Comparative genomics of Butyrivibrio and Pseudobutyrivibrio from the rumen: a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Palmerston North, New Zealand

Determining the role of rumen microbes in plant polysaccharide breakdown is fundamental to understanding digestion, and maximising productivity, in ruminant animals. Rumen bacterial species belonging to the genera Butyrivibrio and Pseudobutyrivibrio are important degraders of plant hemicellulose, an abundant heterogeneous, branched polymer, involved in crosslinking cellulose microfibrils to lignin. To investigate their genes required for hemicellulose degradation, the genomes of 40 Butyrivibrio and 6 Pseudobutyrivibrio strains isolated from the plant-adherent microbiome of New Zealand bovine ruminants, were sequenced, and their CAZyme-encoding genes compared. Within the Butyrivibrio and Pseudobutyrivibrio pan-genomes, respectively, there were a total of 4,421 and 441 glycoside hydrolases, as well as 1,283 and 122 carbohydrate esterases with predicted activities involved in the degradation of the insoluble plant polysaccharides such as xylan and pectin. To examine species differences, the genes of the previously characterised bacterium B. proteoclasticus B316 were compared in detail with those from the newly sequenced B. hungatei MB2003. B316 was found to encode a much more developed polysaccharide-degrading repertoire and it was thus hypothesised that B316 would out-compete MB2003 when grown in co-culture on the insoluble hemicellulose substrate, xylan. To test this hypothesis, the two strains were grown on xylan and pectin, either alone in mono-cultures, or in direct competition in a co-culture. The results showed that MB2003 had little ability to utilise xylan or pectin alone, but was capable of significant growth when co-cultured with B316. This indicates a commensalistic interaction between these species, in which B316 initiates the primary attack on the insoluble substrate, while MB2003 has a secondary role, competing for the released soluble sugars. This work provides the first systematic phenotypic, comparative genomic and functional analysis of ruminal Butyrivibrio and Pseudobutyrivibrio species, which not only defines their conserved features involved in hemicellulose degradation, but is also beginning to differentiate their unique gene complements and growth characteristics that separate them as discrete species.