2 GENETICALLY-DISTINCT AND DIFFERENTIALLY-REGULATED ACONITASES (ACNA AND ACNB) IN ESCHERICHIA-COLI

An acnA mutant of Escherichia coli was constructed by replacing the ch romosomal acnA gene by an internally deleted derivative containing a k an(R) cassette. Southern and Western blotting confirmed that the acnA gene had been replaced by the disrupted gene and that the aconitase A protein was no longer expressed. However, the mutant failed to exhibit the anticipated glutamate auxotrophy and it retained a residual aconi tase activity. This activity was due to an analogous unstable enzyme(s ) designated aconitase B. Studies on the regulation of aconitase A syn thesis using an acnA-lacZ translational fusion showed that the acnA ge ne resembles other citric acid cycle genes in being subject to CRP-med iated catabolite repression and ArcA-mediated anaerobic repression. In addition to being activated by the SoxRS oxidative stress regulatory system, the acnA gene appeared to be activated by the ferric uptake re gulator (Fur). It was concluded that the acnA gene belongs to at least four global regulatory networks, crp, arcA, fur and soxRS. In contras t, the aconitase B activity decreased after exposure to oxidative stre ss and was less affected by anaerobiosis. Comparable studies with the fumarase genes (fumA, B and C) indicated that fumA (encoding the unsta ble aerobic iron-sulphur-containing fumarase) is activated by the ferr ic uptake regulator (Fur) and fumC (encoding the stable fumarase) is a ctivated by the SoxRS oxidative stress regulatory system.