has long intrigued ecologists. The effect was commonly thought to be an artifact of dimensionality reduction, and multiple techniques were developed to unravel this phenomenon and simplify interpretation. Here, we provide evidence that horseshoes arise as a consequence of distance metrics that saturate-a familiar concept in other fields but new to microbial ecology. This saturation property loses information about community dissimilarity, simply because it cannot discriminate between samples that do not share any common features. The phenomenon illuminates niche differentiation in microbial communities and indicates species turnover along environmental gradients. Here we propose a rationale for the observed horseshoe effect from multiple dimensionality reduction techniques applied to simulations, soil samples, and samples from postmortem mice. An in-depth understanding of this phenomenon allows targeting of niche differentiation patterns from high-level ordination plots, which can guide conventional statistical tools to pinpoint microbial niches along environmental gradients. IMPORTANCE The horseshoe effect is often considered an artifact of dimensionality reduction. We show that this is not true in the case for microbiome data and that, in fact, horseshoes can help analysts discover microbial niches across environments.

Microbes are commonly studied as individual species, but they exist as mixed assemblages in nature. At present, we know very little about the spatial organization of the molecules, including natural products that are produced within these microbial networks. Lichens represent a particularly specialized type of symbiotic microbial assemblage in which the component microorganisms exist together. These composite microbial assemblages are typically comprised of several types of microorganisms representing phylogenetically diverse life forms, including fungi, photosymbionts, bacteria, and other microbes. Here, we employed matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry to characterize the distributions of small molecules within a Peltigera lichen. In order to probe how small molecules are organized and localized within the microbial consortium, analytes were annotated and assigned to their respective producer microorganisms using mass spectrometry-based molecular networking and metagenome sequencing. The spatial analysis of the molecules not only reveals an ordered layering of molecules within the lichen but also supports the compartmentalization of unique functions attributed to various layers. These functions include chemical defense (e.g., antibiotics), light-harvesting functions associated with the cyanobacterial outer layer (e.g., chlorophyll), energy transfer (e.g., sugars) surrounding the sun-exposed cyanobacterial layer, and carbohydrates that may serve a structural or storage function and are observed with higher intensities in the non-sun-exposed areas (e.g., complex carbohydrates). IMPORTANCE Microbial communities have evolved over centuries to live symbiotically. The direct visualization of such communities at the chemical and functional level presents a challenge. Overcoming this challenge may allow one to visualize the spatial distributions of specific molecules involved in symbiosis and to define their functional roles in shaping the community structure. In this study, we examined the diversity of microbial genes and taxa and the presence of biosynthetic gene clusters by metagenomic sequencing and the compartmentalization of organic chemical components within a lichen using mass spectrometry. This approach allowed the identification of chemically distinct sections within this composite organism. Using our multipronged approach, various fungal natural products, not previously reported from lichens, were identified and two different fungal layers were visualized at the chemical level.

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry (MS) techniques are well-suited to high-throughput characterization of NP, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social Molecular Networking (GNPS; http://gnps.ucsd.edu), an open-access knowledge base for community-wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS, crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of 'living data' through continuous reanalysis of deposited data.

Dental caries, one of the most globally widespread infectious diseases, is intimately linked to pH dynamics. In supragingival plaque, after the addition of a carbohydrate source, bacterial metabolism decreases the pH which then subsequently recovers. Molecular mechanisms supporting this important homeostasis are poorly characterized in part due to the fact that there are hundreds of active species in dental plaque. Only a few mechanisms (for example, lactate fermentation, the arginine deiminase system) have been identified and studied in detail. Here, we conducted what is to our knowledge, the first full transcriptome and metabolome analysis of a diverse oral plaque community by using a functionally and taxonomically robust in vitro model system greater than 100 species. Differential gene expression analyses from the complete transcriptome of 14 key community members revealed highly varied regulation of both known and previously unassociated pH-neutralizing pathways as a response to the pH drop. Unique expression and metabolite signatures from 400 detected metabolites were found for each stage along the pH curve suggesting it may be possible to define healthy and diseased states of activity. Importantly, for the maintenance of healthy plaque pH, gene transcription activity of known and previously unrecognized pH-neutralizing pathways was associated with the genera Lactobacillus, Veillonella and Streptococcus during the pH recovery phase. Our in vitro study provides a baseline for defining healthy and disease-like states and highlights the power of moving beyond single and dual species applications to capture key players and their orchestrated metabolic activities within a complex human oral microbiome model.

The candidate phylum TM7 is globally distributed and often associated with human inflammatory mucosal diseases. Despite its prevalence, the TM7 phylum remains recalcitrant to cultivation, making it one of the most enigmatic phyla known. In this study, we cultivated a TM7 phylotype (TM7x) from the human oral cavity. This extremely small coccus (200-300 nm) has a distinctive lifestyle not previously observed in human-associated microbes. It is an obligate epibiont of an Actinomyces odontolyticus strain (XH001) yet also has a parasitic phase, thereby killing its host. This first completed genome (705 kb) for a human-associated TM7 phylotype revealed a complete lack of amino acid biosynthetic capacity. Comparative genomics analyses with uncultivated environmental TM7 assemblies show remarkable conserved gene synteny and only minimal gene loss/gain that may have occurred as TM7x adapted to conditions within the human host. Transcriptomic and metabolomic profiles provided the first indications, to our knowledge, that there is signaling interaction between TM7x and XH001. Furthermore, the induction of TNF-α production in macrophages by XH001 was repressed in the presence of TM7x, suggesting its potential immune suppression ability. Overall, our data provide intriguing insights into the uncultivability, pathogenicity, and unique lifestyle of this previously uncharacterized oral TM7 phylotype.

The human oral cavity provides the perfect portal of entry for viruses and bacteria in the environment to access new hosts. Hence, the oral cavity is one of the most densely populated habitats of the human body containing some 6 billion bacteria and potentially 35 times that many viruses. The role of these viral communities remains unclear; however, many are bacteriophage that may have active roles in shaping the ecology of oral bacterial communities. Other implications for the presence of such vast oral phage communities include accelerating the molecular diversity of their bacterial hosts as both host and phage mutate to gain evolutionary advantages. Additional roles include the acquisitions of new gene functions through lysogenic conversions that may provide selective advantages to host bacteria in response to antibiotics or other types of disturbances, and protection of the human host from invading pathogens by binding to and preventing pathogens from crossing oral mucosal barriers. Recent evidence suggests that phage may be more involved in periodontal diseases than were previously thought, as their compositions in the subgingival crevice in moderate to severe periodontitis are known to be significantly altered. However, it is unclear to what extent they contribute to dysbiosis or the transition of the microbial community into a state promoting oral disease. Bacteriophage communities are distinct in saliva compared to sub- and supragingival areas, suggesting that different oral biogeographic niches have unique phage ecology shaping their bacterial biota. In this review, we summarize what is known about phage communities in the oral cavity, the possible contributions of phage in shaping oral bacterial ecology, and the risks to public health oral phage may pose through their potential to spread antibiotic resistance gene functions to close contacts.

Abstract Background Our knowledge of microbial diversity in the human oral cavity has vastly expanded during the last two decades of research. However, much of what is known about the behavior of oral species to date derives from pure culture approaches and the studies combining several cultivated species, which likely does not fully reflect their function in complex microbial communities. It has been shown in studies with a limited number of cultivated species that early oral biofilm development occurs in a successional manner and that continuous low pH can lead to an enrichment of aciduric species. Observations that in vitro grown plaque biofilm microcosms can maintain similar pH profiles in response to carbohydrate addition as plaque in vivo suggests a complex microbial community can be established in the laboratory. In light of this, our primary goal was to develop a robust in vitro biofilm-model system from a pooled saliva inoculum in order to study the stability, reproducibility, and development of the oral microbiome, and its dynamic response to environmental changes from the community to the molecular level. Results Comparative metagenomic analyses confirmed a high similarity of metabolic potential in biofilms to recently available oral metagenomes from healthy subjects as part of the Human Microbiome Project. A time-series metagenomic analysis of the taxonomic community composition in biofilms revealed that the proportions of major species at 3 hours of growth are maintained during 48 hours of biofilm development. By employing deep pyrosequencing of the 16S rRNA gene to investigate this biofilm model with regards to bacterial taxonomic diversity, we show a high reproducibility of the taxonomic carriage and proportions between: 1) individual biofilm samples; 2) biofilm batches grown at different dates; 3) DNA extraction techniques and 4) research laboratories. Conclusions Our study demonstrates that we now have the capability to grow stable oral microbial in vitro biofilms containing more than one hundred operational taxonomic units (OTU) which represent 60-80% of the original inoculum OTU richness. Previously uncultivated Human Oral Taxa (HOT) were identified in the biofilms and contributed to approximately one-third of the totally captured 16S rRNA gene diversity. To our knowledge, this represents the highest oral bacterial diversity reported for an in vitro model system so far. This robust model will help investigate currently uncultivated species and the known virulence properties for many oral pathogens not solely restricted to pure culture systems, but within multi-species biofilms.

In July of 2006 and January of 2008, a total of 671 marine sediment samples were collected at depths from 5 to 2012 m throughout the Fijian islands and selectively processed for the cultivation of marine actinomycetes belonging to the genus Salinispora. The primary objectives were to assess the diversity, distribution and phylogeny of 'S. pacifica', the least well studied of the three species in the genus. Employing a sequential screening method based on antibiotic sensitivity, RFLP patterns, and 16S rRNA and ITS sequence analyses, 42 of 750 isolates with Salinispora-like features were identified as 'S. pacifica'. These strains represent the first report of 'S. pacifica' from Fiji and include 15 representatives of 4 new 'S. pacifica' 16S rRNA sequence types. Among the 'S. pacifica' strains isolated, little evidence for geographical isolation emerged based on 16S, ITS or secondary metabolite biosynthetic gene fingerprinting. The inclusion of isolates from additional collection sites and other Salinispora spp. revealed a high degree of dispersal among 'S. pacifica' populations and phylogenetic support for the delineation of this lineage as a third species.

Polar bears (Ursus maritimus), Eurasian lynx (Lynx lynx) and snow leopards (Panthera uncia) are elusive large carnivores inhabiting snow-covered and remote areas. Their effective conservation and management are challenged by inadequate population information, necessitating development of novel data collection methods. Environmental DNA (eDNA) from snow tracks (footprints in snow) has identified species based on mitochondrial DNA, yet its utility for individual-based analyses remains unsolved due to challenges accessing the nuclear genome. We present a protocol for capturing nuclear eDNA from polar bear, Eurasian lynx and snow leopard snow tracks and verify it through genotyping at a selection of microsatellite markers. We successfully retrieved nuclear eDNA from 87.5% (21/24) of wild polar bear snow tracks, 59.1% (26/44) of wild Eurasian lynx snow tracks, and the single snow leopard sampled. We genotyped over half of all wild polar bear samples (54.2%, 13/24) at five loci, and 11% (9/44) of wild lynx samples and the snow leopard at three loci. Genotyping success from Eurasian lynx snow tracks increased to 24% when tracks were collected by trained rather than untrained personnel. Thirteen wild polar bear samples comprised 11 unique genotypes and two identical genotypes; likely representing 12 individual bears, one of which was sampled twice. Snow tracks show promise for use alongside other non-invasive and conventional methods as a reliable source of nuclear DNA for genetic mark-recapture of elusive and threatened mammals. The detailed protocol we present has utility for broadening end user groups and engaging Indigenous and local communities in species monitoring. [ABSTRACT FROM AUTHOR]

A combination of bromodeoxyuridine (BrdU) immunocapture and terminal restriction fragment length polymorphism (T-RFLP) fingerprinting was used to assess the composition of growing bacteria in Baltic Sea sediments. It was observed that the bacterial and archaeal communities responded differently to pollutant levels.