Project Summary Recent studies suggest that Clostridium scindens is protective against C. difficile infection in vivo. Antibiotic- induced dysbiosis results in altered bile acid profile which is thought to allow C. difficile to grow and cause infection. Prior studies have shown that host bile acids such as taurocholic acid induce C. difficile spore germination in the GI tract; whereas conversion of host bile acids to secondary bile acids such as deoxycholic acid (DCA) by C. scindens prevents C. difficile spore germination and vegetative cell growth. However, a genetic system is currently lacking in C. scindens, representing a barrier to demonstrating causation with respect to DCA formation and inhibition of C. difficile growth and virulence. We have a collection of over a dozen strains of C. scindens strains whose genome sequences are not known. In Aim 1, we propose to sequence the complete genomes of these strains. We have already completed the genome/methylome of C. scindens ATCC 35704T. Determining the ‘pan-genome’ of C. scindens will allow determination of strain variation, and may allow identification of strains more susceptible to genetic manipulation. We will also perform transcriptomic analysis with two C. scindens strains in a newly developed defined medium and identify genes differentially expressed in the presence of bile acids. In Aim 2 we test the hypothesis that the barrier to genetic manipulation of C. scindens is restriction modification (FM) systems. Methylome data has identified extensive m6A modification among conserved sequences in the genome. We will utilize a plasmid artificial modification (PAM) approach to developing a pyrE counter-selectable marker. These studies will provide substantial progress in the understanding of Clostridium scindens biology, bile acid metabolism, and significant progress towards genetic manipulation of C. scindens. This will allow future proposals aimed at testing the hypothesis that DCA formation by C. scindens protects against C. difficile infection in vivo.