Cloning of the ilvA538 gene coding for feedback-hypersensitive threonine deaminase from Escherichia coli K-12.
A variety of experimental results implicate the ilvA gene product, threonine deaminase, as an autoregulatory protein that affects the expression of its own gene and those coding for some related proteins. Some of the most direct evidence comes from the analysis of mutations in the ilvA gene with pleiotropic genetic regulatory effects. The most extensively documented mutation, ilvA538, lowers the expression of and abolishes repression control of the ilvGEDA transcription unit. A pleiotropic effect of the ilvA538 mutation, which may be either incidental or mechanistically related to the loss of repression control, renders threonine deaminase feedback hypersensitive to the inhibition of catalytic activity by the pathway end product, isoleucine. We transferred this mutation to lambda dilv phage and pBR322 derivatives. Direct enzyme assay of the plasmid- and phage-coded ilvA538 gene product in delta ilv hosts confirmed the feedback hypersensitivity of the enzyme product. In conjunction with the ilvG671 (phenotype, ILvG+ Valr; previously designated ilvO671) allele located in cis, high levels of the plasmid and lambda dilv phage-coded mutant enzyme suitable for protein purification were observed. Deletion mapping experiments with lambda dilv phage confirmed that the ilvA538 mutation, and not mutations promoter proximal to ilvD (transcription is from ilvG to ilvA), confer a loss of repression control. These genetic mapping studies indicate, however, that an additional mutation(s) may be present that contributes, at least in part, to the reduced enzyme levels in strains with the ilvA538 mutation.
D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005246:Feedback; D005798:Genes, Bacterial; D009154:Mutation; D010957:Plasmids; D013045:Species Specificity; D013913:Threonine Dehydratase / Q000235:genetics*
D013913:Threonine Dehydratase