The ‘<i>in vivo</i> lifestyle’ of bile acid 7α-dehydroxylating bacteria: comparative genomics, metatranscriptomic, and bile acid metabolomics analysis of a defined microbial community in gnotobiotic mice

Abstract

The formation of secondary bile acids by gut microbes is a current topic of considerable biomedical interest. However, a detailed understanding of the biology of anaerobic bacteria in the genus <i>Clostridium</i> that are capable of generating secondary bile acids is lacking. We therefore sought to determine the transcriptional responses of two prominent secondary bile acid producing bacteria, <i>Clostridium hylemonae</i> and <i>Clostridium hiranonis</i> to bile salts (<i>in vitro</i>) and the cecal environment of gnotobiotic mice. The genomes of <i>C. hylemonae</i> DSM 15053 and <i>C. hiranonis</i> DSM 13275 were closed, and found to encode 3,647 genes (3,584 protein-coding) and 2,363 predicted genes (of which 2,239 are protein-coding), respectively, and 1,035 orthologs were shared between <i>C. hylemonae</i> and <i>C. hiranonis</i>. RNA-Seq analysis was performed in growth medium alone, and in the presence of cholic acid (CA) and deoxycholic acid (DCA). Growth with CA resulted in differential expression (>0.58 log₂FC; FDR < 0.05) of 197 genes in <i>C. hiranonis</i> and 118 genes in <i>C. hylemonae</i>. The bile acid-inducible operons (<i>bai</i>) from each organism were highly upregulated in the presence of CA but not DCA. We then colonized germ-free mice with human gut bacterial isolates capable of metabolizing taurine-conjugated bile acids. This consortium included bile salt hydrolase-expressing <i>Bacteroides uniformis</i> ATCC 8492, <i>Bacteroides vulgatus</i> ATCC 8482, <i>Parabacteroides distasonis</i> DSM 20701, as well as taurine-respiring <i>Bilophila wadsworthia</i> DSM 11045, and deoxycholic/lithocholic acid generating <i>Clostridium hylemonae</i> DSM 15053 and <i>Clostridium hiranonis</i> DSM 13275. Butyrate and iso-bile acid-forming <i>Blautia producta</i> ATCC 27340 was also included. The Bacteroidetes made up 84.71% of 16S rDNA cecal reads, <i>B. wadsworthia</i>, constituted 14.7%, and the clostridia made up <.75% of 16S rDNA cecal reads. Bile acid metabolomics of the cecum, serum, and liver indicate that the synthetic community were capable of functional bile salt deconjugation, oxidation/isomerization, and 7α-dehydroxylation of bile acids. Cecal metatranscriptome analysis revealed expression of genes involved in metabolism of taurine-conjugated bile acids. The <i>in vivo</i> transcriptomes of <i>C. hylemonae</i> and <i>C. hiranonis</i> suggest fermentation of simple sugars and utilization of amino acids glycine and proline as electron acceptors. Genes predicted to be involved in trimethylamine (TMA) formation were also expressed.