Complete Genome Sequence of Human Oral Saccharibacterium "Candidatus Nanosynbacter sp. HMT352" Strain KC1.

"Cand. Nanosynbacter sp. HMT352" strain KC1 is an ectoparasitic saccharibacterium/TM7 that was co-isolated from a human saliva sample with its obligate bacterial host, Schaalia odontolytica. The genome of strain KC1 enables studies of the mechanisms and evolution of interspecies interactions and, for oral species, studies of their potential roles in health and disease.

The microbiome impacts host hybridization and speciation.

Microbial symbiosis and speciation profoundly shape the composition of life's biodiversity. Despite the enormous contributions of these two fields to the foundations of modern biology, there is a vast and exciting frontier ahead for research, literature, and conferences to address the neglected prospects of merging their study. Here, we survey and synthesize exemplar cases of how endosymbionts and microbial communities affect animal hybridization and vice versa. We conclude that though the number of case studies remain nascent, the wide-ranging types of animals, microbes, and isolation barriers impacted by hybridization will likely prove general and a major new phase of study that includes the microbiome as part of the functional whole contributing to reproductive isolation. Though microorganisms were proposed to impact animal speciation a century ago, the weight of the evidence supporting this view has now reached a tipping point.

Living in the endosymbiotic world of Wolbachia: A centennial review.

The most widespread intracellular bacteria in the animal kingdom are maternally inherited endosymbionts of the genus Wolbachia. Their prevalence in arthropods and nematodes worldwide and stunning arsenal of parasitic and mutualistic adaptations make these bacteria a biological archetype for basic studies of symbiosis and applied outcomes for curbing human and agricultural diseases. Here, we conduct a summative, centennial analysis of living in the Wolbachia world. We synthesize literature on Wolbachia's host range, phylogenetic diversity, genomics, cell biology, and applications to filarial, arboviral, and agricultural diseases. We also review the mobilome of Wolbachia including phage WO and its essentiality to hallmark reproductive phenotypes in arthropods. Finally, the Wolbachia system is an exemplar for discovery-based science education using biodiversity, biotechnology, and bioinformatics lessons. As we approach a century of Wolbachia research, the interdisciplinary science of this symbiosis stands as a model for consolidating and teaching the integrative rules of endosymbiotic life.

Genomes of Gut Bacteria from Nasonia Wasps Shed Light on Phylosymbiosis and Microbe-Assisted Hybrid Breakdown.

Phylosymbiosis is a cross-system trend whereby microbial community relationships recapitulate the host phylogeny. In Nasonia parasitoid wasps, phylosymbiosis occurs throughout development, is distinguishable between sexes, and benefits host development and survival. Moreover, the microbiome shifts in hybrids as a rare Proteus bacterium in the microbiome becomes dominant. The larval hybrids then catastrophically succumb to bacterium-assisted lethality and reproductive isolation between the species. Two important questions for understanding phylosymbiosis and bacterium-assisted lethality in hybrids are (i) do the Nasonia bacterial genomes differ from other animal isolates and (ii) are the hybrid bacterial genomes the same as those in the parental species? Here, we report the cultivation, whole-genome sequencing, and comparative analyses of the most abundant gut bacteria in Nasonia larvae, Providencia rettgeri and Proteus mirabilis Characterization of new isolates shows Proteus mirabilis forms a more robust biofilm than Providencia rettgeri and that, when grown in coculture, Proteus mirabilis significantly outcompetes Providencia rettgeri Providencia rettgeri genomes from Nasonia are similar to each other and more divergent from pathogenic, human associates. Proteus mirabilis from Nasonia vitripennis, Nasonia giraulti, and their hybrid offspring are nearly identical and relatively distinct from human isolates. These results indicate that members of the larval gut microbiome within Nasonia are most similar to each other, and the strain of the dominant Proteus mirabilis in hybrids is resident in parental species. Holobiont interactions between shared, resident members of the wasp microbiome and the host underpin phylosymbiosis and hybrid breakdown.IMPORTANCE Animal and plant hosts often establish intimate relationships with their microbiomes. In varied environments, closely related host species share more similar microbiomes, a pattern termed phylosymbiosis. When phylosymbiosis is functionally significant and beneficial, microbial transplants between host species and host hybridization can have detrimental consequences on host biology. In the Nasonia parasitoid wasp genus, which contains a phylosymbiotic gut community, both effects occur and provide evidence for selective pressures on the holobiont. Here, we show that bacterial genomes in Nasonia differ from other environments and harbor genes with unique functions that may regulate phylosymbiotic relationships. Furthermore, the bacteria in hybrids are identical to those in parental species, thus supporting a hologenomic tenet that the same members of the microbiome and the host genome impact phylosymbiosis, hybrid breakdown, and speciation.

Complete Genome Sequence of Human Oral Actinomyces sp. HMT897 Strain ORNL0104, a Host of the Saccharibacterium (TM7) HMT351.

Actinomyces sp. HMT897 strain ORNL0104 was isolated from a human saliva sample and can serve as a host for the ectobiont Saccharibacteria (TM7) HMT351. Its 3.3-Mb chromosome was completely sequenced using PacBio long reads, and it encodes 2,407 proteins and 63 RNAs.

Comparative Genomics Guides Elucidation of Vitamin B12 Biosynthesis in Novel Human-Associated Akkermansia Strains.

Akkermansia muciniphila is a mucin-degrading bacterium found in the gut of most humans and is considered a "next-generation probiotic." However, knowledge of the genomic and physiological diversity of human-associated Akkermansia sp. strains is limited. Here, we reconstructed 35 metagenome-assembled genomes and combined them with 40 publicly available genomes for comparative genomic analysis. We identified at least four species-level phylogroups (AmI to AmIV), with distinct functional potentials. Most notably, we identified genes for cobalamin (vitamin B12) biosynthesis within the AmII and AmIII phylogroups. To verify these predictions, 10 Akkermansia strains were isolated from adults and screened for vitamin B12 biosynthesis genes via PCR. Two AmII strains were positive for the presence of cobalamin biosynthesis genes, while all 9 AmI strains tested were negative. To demonstrate vitamin B12 biosynthesis, we measured the production of acetate, succinate, and propionate in the presence and absence of vitamin supplementation in representative strains of the AmI and AmII phylogroups, since cobalamin is an essential cofactor in propionate metabolism. Results showed that the AmII strain produced acetate and propionate in the absence of supplementation, which is indicative of vitamin B12 biosynthesis. In contrast, acetate and succinate were the main fermentation products for the AmI strains when vitamin B12 was not supplied in the culture medium. Lastly, two bioassays were used to confirm vitamin B12 production by the AmII phylogroup. This novel physiological trait of human-associated Akkermansia strains may affect how these bacteria interact with the human host and other members of the human gut microbiome.IMPORTANCE There is significant interest in the therapeutic and probiotic potential of the common gut bacterium Akkermansia muciniphila However, knowledge of both the genomic and physiological diversity of this bacterial lineage is limited. Using a combination of genomic, molecular biological, and traditional microbiological approaches, we identified at least four species-level phylogroups with differing functional potentials that affect how these bacteria interact with both their human host and other members of the human gut microbiome. Specifically, we identified and isolated Akkermansia strains that were able to synthesize vitamin B12 The ability to synthesize this important cofactor broadens the physiological capabilities of human-associated Akkermansia strains, fundamentally altering our understanding of how this important bacterial lineage may affect human health.

Targeted isolation and cultivation of uncultivated bacteria by reverse genomics.

Most microorganisms from all taxonomic levels are uncultured. Single-cell genomes and metagenomes continue to increase the known diversity of Bacteria and Archaea; however, while 'omics can be used to infer physiological or ecological roles for species in a community, most of these hypothetical roles remain unvalidated. Here, we report an approach to capture specific microorganisms from complex communities into pure cultures using genome-informed antibody engineering. We apply our reverse genomics approach to isolate and sequence single cells and to cultivate three different species-level lineages of human oral Saccharibacteria (TM7). Using our pure cultures, we show that all three Saccharibacteria species are epibionts of diverse Actinobacteria. We also isolate and cultivate human oral SR1 bacteria, which are members of a lineage of previously uncultured bacteria. Reverse-genomics-enabled cultivation of microorganisms can be applied to any species from any environment and has the potential to unlock the isolation, cultivation and characterization of species from as-yet-uncultured branches of the microbial tree of life.

Insights into the Evolution of Host Association through the Isolation and Characterization of a Novel Human Periodontal Pathobiont, Desulfobulbus oralis.

The human oral microbiota encompasses representatives of many bacterial lineages that have not yet been cultured. Here we describe the isolation and characterization of previously uncultured Desulfobulbus oralis, the first human-associated representative of its genus. As mammalian-associated microbes rarely have free-living close relatives, D. oralis provides opportunities to study how bacteria adapt and evolve within a host. This sulfate-reducing deltaproteobacterium has adapted to the human oral subgingival niche by curtailing its physiological repertoire, losing some biosynthetic abilities and metabolic independence, and by dramatically reducing environmental sensing and signaling capabilities. The genes that enable free-living Desulfobulbus to synthesize the potent neurotoxin methylmercury were also lost by D. oralis, a notably positive outcome of host association. However, horizontal gene acquisitions from other members of the microbiota provided novel mechanisms of interaction with the human host, including toxins like leukotoxin and hemolysins. Proteomic and transcriptomic analysis revealed that most of those factors are actively expressed, including in the subgingival environment, and some are secreted. Similar to other known oral pathobionts, D. oralis can trigger a proinflammatory response in oral epithelial cells, suggesting a direct role in the development of periodontal disease.IMPORTANCE Animal-associated microbiota likely assembled as a result of numerous independent colonization events by free-living microbes followed by coevolution with their host and other microbes. Through specific adaptation to various body sites and physiological niches, microbes have a wide range of contributions, from beneficial to disease causing. Desulfobulbus oralis provides insights into genomic and physiological transformations associated with transition from an open environment to a host-dependent lifestyle and the emergence of pathogenicity. Through a multifaceted mechanism triggering a proinflammatory response, D. oralis is a novel periodontal pathobiont. Even though culture-independent approaches can provide insights into the potential role of the human microbiome "dark matter," cultivation and experimental characterization remain important to studying the roles of individual organisms in health and disease.

Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification.

BACKGROUND: Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenic Escherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important component in making biofuels production from lignocellulosic biomass feasible. RESULTS: A butanol dehydrogenase encoding gene from Thermoanaerobacter pseudethanolicus 39E (Teth39_1597), previously shown to have furfural and 5-HMF reducing capabilities, was cloned into a suicide plasmid, pDCW171 and transformed into a lactate dehydrogenase mutant of Caldicellulosiruptor bescii. Integration of the gene into the C. bescii chromosome was verified via PCR amplification and stable expression was observed up to 75°C. Heterologous expression of the NADPH-dependent BdhA enzyme conferred increased resistance of the engineered strain to both furfural and 5-HMF relative to the wild-type and parental strains. Further, when challenged with 15 mM concentrations of either furan aldehyde, the ability to eliminate furfural or 5-HMF from the culture medium was significantly improved in the engineered strain. CONCLUSIONS: A genetically engineered strain of C. bescii (JWCB044) has been constructed that shows both an improved tolerance to furan aldehydes and an improved ability to eliminate furfural and 5-HMF from the culture medium. The work presented here represents the first example of engineering furan aldehyde resistance into a CBP-relevant thermophile and further validates C. bescii as being a genetically tractable microbe of importance for lignocellulosic biofuel production.