Comparative Genomic and Physiological Analysis against Clostridium scindens Reveals Eubacterium sp. c-25 as an Atypical Deoxycholic Acid Producer of the Human Gut Microbiota.
ABSTRACT: The human gut houses bile acid 7α-dehydroxylating bacteria that produce secondary bile acids such as deoxycholic acid (DCA) from host-derived bile acids through enzymes encoded by the bai operon. While recent metagenomic studies suggest that these bacteria are highly diverse and abundant, very few DCA producers have been identified. Here, we investigated the physiology and determined the complete genome sequence of Eubacterium sp. c-25, a DCA producer that was isolated from human feces in the 1980s. Culture experiments showed a preference for neutral to slightly alkaline pH in both growth and DCA production. Genomic analyses revealed that c-25 is phylogenetically distinct from known DCA producers and possesses a multi-cluster arrangement of predicted bile-acid inducible (bai) genes that is considerably different from the typical bai operon structure. This arrangement is also found in other intestinal bacterial species, possibly indicative of unconfirmed 7α-dehydroxylation capabilities. Functionality of the predicted bai genes was supported by the induced expression of baiB, baiCD, and baiH in the presence of cholic acid substrate. Taken together, Eubacterium sp. c-25 is an atypical DCA producer with a novel bai gene cluster structure that may represent an unexplored genotype of DCA producers in the human gut.
Project description:Clostridium sp. strain TO-931 can rapidly convert the primary bile acid cholic acid to a potentially toxic compound, deoxycholic acid. Mixed oligonucleotide probes were used to isolate a gene fragment encoding a putative bile acid transporter from Clostridium sp. strain TO-931. This DNA fragment had 60% nucleotide sequence identity to a known bile acid transporter gene from Eubacterium sp. strain VPI 12708, another bile acid-7alpha-dehydroxylating intestinal bacterium. The DNA (9.15 kb) surrounding the transporter gene was cloned from Clostridium sp. strain TO-931 and sequenced. Within this larger DNA fragment was a 7.9-kb region, containing six successive open reading frames (ORFs), that was encoded by a single 8.1-kb transcript, as determined by Northern blot analysis. The gene arrangement and DNA sequence of the Clostridium sp. strain TO-931 operon are similar to those of a Eubacterium sp. strain VPI 12708 bile acid-inducible operon containing nine ORFs. Several genes in the Eubacterium sp. strain VPI 12708 operon have been shown to encode products required for bile acid 7alpha-dehydroxylation. In Clostridium sp. strain TO-931, genes potentially encoding bile acid-coenzyme A (CoA) ligase, 3alpha-hydroxysteroid dehydrogenase, bile acid 7alpha-dehydratase, bile acid-CoA hydrolase, and a bile acid transporter were similar in size and exhibited amino acid homology to similar gene products from Eubacterium sp. strain VPI 12708 (encoded by baiB, baiA, baiE, baiF, and baiG, respectively). However, no genes similar to Eubacterium sp. strain VPI 12708 biaH or baiI were found in the Clostridium sp. strain TO-931 bai operon, and the two putative Eubacterium sp. strain VPI 12708 genes, baiC and baiD, were arranged in one continuous ORF in Clostridium sp. strain TO-931. Intergene regions showed no significant DNA sequence similarity, but primer extension analysis identified a region 115 bp upstream from the first ORF that exhibited 58% identity to a bai operator/promoter region identified in Eubacterium sp. strain VPI 12708. These results indicate that the gene organization, gene product amino acid sequences, and promoters of the bile acid-inducible operons of Clostridium sp. strain TO-931 and Eubacterium sp. strain VPI 12708 are highly conserved.
Project description:Secondary bile acids, formed by intestinal bacteria, are suggested to play a significant role in cancers of the gastrointestinal tract in humans. Bile acid 7alpha/beta-dehydroxylation is carried out by a few species of intestinal clostridia which harbor a multi-gene bile acid inducible (bai) operon. Several genes encoding enzymes in this pathway have been cloned and characterized. However, no gene product(s) has yet been assigned to the production of 3-oxo-Delta4-cholenoic acid intermediates of cholic acid (CA), chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA). We previously reported that the baiH gene encodes an NADH:flavin oxidoreductase (NADH:FOR); however, the role of this protein in bile acid 7-dehydroxylation is unclear. Homology searches and secondary structural alignments suggest this protein to be similar to flavoproteins which reduce alpha/beta-unsaturated carbonyl compounds. The baiH gene product was expressed in Escherichia coli, purified and discovered to be a stereo-specific NAD(H)-dependent 7beta-hydroxy-3-oxo-Delta4-cholenoic acid oxidoreductase. Additionally, high sequence similarity between the baiH and baiCD gene products suggests the baiCD gene may encode a 3-oxo-Delta4-cholenoic acid oxidoreductase specific for CDCA and CA. We tested this hypothesis using cell extracts prepared from E. coli overexpressing the baiCD gene and discovered that it encodes a stereo-specific NAD(H)-dependent 7alpha-hydroxy-3-oxo-Delta4-cholenoic acid oxidoreductase.
Project description:<h4>Background</h4>Clostridium difficile infection (CDI) is a major cause of hospital-acquired diarrhea. Secondary bile acids were shown to confer resistance to colonization by C. difficile. 7?-dehydroxylation is a key step in transformation of primary to secondary bile acids and required genes have been located in a single bile acid-inducible (bai) operon in C. scindens as well as in C. hiranonis, two Clostridium sp. recently reported to protect against C. difficile colonization.<h4>Aim</h4>To analyze baiCD gene abundance in C. difficile positive and negative fecal samples.<h4>Material & methods</h4>A species-specific qPCR for detecting baiCD genes was established. Fecal samples of patients with CDI, asymptomatic toxigenic C. difficile colonization (TCD), non-toxigenic C. difficile colonization (NTCD), of C. difficile negative (NC) patients, and of two patients before and after fecal microbiota transplantation (FMT) for recurrent CDI (rCDI) were tested for the presence of the baiCD genes.<h4>Results</h4>The prevalence of the baiCD gene cluster was significantly higher in C. difficile negative fecal samples than in samples of patients diagnosed with CDI (72.5% (100/138) vs. 35.9% (23/64; p<0.0001). No differences in baiCD gene cluster prevalence were seen between NC and NTCD or NC and TCD samples. Both rCDI patients were baiCD-negative at baseline, but one of the two patients turned positive after successful FMT from a baiCD-positive donor.<h4>Conclusion</h4>Fecal samples of CDI patients are less frequently baiCD-positive than samples from asymptomatic carriers or C. difficile-negative individuals. Furthermore, we present a case of baiCD positivity observed after successful FMT for rCDI.
Project description:Eubacterium sp. strain VPI 12708 expresses inducible bile acid 7alpha-dehydroxylation activity via a multistep pathway. The genes encoding several of the inducible proteins involved in the pathway have been previously mapped to a bile acid-inducible (bai) operon in Eubacterium sp. strain VPI 12708. We now report the cloning, sequencing, and characterization of the baiG gene, which is part of the bai operon. The predicted amino acid sequence of the BaiG polypeptide shows significant homology to several membrane transport proteins, including sugar and antibiotic resistance transporters, which are members of the major facilitator superfamily. Hydrophilicity plots of BaiG show a high degree of similarity to class K and L TetA proteins from gram-positive bacteria, and, like these classes of TetA proteins, BaiG has 14 proposed transmembrane domains. The baiG gene was cloned into Escherichia coli and shown to confer an energy-dependent bile acid uptake activity. Primary bile acids were preferentially transported into E. coli cells expressing this gene, with at least sevenfold and fourfold increases in the uptake of cholic acid and chenodeoxycholic acid, respectively, over control reactions. Less transport activity was observed with cholylglycine, 7-oxocholic acid, and deoxycholic acid. The transport activity was inhibited by the proton ionophores carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, and nigericin but not by the potassium ionophore valinomycin, suggesting that the transport is driven by the proton motive force across the cell membrane. In summary, we have cloned, sequenced, and expressed a bile acid-inducible bile acid transporter from Eubacterium sp. strain VPI 12708. To our knowledge, this is the first report of the cloning and expression of a gene encoding a procaryotic bile acid transporter.
Project description:Clostridioides difficile is one of the leading causes of antibiotic-associated diarrhea. Gut microbiota-derived secondary bile acids and commensal Clostridia that carry the bile acid-inducible (bai) operon are associated with protection from C. difficile infection (CDI), although the mechanism is not known. In this study, we hypothesized that commensal Clostridia are important for providing colonization resistance against C. difficile due to their ability to produce secondary bile acids, as well as potentially competing against C. difficile for similar nutrients. To test this hypothesis, we examined the abilities of four commensal Clostridia carrying the bai operon (Clostridium scindens VPI 12708, C. scindens ATCC 35704, Clostridium hiranonis, and Clostridium hylemonae) to convert cholate (CA) to deoxycholate (DCA) in vitro, and we determined whether the amount of DCA produced was sufficient to inhibit the growth of a clinically relevant C. difficile strain. We also investigated the competitive relationships between these commensals and C. difficile using an in vitro coculture system. We found that inhibition of C. difficile growth by commensal Clostridia supplemented with CA was strain dependent, correlated with the production of ?2?mM DCA, and increased the expression of bai operon genes. We also found that C. difficile was able to outcompete all four commensal Clostridia in an in vitro coculture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics. Future studies dissecting the regulation of the bai operon in vitro and in vivo and how this affects CDI will be important.IMPORTANCE Commensal Clostridia carrying the bai operon, such as C. scindens, have been associated with protection against CDI; however, the mechanism for this protection is unknown. Herein, we show four commensal Clostridia that carry the bai operon and affect C. difficile growth in a strain-dependent manner, with and without the addition of cholate. Inhibition of C. difficile by commensals correlated with the efficient conversion of cholate to deoxycholate, a secondary bile acid that inhibits C. difficile germination, growth, and toxin production. Competition studies also revealed that C. difficile was able to outcompete the commensals in an in vitro coculture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics.
Project description:The secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), formed by gut microbiota from primary bile acids via a multi-step 7?-dehydroxylation reaction, have wide-ranging effects on host metabolism and play an important role in health and disease. A few 7?-dehydroxylating strains have been isolated, where bile acid-inducible (bai) genes were organized in a gene cluster and encoded major enzymes involved. However, only little is known on diversity and abundance of intestinal bacteria catalysing DCA/LCA formation in the human gut in situ. In this study, we took the opportunity to screen metagenome-assembled genomes (MAGs) from sequence data of stool samples provided by two recent studies along with newly available gut-derived isolates for the presence of the bai gene cluster. We revealed in total 765 and 620 MAGs encoding the potential to form DCA/LCA that grouped into 21 and 26 metagenomic species, respectively. The majority of MAGs (92.4 and 90.3%) were associated with a Ruminococcaceae clade that still lacks an isolate, whereas less MAGs belonged to Lachnospiraceae along with eight new isolates (n total?=?11) that contained the bai genes. Only a few MAGs were linked to Peptostreptococcaceae. Signatures for horizontal transfer of bai genes were observed. This study gives a comprehensive overview of the diversity of bai-exhibiting bacteria in the human gut highlighting the application of metagenomics to unravel potential functions hidden from current isolates. Eventually, isolates of the identified main MAG clade are required in order to prove their capability of 7?-dehydroxylating primary bile acids.
Project description:Primary bile acids are synthesized from cholesterol in the liver, conjugated to either glycine or taurine and secreted into bile. Bile salts undergo enterohepatic circulation several times each day. During this process, they are biotransformed into a variety of metabolites by gut bacteria. The major biotransformation is the 7alpha-dehydroxylation of cholic acid and chenodeoxycholic acid yielding deoxycholic acid and lithocholic acid, respectively. 7alpha-Dehydroxylation is a multi-step pathway. The genes encoding enzymes in this pathway have been identified in two species of "high" activity strains of clostridia. Here, we report the isolation and characterization of a bile acid inducible (bai) operon in Clostridium hylemonae, a "low" activity 7alpha-dehydroxylating strain. The gene organization and sequence of the baiBCDEFGHI operon was highly conserved between C. hylemonae and "high" activity strains. Surprisingly, the baiA gene was missing from the bai operon of C. hylemonae. The baiA gene was isolated using PCR and degenerate oligonucleotide primers. The mRNA start site for the large bai operon was determined and shown to be only 11bp from the initiation codon of the first gene. It was also discovered that allocholic acid (5alpha) induced the bai operon and stimulated the conversion of [24-(14)C] cholic acid to [24-(14)C] allodeoxycholic acid in cultures of C. scindens and C. hylemonae allodeoxycholic acid. Finally, it was discovered that the addition of testosterone to the growth medium markedly increased 7alpha-dehydroxylation of cholic acid in Clostridium scindens and C. hylemonae. We hypothesize that testosterone may be a gratuitous inducer of genes involved in the reductive arm of the bile acid 7alpha-dehydroxylation pathway.
Project description: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 log<sub>2</sub>FC; 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.
Project description:The human bile acid pool composition is composed of both primary bile acids (cholic acid and chenodeoxycholic acid) and secondary bile acids (deoxycholic acid and lithocholic acid). Secondary bile acids are formed by the 7?-dehydroxylation of primary bile acids carried out by intestinal anaerobic bacteria. We have previously described a multistep biochemical pathway in Clostridium scindens that is responsible for bile acid 7?-dehydroxylation. We have identified a large (12 kb) bile acid inducible (bai) operon in this bacterium that encodes eight genes involved in bile acid 7?-dehydroxylation. However, the function of the baiF gene product in this operon has not been elucidated. In the current study, we cloned and expressed the baiF gene in E. coli and discovered it has bile acid CoA transferase activity. In addition, we discovered a second bai operon encoding three genes. The baiK gene in this operon was expressed in E. coli and found to encode a second bile acid CoA transferase. Both bile acid CoA transferases were determined to be members of the type III family by amino acid sequence comparisons. Both bile acid CoA transferases had broad substrate specificity, except the baiK gene product, which failed to use lithocholyl-CoA as a CoA donor. Primary bile acids are ligated to CoA via an ATP-dependent mechanism during the initial steps of 7?-dehydroxylation. The bile acid CoA transferases conserve the thioester bond energy, saving the cell ATP molecules during bile acid 7?-dehydroxylation. ATP-dependent CoA ligation is likely quickly supplanted by ATP-independent CoA transfer.
Project description:Interaction between the dietary fiber and the gut microbes can regulate host bile acid metabolism. This study sought to explore the effects of guar gum combined with pregelatinized waxy maize starch (GCW) in a gestation diet on reproductive performance, gut microbiota composition, and bile acid homeostasis of sows. A total of 61 large white sows were randomly grouped into the control (<i>n</i> = 33) and 2% GCW (<i>n</i> = 28) groups during gestation. GCW diet increased birth-weight of piglets, and decreased the percentage of intrauterine growth restriction (IUGR) piglets. In addition, dietary GCW reduced gut microbial diversity and modulated gut microbial composition in sows on day 109 of gestation. The relative abundance of bile salt hydrolase (BSH) gene-encoding bacteria, <i>Lactobacillus</i> and <i>Bacteroides</i> decreased after GCW administration, whereas no significant difference was observed in the fecal level of total glycine-conjugated and taurine-conjugated bile acids between the two groups. Dietary GCW increased the relative abundance of <i>Ruminococcaceae</i> (one of few taxa comprising 7α-dehydroxylating bacteria), which was associated with elevated fecal deoxycholic acid (DCA) in the GCW group. GCW administration lowered the concentrations of plasma total bile acid (TBA) and 7α-hydroxy-4-cholesten-3-one (C4) (reflecting lower hepatic bile acid synthesis) at day 90 and day 109 of gestation compared with the control diet. Furthermore, the levels of plasma glycoursodeoxycholic acid (GUDCA), tauroursodeoxycholic acid (TUDCA) and glycohyocholic acid (GHCA) were lower in the GCW group compared with the control group. Spearman correlation analysis showed alterations in the composition of the gut microbiota by GCW treatment was associated with improved bile acid homeostasis and reproductive performance of sows. In conclusion, GCW-induced improves bile acid homeostasis during gestation which may enhance reproductive performance of sows.