| The work presented in this Thesis focuses on the SOS responses of Salmonella typhimurium. This area was of interest due to three factors; (1) although this bacterium has been widely employed in the Ames test and related bioassays to detect mutagenic DNA damaging agents, its SOS responses were not well characterized; (2) much confusion existed in the literature as to whether S. typhimurium contained an analog of the umuDC operon, an SOS locus which is required for UV induced mutagenesis in E. coli; and, (3) the sensitivity of this organism to UV was reported to be increased in strains exhibiting altered topoisomerase activities, suggesting a relationship between supercoiling and DNA damage responses in S. typhimurium. Using DNA probes to the E. coli umuD and umuC genes a umuDC analog was cloned from S. typhimurium. At the amino acid level the S. typhimurium umuC gene exhibited approximately 81% identity with E. coli umuC and 55% identity with a plasmid-borne umuDC analog, mucB. This clone also complemented the mutability defect of both umuD and umuC mutants of E. coli and its overexpression caused a pronounced sensitization of the host bacteria to growth at low temperatures. The overall S. typhimurium SOS response was also investigated using lac-operon fusions isolated to DNA damage inducible loci (din-loci) in this bacterium. Detailed characterizations of several din-fusions including one to uvrB and two others to loci closely linked to, but seperate from uvrB were performed. Lastly, effects of supercoiling upon SOS expression were studied. The analysis presented provides the first direct demonstration of an effect of supercoiling upon the inducible expression of DNA damage responses. topA mutants, which exhibit increased levels of DNA supercoiling, were unable to efficiently induce several din-loci in response to MMC. Several SOS lac-operon fusions, including one which was induced greater than 60-fold by exposure to MMC in wild type bacteria, were rendered almost non-inducible in a topA deletion strain. The inducible expression of several other din-loci, including uvrB, was also significantly diminished in topA strains. Evidence suggesting that supercoiling may exert this effect at the level of the LexA operator is presented. |
