Physiological and genomic characteristics of Eubacterium sp. c-25 and their implications on the diversity of deoxycholic acid producers in the human gut [an abstract of entire text]

概要

Bile acids are secreted into the human gut to aid in the digestion of lipids and fat-soluble
vitamins. ..Bile acids are secreted into the human gut to aid in the digestion of lipids and fat-soluble vitamins. A small amount of liver-derived primary bile acids are exposed to the biotransformative actions of the colonic microbiota and result in the formation of secondary bile acids (SBAs) especially through 7α-dehydroxylation, which removes a hydroxy group at the C-7 position of primary bile acids. Deoxycholic acid (DCA) is produced from cholic acid (CA) as the most notable SBA and is known as a regulator of the gut microbiota and has been implicated in diseases such as colonic and hepatic cancer. While several 7α-dehydroxylating bacteria have been identified, they are predominantly members or close relatives of the genus Clostridium and their taxonomic diversity is unknown.
Eubacterium sp. c-25 is a DCA-producing bacteria isolated and briefly studied in the 1980s that is poorly understood in the context of modern-day analytical techniques and knowledge of bile acid transformation. This study aims to characterize the physiology and genome of Eubacterium sp. c-25 against known DCA producers and extrapolate the data to explore the diversity of intestinal DCA producers and identify additional species with the potential to produce 7α-dehydroxylated bile acids.

1. Comparative physiological and genomic characterization of Eubacterium sp. c-25 and
Clostridium scindens
Eubacterium sp. c-25 exhibits a unique chain-like, filamentous morphology that is not found in other DCA producers. In vitro cultures of c-25 showed successful conversion of CA to DCA at a peak rate of ~50%, verifying the strain’s 7α-dehydroxylation ability. It was much lower than reference strains C. scindens G10 and C. scindens ATCC 35704T, however, which were able to rapidly convert 80-90% of CA to DCA in ideal conditions. All three strains xhibited a preference for pH 7 or 8 for maximum growth and DCA production.
The whole genome of c-25 was sequenced and revealed to consist of a 3,042,110 bp circular chromosome containing 2,893 coding sequences. It was found that the c-25 genome contained predicted orthologues of the bile acid-inducible (bai) genes necessary for 7α-dehydroxylation. However, the unusual arrangement of the hypothesized bai genes into multiple clusters differed from the typical bai operon observed in C. scindens and other DCA producers. Several bai genes in c-25, specifically baiB, baiCD, and baiH, were selected for measurement of in vitro expression levels, and it was found that all three were upregulated in the presence of CA substrate, supporting their involvement in 7α-dehydroxylation.

2. Exploration of Eubacterium sp. c-25 phylogeny and identification of additional 7α-dehydroxylating bacteria
The amino acid sequence of BaiB from c-25 was used to search for strains that could share the same bai gene characteristics as c-25 and also produce DCA. Three additional strains were identified possessing genes sharing 74-77% sequence identity: Sporofaciens musculi (obese mouse cecal isolate), Dorea sp. AF36-15AT (human fecal isolate), and Dorea sp. AM58-8 (human fecal isolate). Comparative genomic analyses revealed that these three strains possessed bai genes that were arranged almost identically to c-25. A 16S rDNA phylogenetic tree analysis including these unconfirmed DCA producers suggested that 7α-dehydroxylating bacteria are not restricted to certain evolutionary lineages, but are phylogenetically diverse.
In searching for marker genes that could be reliably indicative of 7α-dehydroxylation ability, it was found that non-DCA producers consistently lacked orthologues of baiE, baiI, and barB. Using BaiE from c-25 and C. scindens G10 as queries, three additional species were detected as potential DCA producers in reference databases: Proteocatella sphenisci (penguin guano isolate), Dorea sp. D27 (human fecal isolate), and Clostridium sp. Marseille-P2538T (human fecal isolate).

In conclusion, this study revealed that Eubacterium sp. c-25 is a phylogenetically unique DCA producer possessing a novel arrangement of bai genes. While experimental evidence of DCA formation in the newly identified, unconfirmed DCA producers is necessary to verify in silico findings, the discovery of c-25 and its shared bai gene arrangement sets a precedent for the plausible existence of other genotypically non-traditional 7α-dehydroxylating bacteria and further implies that DCA producer diversity in the human gut is greater than expected.