The copper-sensitive/temperature-sensitive phenotype of the Escherichia coli cutE mutant has been complemented by cloning wild-type genomic DNA into the plasmid vector pACYC184 and selecting transformants on medium containing 4 mM copper sulfate and chloramphenicol. One of these complementing clones, designated pCUT1, contained a 5.6-kb BamHI fragment. This recombinant plasmid transformed cutE, allowing wild-type growth of transformants on medium containing copper sulfate. Complementation of copper sensitivity was assessed by comparing both cell survival at increased copper levels and the results of 64Cu accumulation assays. An EcoRI subclone, 2.3 kb in size, was also shown to complement cutE when cloned in both medium- and high-copy-number vectors and was completely sequenced. This clone was mapped on the E. coli physical map at 705.70 to 707.80 kb. A series of subclones was constructed from pCUT1 and used to show that the large open reading frame of the translated sequence was essential for complementation. This open reading frame has a potential upstream promoter region, ribosome-binding site, and transcriptional terminator and encodes a putative protein of 512 amino acids that contains a region showing some homology to a putative copper-binding site.

The R factor pMG2 protects Pseudomonas aeruginosa against the lethal effects of ultraviolet (u.v.) and gamma irradiation, and methyl methanesulphonate and N-methyl-N'-nitro-N-nitrosoguanidine treatment. Enhanced survival occurs in strains of uvr+ rec+ (wild-type) genotype and a variety of uvr rec+ type mutants. No protection occurs in a red-type mutant. The plasmid also enhances u.v.-induced mutagenesis. These effects appear to be due to host-cell controlled plasmid-determined DNA repair function(s). Studies on P. aeruginosa strains deficient in DNA polymerase I (polA) suggest that a plasmid-determined repair resynthesis function may be responsible for increased u.v.-survival and enhanced u.v.-mutability in pMG2-containing bacteria.

Thirty-three enteric isolates from Australian (Escherichia coli only) and United Kingdom (U.K.) (Salmonella sp., Citrobacter spp., and E. coli) piggeries were characterized with respect to their copper resistance. The copper resistance phenotypes of four new Australian E. coli isolates were comparable with that of the previously studied E. coli K-12 strain ED8739(pRJ1004), in that the resistance level in rich media was high (up to 18 mM CuS04) and resistance was inducible. Copper resistance was transferable by conjugation from the new Australian isolates to E. coli K-12 recipients. DNA similarity between the new Australian isolates and the pco copper resistance determinant located on plasmid pRJ1004 was strong as measured by DNA-DNA hybridization; however, the copper resistance plasmids were nonidentical as indicated by the presence of restriction fragment length polymorphisms between the plasmids. DNA-DNA hybridization and polymerase chain reaction analysis demonstrated DNA homology between the pco determinant and DNA from the U.K. E. coli, SalmoneUa sp., and Citrobacter freundii isolates. However, the copper resistance level and inducibility were variable among the U.K. strains. Of the U.K. E. coli isolates, 1 demonstrated a high level of copper resistance, 4 exhibited intermediate resistance, and 16 showed a low level of copper resistance; all of these resistances were expressed constitutively. A single U.K. C.freundii isolate had a high level of copper resistance, inducible by subtoxic levels of copper. Transconjugants from one E. coli and one C.freundii donor, with E. coli K-12 strain UB1637 as a recipient, showed copper resistance levels and inducibility of resistance which differed from that expressed from plasmid pRJ1004. We conclude that closely related resistance determinants in nonidentical plasmids are responsible for copper resistance in enteric bacteria isolated at separate geographic locations.

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