Duncan A. Rouch

Molecular genetics and transport analysis of the copper-resistance determinant (pco) from Escherichia coli plasmid pRJ1004

The copper‐resistance determinant (pco) of Escherichia coli plasmid pRJ1004 was cloned and sequenced. Tn1000 transposon mutagenesis identified four complementation groups, mutations in any of which eliminated copper resistance. DNA sequence analysis showed that the four complementation groups contained six open reading frames, designated pcoABCDRS. The protein product sequences derived from the nucleotide sequence show close homology between this copper‐resistance system and the cop system of a plasmid pPT23D of Pseudomonas syringae pv. tomato. The PcoR and PcoS protein sequences show homology to the family of two‐component sensor/responder phosphokinase regulatory systems. A seventh reading frame (pcoE) was identified from DNA sequence data, and lies downstream of a copper‐regulated promoter. Transport assays with 64Cu(II) showed that the resistant cells containing the plasmid had reduced copper accumulation during the log phase of growth, while increased accumulation had previously been observed during stationary phase. Chromosomal mutants defective in cellular copper management were obtained and characterized. In two of these mutants pco resistance was rendered totally inactive, whilst in another two mutants pco complemented the defective genes. These data indicate that plasmid‐borne copper resistance in E. coli is linked with chromosomal systems for copper management.

Copper resistance determinants in bacteria.

Copper is an essential trace element that is utilized in a number of oxygenases and electron transport proteins, but it is also a highly toxic heavy metal, against which all organisms must protect themselves. Known bacterial determinants of copper resistance are plasmid-encoded. The mechanisms which confer resistance must be integrated with the normal metabolism of copper. Different bacteria have adopted diverse strategies for copper resistance, and this review outlines what is known about bacterial copper resistance mechanisms and their genetic regulation.

Induction of bacterial mercury- and copper-responsive promoters: Functional differences between inducible systems and implications for their use in gene-fusions for in vivo metal biosensors

SummaryWe have compared the induction by the cognate metal salts of two promoters responsible for metal-resistance gene expression in bacteria. The mercuric ion resistance promoter, PmerTPAD, of transposon Tn501 and the copper resistance promoter, PpcoE, from plasmid pRJ1004 were separately cloned to express thelacZ gene under the regulation of their normaltrans-acting elements. Thelux genes ofVibrio fischeri were also expressed from PmerTPAD. The induction of PmerTPAD gave a hypersensitive profile, as reported previously: the apparent Hill coefficient was 2.6 when using β-galactosidase activity as a measure oflacZ gene expression. In contrast, the induction of PpcoE was hyposensitive, with an apparent Hill coefficient of 0.63 for induction of β-galactosidase activity, and this may be related to the role of copper as an essential micronutrient. These response profiles suggest that transcriptional fusions of themerTPAD promoter allow the construction of strains that are suitable for detecting threshold levels of mercuric ions, but not for accurate determinations of mercuric ion concentrations across a wide range. In contrast, transcriptional fusions to thepcoE promoter are well suited to determination of the concentrations of copper salts. The comparison of induction profiles of PmerTPAD usinglacZ orlux reporter genes, show different stimulus-response curves, probably due to differing instrument sensitivities. These results have practical implications in the construction of whole cell gene-fusion biosensors for the detection and quantitation of heavy metals.