Characterization of cis-Acting Regulatory Mutations Causing Anaerobic Expression of the sodA Gene in Escherichia coli

Abstract

The biosynthesis of Mn-containing superoxide dismutase is regulated in response to stimuli that affect the redox potential of the cell. To further investigate the mode of regulation of the gene (sodA) encoding this enzyme, cis-acting regulatory mutations in a strain containing a sodA::lacZ gene fusion were studied. The mutant strains expressed β-galactosidase under anaerobic conditions, whereas the wild-type did not. Furthermore, the mutants were not induced in response to the presence of iron chelator, 2,2′-dipyridyl, or to the redox cycling compound, paraquat. The wild-type, however, did respond to these effectors. In vivo cloning was used to isolate the cis-acting regulatory elements from the mutants (NC4 and NC5). Replacement of the wild-type 5′-regulatory region with either of the mutants′ cis-acting regulatory element resulted in the anaerobic expression of active Mn-superoxide dismutase. Sequence and restriction analysis revealed the presence of an IS2 insertion element in the promoter region of one of the mutants (NC5). This insertion caused the displacement of the 5′-regulatory region of sodA and the formation of a functional hybrid promoter consisting of the resident −10 region from sodA and −35 from IS2. The second mutation (from NC4) was similarly analyzed, and an IS5 element was identified. The insertion site of IS5 (in NC4) was 6 bp (5′-TTAATT-3′) upstream from the IS2 site (in NC5). Anaerobic expression of sodA in NC4 was lower than in NC5. This difference was almost eliminated in an arc⁻ background, suggesting that the sequence 5′-TTAATT-3′ might be essential for negative regulation by ArcA.