In vivo complementation and site-specific mutagenesis of the tellurite resistance determinant kilAteIAB from IncPα plasmid RK2Ter

摘要

The IncPα plasmid RK2 carries a cryptic tellurite resistance (Ter) determinant. This determinant from RK2Ter has been previously cloned into a pUC8 plasmid (pDT1558). The Ter determinant identified as the kilA locus comprises an operon of three genes: kilA, telA and telB [also referred to as klaA, klaB and klaC on RK2(Tes)]. Each of the genes was subcloned into the expression vector pJF118EH behind an inducible tac-promotor using PCR. The PCR primers were used to engineer an efficient ribosome-binding site and adjacent sequence to improve protein expression. Expression plasmids were modified by inclusion of different resistance markers for selection during complementation. The tellurite-resistance phenotype was studied with the overexpressing plasmids. The study provides further evidence that all three genes within the kilAteIAB operon are required for cells to show resistance to potassium tellurite. Additionally, site-directed mutagenesis was carried out on the two cysteine residues in TelB. Changing either Cys125 or Cys132 to a Ser or Ala residue decreased the resistance mediated by the operon. These mutants demonstrate the requirement of cysteine residues within TelB for expression of tellurite resistance.