Polysaccharide utilization loci and nutritional specialization in a dominant group of butyrate-producing human colonic Firmicutes

Abstract

Firmicutes and Bacteroidetes are the predominant bacterial phyla colonizing the healthy human large intestine. Whilst both ferment dietary fibre, genes responsible for this important activity have been analysed only in the Bacteroidetes, with very little known about the Firmicutes. This work investigates the carbohydrate-active enzymes (CAZymes) in a group of Firmicutes, Roseburia spp. and Eubacterium rectale, which play an important role in producing butyrate from dietary carbohydrates and in health maintenance. Genome sequences of 11 strains representing E. rectale and four Roseburia spp. were analysed for carbohydrate-active genes. Following assembly into a pan-genome, core, variable and unique genes were identified. The 1840 CAZyme genes identified in the pan-genome were assigned to 538 orthologous groups, of which only 26 were present in all strains, indicating considerable inter-strain variability. This analysis was used to categorize the 11 strains into four carbohydrate utilization ecotypes (CUEs), which were shown to correspond to utilization of different carbohydrates for growth. Many glycoside hydrolase genes were found linked to genes encoding oligosaccharide transporters and regulatory elements in the genomes of Roseburia spp. and E. rectale, forming distinct polysaccharide utilization loci (PULs). Whilst PULs are also a common feature in Bacteroidetes, key differences were noted in these Firmicutes, including the absence of close homologues of Bacteroides polysaccharide utilization genes, hence we refer to Gram-positive PULs (gpPULs). Most CAZyme genes in the Roseburia/E. rectale group are organized into gpPULs. Variation in gpPULs can explain the high degree of nutritional specialization at the species level within this group.

Keywords: Carbohydrate; Lachnospiraceae; Roseburia; comparative genomics; gut microbiota; obligate anaerobe.

Fig. 1.

Growth of Roseburia/E. rectale strains on selected carbohydrates in microtitre plates. (a) Heatmap representing the mean maximum OD₆₅₀ obtained by a strain during growth on a specific carbohydrate (OD₆₅₀ 0.0–1.6). Growth was observed on fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), xylo-oligosaccharide (XOS), amylopectin (AP), amylose (A), 1,3–1,4-β-glucan (B-glu), arabinoxylan (AX), type 1 arabinogalactan (AG1) and inulin (I). No growth was observed for any the 11 strains on β-mannan, xyloglucan, type 2 arabinogalactan, mucin core type 2 or mucin core type 3. Data plotted in graphs are the mean ± sd OD₆₅₀ readings of six replicates of strains grown on (b) 0.5 % arabinoxylan or (c) 0.5 % type 1 arabinogalactan. Full growth data are presented in Table S3.

Fig. 2.

Phylogenetic tree of Roseburia/E. rectale GH13s. Gene names are colour-coded based on KEGG GH annotation. Strongly supported clades (bootstrap ≥ 90) are coloured at their most proximal branch. The branch colour corresponds to the KEGG GH annotation of the genes within the clade. Colour coding is as follows: neopullulanase (EC 3.2.1.135) (light blue), cyclomaltodextrinase (EC 3.2.1.54) (orange), α-glucosidase (EC 3.2.1.3) (brown), α-amylase (EC 3.2.1.1) (red), sucrose phosphorase (EC 3.2.1.7) (pink), oligo-1,6-glucosidase (EC 3.2.1.10) (blue), pullulanase (EC 3.2.1.41) (green), glycogen-debranching enzyme (EC 3.2.1.-) (purple) and malto-oligosyltrehalose trehalohydrolase (EC 3.2.1.141) (gold). Bootstrap values, expressed as a percentage of 1000 replications, are given at the branching nodes. This tree is unrooted and reconstructed using the maximum-likelihood method. The scale bar refers to the number of amino acid differences per position. Clades of core GH13s are indicated by asterisks at their most proximal branch.

Fig. 3.

(a) Principal coordinate analysis of Roseburia/E. rectale strains based on complement of GH families and (b) heatmap showing CUEs. Values of a given GH family or carbohydrate set were taken as the number of these genes each genome possessed. In (a), coordinates were calculated using Kendal τ distance applied to the first five eigenvectors. R. intestinalis strains L1-82, M50/1 and XB6B4 (orange); R. inulinivorans strains A2-194 and L1-83 (blue); R. faecis M72/1 and R. hominis A2-183 (green); and E. rectale strains A1-86, T1-815, M104/1 and ATCC33656 (red) form separate clusters (P < 0.001, non-parametric multivariate ANOVA). In (b), CUEs were determined by complete linkage clustering using Kendall τ (as shown) and Spearman distance (not shown). GH53, an endo-1,4-β-galactanase that cleaves the β-1,4-d-galactosidic linkages in type I arabinogalactans, was assigned to the carbohydrate set ‘Type 1 arabinogalactan’ and is excluded from ‘Xylans and Arabinans’, ‘Pectins’ and ‘Alpha- and Beta-galactosides’. CUE1 consists of R. faecis M72/1 and R. hominis A2-183. CUE2 consists of R. intestinalis strains L1-82, M50/1 and XB6B4. CUE3 consists of E. rectale strains A1-86, T1-815, M104/1 and ATCC33656. CUE4 consists of R. inulinivorans strains A2-194 and L1-83. GH assignment to each carbohydrate set is described in Table S6.

Fig. 4.

Schematic representation of gpPULs concerned with xylan and arabinogalactan utilization. Glycoside hydrolase genes are coloured green. Carbohydrate esterase genes are coloured blue. ABC-transporter system component genes are coloured red. Transcriptional regulator genes are coloured yellow. Uncharacterized transporter genes are coloured black. Hypothetical genes are coloured white. Xylose isomerase genes are coloured grey. Two parallel black bars between genes indicate sections that are separated in the genome sequence. Roseburia/E. rectale strains not represented in the diagram lack an orthologous gpPUL. Genes located vertically to each other are orthologues. Solid blue lines between genes are for easy visual comparison of the genes between species and do not represent real gaps in the genome. Locus tags of gpPULs are listed in Table S7.

Fig. 5.

Schematic representation of gpPULs concerned with fructan utilization. GH genes are coloured green. ABC transporter system component genes are coloured red. Transcriptional regulator genes are coloured yellow. Fructokinase genes are coloured purple. Hypothetical genes are coloured white. Any of the 11 Roseburia/E. rectale strains not represented in the diagram lack an orthologous gpPUL. Genes located vertically to each other are orthologues. The diagonal line through the R. faecis gene represents a frameshift mutation. Locus tags of gpPULs are listed in Table S7.

Fig. 6.

Schematic representation of gpPULs concerned with host-derived carbohydrates. GH genes are coloured green. ABC transporter system component genes are coloured red. The polysaccharide lyase gene is coloured bright yellow. Mucin desulphatase genes are coloured gold. Hypothetical genes are coloured white. Two-component signal transduction component genes consisting of a histidine kinase and a response regulator containing a CheY-like receiver domain and an AraC-like DNA-binding domain are coloured navy. Solid blue lines between genes are for easy visual comparison of the genes between species and do not represent real gaps in the genome. Any of the 11 Roseburia/E. rectale strains not represented in the diagram lack an orthologous gpPUL. Locus tags of gpPULs are listed in Table S7.