IN-VIVO AND IN-VITRO CLONING AND PHENOTYPE CHARACTERIZATION OF TELLURITE RESISTANCE DETERMINANT CONFERRED BY PLASMID PTE53 OF A CLINICAL ISOLATE OF ESCHERICHIA-COLI

A determinant encoding resistance against potassium tellurite (Te-r) w as discovered in a clinical isolate of Escherichia coli strain KL53. T he strain formed typical black colonies on solid LB medium with tellur ite. The determinant was located on a large conjugative plasmid design ated pTE53. Electron-dense particles were observed in cells harboring pTE53 by electron microscopy. X-Ray identification analysis identified these deposits as elemental tellurium and X-ray diffraction analysis showed patterns typical of crystalline structures. Comparison with JCP DS 4-0554 (Joint Committee on Powder Diffraction Standards) reference data confirmed that these crystals were pure tellurium crystals. In co mmon with other characterized Te-r determinants, accumulation studies with radioactively labeled tellurite showed that reduced uptake of tel lurite did not contribute to the resistance mechanism. Tellurite accum ulation rates for E. coli strain AB1157 harboring pTE53 were twice hig her than for the plasmid-free host strain. In addition, no efflux mech anism was detected. The potassium tellurite resistance determinant of plasmid pTE53 was cloned using both in vitro and in vivo techniques in low-copy-number vectors pACYC184 and mini-Mu derivative pPR46. Clonin g of the functional Te-r determinant into high-copy cloning vectors pT Z19R and mini-Mu derivatives pBEf and pJT2 was not successful. During in vivo cloning experiments, clones with unusual ''white colony'' phen otypes were found on solid LB with tellurite. Ali these clones were Mu cts62 lysogens. Their tellurite resistance levels were in the same ord er as the wild type strains. Clones with the ''white'' phenotype had a 3.6 times lower content of tellurium than the tellurite-reducing stra in. Transformation of st ''white'' mutant with a recombinant pACYC184 based Te-r plasmid did not change the phenotype. However, when one clo ne was cured from Mucts62 the ''white'' phenotype reverted to the wild -type ''black'' phenotype. It was suggested that the ''white'' phenoty pe was the result of an insertional inactivation of an unknown chromos omal gene by Mucts62, which reduced the tellurite uptake.