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Cloning and characterization of cutE, a gene involved in copper transport in Escherichia coli
journal contributionposted on 2023-02-07, 03:11 authored by SD Rogers, MR Bhave, Julian MercerJulian Mercer, J Camakaris, BTO Lee
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.
JournalJournal of Bacteriology
PublisherAMER SOC MICROBIOLOGY
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Science & TechnologyLife Sciences & BiomedicineMicrobiologyNUCLEOTIDE-SEQUENCEMERCURY RESISTANCECONSTRUCTIONTRANSCRIPTIONBINDINGPROTEINMERRORGANIZATIONCHROMOSOMEEXPRESSIONAcetyltransferasesAmino Acid SequenceBacterial ProteinsBase SequenceBiological TransportCloning, MolecularCopperDNA, BacterialEscherichia coliEscherichia coli ProteinsGenes, BacterialGenetic Complementation TestMembrane Transport ProteinsMolecular Sequence DataRestriction MappingTemperatureGeneticsBiotechnologyBiological SciencesAgricultural and Veterinary SciencesMedical and Health Sciences