Properties of Arsenite Efflux Permeases (Acr3) from Alkaliphilus metalliredigens and Corynebacterium glutamicum

Members of the Acr3 family of arsenite permeases confer resistance to trivalent arsenic by extrusion from cells, with members in every phylogenetic domain. In this study bacterial Acr3 homologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum were cloned and expressed in Esch e richia coli. Modification of a single cysteine residue that is conserved in all analyzed Acr3 homologues resulted in loss of transport activity, indicating that it plays a role in Acr3 function. The results of treatment with thiol reagents suggested that the conserved cysteine is located in a hydrophobic region of the permease. A scanning cysteine accessibility method was used to show that Acr3 has 10 transmembrane segments, and the conserved cysteine would be predicted to be in the fourth transmembrane segment. Members of the Acr3 family of arsenite permeases confer resistance to trivalent arsenic by extrusion from cells, with members in every phylogenetic domain. In this study bacterial Acr3 homologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum were cloned and expressed in Esch e richia coli. Modification of a single cysteine residue that is conserved in all analyzed Acr3 homologues resulted in loss of transport activity, indicating that it plays a role in Acr3 function. The results of treatment with thiol reagents suggested that the conserved cysteine is located in a hydrophobic region of the permease. A scanning cysteine accessibility method was used to show that Acr3 has 10 transmembrane segments, and the conserved cysteine would be predicted to be in the fourth transmembrane segment. Arsenic is a carcinogen that ranks first on the Superfund List of Hazardous Substances (www. atsdr. cdc. gov). As a consequence of its environmental ubiquity, nearly every organism, from bacteria to humans, has genes that confer resistance to arsenic (1Bhattacharjee H. Rosen B. P. Nies D. H. Silver S. Molecular Microbiology of Heavy Metals. Springer-Verlag, Inc. , New York2007: 205-219Google Scholar). The most common mechanism of arsenite resistance is efflux from cells catalyzed by members of three unrelated families of transporters. Homologues of the Mrp members of the ATP-binding cassette superfamily catalyze ATP-dependent pumping of As (III) -thiol complexes out of the cytosol. These include Mrp1 and Mrp2 in mammals that extrude As (GS) 3 into blood or bile (2Deeley R. G. Westlake C. Cole S. P. Physiol. Rev. 2006; 86: 849-899Crossref PubMed Scopus (625) Google Scholar), Ycf1p in yeast that extrudes As (GS) 3 into the vacuole (3Ghosh M. Shen J. Rosen B. P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5001-5006Crossref PubMed Scopus (339) Google Scholar), and PgpA in Leishmania that extrudes the As (III) -trypanothione complex into intracellular compartments (4Légaré D. Richard D. Mukhopadhyay R. Stierhof Y. D. Rosen B. P. Haimeur A. Papadopoulou B. Ouellette M. J. Biol. Chem. 2001; 276: 26301-26307Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). These pumps are generalized resistance pumps and are not specific for arsenite. In contrast, ArsB, the first identified member of the second family of arsenite efflux proteins, has the physiological role of conferring resistance to inorganic As (III) and Sb (III) (5Chen C. M. Misra T. K. Silver S. Rosen B. P. J. Biol. Chem. 1986; 261: 15030-15038Abstract Full Text PDF PubMed Google Scholar, 6Tisa L. S. Rosen B. P. J. Biol. Chem. 1990; 265: 190-194Abstract Full Text PDF PubMed Google Scholar). The best characterized member of the ArsB family is that encoded by the arsRDABC operon of the conjugative R-factor R773 of Escherichia coli. ArsB is widespread in bacteria and archaea. It has 12 membrane-spanning segments (7Wu J. Tisa L. S. Rosen B. P. J. Biol. Chem. 1992; 267: 12570-12576Abstract Full Text PDF PubMed Google Scholar), which is similar to members of the Major Facilitator Superfamily (8Marger M. D. Saier Jr. , M. H. Trends Biochem. Sci. 1993; 18: 13-20Abstract Full Text PDF PubMed Scopus (753) Google Scholar). It transports As (III) but has higher affinity for Sb (III). ArsB is an antiporter that catalyzes the exchange of trivalent metalloid for protons, coupling arsenite efflux to the electrochemical proton gradient (9Meng Y. L. Liu Z. Rosen B. P. J. Biol. Chem. 2004; 279: 18334-18341Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). The third arsenic resistance transporter is Acr3, which is a member of the BART (bile/arsenite/riboflavin transporter) superfamily and includes members found in bacteria, archaea, and fungi and is more widely distributed than members of the ArsB family (10Mansour N. M. Sawhney M. Tamang D. G. Vogl C. Saier Jr. , M. H. FEBS J. 2007; 274: 612-629Crossref PubMed Scopus (59) Google Scholar) (supplemental Fig. 1). Homologues have recently been identified in plant (Pteris vittata, NCBI accession number ACN65413) and animal genomes (Danio rerio, NCBI accession number XP₀01921075). Unfortunately, the literature is confused by the fact that many members of the Acr3 family are annotated as ArsB even though they exhibit no significant sequence similarity to ArsB. The first identified member of this family is encoded by the ars operon of the skin (sigK intervening) element in the chromosome of Bacillus subtilis (11Sato T. Kobayashi Y. J. Bacteriol. 1998; 180: 1655-1661Crossref PubMed Google Scholar). The membrane topology of the B. subtilis Acr3 was recently investigated using translational fusions, but the results could not distinguish between 8 and 10 transmembrane-spanning segments (TMs) 2The abbreviations used are: TMtransmembrane-spanning segmentSCAMscanning cysteine accessibility methodLBLuria brothAMS4-acetamido-4′-maleimidylstilbene-2-2′disulfonic acidBMbiotin-PE-maleimide (N′-2-N-maleimido) ethyl-N-piperazinyl-d-biotinamide hydrochloride. (12Aaltonen E. K. Silow M. Biochim. Biophys. Acta. 2008; 1778: 963-973Crossref PubMed Scopus (27) Google Scholar). Fungal members of this family include the Saccharomyces cerevisiae Acr3p metalloid efflux protein (3Ghosh M. Shen J. Rosen B. P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5001-5006Crossref PubMed Scopus (339) Google Scholar, 13Bobrowicz P. Wysocki R. Owsianik G. Goffeau A. Ułaszewski S. Yeast. 1997; 13: 819-828Crossref PubMed Scopus (187) Google Scholar). Interestingly, yeast Acr3p appears to be selective for As (III) over Sb (III), which is surprising considering the similarity in chemical properties between the two metalloids. The properties of a more distant homologue from Shewanella oneidensis was examined recently (14Xia X. Postis V. L. Rahman M. Wright G. S. Roach P. C. Deacon S. E. Ingram J. C. Henderson P. J. Findlay J. B. Phillips S. E. McPherson M. J. Baldwin S. A. Mol. Membr. Biol. 2008; 25: 691-705Crossref PubMed Scopus (18) Google Scholar). The S. oneidensis homologue confers resistance to arsenate but not arsenite. Similarly, the purified protein binds arsenate, not arsenite, indicating that this protein is not an Acr3 orthologue. transmembrane-spanning segment scanning cysteine accessibility method Luria broth 4-acetamido-4′-maleimidylstilbene-2-2′disulfonic acid biotin-PE-maleimide (N′-2-N-maleimido) ethyl-N-piperazinyl-d-biotinamide hydrochloride. Here we examined the properties of Acr3 orthologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum (supplemental Fig. 1). A. metalliredigens is a borate-tolerant Gram-positive alkaliphile and strict anaerobe that uses reduction of metals as electron acceptors (15Ye Q. Roh Y. Carroll S. L. Blair B. Zhou J. Zhang C. L. Fields M. W. Appl. Environ. Microbiol. 2004; 70: 5595-5602Crossref PubMed Scopus (124) Google Scholar). It is a novel metal-reducing bacterium that is distantly related to other commonly studied iron-reducing microorganisms. The genome of A. metalliredigens QYMF (NCBI accession number NC₀09633) contains two novel ars operons, arsR1Bacr3–1D1A1–1A1–2 and arsR2CBacr3–2D2A2–1A2–2. The two genes for the AmAcr3s were designated arsacr3 because they are both in ars operons and are controlled by ArsR repressors, even though they are not homologues of ArsB. Interestingly, both ars operons have genes for ArsD and two genes corresponding to the two homologous halves of ArsA, which we designate AmArsA1 and AmArsA2. ArsD is an arsenic chaperone that transfers As (III) to ArsA (16Lin Y. F. Walmsley A. R. Rosen B. P. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 15617-15622Crossref PubMed Scopus (140) Google Scholar), which then interacts with ArsB to extrude As (III) from the cells in an ATP-dependent manner (6Tisa L. S. Rosen B. P. J. Biol. Chem. 1990; 265: 190-194Abstract Full Text PDF PubMed Google Scholar, 17Dey S. Dou D. Rosen B. P. J. Biol. Chem. 1994; 269: 25442-25446Abstract Full Text PDF PubMed Google Scholar, 18Dey S. Dou D. Tisa L. S. Rosen B. P. Arch. Biochem. Biophys. 1994; 311: 418-424Crossref PubMed Scopus (50) Google Scholar). Whether or how Acr3 can replace ArsB in this process is a question of considerable interest. C. glutamicum is a Gram-positive soil bacterium that is used for commercial production of glutamate, lysine, and other amino acids, nucleotides, and vitamins and from which the genome sequence has been described (NCBI accession number NC₀06958). It is highly arsenic-resistant and has three genes encoding Acr3 homologues (19Ordóñez E. Letek M. Valbuena N. Gil J. A. Mateos L. M. Appl. Environ. Microbiol. 2005; 71: 6206-6215Crossref PubMed Scopus (127) Google Scholar). Two of the homologues are in ars operons regulated by ArsRs (arsR1Bacr3–1C1C1′ and arsR2Bacr3–2arsC2) and a third orphan gene (arsBacr3–3) that is not in an operon and may not be expressed to the same extent as the other two. (Again, the genes were misnamed arsB even though they encode Acr3 homologues. ) The genes for AmAcr3 and CgAcr3 from the ars1 operons of the respective species were cloned and expressed in the arsenite-hypersensitive E. coli strain AW3110, in which the chromosomal arsRBC operon had been deleted (20Carlin A. Shi W. Dey S. Rosen B. P. J. Bacteriol. 1995; 177: 981-986Crossref PubMed Scopus (310) Google Scholar). Both conferred resistance to arsenite but not arsenate or antimonite. Examination of the sequence of Acr3 homologues from many species indicates that there is conserved cysteine residues, Cys138 in AmAcr3 and Cys129 in CgAcr3 (supplemental Fig. 1). Those and other nonconserved cysteine residues were changed by mutagenesis, and substitution of only Cys138 in AmAcr3 and Cys129 in CgAcr3 led to loss of function, suggesting that the conserved cysteine residue participates in As (III) transport. A scanning cysteine accessibility method (SCAM) (21Bogdanov M. Zhang W. Xie J. Dowhan W. Methods. 2005; 36: 148-171Crossref PubMed Scopus (116) Google Scholar) was used to determine the transmembrane topology of AmAcr3. SCAM analysis is preferable to the use of gene fusions because there are minimal structural changes in the membrane protein, and the sidedness of inserted cysteines can be unambiguously determined with maleimide reagents of differing membrane permeability. A series of single cysteine mutants of AmAcr3 was constructed and the reactivity of each cysteine residue assayed. The results unambiguously demonstrate that Acr3 has 10 TMs. Strains and plasmids used are given in supplemental Tables 1 and 2. E. coli cells were grown in Luria-Bertani (LB) medium (22Sambrook J. Fritsch E. F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) at 37 °C supplemented with 100 μg/ml ampicillin, as required. Bacterial growth was monitored by measuring the A600 nm. The thiol reagents iodoacetamide, p-chloromercuribenzoate, 5, 5′-dithiobis- (2-nitrobenzoic acid), and methyl methanethiosulfonate were from Sigma. N-Ethylmaleimide was from Pierce. Biotin-PE-maleimide (N′-2-N-maleimido) ethyl-N-piperazinyl-d-biotinamide hydrochloride (BM), and 4-acetamido-4′-maleimidylstilbene-2, 2′-disulfonic acid (AMS) were from Molecular Probes (Eugene, OR). Other reagents were obtained from commercial sources. The acr3 genes were cloned by PCR using Pfu DNA polymerase according to the manufacturer's directions (Stratagene, La Jolla, CA). The oligonucleotide primers for PCR are given in supplemental Table 3. For of acr3 from A. metalliredigens in E. in which the gene is the of the and has the sequence for a was DNA from A. metalliredigens QYMF was by W. Fields (15Ye Q. Roh Y. Carroll S. L. Blair B. Zhou J. Zhang C. L. Fields M. W. Appl. Environ. Microbiol. 2004; 70: 5595-5602Crossref PubMed Scopus (124) Google Scholar). the the DNA was and with and the DNA was into A similar was used to the gene using chromosomal DNA from C. glutamicum strain (19Ordóñez E. Letek M. Valbuena N. Gil J. A. Mateos L. M. Appl. Environ. Microbiol. 2005; 71: 6206-6215Crossref PubMed Scopus (127) Google Scholar) and the corresponding primers in supplemental Table 3. The was into and The gene was then using the and the was and and into in acr3 genes from both were using a (Stratagene, La Jolla, using the primers in supplemental Table The of the was by DNA Cys129 and in CgAcr3 were changed to and and Cys138 in AmAcr3 were changed to and For the by mutagenesis, the AmAcr3 was used as the and single cysteine from the were by PCR E. 1998; PubMed Scopus Google Scholar) (supplemental Table For arsenite resistance of E. coli strain plasmids were grown in medium and into medium the of arsenite or at 37 °C with of growth at 37 A600 was SCAM analysis of the topology of AmAcr3 was by cysteines with as described (21Bogdanov M. Zhang W. Xie J. Dowhan W. Methods. 2005; 36: 148-171Crossref PubMed Scopus (116) Google Scholar) with a genes were into cells of E. coli strain which and grown in medium at 37 into and with for the A600 the cells were with a of cysteines in the the thiol was to the to a of for 10 at of was to cysteine residues in the cytosol. is and can with thiol on of the but cysteine residues in hydrophobic are not The was 10 by 1 of the same The cells were with A and in of a of 1 and The cells were by a single a at and of of was at for to the was at for 1 and the membrane obtained was in 1 of to the The was for 1 at which the was by at for 1 The was with of and with for 1 at The were with of and then in of PubMed Scopus Google Scholar) and for 10 was on AmAcr3 was by using and an For were grown to A600 1 at 37 °C with in The cells were and in a of and 1 at a of A600 the transport arsenite or was to 1 of were at the with of and For in which the cells were with of grown cells were and at A600 1 with and in of the same The cells were then at 37 and an of the same was 10 was The cells were by and in the at a of A600 reagents were as for 10 the transport by of 100 arsenite. The were with of at °C for to to and with of to a of of Arsenic and were by were in the of in using arsenic and N. Both C. glutamicum and A. metalliredigens have two operons acr3 and all exhibit considerable similarity with other Acr3 homologues (supplemental Fig. 1). is that cysteine Cys138 in AmAcr3 and Cys129 in is conserved in all homologues acr3 gene was cloned from each and expressed in E. coli strain Both CgAcr3 A and and AmAcr3 not were in arsenite resistance and to the intracellular of which the to catalyze CgAcr3 and AmAcr3 not could confer resistance or extrude Sb (III). is with the that yeast confers resistance to As (III) but not Sb (III) (3Ghosh M. Shen J. Rosen B. P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5001-5006Crossref PubMed Scopus (339) Google Scholar, R. P. Ułaszewski S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). is a considering the chemical of the two trivalent metalloids. that ArsB extrudes both trivalent and confers resistance to both indicating that there be a between ArsB and are three cysteines in AmAcr3 and was to or single mutants and the and the The of each cysteine residue was analyzed by the to confer As (III) resistance in the arsenite-hypersensitive E. coli strain and to the intracellular of As (III) The show that are for AmAcr3 function. In contrast, cells mutants and were to and to extrude arsenite, suggesting that conserved Cys138 plays a role in Acr3 function. the conserved cysteine is for Acr3 function, Cys129 and in CgAcr3 were to and and the of the on resistance and transport was assayed. with the results with only cells the Cys129 mutants of CgAcr3 arsenite and loss of transport activity, the that the conserved cysteine plays a role in Acr3 function. the for the conserved cysteine in more of As (III) was in cells of CgAcr3 and the which has only conserved Cys129 with thiol reagents The cells were first with to the membrane and then with 5, 5′-dithiobis- (2-nitrobenzoic or which are or the thiol A. 1998; PubMed Scopus Google Scholar, M. A. 1999; 274: PubMed Scopus Google Scholar, N. S. S. T. S. A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). in cells CgAcr3 and the was not by 5, 5′-dithiobis- (2-nitrobenzoic or In contrast, the As (III) Similarly, was by the reagents methyl methanethiosulfonate and not As a arsenite in cells of E. coli ArsB, which has no Y. Dey S. Rosen B. P. J. Bacteriol. PubMed Google Scholar), was by of the thiol These results first that the conserved cysteine in Acr3 participates in As (III) and second that it may be located in a hydrophobic transmembrane a by the analysis ArsB has 12 segments (7Wu J. Tisa L. S. Rosen B. P. J. Biol. Chem. 1992; 267: 12570-12576Abstract Full Text PDF PubMed Google Scholar), Acr3 has been predicted to have only 10 (12Aaltonen E. K. Silow M. Biochim. Biophys. Acta. 2008; 1778: 963-973Crossref PubMed Scopus (27) Google Scholar). for AmAcr3 from three analysis and These two with and the other with 10 and It is that in all predicted the conserved residue Cys138 is located in a transmembrane analysis of the B. subtilis Acr3 using translational fusions could not distinguish between 8 and 10 transmembrane segments (12Aaltonen E. K. Silow M. Biochim. Biophys. Acta. 2008; 1778: 963-973Crossref PubMed Scopus (27) Google Scholar). In this SCAM (21Bogdanov M. Zhang W. Xie J. Dowhan W. Methods. 2005; 36: 148-171Crossref PubMed Scopus (116) Google Scholar) was used to determine the transmembrane topology of AmAcr3. with the of which has only single mutants only a single cysteine residue were constructed all predicted hydrophobic and Cys138 is for function, but as it not with of the SCAM and the in the for SCAM The of each in E. coli was examined by of the mutants was and were expressed at than to confer resistance to arsenite was determined for the each conferred mutants as and were at higher of arsenite. more than the of or the fact that they indicates that they have an topology similar to that of the which for SCAM determine topology using the cells were first with and the Acr3 were with of the with were by with is and with cysteine on of the membrane but not with cysteines in TMs. The the mutants with or and mutants with cysteine residues and not with indicating that they are in a or not to that AmAcr3 the with indicating that of the three cysteine residues and conserved are and are in transmembrane The mutants that with were analyzed by with which is and with cysteine The mutants were then with which would with the cysteine residues that are in the cytosol. of mutants and indicating that residues are to the The other 10 residues and with even indicating that the residues in mutants are most to the cytosol. These results are with a of 10 with the and in the that this the conserved cysteine residue is located in and second that is in a The may not have to for of and which may its to confer arsenite of AmAcr3. The of AmAcr3 is on the SCAM from Fig. The residues were to residues to both and The conserved Cys138 is located in As a of the environmental of of have and efflux is of the more common of the most widespread arsenic efflux is Acr3, which is found in members of every is the mechanism of Acr3 transport. In this study we examined the transport properties of two Acr3 permeases from two bacteria, the iron-reducing A. metalliredigens and a soil C. Both have two ars operons, each an acr3 and both are acr3 gene from each was cloned and expressed in a strain of E. coli in which the chromosomal arsRBC operon had been deleted and is Both acr3 genes the arsenite and both catalyzed arsenite efflux from cells, the first in analysis of Acr3 transport The first is that Acr3 can distinguish between As (III) and Sb (III) 1). had been from the of yeast but R. P. Ułaszewski S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). metalloid this is Both As (III) and Sb (III) are metals that to two or three cysteines in other arsenic resistance proteins, ArsA X. H. Rosen B. P. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), ArsD Y. F. J. Rosen B. P. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar), and three ArsRs J. J. Rosen B. P. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, W. J. Rosen B. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. S. Gil J. A. Mateos L. M. Rosen B. P. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). ArsB, which uses no cysteines in its is not selective for As (III) over Sb (III) Y. Dey S. Rosen B. P. J. Bacteriol. PubMed Google Scholar). Interestingly, a related arsenic resistance protein from a S. transports arsenate and not As (III) or Sb (III) (14Xia X. Postis V. L. Rahman M. Wright G. S. Roach P. C. Deacon S. E. Ingram J. C. Henderson P. J. Findlay J. B. Phillips S. E. McPherson M. J. Baldwin S. A. Mol. Membr. Biol. 2008; 25: 691-705Crossref PubMed Scopus (18) Google Scholar). Other members of the BART superfamily transport a of bile and other (10Mansour N. M. Sawhney M. Tamang D. G. Vogl C. Saier Jr. , M. H. FEBS J. 2007; 274: 612-629Crossref PubMed Scopus (59) Google Scholar). The for in members of this superfamily is not that the As (III) from other superfamily members appears to be a conserved cysteine residue (supplemental Fig. 1). Modification of this by or chemical to loss of transport A is that the conserved cysteine is in As (III) residue is located in the of a transmembrane segment and it is to that thiol is in though single trivalent arsenic with that As (III) on of the the affinity would of As (III) from the on the other of the Acr3 and ArsB are similar in that both are that extrude As (III) from they not only in but in transmembrane ArsB has 12 (7Wu J. Tisa L. S. Rosen B. P. J. Biol. Chem. 1992; 267: 12570-12576Abstract Full Text PDF PubMed Google Scholar). analysis of Acr3 suggested or 10 and analysis of the B. subtilis Acr3 8 or 10 (12Aaltonen E. K. Silow M. Biochim. Biophys. Acta. 2008; 1778: 963-973Crossref PubMed Scopus (27) Google Scholar). gene fusions were used in that the results the topology of membrane and not the permease. The use of SCAM for of and membrane (21Bogdanov M. Zhang W. Xie J. Dowhan W. Methods. 2005; 36: 148-171Crossref PubMed Scopus (116) Google Scholar). this Acr3 was to have 10 The of the conserved cysteine residue in is with a role in As (III) that arsenite efflux Acr3 is to the and B. P. in but how is not ArsB is an antiporter in which is for and the is by the It is that Acr3 is that Acr3 is a that transports the arsenite to the membrane which is in cells and in membrane The of is a would be in the of E. coli and even more in the of In contrast, the of is at the of the E. coli there would be no in between the two trivalent the of Acr3 to between As (III) and Sb (III). the mechanism of Acr3 a more W. for A. metalliredigens QYMF chromosomal with