GENETIC DEFINITION OF THE ESCHERICHIA-COLI ZWF SOXBOX, THE DNA-BINDING SITE FOR SOXS-MEDIATED INDUCTION OF GLUCOSE-6-PHOSPHATE-DEHYDROGENASE IN RESPONSE TO SUPEROXIDE

In Escherichia coli K-12, transcription of zwf, the gene for glucose 6-phosphate dehydrogenase, is subject to growth rate-dependent regulation and is activated by SoxS in response to superoxide stress. To define genetically the site of SoxS activation, we undertook a detailed deletion analysis of the zwf promoter region. Using specifically targeted 5' and 3' deletions of zwf sequences, we localized the SoxS activation site to a 21-bp region upstream of the zwf promoter. This minimal ''soxbox'' was able to confer paraquat inducibility when placed upstream of a normally unresponsive gnd-lacZ protein fusion. In addition, we used these findings as the basis for resecting unnecessary sequences from the region upstream of the promoters of two other SoxS-regulated genes, sodA and nfo. Like the zwf soxbox, the regions required for SoxS activation of sodA and nfo appear to lie just upstream of or overlap the -35 hexamers of the corresponding promoters. Importantly, the sequence boundaries established here by deletion analysis agree with the primary SoxS recognition sites of zwf, sodA, and nfo that we previously identified in vitro by gel mobility shift and DNase I protection assays with a purified MalE-SoxS fusion protein.