Project Summary The composition of microorganisms that reside in the human intestine (gut microbiota) has a significant impact on human health. Despite recent advances in understanding the role of the gut microbiota in health, there remains a need for effective mechanisms of manipulating the gut microbiota to treat and prevent disease. One potential target for manipulation of the gut community is the corrinoid family of cofactors (vitamin B12 and related cobalt-containing modified tetrapyrroles), which are required by the majority of bacteria in the gut, though they are produced only by a fraction of the population. A variety of structural forms of corrinoids exist that are not functionally equivalent, yet little is known about the link between bacterial genome sequences and adaptations for the use of particular corrinoids. The goal of this proposal is to determine the relationship between genome sequence and corrinoid preferences in gut microbes. This goal will be accomplished by examining bacterial corrinoid preferences at the molecular, organismal, and community levels. Corrinoid specificity of homologs of a model corrinoid-dependent enzyme will be dissected by a combination of biochemical, genetic, and bioinformatic approaches. In parallel, corrinoid specificity of riboswitch RNAs, which are present in the majority of genomes in the human microbiome, will be examined by using an in vitro fluorescence-based binding assay and in vivo reporter assays. These data will be used to predict sequence signatures of corrinoid specificity and test these predictions by targeted mutagenesis. Additionally, corrinoid preferences will be examined in cultured human gut bacteria by measuring growth under corrinoid-requiring conditions using a high-throughput robotics platform. This work will lead to an improved ability to interpret genome sequence information and provide a detailed characterization of metabolic processes critical to the majority of human gut microbes. The results will promote the development of novel strategies to prevent and treat diseases associated with the gut microbiota.