Metabolic niche of a prominent sulfate-reducing human gut bacterium

Abstract

Sulfate-reducing bacteria (SRB) colonize the guts of ∼50% of humans. We used genome-wide transposon mutagenesis and insertion-site sequencing, RNA-Seq, plus mass spectrometry to characterize genetic and environmental factors that impact the niche of Desulfovibrio piger, the most common SRB in a surveyed cohort of healthy US adults. Gnotobiotic mice were colonized with an assemblage of sequenced human gut bacterial species with or without D. piger and fed diets with different levels and types of carbohydrates and sulfur sources. Diet was a major determinant of functions expressed by this artificial nine-member community and of the genes that impact D. piger fitness; the latter includes high- and low-affinity systems for using ammonia, a limiting resource for D. piger in mice consuming a polysaccharide-rich diet. Although genes involved in hydrogen consumption and sulfate reduction are necessary for its colonization, varying dietary-free sulfate levels did not significantly alter levels of D. piger, which can obtain sulfate from the host in part via cross-feeding mediated by Bacteroides-encoded sulfatases. Chondroitin sulfate, a common dietary supplement, increased D. piger and H2S levels without compromising gut barrier integrity. A chondroitin sulfate-supplemented diet together with D. piger impacted the assemblage's substrate utilization preferences, allowing consumption of more reduced carbon sources and increasing the abundance of the H2-producing Actinobacterium, Collinsella aerofaciens. Our findings provide genetic and metabolic details of how this H2-consuming SRB shapes the responses of a microbiota to diet ingredients and a framework for examining how individuals lacking D. piger differ from those who harbor it.

Keywords: artificial human gut microbiota/microbiome; determinants of microbial fitness; hydrogen sulfide; hydrogenotrophs; microbial foodwebs.

Fig. 1.

Genetic identification of determinants important for D. piger acquisition of nitrogen and sulfate in the gut. (A) Venn diagram of the number of D. piger fitness determinants identified by INSeq analysis of fecal microbiota obtained 7 d after introduction of the D. piger mutant library into mice harboring the eight-member consortium of human gut bacterial species and fed the LF/HPP or HF/HS diets (threshold criteria for significant fitness effect; output–input ratio of mutant strain <0.3; p_adj < 0.05) (n = 4 mice/diet). (B) Ammonia assimilation genes identified by INSeq analysis of fecal and cecal microbiota whose fitness effects exhibit diet specificity as well as specificity for cecal versus fecal microbiota. The representation of the indicated mutant locus in the output population is compared with representation in the input library: *P_adj < 0.05; **P_adj < 0.001 (negative binomial test from DESeq package; SI Methods). Significance of the difference observed in fecal samples obtained from mice on the LF/HPP versus HF/HS diets; #, P_adj < 0.001. See C for color code. (C) Ammonia levels in fecal and cecal samples collected from mice colonized with the nine-member artificial community containing wild-type D. piger and fed the LF/HPP versus HF/HS diets. Mean values ± SEM are plotted. *P < 0.05 (Student t test). (D) In vitro test of sulfate cross-feeding. Left y-axis plots D. piger growth (final OD₆₀₀) in filter-sterilized medium harvested from cultures of the B. thetaiotaomicron sulfatase maturation mutant (Δbt0238) or the isogenic wild-type (WT) strain after their growth in minimal medium with chondroitin sulfate or fructose. H₂S levels during D. piger growth in B. thetaiotaomicron–conditioned medium are plotted on the right y-axis. Mean values ± SEM are shown (n = 3 replicates per condition; representative of two independent experiments). (E) Quantitative PCR analysis of D. piger levels in the feces of mice co-colonized with either wild-type or Δbt0238 B. thetaiotaomicron. Mean values ± SEM are plotted (n = 3–5 mice/time point/group). *P < 0.05 (Student t test).

Fig. 2.

Impact of D. piger on the artificial human gut microbiota and host. (A) Bacterial species from the eight-member artificial community that showed significant changes in abundance in the fecal microbiota when D. piger was present versus absent. Mice (n = 19–20/treatment group; three independent experiments) were fed the HF/HS diet supplemented with 3% chondroitin sulfate; *P < 0.05 (Mann–Whitney test). (B) GC-MS and UPLC-MS (*) analysis of cecal contents from the mice described in A. Metabolites that were significantly changed when D. piger was present in mice consuming the HF/HS diet supplemented with chondroitin sulfate are listed. Normalized MS peak areas were mean centered and unit variance scaled. Scores ± SEM are plotted (P < 0.05, Student t test). (C) Microbial RNA-Seq analysis of the fecal metatranscriptome in response to colonization with D. piger. The heat map shows selected ECs encoded by mRNA that were differentially represented between the two conditions [fold-change <–2 or >2; P < 0.01, posterior probability of differential expression (PPDE) > 0.95]. Each column represents a different mouse in the indicated treatment group sampled 14 d after colonization. The maximal relative expression across a row is red; the minimum is green. (D and E) Targeted GC-MS analysis of cecal short chain fatty acid and H₂S levels [n = 19–20 mice; mean values ± SEM are plotted; *P < 0.05 (Student t test)].