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dc.contributor.advisorTannock, Gerald
dc.contributor.advisorO'Toole, Paul
dc.contributor.authorWilson, Charlotte Mary
dc.date.available2011-07-11T20:55:45Z
dc.date.copyright2011
dc.identifier.citationWilson, C. M. (2011). Lactobacillus dialogues: the impact of cell signalling on gene expression and ecological behaviour of a gut commensal (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/1754en
dc.identifier.urihttp://hdl.handle.net/10523/1754
dc.description.abstractQuorum-sensing is a system where genes are regulated in response to changing cell density by the synthesis, release and detection of a signalling molecule to facilitate the coordination of group behaviour such as in the development of a biofilm. Studies in Vibrio, Salmonella and Escherichia identified the autoinducer-2 (AI-2) signalling molecule produced by LuxS, an enzyme widely distributed among bacterial species. The extensive taxonomic distribution of luxS suggested a universal signalling role for AI-2 with potential for interspecies microbial communication. The identification of loss of quorum-sensing phenotypes when luxS is mutated is complicated however by the dual role of LuxS in both the production of AI-2 and catalysis of a reaction in the activated methyl cycle (AMC). Previously a luxS mutant strain of Lactobacillus reuteri 100-23, an autochthonous inhabitant of the mouse gastrointestinal (GI) tract, was developed and found to have altered biofilm formation, lower cellular ATP and a reduced ability to compete with a closely related species in the GI tract of mice. It was hypothesised that loss of quorum-sensing ability affected the phenotype of L. reuteri 100-23. Cells of wild type and luxS mutant L. reuteri 100-23 were harvested from log phase and stationary growth phases, when extracellular amounts of AI-2 were highest and near baseline, respectively. Transcriptome analysis, using a microarray representing the genome of strain 100-23, identified altered gene regulation in the metabolic pathways of methionine biosynthesis, cysteine biosynthesis, and pyrimidine biosynthesis when luxS was mutated. Two prophage regions were down-regulated in the mutant relative to the wild type and circularised phage DNA could be detected in the wild type. The regulation of two genes encoding proteins with LPXTG motifs was affected by luxS mutation during growth in laboratory culture. The expression pattern of one of these genes was similarly affected when the mutant formed in vitro biofilms in a bioreactor and during colonisation of the mouse stomach. Stress response genes were induced in all conditions analysed when luxS was mutated. The metabolite profile measured by GC-MS supported the cellular stress response induced by luxS mutation, and an accumulation of amino acids in the mutant relative to the wild type suggested a redirection of metabolic pathways to compensate for loss of LuxS. Further studies investigated important colonisation features of L. reuteri 100-23 by transcriptome comparison of L. reuteri 100-23 harvested from the mouse stomach to L. reuteri 100-23 harvested from broth culture. Genes involved in the acid tolerance response including glutamate decarboxylase and the urease enzyme were substantially up-regulated during gut colonisation. The urease enzyme was functional and a knockout of the main subunit of the enzyme, UreC, resulted in a strain unable to use urea and with a reduced tolerance to acid. This strain was severely compromised during colonisation of the mouse and constituted less than 1% of the total Lactobacillus population in the stomach, jejunum and caecum seven days after co-inoculation with the wild type strain. Under the conditions analysed in this study, the effects of AI-2-mediated quorum-sensing in L. reuteri 100-23 were negligible and may be limited to the induction of prophage. The majority of transcriptome effects were attributable to the metabolic disturbances caused by loss of a complete AMC. A functional urease enzyme was found to be essential for the successful colonisation of the mouse stomach by L. reuteri 100-23 and in the ability of this organism to compete and persist in the mouse GI tract.en_NZ
dc.language.isoenen_NZ
dc.publisherUniversity of Otago
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dc.subjectLactobacillusen_NZ
dc.subjectQuorum sensingen_NZ
dc.titleLactobacillus dialogues: the impact of cell signalling on gene expression and ecological behaviour of a gut commensalen_NZ
dc.typeThesis
dc.date.updated2011-07-08T03:14:23Z
thesis.degree.disciplineMicrobiology and Immunologyen_NZ
thesis.degree.nameDoctor of Philosophyen_NZ
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral Theses
otago.interloanyesen_NZ
otago.openaccessAbstract Only
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