Genome sequencing of a large number Firmicute species has recently been completed, including some of the highly oxygen-sensitive butyrate-producing bacteria, belonging to the Lachnospiraceae and Ruminococcaceae families, which have been isolated at the Rowett Institute of Nutrition and Health. However, detailed knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of detailed genomic annotation and pathway analysis, and lack of genetic manipulation techniques. Therefore, the aim of this work was the genomic analysis of the carbohydrate-utilisation and motility genes, and establishment of genetic manipulation techniques for a selected group of these bacteria, specifically the Roseburia/Eubacterium rectale group and Faecalibacterium prausnitzii. This involved the establishment of a Roseburia/E. rectale pan-genome consisting of genome sequences from eleven strains (three of which are first introduced in this work), representing five species. 1840 Carbohydrate-active enzymes (CAZymes), 932 of which were glycoside hydrolases (GHs), were identified in this pan-genome. The GH complement of each strain was used to predict dietary niches of these bacteria in the human colon. The members of the Roseburia/E. rectale group were predicted to have the core capacity to utilise starch, with specific members possessing specialised dietary niches. The motility loci of selected members of the Roseburia/E. rectale group were annotated, and the gene orders of these loci were highly conserved between different members of the group. The motility of these bacteria was shown to be affected by the carbon source utilised for growth. This was followed by the design of methods to allow the transfer of autonomously-replicating plasmids into Roseburia/E. rectale species. The modular plasmids pMTL83151 and pMTL82151 were transferred from an E. coli donor into Roseburia inulinivorans A2-194. pMTL83151 could also be transferred into Eubacterium rectale A1-86 and T1-815. This technique has enabled the heterologous expression of a β-(1,3-1,4)-glucanase enzyme in R. inulinivorans A2-194 and E .rectale T1-815.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:635974 |
| Date | January 2014 |
| Creators | Sheridan, Paul O. |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=223921 |
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