Characterization of regulatory mutations causing anaerobic derepression

The genetic loci leading to anaerobic derepression of a sodA : : lac2 protein fusion in a UV-generated mutant strain (UV14) of Escherichia coli were identified. The mutant (UV14) was found to harbour two altered loci: one is in the trans-regulatory genefnv (fumarate nitrate reduction) where leucine-129 was changed to glutamine (finrl4), and the second (sodAl4) is in the promoter region (cis) of the sodA gene apparently affecting the binding of the Fur (ferric uptake regulation) protein. Introduction of an fnr’ gene into W 1 4 restored anaerobic repression of sodA : : lac2 and restored the ability of the cells to reduce nitrate. However, when either the fnrl4 or the sodAl4 mutation was introduced into an otherwise wild-type background, only slight anaerobic derepression of sodA was observed. When both the cisand trans-acting mutations (i.e. sodAl4 and fnrl4) were combined simultaneously in an otherwise wild-type background, the specific activity of sodA : : Zuc2 expression was comparable to that of the original mutant strain (UV14). Furthermore, a genetically confirmed fur fnr double mutant was also similarly derepressed in anaerobic sodA::lucZ expression. The data presented suggest that the cis-mutation in UV14 (sodA14) affects the Fur-binding site in the sodA promoter, while having no effect on Fnr or Arc mediated repression. Also, a second putative Fnr-binding site that straddles the ribosomal binding-site was identified in the sodA gene.