METAL RESISTANCE AND PLASMID DNA IN THIOBACILLUS-FERROOXIDANS

The minimal inhibitory concentrations of copper and nickel were determ ined for each of fifteen isolates of T. ferrooxidans native to a Cu/Ni tailings environment. Ten isolates were inhibited by 160 mM Cu2+ or l ess, and ten were inhibited by 160 mM Ni2+ or less. The isolates were screened for plasmid DNA using an alkaline lysis method and CCC plasmi d forms were confirmed using the Hintermann technique. Two isolates we re found to be devoid of plasmid DNA, and only one isolate contained m ore than two plasmids. Variability existed in plasmid size, although t he majority were larger than the standard pBR322 (4.3 kbp). One plasmi d was selected for further analysis using restriction endonucleases. E coRI, HindIII and KpnI all cleaved the plasmid in two locations, and P stI cleaved the plasmid in six locations. PstI-digested fragments of t he plasmid were ligated into pBR322, and the recombinant plasmids were transformed into Escherichia coli ATCC 8739. Four genetically-differe nt transformants resulted, and each was grown in media containing 2.0 mM Cu2+ and compared to the growth of a control under similar conditio ns. There was no conferred copper resistance in E. coli, although one recombinant plasmid appeared to decrease the tolerance for E. coli ATC C 8739 to Cu2+.