The glycobiome of the rumen bacterium Butyrivibrio proteoclasticus B316(T) highlights adaptation to a polysaccharide-rich environment

Abstract

Determining the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals. Butyrivibrio proteoclasticus B316(T) is a gram-positive, butyrate-forming rumen bacterium with a key role in plant polysaccharide degradation. The 4.4 Mb genome consists of 4 replicons; a chromosome, a chromid and two megaplasmids. The chromid is the smallest reported for all bacteria, and the first identified from the phylum Firmicutes. B316 devotes a large proportion of its genome to the breakdown and reassembly of complex polysaccharides and has a highly developed glycobiome when compared to other sequenced bacteria. The secretion of a range of polysaccharide-degrading enzymes which initiate the breakdown of pectin, starch and xylan, a subtilisin family protease active against plant proteins, and diverse intracellular enzymes to break down oligosaccharides constitute the degradative capability of this organism. A prominent feature of the genome is the presence of multiple gene clusters predicted to be involved in polysaccharide biosynthesis. Metabolic reconstruction reveals the absence of an identifiable gene for enolase, a conserved enzyme of the glycolytic pathway. To our knowledge this is the first report of an organism lacking an enolase. Our analysis of the B316 genome shows how one organism can contribute to the multi-organism complex that rapidly breaks down plant material in the rumen. It can be concluded that B316, and similar organisms with broad polysaccharide-degrading capability, are well suited to being early colonizers and degraders of plant polysaccharides in the rumen environment.

Figure 1. Genome atlas highlighting the glycobiome of Butyrivibrio proteoclasticus B316.

The figure shows the four replicons that make up the B316 genome and the location of the genes predicted to encode proteins involved in polysaccharide degradation and exopolysaccharide biosynthesis. The four rRNA operons on the main chromosome and two on BPc2 are also shown. The colour coding of the genomic features in circle 2 represent different Clusters of Orthologous Groups (COG) categories.

Figure 2. Comparison of bacterial glycobiomes.

Comparative analysis of the glycoside hydrolase and glycosyl transferase complement of B316 against bacterial genomes included in the CAZy database (n = 960).

Figure 3. Overview of carbohydrate metabolism in B. proteoclasticus B316.

The enolase catalysed reaction is shown in red as neither the gene nor the enzyme activity was present in B316. Abbreviations: DHAP, dihydroxyacetone phosphate; DKI, 5-keto-4-deoxyuronate; DKII, 2,5-diketo-3-deoxygluconate; GAP, glyceraldehyde-3-phosphate; 3HB-CoA, 3-hydroxybutanoyl-CoA; KDG, 2-keto-3-deoxygluconate; KDGP, 2-keto-3-deoxygluconate phosphate; PEP, phosphoenolpyruvate.

Figure 4. Enolase from Butyrivibrio and Pseudobutyrivibrio.

A, PCR amplification of the enolase gene from Butyrivibrio and Pseudobutyrivibrio cultures using primers designed from the B. crossotus genome sequence. B, Enolase enzyme activity of Butyrivibrio and Pseudobutyrivibrio cultures.