Identification of the Sequences Recognized by the Bacillus subtilis Response Regulator YrkP

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The Bacillus subtilis yrkP gene encodes a response regulator of a two-component regulatory system of unknown function. A previous DNA microarray experiment suggested that multicopy yrkP greatly enhanced the expression of yrkN, the ykcBC operon, and yrkO, which encodes a putative transporter. Here, lacZ fusion analysis confirmed these results and also revealed that YrkP autoregulates the putative yrkPQR operon, indicating that yrkPQR and yrkO form a divergon structure. In addition, real-time PCR analysis revealed that transcription of yrkO, yrkN, and ykcBC was significantly reduced in the yrkP strain. Hence, YrkP positively regulates the expression of these genes. Gel retardation analyses showed that YrkP bound to the promoter regions of yrkO, yrkN, and ykcB, albeit with lower binding affinities to the latter two promoters. The in vitro binding of YrkP to the promoter region of the yrkPQR and yrkO divergon was then analyzed by DNase I footprinting analysis. This revealed that YrkP recognizes three regions containing single-motifs or a direct repeat of the ten-base sequence [T/G]TCA[T/C]AAATT. lacZ fusion analysis of deleted and mutagenized promoter regions of yrkO and yrkPQR divergon confirmed that the three YrkP-binding regions are needed for the YrkP-mediated activation of yrkO and/or yrkPQR.
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