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In vivo complementation and site-specific mutagenesis of the tellurite resistance determinant kilAteIAB from IncP plasmid RK2Te^r

Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
Department of Medical Microbiology and Infectious Diseases, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

^*Author for correspondence: Diane E. Taylor Tel: +1 403 492 4777. Fax: +1 403 492 7521.

ABSTRACT

The IncP plasmid RK2 carries a cryptic tellurite resistance (Te^r) determinant. This determinant from RK2Te^r has been previously cloned into a pUC8 plasmid (pDT1558). The Te^r 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(Te^s)]. 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.

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