In vivo complementation and site-specific mutagenesis of the tellurite resistance determinant kilAtelAB from IncP alpha plasmid RK2Ter.
The IncP alpha 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 kilAtelAB 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.
Turner
R J
RJ
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Weiner
J H
JH
Taylor
D E
DE
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003545:Cysteine; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon; D016133:Polymerase Chain Reaction; D011815:R Factors / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D013691:Tellurium / Q000494:pharmacology*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C067119:kilA protein, E coli; C067121:KlaA protein, E coli; C067122:telB protein, bacterial; C026899:potassium tellurate(IV); D003545:Cysteine; D013691:Tellurium