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Genomic sequencing-based mutational enrichment analysis identifies motility genes in a genetically intractable gut microbe

Significance There is broad interest in the role microbial communities play in human health. Although DNA-sequencing technologies enabled a broad assessment of microbial diversity and genomic content, our understanding of the molecular mechanisms underlying microbe–microbe and microbe–host interactions has proceeded much more slowly because only a small fraction of microbes are amenable to molecular genetic manipulation. We describe a method, independent of recombinant DNA tools, to perform genetic analysis in any cultivatable microbial species. We identified determinants of motility in a member of the vertebrate microbiome, the Firmicutes Exiguobacterium acetylicum, and experimentally determined a role for motility in animal colonization by this previously uncharacterized commensal bacteria that is important for host nutrient homeostasis. A major roadblock to understanding how microbes in the gastrointestinal tract colonize and influence the physiology of their hosts is our inability to genetically manipulate new bacterial species and experimentally assess the function of their genes. We describe the application of population-based genomic sequencing after chemical mutagenesis to map bacterial genes responsible for motility in Exiguobacterium acetylicum, a representative intestinal Firmicutes bacterium that is intractable to molecular genetic manipulation. We derived strong associations between mutations in 57 E. acetylicum genes and impaired motility. Surprisingly, less than half of these genes were annotated as motility-related based on sequence homologies. We confirmed the genetic link between individual mutations and loss of motility for several of these genes by performing a large-scale analysis of spontaneous suppressor mutations. In the process, we reannotated genes belonging to a broad family of diguanylate cyclases and phosphodiesterases to highlight their specific role in motility and assigned functions to uncharacterized genes. Furthermore, we generated isogenic strains that allowed us to establish that Exiguobacterium motility is important for the colonization of its vertebrate host. These results indicate that genetic dissection of a complex trait, functional annotation of new genes, and the generation of mutant strains to define the role of genes in complex environments can be accomplished in bacteria without the development of species-specific molecular genetic tools.