Cloning and characterization of cutE, a gene involved in copper transport in Escherichia coli

Abstract

The copper-sensitive/temperature-sensitive phenotype of the Escherichia coli cutE mutant has been complemented by cloning wild-type genomic DNA into the plasmid vector pACYC184 and selecting transformants on medium containing 4 mM copper sulfate and chloramphenicol. One of these complementing clones, designated pCUT1, contained a 5.6-kb BamHI fragment. This recombinant plasmid transformed cutE, allowing wild-type growth of transformants on medium containing copper sulfate. Complementation of copper sensitivity was assessed by comparing both cell survival at increased copper levels and the results of 64Cu accumulation assays. An EcoRI subclone, 2.3 kb in size, was also shown to complement cutE when cloned in both medium- and high-copy-number vectors and was completely sequenced. This clone was mapped on the E. coli physical map at 705.70 to 707.80 kb. A series of subclones was constructed from pCUT1 and used to show that the large open reading frame of the translated sequence was essential for complementation. This open reading frame has a potential upstream promoter region, ribosome-binding site, and transcriptional terminator and encodes a putative protein of 512 amino acids that contains a region showing some homology to a putative copper-binding site.