Cloning andCharacterization ofcutE, aGeneInvolved in Copper Transport inEscherichia coli

phenotype oftheEscherichia coli cutEmutanthasbeencomplemented bycloning wild-type genomic DNAinto theplasmid vector pACYC184 andselecting transformants on mediumcontaining 4mM copper sulfate andchloramphenicol. Oneofthese complementing clones, designated pCUT1,contained a5.6-kb BamHIfragment. Thisrecombinant plasmid transformed cutE, allowing wild-type growth oftransformants onmediumcontaining copper sulfate. Complementation ofcopper sensitivity was assessed bycomparing bothcell survival atincreased copper levels andtheresults of"Cuaccumulation assays. AnEcoRIsubclone, 2.3kbinsize, wasalso showntocomplement cutEwhencloned inbothmedium- and high-copy-number vectors andwascompletely sequenced. Thisclone wasmapped ontheE.coli physical map at705.70 to707.80 kb.Aseries ofsubclones wasconstructed frompCUT1andusedtoshowthat thelarge open reading frameofthetranslated sequence wasessential forcomplementation. Thisopenreading framehasa potential upstream promoter region, ribosome-binding site, andtranscriptional terminator andencodes a putative protein of512aminoacids that contains aregion showing somehomology toaputative copper-binding site.