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Pb(II)-translocating P-type ATPases

1998; Elsevier BV; Volume: 273; Issue: 49 Linguagem: Inglês

10.1074/jbc.273.49.32614

1083-351X

Christopher Rensing, Yan V. Sun, Bharati Mitra, Barry P. Rosen,

Drug Transport and Resistance Mechanisms

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 colihomologue 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). 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 colihomologue 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). polymerase chain reaction 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. 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 (1Rensing C. Rosen B.P. Molecular Biology and Toxicology of Metals.in: Koropatnik D.J. Zalups R.K. Taylor and Francis, London1998Google Scholar, 2Rosen B.P. Metalloproteins.in: Higgins S.J. Ballou D.P. 34. Portland Press, Ltd., London1998Google Scholar) 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 (3Dey S. Rosen B.P. Georgopapadakou N.H. Drug Transport in Antimicrobial and Anticancer Chemotherapy. Marcel Dekker, Inc., New York1995: 103-132Google Scholar). 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 fromEnterococcus hirae, and eukaryotic Cu(I) homeostasis proteins such as the Menkes and Wilson disease-associated proteins (1Rensing C. Rosen B.P. Molecular Biology and Toxicology of Metals.in: Koropatnik D.J. Zalups R.K. Taylor and Francis, London1998Google Scholar,4Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (415) Google Scholar, 5Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (415) Google Scholar). ZntA has been shown to catalyze ATP-dependent transport of Zn(II) and Cd(II) (6Rensing C. Mitra B. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14326-14331Crossref PubMed Scopus (340) Google Scholar), and CadA has been shown to transport Cd(II) (7Tsai K.J. Yoon K.P. Lynn A.R. J. Bacteriol. 1992; 174: 116-121Crossref PubMed Scopus (83) Google Scholar). Both have been shown to confer resistance to cadmium and zinc ions (8Nucifora G. Chu L. Misra T.K. Silver S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3544-3548Crossref PubMed Scopus (288) Google Scholar, 9Beard S.J. Hashim R. Membrillo-Hernandez J. Hughes M.N. Poole R.K. Mol. Microbiol. 1997; 25: 883-891Crossref PubMed Scopus (158) Google Scholar, 10Rensing, C., Mitra, B., and Rosen, B. P. (1998) Biochem. Cell Biol., in pressGoogle Scholar). The pI258 cadCA operon is regulated at the transcriptional level by the product of thecadC gene, which encodes the 122-residue CadC repressor (11Yoon K.P. Silver S. J. Bacteriol. 1991; 173: 7636-7642Crossref PubMed Google Scholar, 12Corbisier P. Ji G. Nuyts G. Mergeay M. Silver S. FEMS Microbiol. Lett. 1993; 110: 231-238Crossref PubMed Scopus (1) Google Scholar, 13Endo G. Silver S. J. Bacteriol. 1995; 177: 4437-4441Crossref PubMed Google Scholar).In this report, we show that CadC repression of the cadpromoter is relieved upon addition of soft metals, with the order of effectiveness Pb(II) > Cd(II) > Zn(II). In E. coli Zn(II) responsiveness could be observed only in a zntA-disrupted strain. The zntA-disrupted strain of E. coliexhibited hypersensitivity to Pb(II) that was complemented bycadA, 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 orcadA exhibited ATP-dependent65Zn(II) accumulation. Since no radioisotopes of Pb(II) are available, direct transport of Pb(II) was not assayed. However, Pb(II) inhibited 65Zn(II) transport, indicating that Pb(II) is a substrate of the two P-type ATPases. These results support the concept that ZntA and CadA are Pb(II) pumps with physiological functions that include to provide resistance to environmental lead.DISCUSSIONIn humans chronic exposure to low levels of lead may cause neurological, reproductive, and developmental problems. Lead exposure is especially harmful to children, and nearly one million American children below the age of 5 years have blood-lead levels that exceed those considered as elevated by the Centers for Disease Control and Prevention (22Environmental Protection Agency Federal Register. 1998; 63: 30301-30356Google Scholar). Even though lead affects virtually every organ and tissue in the body, little is known about the routes of lead ion uptake and extrusion. Even less is known about Pb(II)-regulated gene transcription, and there are no genetic markers for lead exposure. We have undertaken a study of Pb(II)-responsive genes and transporters. The CadC repressor and CadA/ZntA pumps represent the first proteins demonstrated to have a physiological function that includes providing the host organism with a protective response to environmental lead stress.CadA and ZntA are members of the superfamily of P-type cation-translocating ATPases, but belong to a group of soft metal transporters that includes bacterial enzymes such as the CopA Cu(I) pump (23Odermatt A. Suter H. Krapf R. Solioz M. Ann. N. Y. Acad. Sci. 1992; 671: 484-486Crossref PubMed Scopus (81) Google Scholar), the yeast CCC2 Cu(I) pump (24Fu D. Beeler T.J. Dunn T.M. Yeast. 1995; 11: 283-292Crossref PubMed Scopus (142) Google Scholar), and the human Cu(I)-transporting ATPases such as MNK (25Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1208) Google Scholar) and WND (26Bull P.C. Thomas G.R. Rommens J.M. Forbes J.R. Cox D.W. Nat. Genet. 1993; 5: 327-337Crossref PubMed Scopus (1686) Google Scholar). The soft metal pumps can be further subdivided into the Cu(I)/Ag(I)-translocating ATPases and the Zn(II)/Cd(II)/Pb(II) ATPases (1Rensing C. Rosen B.P. Molecular Biology and Toxicology of Metals.in: Koropatnik D.J. Zalups R.K. Taylor and Francis, London1998Google Scholar). While none of these proteins has yet been demonstrated to catalyze ATP hydrolysis, several have been shown to have properties consistent with being cation-translocating ATPases. First, transport requires ATP and is inhibited by orthovanadate, a classical inhibitor of P-type ATPases (5Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (415) Google Scholar). Second, P-type ATPases form a β-acylphosphate intermediate, and several soft metal pumps have been shown to form these intermediates (27Tsai K.J. Linet A.L. Arch. Biochem. Biophys. 1993; 305: 267-270Crossref PubMed Scopus (40) Google Scholar, 28Solioz M. Camakaris J. FEBS Lett. 1997; 412: 165-168Crossref PubMed Scopus (21) Google Scholar).CadA had been shown to catalyze ATP-coupled, vanadate-sensitive Cd(II) transport (7Tsai K.J. Yoon K.P. Lynn A.R. J. Bacteriol. 1992; 174: 116-121Crossref PubMed Scopus (83) Google Scholar) and to form a phosphoenzyme intermediate (27Tsai K.J. Linet A.L. Arch. Biochem. Biophys. 1993; 305: 267-270Crossref PubMed Scopus (40) Google Scholar). ZntA has been shown to transport both Zn(II) and Cd(II) in an ATP-requiring, vanadate-sensitive reaction (6Rensing C. Mitra B. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14326-14331Crossref PubMed Scopus (340) Google Scholar). In this paper we report that CadA also transports Zn(II). Despite the unavailability of lead isotopes for direct transport assays, the results described here are consistent with Pb(II) transport catalyzed by members of this subgroup of P-type ATPases. First, Pb(II) induces expression of CadA (Fig. 1). Second, both ZntA and CadA confer Pb(II) resistance (Fig. 2). Third, Pb(II) inhibits 65Zn(II) transport by both ZntA and CadA (Fig. 3, A and B). Fourth, inhibition of ZntA activity by Pb(II) was essentially identical to inhibition by Cd(II), a known pump substrate (Fig. 3 C).Copper pumps are widely distributed in nature, and genetic diseases such as Menkes and Wilsons result from mutations in the genes for these pumps (25Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1208) Google Scholar, 26Bull P.C. Thomas G.R. Rommens J.M. Forbes J.R. Cox D.W. Nat. Genet. 1993; 5: 327-337Crossref PubMed Scopus (1686) Google Scholar). We predict that Zn(II)/Cd(II)/Pb(II) P-type ATPases exist in humans, and it is not unreasonable to expect that there are diseases related to defects in the genes for these pumps. Elucidation of these bacterial model systems may also lead to the development of biomarkers for lead exposure and susceptibility in humans. 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 (1Rensing C. Rosen B.P. Molecular Biology and Toxicology of Metals.in: Koropatnik D.J. Zalups R.K. Taylor and Francis, London1998Google Scholar, 2Rosen B.P. Metalloproteins.in: Higgins S.J. Ballou D.P. 34. Portland Press, Ltd., London1998Google Scholar) 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 (3Dey S. Rosen B.P. Georgopapadakou N.H. Drug Transport in Antimicrobial and Anticancer Chemotherapy. Marcel Dekker, Inc., New York1995: 103-132Google Scholar). 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 fromEnterococcus hirae, and eukaryotic Cu(I) homeostasis proteins such as the Menkes and Wilson disease-associated proteins (1Rensing C. Rosen B.P. Molecular Biology and Toxicology of Metals.in: Koropatnik D.J. Zalups R.K. Taylor and Francis, London1998Google Scholar,4Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (415) Google Scholar, 5Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (415) Google Scholar). ZntA has been shown to catalyze ATP-dependent transport of Zn(II) and Cd(II) (6Rensing C. Mitra B. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14326-14331Crossref PubMed Scopus (340) Google Scholar), and CadA has been shown to transport Cd(II) (7Tsai K.J. Yoon K.P. Lynn A.R. J. Bacteriol. 1992; 174: 116-121Crossref PubMed Scopus (83) Google Scholar). Both have been shown to confer resistance to cadmium and zinc ions (8Nucifora G. Chu L. Misra T.K. Silver S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3544-3548Crossref PubMed Scopus (288) Google Scholar, 9Beard S.J. Hashim R. Membrillo-Hernandez J. Hughes M.N. Poole R.K. Mol. Microbiol. 1997; 25: 883-891Crossref PubMed Scopus (158) Google Scholar, 10Rensing, C., Mitra, B., and Rosen, B. P. (1998) Biochem. Cell Biol., in pressGoogle Scholar). The pI258 cadCA operon is regulated at the transcriptional level by the product of thecadC gene, which encodes the 122-residue CadC repressor (11Yoon K.P. Silver S. J. Bacteriol. 1991; 173: 7636-7642Crossref PubMed Google Scholar, 12Corbisier P. Ji G. Nuyts G. Mergeay M. Silver S. FEMS Microbiol. Lett. 1993; 110: 231-238Crossref PubMed Scopus (1) Google Scholar, 13Endo G. Silver S. J. Bacteriol. 1995; 177: 4437-4441Crossref PubMed Google Scholar). In this report, we show that CadC repression of the cadpromoter is relieved upon addition of soft metals, with the order of effectiveness Pb(II) > Cd(II) > Zn(II). In E. coli Zn(II) responsiveness could be observed only in a zntA-disrupted strain. The zntA-disrupted strain of E. coliexhibited hypersensitivity to Pb(II) that was complemented bycadA, 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 orcadA exhibited ATP-dependent65Zn(II) accumulation. Since no radioisotopes of Pb(II) are available, direct transport of Pb(II) was not assayed. However, Pb(II) inhibited 65Zn(II) transport, indicating that Pb(II) is a substrate of the two P-type ATPases. These results support the concept that ZntA and CadA are Pb(II) pumps with physiological functions that include to provide resistance to environmental lead. DISCUSSIONIn humans chronic exposure to low levels of lead may cause neurological, reproductive, and developmental problems. Lead exposure is especially harmful to children, and nearly one million American children below the age of 5 years have blood-lead levels that exceed those considered as elevated by the Centers for Disease Control and Prevention (22Environmental Protection Agency Federal Register. 1998; 63: 30301-30356Google Scholar). Even though lead affects virtually every organ and tissue in the body, little is known about the routes of lead ion uptake and extrusion. Even less is known about Pb(II)-regulated gene transcription, and there are no genetic markers for lead exposure. We have undertaken a study of Pb(II)-responsive genes and transporters. The CadC repressor and CadA/ZntA pumps represent the first proteins demonstrated to have a physiological function that includes providing the host organism with a protective response to environmental lead stress.CadA and ZntA are members of the superfamily of P-type cation-translocating ATPases, but belong to a group of soft metal transporters that includes bacterial enzymes such as the CopA Cu(I) pump (23Odermatt A. Suter H. Krapf R. Solioz M. Ann. N. Y. Acad. Sci. 1992; 671: 484-486Crossref PubMed Scopus (81) Google Scholar), the yeast CCC2 Cu(I) pump (24Fu D. Beeler T.J. Dunn T.M. Yeast. 1995; 11: 283-292Crossref PubMed Scopus (142) Google Scholar), and the human Cu(I)-transporting ATPases such as MNK (25Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1208) Google Scholar) and WND (26Bull P.C. Thomas G.R. Rommens J.M. Forbes J.R. Cox D.W. Nat. Genet. 1993; 5: 327-337Crossref PubMed Scopus (1686) Google Scholar). The soft metal pumps can be further subdivided into the Cu(I)/Ag(I)-translocating ATPases and the Zn(II)/Cd(II)/Pb(II) ATPases (1Rensing C. Rosen B.P. Molecular Biology and Toxicology of Metals.in: Koropatnik D.J. Zalups R.K. Taylor and Francis, London1998Google Scholar). While none of these proteins has yet been demonstrated to catalyze ATP hydrolysis, several have been shown to have properties consistent with being cation-translocating ATPases. First, transport requires ATP and is inhibited by orthovanadate, a classical inhibitor of P-type ATPases (5Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (415) Google Scholar). Second, P-type ATPases form a β-acylphosphate intermediate, and several soft metal pumps have been shown to form these intermediates (27Tsai K.J. Linet A.L. Arch. Biochem. Biophys. 1993; 305: 267-270Crossref PubMed Scopus (40) Google Scholar, 28Solioz M. Camakaris J. FEBS Lett. 1997; 412: 165-168Crossref PubMed Scopus (21) Google Scholar).CadA had been shown to catalyze ATP-coupled, vanadate-sensitive Cd(II) transport (7Tsai K.J. Yoon K.P. Lynn A.R. J. Bacteriol. 1992; 174: 116-121Crossref PubMed Scopus (83) Google Scholar) and to form a phosphoenzyme intermediate (27Tsai K.J. Linet A.L. Arch. Biochem. Biophys. 1993; 305: 267-270Crossref PubMed Scopus (40) Google Scholar). ZntA has been shown to transport both Zn(II) and Cd(II) in an ATP-requiring, vanadate-sensitive reaction (6Rensing C. Mitra B. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14326-14331Crossref PubMed Scopus (340) Google Scholar). In this paper we report that CadA also transports Zn(II). Despite the unavailability of lead isotopes for direct transport assays, the results described here are consistent with Pb(II) transport catalyzed by members of this subgroup of P-type ATPases. First, Pb(II) induces expression of CadA (Fig. 1). Second, both ZntA and CadA confer Pb(II) resistance (Fig. 2). Third, Pb(II) inhibits 65Zn(II) transport by both ZntA and CadA (Fig. 3, A and B). Fourth, inhibition of ZntA activity by Pb(II) was essentially identical to inhibition by Cd(II), a known pump substrate (Fig. 3 C).Copper pumps are widely distributed in nature, and genetic diseases such as Menkes and Wilsons result from mutations in the genes for these pumps (25Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1208) Google Scholar, 26Bull P.C. Thomas G.R. Rommens J.M. Forbes J.R. Cox D.W. Nat. Genet. 1993; 5: 327-337Crossref PubMed Scopus (1686) Google Scholar). We predict that Zn(II)/Cd(II)/Pb(II) P-type ATPases exist in humans, and it is not unreasonable to expect that there are diseases related to defects in the genes for these pumps. Elucidation of these bacterial model systems may also lead to the development of biomarkers for lead exposure and susceptibility in humans. In humans chronic exposure to low levels of lead may cause neurological, reproductive, and developmental problems. Lead exposure is especially harmful to children, and nearly one million American children below the age of 5 years have blood-lead levels that exceed those considered as elevated by the Centers for Disease Control and Prevention (22Environmental Protection Agency Federal Register. 1998; 63: 30301-30356Google Scholar). Even though lead affects virtually every organ and tissue in the body, little is known about the routes of lead ion uptake and extrusion. Even less is known about Pb(II)-regulated gene transcription, and there are no genetic markers for lead exposure. We have undertaken a study of Pb(II)-responsive genes and transporters. The CadC repressor and CadA/ZntA pumps represent the first proteins demonstrated to have a physiological function that includes providing the host organism with a protective response to environmental lead stress. CadA and ZntA are members of the superfamily of P-type cation-translocating ATPases, but belong to a group of soft metal transporters that includes bacterial enzymes such as the CopA Cu(I) pump (23Odermatt A. Suter H. Krapf R. Solioz M. Ann. N. Y. Acad. Sci. 1992; 671: 484-486Crossref PubMed Scopus (81) Google Scholar), the yeast CCC2 Cu(I) pump (24Fu D. Beeler T.J. Dunn T.M. Yeast. 1995; 11: 283-292Crossref PubMed Scopus (142) Google Scholar), and the human Cu(I)-transporting ATPases such as MNK (25Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1208) Google Scholar) and WND (26Bull P.C. Thomas G.R. Rommens J.M. Forbes J.R. Cox D.W. Nat. Genet. 1993; 5: 327-337Crossref PubMed Scopus (1686) Google Scholar). The soft metal pumps can be further subdivided into the Cu(I)/Ag(I)-translocating ATPases and the Zn(II)/Cd(II)/Pb(II) ATPases (1Rensing C. Rosen B.P. Molecular Biology and Toxicology of Metals.in: Koropatnik D.J. Zalups R.K. Taylor and Francis, London1998Google Scholar). While none of these proteins has yet been demonstrated to catalyze ATP hydrolysis, several have been shown to have properties consistent with being cation-translocating ATPases. First, transport requires ATP and is inhibited by orthovanadate, a classical inhibitor of P-type ATPases (5Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (415) Google Scholar). Second, P-type ATPases form a β-acylphosphate intermediate, and several soft metal pumps have been shown to form these intermediates (27Tsai K.J. Linet A.L. Arch. Biochem. Biophys. 1993; 305: 267-270Crossref PubMed Scopus (40) Google Scholar, 28Solioz M. Camakaris J. FEBS Lett. 1997; 412: 165-168Crossref PubMed Scopus (21) Google Scholar). CadA had been shown to catalyze ATP-coupled, vanadate-sensitive Cd(II) transport (7Tsai K.J. Yoon K.P. Lynn A.R. J. Bacteriol. 1992; 174: 116-121Crossref PubMed Scopus (83) Google Scholar) and to form a phosphoenzyme intermediate (27Tsai K.J. Linet A.L. Arch. Biochem. Biophys. 1993; 305: 267-270Crossref PubMed Scopus (40) Google Scholar). ZntA has been shown to transport both Zn(II) and Cd(II) in an ATP-requiring, vanadate-sensitive reaction (6Rensing C. Mitra B. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14326-14331Crossref PubMed Scopus (340) Google Scholar). In this paper we report that CadA also transports Zn(II). Despite the unavailability of lead isotopes for direct transport assays, the results described here are consistent with Pb(II) transport catalyzed by members of this subgroup of P-type ATPases. First, Pb(II) induces expression of CadA (Fig. 1). Second, both ZntA and CadA confer Pb(II) resistance (Fig. 2). Third, Pb(II) inhibits 65Zn(II) transport by both ZntA and CadA (Fig. 3, A and B). Fourth, inhibition of ZntA activity by Pb(II) was essentially identical to inhibition by Cd(II), a known pump substrate (Fig. 3 C). Copper pumps are widely distributed in nature, and genetic diseases such as Menkes and Wilsons result from mutations in the genes for these pumps (25Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1208) Google Scholar, 26Bull P.C. Thomas G.R. Rommens J.M. Forbes J.R. Cox D.W. Nat. Genet. 1993; 5: 327-337Crossref PubMed Scopus (1686) Google Scholar). We predict that Zn(II)/Cd(II)/Pb(II) P-type ATPases exist in humans, and it is not unreasonable to expect that there are diseases related to defects in the genes for these pumps. Elucidation of these bacterial model systems may also lead to the development of biomarkers for lead exposure and susceptibility in humans. We thank Professor Simon Silver of the University of Illinois School of Medicine for plasmid pYPK11 and for valuable discussions. We thank Dr. Kan-Jen Tsai of Chung Shan Medical and Dental College for plasmid pKJ3.