MOLECULAR-GENETICS OF A CHROMOSOMAL LOCUS INVOLVED IN COPPER TOLERANCE IN ESCHERICHIA-COLI K-12

The cutA locus, presumably involved in copper tolerance in Escherichia coli, was characterized by a mutation leading to copper sensitivity. Copper-accumulation measurements with radioactive Cu-64(2+) showed increased uptake by cutA copper-sensitive mutant cells, and reduced uptake when the cutA mutation was complemented in trans. The locus was mapped using complementation of the cutA mutant to partial copper tolerance with wild-type chromosomal fragments. The 3.2 kb DNA region involved in cotA was sequenced and analysed, revealing three significant open reading frames, none of which had been previously published. The products of all three open reading frames were identified, when synthesized with the T7 phage promoter expression system, as polypeptides of about 50 kDa, 24 kDa, and 13 kDa, consistent with the sizes predicted from the DNA sequences. The 50 kDa and 24 kDa polypeptides were found in the bacterial inner membrane, and the 13 kDa polypeptide with the cytoplasmic fraction. In addition to being required for copper tolerance, cutA affects tolerance levels to zinc, nickel, cobalt and cadmium salts. Transcriptional fusions of cutA with the lux operon showed induction by copper, zinc, nickel, cobalt and, to a lesser extent, cadmium, manganese and silver salts.