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 pR J1004 was cloned and sequenced. Tn1000 transposon mutagenesis identifi ed four complementation groups, mutations in any of which eliminated c opper resistance. DNA sequence analysis showed that the four complemen tation groups contained six open reading frames, designated pco-ABCDRS . The protein product sequences derived from the nucleotide sequence s how close homology between this copper-resistance system and the cop s ystem of a plasmid pPT23D of Pseudomonas syringae pv. tomato. The PcoR and PcoS protein sequences show homology to the family of two-compone nt sensor/responder phosphokinase regulatory systems. A seventh readin g frame (pcoE) was identified from DNA sequence data, and lies downstr eam of a copper-regulated promoter. Transport assays with Cu-64(II) sh owed that the resistant cells containing the plasmid had reduced coppe r accumulation during the log phase of growth, while increased accumul ation had previously been observed during stationary phase. Chromosoma l mutants defective in cellular copper management were obtained and ch aracterized. In two of these mutants pco resistance was rendered total ly inactive, whilst in another two mutants pco complemented the defect ive genes. These data indicate that plasmid-borne copper resistance in E. coli is linked with chromosomal systems for copper management.