CONSTRUCTION AND PROPERTIES OF ACONITASE MUTANTS OF ESCHERICHIA-COLI

Escherichia coli contains two genes (acnA and acnB) encoding aconitase activities. An acnB mutant was engineered by replacing the chromosoma l acnB gene by an internally deleted derivative containing a tet(R) ca ssette. An acnAB double mutant was then made by transducing a previous ly constructed acnA:: kan(R) mutation into the acnB::tet(R) strain. We stern blotting confirmed that the AcnA and AcnB proteins were no longe r produced by the corresponding mutants and PCR analysis showed that t he chromosomal acnB gene had been replaced by the disrupted gene. Aero bic and anaerobic growth in glucose minimal medium were impaired but n ot abolished by the acnB mutation, indicating that the lesion is parti ally complemented by the acnA(+) gene, and growth was enhanced by glut amate. The acnAB double mutant would not grow on unsupplemented glucos e minimal medium and although it responded to glutamate like a typical auxotroph under anaerobic conditions, under aerobic conditions no res ponse to glutamate was observed before it was over-grown by 'revertant s' lacking citrate synthase (acnAB gltA). The acnAB double mutant reta ined a low but significant aconitase activity (less than or equal to 5 % of wildtype), designated AcnC. Enzymological and regulatory studies with acn-lacZ fusions indicated that AcnB is the major aconitase, whic h is synthesized earlier in the growth cycle than AcnA, and subject to catabolite and anaerobic repression.