COPPER-INDUCIBLE TRANSCRIPTIONAL REGULATION AT 2 PROMOTERS IN THE ESCHERICHIA-COLI COPPER RESISTANCE DETERMINANT PCO

The pco determinant of Escherichia coil plasmid pRJ1004 encodes induci ble resistance to the trace element copper. The identification of two copper-dependent transcriptional initiation regions within pco that ea ch contain a similar upstream hyphenated dyad motif is described. Dele tion constructs showed that this 'copper box' motif was essential for copper-inducible activity at both pco promoters, PpcoA and PpcoE. The placement of the motif differs in the two promoters, and PpcoA contain s an extended -10 nonamer typical of promoters for which RNA polymeras e does not bind specifically to -35 sequences. PpcoE does not contain this motif and is the more strongly expressed promoter. The transcript from PpcoA contains the pcoABCDRS genes, while PpcoE expresses only p coE. The induction profiles for PpcoA- and PpcoE-lacZ fusions were fla ttened sigmoidal curves with a gradual response to increasing copper c oncentration. On high-copy-number plasmids, zinc was found also to ind uce transcription from both promoters in vivo. Both promoters showed i nducible activity in the absence of pcoRS, the plasmid-borne two-compo nent regulatory system, indicating that a second trans-acting regulato ry system is present on the chromosome. The pcoR product showed repres sor action in the absence of pcoS, while still allowing induction, sug gesting the chromosome encoded a similar two-component system to pco. TnphoA insertion mutagenesis identified chromosomal genes which affect ed promoter expression, including ptsH, ptsI (sugar phosphotransferase system) and cya (adenylate cyclase). The results support that idea th at pco-encoded copper resistance is an auxiliary mechanism for handlin g copper, the regulation of which is integrated with the chromosomal r egulation of cellular copper metabolism.