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The cloning and DNA sequence of the gene for the glutathione‐regulated potassium‐efflux system KefC of Escherichia coli
Author(s) -
Munro A. W.,
Ritchie G. Y.,
Lamb A. J.,
Douglas R. M.,
Booth I. R.
Publication year - 1991
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1991.tb00731.x
Subject(s) - biology , nucleic acid sequence , gene , microbiology and biotechnology , peptide sequence , open reading frame , biochemistry , escherichia coli , structural gene , molecular cloning , gene product , gene expression
Summary The kefC gene of Escherichia coli encodes a potassium‐efflux system that is regulated by glutathione metabolites. The close proximity of the E. coli kefC gene to the folA gene, encoding dihydrofolate reductase, has been utilized to clone the structural gene for the system from a Clarke‐Carbon plasmid. The cloned gene has been refined to a region of DNA approximately 2.1 kb in length using exonuclease III‐generated deletions and random Muc/M1734 ( IacZ ) insertions. The direction of transcription has been deduced from the orientation of the Mu insertions in the cloned DNA. A hybrid protein consisting of approximately two thirds of the KefC protein fused to β‐galactosidase has been shown to be membrane‐located. The DNA sequence of the gene has been determined and an open reading frame of 1.86kb has been located which could encode a protein of 620 amino acids (7901 0Da). Using the T7 expression system a membrane protein, of apparent molecular mass 55–60 kDa, has been shown to be encoded by the kefC gene. The predicted protein sequence shows a highly hydrophobic amino‐terminus and a strongly hydrophilic carboxy‐terminus, Comparison of the amino acid sequence of the kefC gene product with those of two glutathione‐utilizing enzymes, glyoxalase and dehalogenase, has revealed some similarities.