Genomic SELEX Search for Target Promoters under the Control of the PhoQP-RstBA Signal Relay Cascade

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Abstract
RstBA, a two-component regulatory system ofEscherichia coliwith an unidentified regulatory function, is under the control of a Mg2+-sensing PhoQP two-component system. In order to identify the network of transcription regulation downstream of RstBA, we isolated a set of RstA-binding sequences from theE. coligenome by using the genomic SELEX system. A gel mobility shift assay indicated the binding of RstA to two SELEX DNA fragments, one including the promoter region ofasr(acid shock RNA) and another including the promoter forcsgD(a regulator of the curli operon). Using a DNase I footprinting assay, we determined the RstA-binding sites (RstA boxes) with the consensus sequence TACATNTNGTTACA. Transcription of theasrgene was induced 10- to 60-fold either in low-pH (pH 4.5) LB medium or in low-phosphate minimal medium as detected by promoter assay. The acid-induced in vivo transcription ofasrwas reduced after the deletion ofrstA. In vivo transcription of theasrpromoter was observed only in the presence of RstA. In agreement with the PhoQP-RstBA network, the addition of Mg2+led to a severe reduction of theasrpromoter activity, and the disruption ofphoPalso reduced theasrpromoter activity, albeit to a lesser extent. These observations altogether indicate that RstA is an activator ofasrtranscription. In contrast, transcription ofcsgDwas repressed by overexpression of RstA, indicating that RstA is a repressor forcsgD. With these data taken together, we conclude that the expression of bothasrandcsgDis under the direct control of the PhoQP-RstBA signal relay cascade.