| Summary: | Thesis (Ph.D.)--Memorial University of Newfoundland, 1984. Medicine Bibliography: leaves 200-223. The putrescine biosynthetic enzyme, ornithine decarboxylase (E.C. 4.1.1.17), is negatively regulated by cAMP in Escherichia coli. The specific activity of ornithine decarboxylase was determined in crude extracts prepared from wild-type strains of E. coli and from strains carrying a mutation in the adenylate cyclase (E.C. 4.6.1.1) structural gene (cya). These strains were grown with various carbon sources in the presence and absence of cAMP. In wild-type strains ornithine decarboxylase activity was was less after growth on glycerol than after growth on glucose. When cya strains were grown on glucose or glycerol, ornithine decarboxylase activity was the same. Addition of 1 mM cAMP to a glucose-based medium repressed ornithine decarboxylase activity by approximately 50% in both wild-type and cya strains. Furthermore, cAMP exerts its negative control through the cAMP receptor protein (CRP), because a strain carrying a lesion in the crp structural gene failed to exhibit repressed ornithine decarboxylase activity in response to elevated cAMP. These results suggested that negative regulation of ornithine decarboxylase is exerted at the level of transcription. Negative control was shown not to be mediated by a cAMP-induced repressor because synthesis of ornithine decarboxylase in E. coli minicells, and in a cell-free protein synthesizing system, was repressed by cAMP. Repression of ornithine decarboxylase synthesis by cAMP in vitro required functional CRP as evidenced by the lack of repression in a CRP deficient reaction system. The tetracycline resistance of E. coli wild-type and cya strains harbouring a tetracycline resistance gene (tet) under control of the speC promoter was repressed by cAMP. Cyclic AMP had no effect, on the tetracycline resistance of crp strains bearing the speC:tet gene fusion, nor on wild-type strains bearing a normal tet gene. These results indicate that cAMP and its receptor protein interact with the speC promoter region to facilitate negative transcriptional control of the speC gene. Negative control of speC transcription by cAMP was confirmed by analysis of steady-state levels of ornithine decarboxylase mRNA in cya and crp strains of E. coli. Ornithine decarboxylase mRNA levels were repressed approximately 80% when cya strains were grown in the presence of 2 mM cAMP. No repression of ornithine decarboxylase mRNA levels was seen in a crp strain cultured in the presence of cAMP.
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