Human gut microbes use multiple transporters to distinguish vitamin B₁₂ analogs and compete in the gut

Abstract

Genomic and metagenomic sequencing efforts, including human microbiome projects, reveal that microbes often encode multiple systems that appear to accomplish the same task. Whether these predictions reflect actual functional redundancies is unclear. We report that the prominent human gut symbiont Bacteroides thetaiotaomicron employs three functional, homologous vitamin B₁₂ transporters that in at least two cases confer a competitive advantage in the presence of distinct B₁₂ analogs (corrinoids). In the mammalian gut, microbial fitness can be determined by the presence or absence of a single transporter. The total number of distinct corrinoid transporter families in the human gut microbiome likely exceeds those observed in B. thetaiotaomicron by an order of magnitude. These results demonstrate that human gut microbes use elaborate mechanisms to capture and differentiate corrinoids in vivo and that apparent redundancies observed in these genomes can instead reflect hidden specificities that determine whether a microbe will colonize its host.

Figure 1. Widespread redundancies in B₁₂ transport in human gut microbial genomes

(A) Over 80% of sequenced human gut microbial species (260/313) encode B₁₂-dependent genes or riboswitches. (B) Most of these 260 species lack the genes required to synthesize B₁₂ de novo and rely on transport to meet their B₁₂ requirements. Species that encode B₁₂ transport are shown in orange and those that possess partial or complete B₁₂ biosynthetic pathways are marked in green; species with both of these capabilities are designated with a hatched pattern. (C) The majority of human gut Bacteroidetes encode multiple B₁₂ transporters within their genomes. See also Figure S1.

Figure 2. The prominent human gut symbiont B. thetaiotaomicron encodes three functional B₁₂ acquisition systems

(A) Genetic organization of the canonical btuBFCD B₁₂ transporter in E. coli and the three B₁₂ transport systems in B. thetaiotaomicron. (B) Each B. thetaiotaomicron BtuB homolog supports doubling times equivalent to the wildtype strain in conditions that require B₁₂ transport. Mean doubling times and standard deviations of triplicate cultures are shown. Wt, wildtype; Met, methionine. See also Figure S2.

Figure 3. Competition assays reveal functional specificity for predicted redundant transporters in vitro

(A) A B. thetaiotaomicron strain encoding only BtuB1 is dramatically outcompeted in competition with the wildtype parent in conditions in which B₁₂ transport is required for growth, while a strain encoding only BtuB2 shows no competitive defect in these conditions and BtuB3 provides an intermediate phenotype. The slight competitive defect observed in methionine-replete conditions is complemented by expression of the deleted genes in trans. Wildtype, mutant and complemented strains were inoculated at an initial ratio of 1:8:1, passaged by daily 1:1,000 dilution for five days, and strain abundances determined by qPCR. Normalized mean ratios and standard deviations of mutant and complemented strains to the wildtype from triplicate cultures are shown. Asterisks indicate significant differences (t-test, * p < 0.05 of Log10 transformed data). (B) Direct competition of B. thetaiotaomicron strains encoding only BtuB1 or BtuB3 reproduces functional preferences observed in wildtype-mutant competition assays in media with methionine or B₁₂. Corrinoid structure also determines the relative fitness of these strains. Strains were inoculated at an initial ratio of 1:1, passaged, and quantified as above and mean percentages and standard deviations of each strain across triplicate cultures are shown. (C) Molecular structure of vitamin B₁₂ (cyanocobalamin) and the lower ligands of other corrinoids purified and examined in this study; abbreviations describe the corresponding cobamides (Cba) (Allen and Stabler, 2008; Renz, 1999). Dashed lines outline the structural precursor cobinamide (Cbi) and the lower ligand, which varies between corrinoids. See also Figure S3.

Figure 4. Functional specificity of homologous transporters determines microbial fitness in the mammalian gut

Population dynamics of B. thetaiotaomicron wild type (black squares), ΔbtuB2 mutant (red circles), and complemented mutant strains (blue triangles) in gnotobiotic Swiss Webster mice are shown (A) in fecal samples over time (n=10/group) and (B) along the gastrointestinal tract (n=5/group). Strain abundances during in vivo competition between B. thetaiotaomicron strains encoding only BtuB1 (purple squares) or BtuB3 (green squares) in gnotobiotic Swiss Webster mice are shown over time (C) and along the length of the gut (D) (n=9/group). Asterisks indicate significant differences in ΔbtuB2 (A and B) or ΔbtuB2 ΔbtuB3 (C and D) mutant abundances in mice consuming a B₁₂-replete diet (closed symbols and solid lines) compared to mice consuming a B₁₂-deplete diet (open symbols and dashed lines; t-test * p < 0.05, ** p < 0.01). The limit of detection is shown with a dashed grey line; n.s., not significant; n.d., not detected. Genomic DNA was extracted and qPCR performed on the luminal contents of the proximal (Si-1), medial (Si-2) and distal (Si-3) small intestine, the cecum (Ce), colon (Co) and feces (Fc). See also Figure S4.

Figure 5. Human gut microbes likely encode at least 27 distinct corrinoid transporter families

(A) Amino acid sequence identity from >80% of the length aligned between BtuB orthologs from B. thetaiotaomicron (BtuB1-BtuB3), E. coli K-12 (BtuB-Ec), and S. typhimurium LT2 (BtuB-St). (B) Rarefaction analysis on BtuB protein families as defined by a 50% amino acid identity cutoff. The mean number of BtuB families observed as increasing numbers of genomes are sampled is shown as a bold line; standard deviations from 100 permutations are shown with shading. See also Figure S5.

Figure 6. Duplication, diversification and horizontal transfer of corrinoid transporters among human gut microbes

(A) Phylogenetic relationships between human gut microbial species and between BtuB sequences were reconstructed by maximum likelihood. Terminal branches on the BtuB phylogeny have been collapsed and labeled according to their placement into the 27 families (A-AA) predicted by rarefaction analysis. Species distributions of representative BtuB families with evidence of recent horizontal gene transfer are indicated with shaded lines. BtuB sequences present in representative species are shown in black (Bs, B. stercoris; Bc, B. caccae; Ah, Alistipes sp. HGB5; Yb, Yersinia bercovieri; Ck, Citrobacter koseri; Ec, E. coli; St, S. typhimurium) and red (Bt, B. thetaiotaomicron). Internal nodes with bootstrap support ≥ 75% are shown as dark gray circles and colored regions correspond to the indicated bacterial phylum or class. (B) The corrinoid transport system encoded in locus 1 requires proteins encoded in the other corrinoid transport systems for function. B. thetaiotaomicron strains encoding only locus 1 are not viable in conditions requiring B₁₂ transport for growth, while strains encoding locus 1 and btuFCD genes from locus 2 or locus 3 grow at wildtype rates in conditions that require B₁₂ transport. Colors correspond to the key and mean doubling times and standard deviations from triplicate cultures are shown. (C) Predicted routes of corrinoid transport in mutant strains described in (B). In the wildtype strain, B₁₂ can be transported across the outer membrane by any of the three BtuB homologs and cross the inner membrane through either of the two BtuFCD systems. Because locus 1 does not encode its own btuFCD genes, a strain lacking locus 2 and locus 3 is not viable in B₁₂ dependent conditions. The btuFCD genes from locus 2 or locus 3 restore B₁₂-dependent growth to this strain, suggesting that B₁₂ transported through the outer membrane via BtuB1 can enter the cytoplasm via BtuFCD homologs encoded in the other two corrinoid transporter loci. See also Figure S6.

Figure 7. Many genes not previously associated with vitamin B₁₂ are encoded downstream of predicted B₁₂ riboswitches

(A) B₁₂ riboswitches were identified across 313 human gut microbial genomes; B₁₂ related ORFs are listed in Table S3 and include transport, biosynthesis, and dependent proteins and their B₁₂-independent isoenzymes. (B) A large fraction of predicted B₁₂ riboswitch-regulated operons in sequenced human gut microbial species do not contain any genes directly (B₁₂ biosynthesis, transport, or dependency) or indirectly (B₁₂-independent isozymes) related to corrinoid utilization. 882 predicted B₁₂ riboswitch-regulated operons from 234 species are shown.