Alisha G. Campbell

The composition of the human microbiota is recognized as an important factor in human health and disease. Many of our cohabitating microbes belong to phylum-level divisions for which there are no cultivated representatives and are only represented by small subunit rRNA sequences. For one such taxon (SR1), which includes bacteria with elevated abundance in periodontitis, we provide a single-cell genome sequence from a healthy oral sample. SR1 bacteria use a unique genetic code. In-frame TGA (opal) codons are found in most genes (85%), often at loci normally encoding conserved glycine residues. UGA appears not to function as a stop codon and is in equilibrium with the canonical GGN glycine codons, displaying strain-specific variation across the human population. SR1 encodes a divergent tRNA(Gly)UCA with an opal-decoding anticodon. SR1 glycyl-tRNA synthetase acylates tRNA(Gly)UCA with glycine in vitro with similar activity compared with normal tRNA(Gly)UCC. Coexpression of SR1 glycyl-tRNA synthetase and tRNA(Gly)UCA in Escherichia coli yields significant β-galactosidase activity in vivo from a lacZ gene containing an in-frame TGA codon. Comparative genomic analysis with Human Microbiome Project data revealed that the human body harbors a striking diversity of SR1 bacteria. This is a surprising finding because SR1 is most closely related to bacteria that live in anoxic and thermal environments. Some of these bacteria share common genetic and metabolic features with SR1, including UGA to glycine reassignment and an archaeal-type ribulose-1,5-bisphosphate carboxylase (RubisCO) involved in AMP recycling. UGA codon reassignment renders SR1 genes untranslatable by other bacteria, which impacts horizontal gene transfer within the human microbiota.

The mammalian gut harbors complex and variable microbial communities, across both host phylogenetic space and conspecific individuals. A synergy of host genetic and environmental factors shape these communities and account for their variability, but their individual contributions and the selective pressures involved are still not well understood. We employed barcoded pyrosequencing of V1-2 and V4 regions of bacterial small subunit ribosomal RNA genes to characterize the effects of host genetics and environment on cecum assemblages in 10 genetically distinct, inbred mouse strains. Eight of these strains are the foundation of the Collaborative Cross (CC), a panel of mice derived from a genetically diverse set of inbred founder strains, designed specifically for complex trait analysis. Diversity of gut microbiota was characterized by complementing phylogenetic and distance-based, sequence-clustering approaches. Significant correlations were found between the mouse strains and their gut microbiota, reflected by distinct bacterial communities. Cohabitation and litter had a reduced, although detectable effect, and the microbiota response to these factors varied by strain. We identified bacterial phylotypes that appear to be discriminative and strain-specific to each mouse line used. Cohabitation of different strains of mice revealed an interaction of host genetic and environmental factors in shaping gut bacterial consortia, in which bacterial communities became more similar but retained strain specificity. This study provides a baseline analysis of intestinal bacterial communities in the eight CC progenitor strains and will be linked to integrated host genotype, phenotype and microbiota research on the resulting CC panel.

Many microbial phyla that are widely distributed in open environments have few or no representatives within animal-associated microbiota. Among them, the Chloroflexi comprises taxonomically and physiologically diverse lineages adapted to a wide range of aquatic and terrestrial habitats. A distinct group of uncultured chloroflexi related to free-living anaerobic Anaerolineae inhabits the mammalian gastrointestinal tract and includes low-abundance human oral bacteria that appear to proliferate in periodontitis. Using a single-cell genomics approach, we obtained the first draft genomic reconstruction for these organisms and compared their inferred metabolic potential with free-living chloroflexi. Genomic data suggest that oral chloroflexi are anaerobic heterotrophs, encoding abundant carbohydrate transport and metabolism functionalities, similar to those seen in environmental Anaerolineae isolates. The presence of genes for a unique phosphotransferase system and N-acetylglucosamine metabolism suggests an important ecological niche for oral chloroflexi in scavenging material from lysed bacterial cells and the human tissue. The inferred ability to produce sialic acid for cell membrane decoration may enable them to evade the host defence system and colonize the subgingival space. As with other low abundance but persistent members of the microbiota, discerning community and host factors that influence the proliferation of oral chloroflexi may help understand the emergence of oral pathogens and the microbiota dynamics in health and disease states.

The bacterial microbiota of plants is diverse, with 1000s of operational taxonomic units (OTUs) associated with any individual plant. In this work, we used phenotypic analysis, comparative genomics, and metabolic models to investigate the differences between 19 sequenced Pseudomonas fluorescens strains. These isolates represent a single OTU and were collected from the rhizosphere and endosphere of Populus deltoides. While no traits were exclusive to either endosphere or rhizosphere P. fluorescens isolates, multiple pathways relevant for plant-bacterial interactions are enriched in endosphere isolate genomes. Further, growth phenotypes such as phosphate solubilization, protease activity, denitrification and root growth promotion are biased toward endosphere isolates. Endosphere isolates have significantly more metabolic pathways for plant signaling compounds and an increased metabolic range that includes utilization of energy rich nucleotides and sugars, consistent with endosphere colonization. Rhizosphere P. fluorescens have fewer pathways representative of plant-bacterial interactions but show metabolic bias toward chemical substrates often found in root exudates. This work reveals the diverse functions that may contribute to colonization of the endosphere by bacteria and are enriched among closely related isolates.