Comparative genomics of the genus Roseburia reveals divergent biosynthetic pathways that may influence colonic competition among species

Abstract

Roseburia species are important denizens of the human gut microbiome that ferment complex polysaccharides to butyrate as a terminal fermentation product, which influences human physiology and serves as an energy source for colonocytes. Previous comparative genomics analyses of the genus Roseburia have examined polysaccharide degradation genes. Here, we characterize the core and pangenomes of the genus Roseburia with respect to central carbon and energy metabolism, as well as biosynthesis of amino acids and B vitamins using orthology-based methods, uncovering significant differences among species in their biosynthetic capacities. Variation in gene content among Roseburia species and strains was most significant for cofactor biosynthesis. Unlike all other species of Roseburia that we analysed, Roseburia inulinivorans strains lacked biosynthetic genes for riboflavin or pantothenate but possessed folate biosynthesis genes. Differences in gene content for B vitamin synthesis were matched with differences in putative salvage and synthesis strategies among species. For example, we observed extended biotin salvage capabilities in R. intestinalis strains, which further suggest that B vitamin acquisition strategies may impact fitness in the gut ecosystem. As differences in the functional potential to synthesize components of biomass (e.g. amino acids, vitamins) can drive interspecies interactions, variation in auxotrophies of the Roseburia spp. genomes may influence in vivo gut ecology. This study serves to advance our understanding of the potential metabolic interactions that influence the ecology of Roseburia spp. and, ultimately, may provide a basis for rational strategies to manipulate the abundances of these species.

Keywords: B vitamin biosynthesis; Lachnospiraceae; Roseburia; amino acid biosynthesis; butyrate synthesis; comparative genomics.

Fig. 1.

Phylogenetic trees of the family Lachnospiraceae . (a) Full-length 16S rRNA gene maximum-likelihood tree from 1000 bootstrap replicates is shown. Genera other than Roseburia were collapsed to simplify visualization of the tree. The fully expanded tree including accession numbers can be found in Fig. S1. (b) The 18-gene concatenated maximum-likelihood tree from 100 bootstrap replicates is shown with collapsed nodes and the fully expanded tree can be found in Fig. S2. (Bootstrap scores >70 are reported). (c) Roseburia core genome(s) displaying the overlapping orthologous protein encoding genes among the species evaluated. (d) Pairwise comparison of orthologies among Roseburia sp. Rows are coloured according to the species and numbers along the diagonal represent the core genome for a given species.

Fig. 2.

Metabolic pathways of various carbohydrate mono-, di-, and poly-saccharides in Roseburia . Each node represents an intermediate compound and each oval represents the metabolic pathway that the metabolite(s) are shuttled to during metabolism. Cofactors are not shown. uxaC, glucuronate isomerase; uxaB, tagaturonate reductase; uxaA, altronate hydrolase; kdgK, 2-dehydro-3-deoxygluconokinase; eda, 2-dehydro-3-deoxyphosphogluconate aldolase/(4S)-4-hydroxy-2-oxoglutarate aldolase; galM, aldose 1-epimerase; galK, galactokinase; galT=UDPglucose-hexose-1-phosphate uridylyltransferase; glf, UDP-galactopyranose mutase, pgm, phosphoglucomutase; glgP, glycogen phosphorylase; amyA, alpha-amylase; malL, oligo-1,6-glucosidase; malZ, alpha-glucosidase; glk, glucokinase; mgp, beta-1,4-mannooligosaccharide phosphorylase; mep, mannobiose 2-epimerase; mp2, 4-O-beta-d-mannosyl-d-glucose phosphorylase; pgm*, bifunctional phosphoglucomutase/phosphomannomutase; mpi, mannose 6-phosphate isomerase; pfkB, 6-phosphofructokinase 2; fruA, PTS fructose-specific enzyme IIABC component; fruK, 1-phosphofructokinase; xylA, xylose isomerase; xylB, xylulokinase; araA, L-arabinose isomerase; araB, L-ribulokinase; araD, L-ribulose-5-phosphate 4-epimerase; rhaA, L-rhamnose isomerase; rhaB, rhamnulokinase; rhaD, rhamnulose-1-phosphate aldolase; tpiA, triosephosphate isomerase; tktA/B, transketolase 1/2; xfp, xylulose-5-phosphate/fructose-6-phosphate phosphoketolase; rpe, ribulose-phosphate 3-epimerase; pgi, glucose-6-phosphate isomerase; pfp, diphosphate-dependent phosphofructokinase; pfkA, 6-phosphofructokinase 1; fbp, fructose-1,6-bisphosphatase I; fbaA, fructose-1,6-bisphosphate aldolase; rpiB, ribose 5-phosphate isomerase B; prsA, ribose-phosphate pyrophosphokinase; mannose₂, mannobiose; glucose-6P, glucose-6 phosphate; fructose-1,6P₂, fructose-6 phosphate; fructose-1,6P₂, fructose-1,6 bisphosphate; glyceraldehyde-3P, glyceraldehyde-3 phosphate; riboulose-5P, ribulose 5-phosphate.

Fig. 3.

Central metabolism of Roseburia species, including the dominant fermentation pathways and electron transferring complexes. Although Roseburia can generate ATP via substrate-level phosphorylation of acetyl-phosphate to acetate, they are net consumers of acetate and thus flux through this pathway is low (indicated by the grey reaction arrows). Instead, Roseburia spp. appear to derive their ATP almost exclusively through ‘oxidative phosphorylation’ via an F-type ATPase via the proton gradient generated by an H⁺-translocating rnf complex that also recycles ferredoxins/flavodoxins. The FeFe group B hydrogenase also regenerates oxidized ferredoxin/flavodoxin while generating H₂ (shown as H₂ formation). Gene symbols: ldhL/ldhD, L-/D-lactate dehydrogenase; porAB, pyruvate ferredoxin oxidoreductase alpha/beta subunit; pflD, formate C-acetyltransferase; pta, phosphate acetyltransferase; ackA, acetate kinase; thl, atoB-like thiolase (acetyl-CoA acetyltransferase); bhbD, β-hydroxyacyl-CoA dehydrogenase; cro, crotonyl-CoA hydratase; bcd, butyryl-CoA dehydrogenase; EtfAB, electron transfer flavoprotein alpha and beta-subunit; butCoAT, butyryl-CoA : acetate-CoA transferase; rnfABCDEG, Na⁺/H⁺-translocating ferredoxin : NAD⁺ oxidoreductase subunits A–G; hyd, FeFe hydrogenase.

Fig. 4.

Histidine biosynthetic pathway and operon structure. The histidine biosynthetic pathway (top) not only synthesizes l-histidine from 5-phospho-d-ribose α-1-pyrophosphate (PRPP), but also the precursor of purine biosynthesis, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Across the bacterial kingdom, the histidine operon structure is paraphyletic, displaying similar organization among closely related species, while more distant ancestors have varying organizations. To demonstrate these similarities, the operon organizations of various representative microbes across different phyla and classes are shown (bottom). Gene names: HisA, phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase; HisBd, imidazoleglycerol-phosphate dehydratase; HisBpx, histidinol-phosphatase; HisC, histidinol-phosphate aminotransferase; HisD, histidinol dehydrogenase; HisF, imidazole glycerol-phosphate synthase cyclase subunit; HisG, ATP phosphoribosyltransferase; HisH, glutamine amidotransferase; HisEI, phosphoribosyl ATP pyrophosphohydrolase/phosphoribosyl-AMP cyclohydrolase; HisZ, ATP phosphoribosyltransferase regulatory subunit. The chromosome number where the gene is located is displayed for eukaryotes. Genes in the other biosynthetic operons are denoted as follows: ▲, tyrosine/phenylalanine biosynthesis; *, tryptophan biosynthesis; ■, riboflavin biosynthesis; ○, glutamine biosynthesis; ♦, cystine biosynthesis

Fig. 5.

Vitamin synthesis and transport in Roseburia species. Rectangles represent biosynthetic pathways and circles represent transporters that were predicted by the species indicated. Each shape is coloured to represents a particular B vitamin that we predict can be synthesized or transported. The ThiT, RibU, PanT, PdxU2, BioY and FolT genes are energy coupling factor (ECF)–type transporters for thiamine, riboflavin, pantothenate, pyridoxine, biotin and folate, respectively, while BtuCDF is an ATP-binding cassette (ABC) transporter with a substrate-specific domain for cobalamin transport.

Fig. 6.

Overview of Roseburia ’s metabolic and biosynthetic capabilities based on this analysis.