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The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
Author for correspondence: John K. Guest. Tel: +44 114 222 4406/3. Fax: + 44 114 2728697 e-mail: J.R.Guest@Sheffield.ac.uk
ABSTRACT
Escherichia coli contains two genes (acnA and acnB) encoding aconitase activities. An acnB mutant was engineered by replacing the chromosomal acnB gene by an internally deleted derivative containing a tetR cassette. An acnB double mutant was then made by transducing a previously constructed acnA::kanR mutation into the acnB::tetR strain. Western blotting confirmed that the AcnA and AcnB proteins were no longer produced by the corresponding mutants and PCR analysis showed that the chromosomal acnB gene had been replaced by the disrupted gene. Aerobic and anaerobic growth in glucose minimal medium were impaired but not abolished by the acnB mutation, indicating that the lesion is partially complemented by the acnA+ gene, and growth was enhanced by glutamate. The acnAB double mutant would not grow on unsupplemented glucose minimal medium and although it responded to glutamate like a typical auxotroph under anaerobic conditions, under aerobic conditions no response to glutamate was observed before it was over-grown by ‘revertants’ lacking citrate synthase (acnAB gltA). The acnAB double mutant retained a low but significant aconitase activity (<5% of wild-type), designated AcnC. Enzymological and regulatory studies with acn-lacZ fusions indicated that AcnB is the major aconitase, which is synthesized earlier in the growth cycle than AcnA, and subject to catabolite and anaerobic repression.
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