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

Abstract

Three distinct clones from a Salmonella typhimurium genomic library were identified which suppressed the copper-sensitive (Cu(s)) phenotype of cutF mutants of Escherichia coli. One of these clones, pCUTFS2, also increased the copper tolerance of cutA, -C, and -E mutants, as well as that of a lipoprotein diacylglyceryl transferase (lgt) mutant of E. coli. Characterization of pCUTFS2 revealed that the genes responsible for suppression of copper sensitivity (scs) reside on a 4.36-kb DNA fragment located near 25.4 min on the S. typhimurium genome. Sequence analysis of this fragment revealed four open reading frames (ORF120, ORF627, ORF207, and ORF168) that were organized into two operons. One operon consisted of a single gene, scsA (ORF120), whereas the other operon contained the genes scsB (ORF627), scsC (ORF207), and scsD (ORF168). Comparison of the deduced amino acid sequences of the predicted gene products showed that ScsB, ScsC, and ScsD have significant homology to thiol-disulfide interchange proteins (CutA2, DipZ, CycZ, and DsbD) from E. coli and Haemophilus influenzae, to an outer membrane protein (Com1) from Coxiella burnetii, and to thioredoxin and thioredoxin-like proteins, respectively. The two operons were subcloned on compatible plasmids, and complementation analyses indicated that all four proteins are required for the increased copper tolerance of E. coli mutants. In addition, the scs locus also restored lipoprotein modification in lgt mutants of E. coli. Sequence analyses of the S. typhimurium scs genes and adjacent DNAs revealed that the scs locus is flanked by genes with high homology to the cbpA (predicted curved DNA-binding protein) and agp (acid glucose phosphatase) genes of E. coli located at 22.90 min (1,062.07 kb) and 22.95 min (1,064.8 kb) of the E. coli chromosome, respectively. However, examination of the E. coli chromosome revealed that these genes are absent at this locus and no evidence has thus been obtained for the occurrence of the scs locus elsewhere on the genome.