Amber mutants of the -ketoglutarate dehydrogenase gene of Escherichia coli K12.

Abstract

SUMMARY Of 21 suc mutants of Escherichia coli k12 five were shown to be amber mutants by their sensitivity to amber suppressors. In addition to lacking activity for the overall Α-ketoglutarate dehydrogenase complex and the Α-ketoglutarate dehydrogenase component (E1), the amber mutants had little or no dihydrolipoyl trans-succinylase (E2) and 20 to 30% less dihydrolipoyl dehydrogenase (E3) than the parental strain. In common with non-suppressible suc mutants they also had reduced amounts of succinylCoA synthetase and succinate dehydrogenase. Fine structure mapping by Pi-transduction showed that some of the suc-amber sites were located in the sucA gene. It is concluded that expression of the suc region is polarized from sucA to sucB, i.e. synthesis of the E1 component precedes that of the E2 component. Double amber mutants with lesions in the pyruvate and Α-ketoglutarate dehydrogenase genes (aceE, sucA) were constructed but their E3 activity was never less than 30% of that of the parental strain. The results are consistent with the existence of separate genes for the dihydrolipoyl dehydrogenase component of the pyruvate and Α-ketoglutarate dehydrogenase complexes, but other possibilities could not be ruled out. Further studies on the positions of the four closely-linked tricarboxylic acid cycle genes specifying citrate synthase (gltA), succinate dehydrogenase (sdh) and two components of the Α-ketoglutarate dehydrogenase complex (sucA and sucB) indicate the relative order: gltA ‥sdh ‥sucA.sucB ‥tolII …gal.