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Title: A genetic approach to heat resistance in Salmonella species
Author: Moore, Stephen Jon
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1992
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The aim of this project was to develop the genetics of heat tolerance in the mesophilic food-poisoning bacterium Salmonella typhimurium strain SA2009. Since this required the isolation of mutants having a reduced or increased resistance to heat it was necessary to establish the heat resistance of strain SA2009 in liquid growth medium. Survival curves initially showed a great deal of variability when exponentially-growing bacteria were heated but by careful control of a number of important parameters such as the precise stage of growth and the volume of preheated medium into which they were dispersed the variability was removed. Three methods were used in an attempt to generate mutants. Two involved a search for heat-sensitive mutants by the induction of mutagenesis, viz treatment of exponentially-growing bacteria with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine and the use of transposons which by insertion into genes disrupt their function. The third method selected for spontaneous (or heat-induced) heat-resistant mutants of S.typhimurium by repeated cycles of heating cultures to reduce substantially the number of viable bacteria and then allowing the survivors to give rise to a culture which was heat treated etc. None of these methods provided mutants with altered heat tolerance. During these experiments an anomaly between the survival of putative mutants in liquid media and on the surface of agar plates was observed. Apparently-sensitive organisms detected on the surface of agar plates gave heat survival curves identical to that of the wild type. An alternative strategy was to attempt to transfer the gene(s) responsible for the extreme heat resistance of Salmonella senftenberg strain 775W into a wild type strain of S.typhimurium and Escherichia coli. A gene library of S.senftenberg 775W DNA was constructed in the positive selection plasmid pUN121. A culture of the restriction/modification deficient E.coli, strain MM294 was transformed with the plasmid library and cycled through several heat treatments. Two clones of E.coli were isolated that had greatly increased heat resistance similar to that of S.senftenberg 775W.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available