Regulation of Transcription of themph(A) Gene for Macrolide 2′-Phosphotransferase I inEscherichia coli: Characterization of the Regulatory GenemphR(A)

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Abstract
The synthesis of macrolide 2′-phosphotransferase I [Mph(A)], which inactivates erythromycin, is inducible by erythromycin. The expression of high-level resistance to erythromycin requires themph(A) and mrx genes, which encode Mph(A) and an unidentified protein, respectively. We have studied themphR(A) gene, which regulates the inducible expression ofmph(A). An analysis of the synthesis of Mph(A) in minicells and results of a complementation test indicated thatmphR(A) is located downstream from mrx and that its product, MphR(A), represses the production of Mph(A). DNA sequencing indicated that the mph(A), mrx, andmphR(A) genes exist as a cluster that begins withmph(A) and that the deduced amino acid sequence of MphR(A) can adopt an α-helix–turn–α-helix structure. To study the regulation of gene expression by MphR(A), we performed Northern blotting and primer extension. A transcript of 2.9 kb that corresponded to the transcript of mph(A) through mphR(A) was detected, and its level was elevated upon exposure of cells to erythromycin. Gel mobility shift assays and DNase I footprinting indicated that MphR(A) binds specifically to the promoter region ofmph(A), and the amount of DNA shifted as a results of the binding of MphR(A) decreased as the concentration of erythromycin was increased. These results indicate that transcription of themph(A)-mrx-mphR(A) operon is negatively regulated by the binding of a repressor protein, MphR(A), to the promoter of the mph(A) gene and is activated upon inhibition of binding of MphR(A) to the promoter in the presence of erythromycin.