In the natural environment, bacteria spend the majority of their existence in periods of starvation. While some Gram positive bacteria, such as Bacillus subtilis respond to this stress by forming highly heat-resistant spores, others, such as E. coli, enter a senescent state known as stationary phase. The stationary phase response is characterized by an increased cellular resistance to stresses associated with dormancy such as heat-shock, oxidative stress and osmotic challenge. These confer the cells the ability to survive prolonged periods of nutrient deprivation. The expression of these genes is regulated in part by os, the stationary phase-specific, alternative sigma factor, encoded by the gene rpoS. Several methods have been used to identify these genes including two-dimensional gel electrophoresis (the expression of more than 30 proteins is absent in a rpoS mutant) and the identification of mutants possessing lacZ fusions that respond to a given related stimulus, such as carbon starvation. On the basis of these methods, the os-regulon is predicted to be quite large. However, only fifteen or so genes have been characterized to date. To identify other os-regulated functions, we have introduced a rpoS::Tn10 null allele into a library of 5000 random lacZ fusion mutants and determined the effects of the null allele upon lacZ expression. One-hundred and five mutants were isolated and the os-dependent phenotype confirmed by complementing the rpoS function. Seventy-three strains were complemented, four were not and complementation results were not obtained for twenty-eight strains. Growth-phase-expression assays performed on the strains demonstrated induction of fusions at the onset of stationary phase, and this induction was reduced or abolished in an isogenic rpoS mutant. Furthermore, each mutant exhibited its own characteristic pattern of induction, consistent with the diversity of factors regulated by os. Identification of selected os-dependent functions was accomplished by sequencing the fusion junctions and comparing the sequence obtained with published E. coli sequence databanks. Results show that six fusions map to open reading frames encoding proteins of unknown function and two do not map to any known region in the E. coli chromosome. One fusion maps to a gene encoding a putative lysine decarboxylase. This enzyme is implicated in the maintenance of medium pH and generation of carbon dioxide for cellular growth. The other maps to a recently identified talB gene. talB encodes a transaldolase protein involved in the pentose phosphate cycle, which generates precursors of nucleic acids, aromatic amino acids, and vitamins. / Thesis / Master of Science (MS)

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