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 inc reased uptake by cutA copper-sensitive mutant cells, and reduced uptak e when the cutA mutation was complemented in trans. The locus was mapp ed using complementation of the cutA mutant to partial copper toleranc e 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 produ cts of all three open reading frames were identified, when synthesized with the T7 phage promoter expression system, as polypeptides of abou t 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 t he bacterial inner membrane, and the 13 kDa polypeptide with the cytop lasmic fraction. In addition to being required for copper tolerance, c utA 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, man ganese and silver salts.