Abstract
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 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.
Highlights
Genetic Definition of the Escherichia coli zwf ‘‘Soxbox,’’ the DNA Binding Site for SoxS-Mediated Induction of Glucose
Unlike gnd, zwf expression is transcriptionally activated by SoxS during episodes of oxidative stress induced by exposure of E. coli to superoxide-generating agents such as paraquat [17, 18, 39]
Our initial approach to delineating the cis-acting site in zwf that is involved in the soxRS-mediated response to oxidative stress was based on the observation that a zwf-lacZ operon fusion, DR52 [26], was regulated by soxRS at the transcriptional level [18]
Summary
Genetic Definition of the Escherichia coli zwf ‘‘Soxbox,’’ the DNA Binding Site for SoxS-Mediated Induction of Glucose. Taking advantage of PCR and recombinant DNA techniques, we made specific 5Ј and 3Ј deletions of the zwf promoter region and examined the effects of these deletions on the paraquat inducibility of lacZ protein fusions in vivo.
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