Investigating the metabolic function of the rumen microbiome

Abstract

The enormity of bacterial diversity is such that taxonomy is necessary for classification. Taxonomy has historically used an array of different classification techniques, from GC content, to morphology, biochemical capability, and more recently,16S rDNA and whole genome analyses. Currently, there is no standard approach to placing a bacterium taxonomically, leading to a large degree of subjectivity.

Average nucleotide identity (ANI) analyses of 71 culture-based genomes and 64
metagenome-assembled genomes revealed vast genetic diversity, with the genomes clustering into 59 genera and 75 species. Where these species formed groups of four or more genomes, pangenomic analyses were carried out on both these groups, and the original taxa they had been assigned to (Butyrivibrio fibrisolvens, Butyrivibrio proteoclasticus, Butyrivibrio hungatei, Pseudobutyrivibrio ruminis, Pseudobutyrivibrio xylanivorans, and an additional unnamed Butyrivibrio sp group). Core genes only formed 0.66-8.50% of the genomes of the original taxa, revealing a wealth of accessory genes. In contrast, the core genes for the groups formed by ANIm analysis composed 38.48-76.67% of the genomes. Many of the accessory genes were identified as carbohydrate active enzymes, belonging to glycosyl hydrolase families 2, 3, 5, 13, and 43. Cloning and expression of a selection of these genes was attempted, but success was limited due to time constraints.

The culture-based genomes were bioprospected for linoleate isomerases, and putative enzymes identified. Two of these were selected for expression and purification. The resulting proteins were incubated with linoleic acid and the incubations analysed via gas chromatography. Whilst peaks could be seen at appropriate retention times, it is inconclusive as to whether this is as a result of these proteins catalysing the breakdown of linoleic acid. This thesis provides the largest pangenomic study on Butyrivibrio and Pseudobutyrivibrio to date. As such, it provides novel insight into the functional and genomic capacity of these dominant rumen bacteria.

Date of Award	Dec 2022
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Department for the Economy
Supervisor	Sharon Huws (Supervisor), Tim Skvortsov (Supervisor) & Nigel Scollan (Supervisor)