The copA gene product, a putative copper-translocating P-type ATPase, has been shown to be involved in copper resistance in Escherichia coli. The copA gene was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain was more sensitive to copper salts but not to salts of other metals, suggesting a role in copper homeostasis. The coppersensitive phenotype could be rescued by complementation by a plasmid carrying copA from E. coli or copB from Enterococcus hirae. Expression of copA was induced by salts of copper or silver but not zinc or cobalt. Everted membrane vesicles from cells expressing copA exhibited ATP-coupled accumulation of copper, presumably as Cu(I). The results indicate that CopA is a Cu(I)-translocating efflux pump that is similar to the copper pumps related to Menkes and Wilson diseases and provides a useful prokaryotic model for these human diseases.soft metal resistance ͉ Menkes ͉ Wilson disease

The first Zn(II)-translocating P-type ATPase has been identified as the product of o732, a potential gene identified in the sequencing of the Escherichia coli genome. This gene, termed zntA, was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain exhibited hypersensitivity to zinc and cadmium salts but not salts of other metals, suggesting a role in zinc homeostasis in E. coli. Everted membrane vesicles from a wild-type strain accumulated 65 Zn(II) and 109 Cd(II) by using ATP as an energy source. Transport was sensitive to vanadate, an inhibitor of P-type ATPases. Membrane vesicles from the zntA::kan strain did not accumulate those metal ions. Both the sensitive phenotype and transport defect of the mutant were complemented by expression of zntA on a plasmid.

ZntA, a soft metal-translocating P1-type ATPase from Escherichia coli, confers resistance to Pb(II), Cd(II), and Zn(II). ZntA was expressed as a histidyl-tagged protein, solubilized from membranes with Triton X-100, and purified to homogeneity. The soft metal-dependent ATP hydrolysis activity of purified ZntA was characterized. The activity was specific for Pb(II), Cd(II), Zn(II), and Hg(II), with the highest activity obtained when the metals were present as thiolate complexes of cysteine or glutathione. The maximal ATPase activity of ZntA was ϳ3 mol/(mg⅐min) obtained with the Pb(II)-thiolate complex. In the absence of thiolates, Cd(II) inhibits ZntA above pH 6, whereas the Cd(II)-thiolate complexes stimulate activity, suggesting that a metal-thiolate complex is the true substrate in vivo. These results are consistent with the physiological role of ZntA as mediator of resistance to toxic concentrations of the divalent soft metals, Pb(II), Cd(II), and Zn(II), by ATP-dependent efflux. Our results confirm that ZntA is the first Pb(II)-dependent ATPase discovered to date.

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 65 Zn(II) in everted membrane vesicles of E. coli catalyzed by either of these two P-type ATPase superfamily members is inhibited by Pb(II).Exposure to environmental sources of lead is a serious public health concern. In humans chronic lead exposure produces neurotoxicity, anemia, and kidney damage, and acute lead toxicity can be fatal. Neither the specific lead transporters nor the regulatory elements that control the expression of the transporter genes have been identified. As models for human metal toxicity, we have been characterizing transporters for toxic metals and their genetic regulation (1, 2) and report here the identification of two P-type ATPases that are responsible for Pb(II) extrusion and resistance in bacteria.Bacterial metal ion resistance probably arose early in evolution due to widespread geological occurrence of metals. Bacterial cells have chromosomally and plasmid-encoded mechanisms for extrusion of antimicrobial substances, including toxic soft metals (3). While the ionic forms of some of these metals such as zinc and copper are essential for all organisms, all of these ions are toxic in excess. 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). ZntA has been shown to catalyze ATP-dependent transport of Zn(II) and Cd(II) (6), and CadA has been shown to transport Cd(II) (7). Both have been shown to confer resistance to cadmium and zinc ions (8 -10). The pI258 cadCA operon is regulated at the transcriptional level by the product of the cadC gene, which encodes the 122-residue CadC repressor (11-13).In this report, we show that CadC repression of the cad promoter is relieved upon addition of soft metals, with the order of effectiveness Pb(II) Ͼ Cd(II) Ͼ Zn(II). In E. coli Zn(II) responsiveness could be observed only in a zntA-disrupted strain. 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. Everted membrane vesicles from cells expressing either zntA or cadA exhibited ATPdependent 65 Zn(II) accumulation. Since no radioisotopes of ...

In a search for genes responsible for the accumulation of antimonite in Escherichia coli, TnphoA was used to create a pool of random insertional mutants, from which one antimonite-resistant mutant was isolated. Sequence analysis showed that the TnphoA insertion was located in the glpF gene, coding for the glycerol facilitator GlpF. The mutant was shown to be defective in polyol transport by GlpF. These results suggest that in solution Sb(III) is recognized as a polyol by the glycerol facilitator.Resistance to antimonite [Sb(III)], arsenite [As(III)], and arsenate [As(V)] is encoded by both plasmid-borne and chromosomal arsenical resistance (ars) operons (2, 3, 10). These operons encode transport systems that extrude the toxic metalloids, thus lowering the intracellular concentration and producing resistance (2,8,13). Arsenate is accumulated by both the Pit and Pst phosphate transport systems, although the Pit system may be responsible for the majority of uptake (13). The pathways for antimonite and arsenite accumulation have not been defined. In an attempt to identify the cellular transporters for the metalloid salts, Escherichia coli AW3110 (2) was subjected to random TnphoA mutagenesis (7).Random TnphoA-mediated mutagenesis to obtain Sb(III)-resistant mutant. Strains, plasmids, and phage used in this study are given in Table 1. E. coli AW3110, which lacks the chromosomal ars operon (2), was infected with b221 rex::TnphoA cI857 (7). Cells were plated on Luria-Bertani (LB) agar containing 1 mM either potassium antimonyl tartrate or sodium arsenite plus 35 g of kanamycin/ml. One mutant was obtained on antimonite-containing agar. No arsenite-resistant mutants were isolated. Colonies of the antimoniteresistant strain, OSBR1, were white on plates containing 20 g of 5-bromo-4-chloro-3-indolyl phosphate (XP)/ml. This could indicate an intracellular localization of the alkaline phosphatase portion of the moiety, an out-of-frame fusion, or fusion with the reading frames in the opposite orientation.Antimonite resistance is due to a single TnphoA insertion. To determine whether the mutant strain carried the TnphoA insertion in a single locus, the kanamycin resistance phenotype was transduced back into strain AW3110 by generalized transduction with P1 phage. All transductants were Sb(III) resistant. Southern blot hybridization was performed with BamHIdigested genomic DNA of OSBR1, with DNA from AW3110 as a control, by using a 485-bp TnphoA-specific probe. The result of the Southern blot confirmed the existence of only a single TnphoA insertion (data not shown).The TnphoA insertion is located in the glpF gene. Since there is no BamHI site between the site of fusion in TnphoA and the kanamycin phosphotransferase gene, and there is a BamHI site immediately following the 3Ј end of the kanamycin phosphotransferase gene (5), chromosomal DNA of OSBR1 was digested with BamHI. The portion of DNA proximal to the fusion junction was cloned into the unique BamHI site of pUC18; the transformed colonies were screened for Km r . The resu...