Although it is well established that synthesis of the biodegradative threonine dehydratase of Escherichia coli requires anaerobic conditions and a medium rich in amino acids but lacking glucose, the precise nature of the inducer(s) or repressor(s) is still unclear. This dissertation is a report of research directed at elucidating the regulation of synthesis of threonine dehydratase by a genetic and biochemical characterization of mutants that are affected in induction of the enzyme. Because conditions selective for mutants that lack threonine dehydratase are not known, a direct enzyme assay was used to isolate mutant strains. Variants of E. coli that produce minimal levels of the enzyme (70% cotransduction) to malB. When cultivated anaerobically in TYE (an amino acid-rich medium) the revertant grew more rapidly, and to a greater cell density than the mutant. However, the addition of 10 mM NO(,3) to the TYE medium improved the growth of MB201 relative to MB202. More significantly, threonine dehydratase was induced by the mutant strain grown in the presence of nitrate and the enzyme produced was identical, by several enzymological and immunological criteria, to that of the revertant and wild type strains (cultivated in the absence of nitrate), implying that the structural gene for the dehydratase in the mutant is intact. The addition of 5 mM nitrite or 40 mM fumarate to the TYE medium also promoted enzyme synthesis in the tdcI('-) strain, suggesting that induction is facilitated by the presence of an exogenous electron acceptor. D-serine deaminase and tryptophanase levels were similar in strains MB201 and MB202, indicating that the mutation in MB201 did not influence all catabolic enzymes. In addition, the levels of fumarate reductase and cytochrome c(,552) were comparable in the mutant and revertant, suggesting that the mutated gene does not generally affect proteins induced under anaerobic conditions. Strain MB201, which exhibited a reduced growth rate in TYE anaerobically, also grew slower (than the revertant, MB202) in aerated glucose minimal medium and did not grow in this medium anaerobically. However, the mutant was able to grow on fructose anaerobically, suggesting that it lacked phosphoglucose isomerase activity--a conclusion confirmed by direct assay of the enzyme. When a malB derivative of MB201 was transduced to malB('+) using bacteriophage P1 grown on MB202, tdcI and pgi (a locus at 91 min which encodes phosphoglucose isomerase) cosegregated in 50/50 transductants. Thus it appears likely that tdcI is identical with pgi; however, in the context of other strain backgrounds, a pgi mutation did not prevent induction of threonine dehydratase. Although the mechanism by which nitrate and the pgi locus affect the induction of threonine dehydratase in strain MB201 is not clear, it is possible that a mutation in pgi results in the accumulation of a metabolite which represses the enzyme, and nitrate allows removal of the putative repressor.Ph.D.MicrobiologyUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/158311/1/8116298.pd
