Regulation of transcription of the mph(A) gene for macrolide 2 '-phosphotransferase I in Escherichia coli: Characterization of the regulatory gene mphR(A)

The synthesis of macrolide 2'-phosphotransferase I [Mph(A)], which inactiva tes erythromycin, is inducible by erythromycin, The expression of high-leve l resistance to erythromycin requires the mph(A) and mrx genes, which encod e Mph(A) and an unidentified protein, respectively. We have studied the mph R(A) gene, which regulates the inducible expression of mph(A). An analysis of the synthesis of Mph(A) in minicells and results of a complementation te st indicated that mphR(A) is located downstream from mrx and that its produ ct, MphR(A), represses the production of Mph(A). DNA sequencing indicated t hat the mph(A), mrx, and mphR(A) genes exist as a cluster that begins with mph(A) and that the deduced amino acid sequence of MphR(A) can adopt an alp ha-helix-turn-alpha-helix structure. To study the regulation of gene expres sion by MphR(A), we performed Northern blotting and primer extension. A tra nscript of 2.9 kb that corresponded to the transcript of mph(A) through mph R(A) was detected, and its level was elevated upon exposure of cells to ery thromycin. Gel mobility shift assays and DNase I footprinting indicated tha t MphR(A) binds specifically to the promoter region of mph(A), and the amou nt of DNA shifted as a results of the binding of MphR(A) decreased as the c oncentration of erythromycin was increased. These results indicate that tra nscription of the mph(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.