No description available.

No description available.

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.

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

Previous genetic evidence suggested that RecA was required in SOS mutagenesis for its regulatory role and perhaps some other nonregulatory role (Mount, 1977; Blanco et al., 1982). I undertook a genetic study which confirmed the above studies and provided further evidence that RecA protein appeared to have a dual "role in mutagenesis; first, the cleavage of LexA repressor for the derepression of specific SOS genes and second, one or more additional role(s). For these studies a new phage mutagenesis assay was developed which allows rapid scoring of SOS mutagenesis in a large number of host mutants. I next conducted a genetic analysis to determine if the newly defined RecA mutagenesis function was separable by mutation from the numerous other phenotypes which are known to be influenced by RecA protein. From the study of recA mutants it appears that the RecA mutagenesis function(s) is genetically separable from the following RecA phenotypes: LexA cleavage, lambda cI repressor cleavage, UV resistance and homologous recombination. In addition, I discovered that the LexA cleavage function and lambda cI cleavage function is also separable. I also studied in some detail the novel genetic properties that I uncovered for recA432 mutant strains. recA432 was defined as a mutagenesis defective allele (Kato and Shinoura, 1977). LexA cleavage in recA432 cells was more easily induced that in recA⁺ cells, causing lethal filamentation of these mutant cells even at very low UV doses. I concluded that the basis for the Mut⁻ phenotype was this strain's propensity to lethally filament, which complicated the detection of mutant cells. In another set of experiments, I examined the regulatory requirements for SOS mutagenesis and Weigle phage-reactivation; I wanted to determine which SOS operons must be derepressed for this process. lexA(Ind⁻) mutant cells are defective in mutagenesis because they cannot derepress specific SOS genes required in this process. I found that the selective derepression of umuDC was sufficient to restore mutagenesis to these lexA(Ind⁻) mutants; however, derepression of umuDC and recA was required for phage reactivation.

This report describes the development and use of an expression vector system based on the single-stranded DNA bacteriophage M13. A derivative of M13mp8, designated M13mp8/P, was prepared in which the promoter and N-terminal codons of bacterial genes may be fused to a portion of β-galactosidase, resulting in an easily scorable phenotype. Because transcription from the inserted promoter remains responsive to the host regulatory system, it is simple to screen mutagenized phage for isolates with aberrant regulatory phenotypes, and to determine the mutational changes by dideoxy sequence analysis. The feasibility of this method was demonstrated by identification of a large number of mutations in the regulatory regions of two genes, recA and lexA. Base substitutions that altered the phenotype of recombinant phage were identified both in the single LexA repressor binding site of recA and in the two binding sites of lexA, as well as in other sites that likely affect translational efficiency. My results suggest that this method will be generally useful for mutational analysis of transcriptional and translational regulatory elements. The mutants that were isolated by the above approach were used to investigate the specificity of LexA protein binding by quantifying the repressibility of a several mutant recA and lexA operator/promoter regions fused to the E. coli galactokinase (galK) gene. The results of this analysis indicated that two sets of four nucleotides (terminal nucleotide contacts), one set at each extreme end of the operator, are most critical for repressor binding. In addition, our results indicate that the repressor-operator interaction is symmetric in nature, in that mutations at symmetrically equivalent positions in the recA operator had comparable effects on repressibility. The inferred symmetry of the interaction justified the reevaluation of the consensus sequence by half-site comparison, which yielded the half-site consensus: (5') CTGTATAT. Although the first four positions of this half-site sequence have the greatest effect on LexA repressor binding, the last four are well conserved among binding sites and appear to modulate repressor affinity. The role of the terminal nucleotide contacts and the mechanism by which the internal sequences affect repressor binding is discussed.

No description available.