Fong S T, Camakaris J, Lee B T
Department of Genetics, University of Melbourne, Parkville, Victoria, Australia.
Mol Microbiol. 1995 Mar;15(6):1127-37. doi: 10.1111/j.1365-2958.1995.tb02286.x.
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 64Cu2+ 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 cutA 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.
推测与大肠杆菌铜耐受性有关的cutA基因座，其特征是发生了导致铜敏感性的突变。用放射性64Cu2+进行的铜积累测量表明，cutA铜敏感突变体细胞的铜摄取增加，而当cutA突变通过反式互补时铜摄取减少。利用野生型染色体片段对cutA突变体进行部分铜耐受性互补来定位该基因座。对涉及cutA的3.2 kb DNA区域进行测序和分析，发现了三个重要的开放阅读框，之前均未发表过。当用T7噬菌体启动子表达系统合成时，所有三个开放阅读框的产物都被鉴定为约50 kDa、24 kDa和13 kDa的多肽，与从DNA序列预测的大小一致。发现50 kDa和24 kDa的多肽存在于细菌内膜中，而13 kDa的多肽存在于细胞质部分。除了对铜耐受性是必需的之外，cutA还影响对锌、镍、钴和镉盐的耐受水平。cutA与lux操纵子的转录融合显示受铜、锌、镍、钴以及程度较轻的镉、锰和银盐的诱导。
