In vivo andin vitro cloning and phenotype characterization of tellurite resistance determinant conferred by plasmid pTE53 of a clinical isolate ofEscherichia coli

• Published in: Folia microbiologica 1998-12, Vol.43 (6), p.589-599
• Main Authors: Burian, J., Tu, Nguyen, Kl'učár, L'., Guller, L., Lloyd-Jones, G., Stuchlík, S., Fejdi, P., Siekel, P., Turna, J.
• Format: Article
• Language: English
• Subjects: Accumulation; Bacteria; Cloning; Cloning vectors; Colonies; Crystals; Diffraction patterns; Efflux; Electron microscopy; Genotype & phenotype; In vivo methods and tests; Inactivation; Lysogens; Phenotypes; Potassium; Potassium tellurite; Tellurite; Tellurium; Tellurium dioxide; X-ray diffraction
• ISSN: 0015-5632; 1874-9356
• DOI: 10.1007/BF02816374

A determinant encoding resistance against potassium tellurite (Ter) was discovered in a clinical isolate ofEscherichia coli strain KL53. The strain formed typical black colonies on solid LB medium with tellurite. The determinant was located on a large conjugative plasmid designated 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 JCPDS 4-0554 (Joint Committee on Powder Diffraction Standards) reference data confirmed that these crystals were pure tellurium crystals. In common with other characterized Ter determinants, accumulation studies with radioactively labeled tellurite showed that reduced uptake of tellurite did not contribute to the resistance mechanism. Tellurite accumulation rates forE. coli strain AB1157 harboring pTE53 were twice higher than for the plasmid-free host strain. In addition, no efflux mechanism was detected. The potassium tellurite resistance determinant of plasmid pTE53 was cloned using bothin vitro andin vivo techniques in low-copy-number vectors pACYC184 and mini-Mu derivative pPR46. Cloning of the functional Ter determinant into high-copy cloning vectors pTZ19R and mini-Mu derivatives pBEf and pJT2 was not successful. Duringin vivo cloning experiments, clones with unusual “white colony” phenotypes were found on solid LB with tellurite. All these clones were Mucts62 lysogens. Their tellurite resistance levels were in the same order as the wild type strains. Clones with the “white” phenotype had a 3.6 times lower content of tellurium than the tellurite-reducing strain. Transformation of a “white” mutant with a recombinant pACYC184 based Ter plasmid did not change the phenotype. However, when one clone was cured from Mucts62 the “white” phenotype reverted to the wild-type “black” phenotype. It was suggested that the “white” phenotype was the result of an insertional inactivation of an unknown chromosomal gene by Mucts62, which reduced the tellurite uptake.