Genomic SELEX search for target promoters under the control of the PhoQP-RstBA signal relay cascade.
RstBA, a two-component regulatory system of Escherichia coli with an unidentified regulatory function, is under the control of a Mg(2+)-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 the E. coli genome 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 of asr (acid shock RNA) and another including the promoter for csgD (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 the asr gene 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 of asr was reduced after the deletion of rstA. In vivo transcription of the asr promoter was observed only in the presence of RstA. In agreement with the PhoQP-RstBA network, the addition of Mg(2+) led to a severe reduction of the asr promoter activity, and the disruption of phoP also reduced the asr promoter activity, albeit to a lesser extent. These observations altogether indicate that RstA is an activator of asr transcription. In contrast, transcription of csgD was repressed by overexpression of RstA, indicating that RstA is a repressor for csgD. With these data taken together, we conclude that the expression of both asr and csgD is under the direct control of the PhoQP-RstBA signal relay cascade.
Ogasawara
Hiroshi
H
Hosei University, Department of Frontier Bioscience, Kajino-cho 3-7-2, Koganei, Tokyo 184-8584, Japan.
Hasegawa
Akiko
A
Kanda
Emi
E
Miki
Takenori
T
Yamamoto
Kaneyoshi
K
Ishihama
Akira
A
D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D052156:SELEX Aptamer Technique; D015398:Signal Transduction / Q000235:genetics* / Q000502:physiology; D014158:Transcription, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins