Abstract
The cad operon of Staphylococcus aureus plasmid pI258, which confers cadmium resistance, encodes a transcriptional regulator, CadC, and CadA, an ATP-coupled Cd(II) pump that is a member of the superfamily of cation-translocating P-type ATPases. The Escherichia coli homologue of CadA, termed ZntA, is a Zn(II)/Cd(II) pump. The results described in this paper support the hypothesis that ZntA and CadA are Pb(II) pumps. First, CadC is a metal-responsive repressor that responds to soft metals in the order Pb>Cd>Zn. Second, both CadA and ZntA confer resistance to Pb(II). Third, transport of 65Zn(II) in everted membrane vesicles of E. coli catalyzed by either of these two P-type ATPase superfamily members is inhibited by Pb(II).
Highlights
Bacterial metal ion resistance probably arose early in evolution due to widespread geological occurrence of metals
ZntA from Escherichia coli and CadA from plasmid pI258 of Staphylococcus aureus are both members of the superfamily of P-type cation-translocating ATPases but belong to a subgroup of soft metal transporters that includes CopA, a Cu(I) pump from Enterococcus hirae, and eukaryotic Cu(I) homeostasis proteins such as the Menkes and Wilson disease-associated proteins [1, 4, 5]
The zntA-disrupted strain of E. coli exhibited hypersensitivity to Pb(II) that was complemented by cadA, indicating that both soft metal-translocating P-type ATPases are essential for Pb(II) resistance in bacteria
Summary
Cells were grown overnight, diluted 50-fold in the same medium containing metal ion salts, and incubated for 24 h at 37 °C with shaking. A 121-base pair fragment from plasmid pYPK11 containing the pI258 cad operator/promoter was amplified by PCR. The fragment was ligated into plasmid pMLB1034 that had been digested with EcoRI and BamHI, generating plasmid pYS2, in which a lacZ gene is controlled by the cad operator/promoter. In several steps the pI258 cadC gene was amplified by PCR from plasmid pYPK11 and cloned as a 0.5-kilobase pair fragment into plasmid pACYC184 under control of the T7 promoter. Overnight LB ϩ 2% glucose cultures of E. coli strains BL21(DE3) or BL21(DE3) zntA::km harboring compatible plasmids pYS2 and pYSC1 were diluted 20-fold into a low phosphate minimal medium [14] containing 2% glucose plus the appropriate antibiotics. Pb(OAc), ZnSO4, Cd(OAc), HgCl2, NaAsO2, Bi(NO3), CuSO4, NiCl2, or potassium anti-
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
