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.

Depletion of Apolipoprotein N-Acyltransferase Causes Mislocalization of Outer Membrane Lipoproteins in Escherichia coli

“…NlpE (52)), rather than to direct copper-Lnt interactions. The coding sequence of the mutant lnt (cutE) gene amplified from the copper-sensitive E. coli mutant reported by Rogers et al (22) was found to be devoid of mutations (data not shown). This suggests that the mutation in this strain is not in lnt itself but might be in the upstream ybeX promoter and, consequently, might diminish lnt expression.…”

“…Mutations in the lnt (cutE/actA) genes have been reported to confer copper sensitivity (22,51). This is an interesting observation, especially in view of the presence of a putative copperbinding site, HXXMXXM (amino acids 425-431; HFQMARM in E. coli Lnt (22)), in the large, well conserved periplasmic loop of Lnt Ec between transmembrane segments 5 and 6.…”

“…Ec Is Essential in E. coli-To study the consequences of Lnt Ec depletion in E. coli, a conditional mutant was constructed in which the chromosomal lnt Ec gene (originally called cutE (22), see Introduction) is expressed only from the arabinoseinducible AraC-dependent promoter p araB . The lnt Ec gene is located in an operon downstream from the ybeX gene, whose function is unknown.…”

“…Typhimurium through studies of a temperature-sensitive mutant in which apoLpp (lacking the N-acyl group) accumulated at the nonpermissive temperature (21). The lnt gene E. coli (initially called cutE and referred to here as lnt Ec to reflect its known function and to distinguish it from the S. enterica gene, lnt Se ) was independently identified in studies of a copper-sensitive mutant (22).…”

A Salmonella typhimurium genetic locus which confers copper tolerance on copper-sensitive mutants of Escherichia coli

“…In addition, a regulatory gene, cutR, was identified (6). The cutA locus (13) and the cutC (16), cutE (18,31,32), and cutF (16) genes have been cloned and sequenced, and they are located at 94 min (4,360.5 kb), 42.18 min (1,956.54 kb), 14.84 min (688.56 kb), and 4.64 min (215.26 kb) on the E. coli chromosome, respectively (5). The cutA locus consists of two operons; one operon contains a single open reading frame (ORF) that encodes a cytoplasmic protein of 13 kDa (CutA1), and the other operon is comprised of two genes encoding 50-kDa (CutA2) and 24-kDa (CutA3) inner membrane proteins (13).…”