A Novel Cyanobacterial SmtB/ArsR Family Repressor Regulates the Expression of a CPx-ATPase and a Metallothionein in Response to Both Cu(I)/Ag(I) and Zn(II)/Cd(II)

Abstract

A novel SmtB/ArsR family metalloregulator, denoted BxmR, has been identified and characterized from the cyanobacterium Oscillatoria brevis. Genetic and biochemical evidence reveals that BxmR represses the expression of both bxa1, encoding a CPx-ATPase metal transporter, as well as a divergently transcribed operon encoding bxmR and bmtA, a heavy metal sequestering metallothionein. Derepression of the expression of all three genes is mediated by both monovalent (Ag(I) and Cu(I)) and divalent (Zn(II) and Cd(II)) heavy metal ions, a novel property among SmtB/ArsR metal sensors. Electrophoretic gel mobility shift experiments reveal that apoBxmR forms multiple resolvable complexes with oligonucleotides containing a single 12-2-12 inverted repeat derived from one of the two operator/promoter regions with similar apparent affinities. Preincubation with either monovalent or divalent metal ions induces disassembly of both the BxmR-bxa1 and BxmR-bxmR/bmtA operator/promoter complexes. Interestingly, the temporal regulation of expression of bxa1 and bmtA mRNAs is different in O. brevis with bxa1 induced first upon heavy metal treatment, followed by bmtA/bxmR. A dynamic interplay among Bxa1, BmtA, and BxmR is proposed that maintains metal homeostasis in O. brevis by balancing the relative rates of metal storage and efflux of multiple heavy metal ions. A novel SmtB/ArsR family metalloregulator, denoted BxmR, has been identified and characterized from the cyanobacterium Oscillatoria brevis. Genetic and biochemical evidence reveals that BxmR represses the expression of both bxa1, encoding a CPx-ATPase metal transporter, as well as a divergently transcribed operon encoding bxmR and bmtA, a heavy metal sequestering metallothionein. Derepression of the expression of all three genes is mediated by both monovalent (Ag(I) and Cu(I)) and divalent (Zn(II) and Cd(II)) heavy metal ions, a novel property among SmtB/ArsR metal sensors. Electrophoretic gel mobility shift experiments reveal that apoBxmR forms multiple resolvable complexes with oligonucleotides containing a single 12-2-12 inverted repeat derived from one of the two operator/promoter regions with similar apparent affinities. Preincubation with either monovalent or divalent metal ions induces disassembly of both the BxmR-bxa1 and BxmR-bxmR/bmtA operator/promoter complexes. Interestingly, the temporal regulation of expression of bxa1 and bmtA mRNAs is different in O. brevis with bxa1 induced first upon heavy metal treatment, followed by bmtA/bxmR. A dynamic interplay among Bxa1, BmtA, and BxmR is proposed that maintains metal homeostasis in O. brevis by balancing the relative rates of metal storage and efflux of multiple heavy metal ions. Transition metal ions such as zinc, copper, iron, and manganese are essential trace elements that play integral catalytic functions in myriad metalloenzymes and electron transfer in all organisms (1Luchinat C. Sola M. 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Another mechanism involves the specific chelation of metal ions by intracellular chelators, e.g. metallothioneins (MTs), 1The abbreviations used are: MTs, metallothioneins; O/P, operator/promoter; EMSA, electrophoretic mobility shift analysis; DIG, digoxigenin; ORFs, open reading frames; RT, reverse transcriptase; rQRTPCR, real time quantification RT-PCR; IRE, inverted repeat element; dsDNA, double-stranded DNA. now known to be widely distributed in nature (13Robinson N.J. Whitehall S.K. Cavet K.S. Adv. Microbiol. Physiol. 2001; 44: 183-213Crossref PubMed Scopus (97) Google Scholar, 22Zhang B. Georgiev O. Hagmann M. Günes C. Cramer M. Faller P. Vasak M. Schaffner W. Mol. Cell. Biol. 2003; 23: 8471-8485Crossref PubMed Scopus (208) Google Scholar, 23Blindauer C.A. Harrison M.D. Robinson A.K. Parkinson J.A. Bowness P.W. Sadler P.J. Robinson N.J. Mol. Microbiol. 2002; 45: 1421-1432Crossref PubMed Scopus (147) Google Scholar). 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M. 2003; PubMed Scopus Google Scholar). reveal that a identified SmtB/ArsR BxmR, represses the expression of two distinct transcription one encoding and the a divergently transcribed operon encoding the BmtA, and the the of metal homeostasis is by both monovalent and and divalent (Zn(II) and Cd(II)) heavy metal ions. is by the of of these metals to BxmR, in the affinity of the for the operator/promoter that the homeostasis governing storage and of multiple heavy metal ions in be mediated by a that a a metal transporter, and a to homeostasis in Biometals. 2001; 14: PubMed Scopus Google Scholar). and cyanobacterium O. brevis in of as T. S. M. M. J. 2002; PubMed Scopus Google Scholar). to of for or transcriptional heavy metals to the from and to E. for the and for as T. S. M. M. J. 2002; PubMed Scopus Google Scholar). of of as T. S. M. M. J. 2002; PubMed Scopus Google to the or of bxa1 or bmtA and the for as T. S. M. M. J. 2002; PubMed Scopus Google Scholar). used for and of the from O. with of and in of containing and for and three with A and by and in A and by with with and the and in A storage of a with as a and of and used to bxmR O. brevis as a for Two these a and a reverse E. with the by the the expression with and the of the by with with the E. for and with for by and with containing by with and BxmR containing from the by a and with to the and to the and BxmR to be as by a of the gel of BxmR the to Electrophoretic for and by the in A and of with and and experiments a shift to the or BxmR the or a O. brevis with of or in a containing of for cell in a of of metals by of either or and to the for and complexes and to a the with the of or complexes with of known of the gel and to of the In of of bmtA, and bxa1 by the experiments to the metal of O. brevis in of a containing or or or and or for for the experiments to the of metal or to the of O. brevis as for and and for as quantification of concentrations induced by heavy metal ions by as T. S. M. M. J. 2002; PubMed Scopus Google Scholar, T. S. M. M. 2003; PubMed Scopus Google Scholar). of the of specific by and T. S. M. M. 2003; PubMed Scopus Google Scholar). of bxa1 in E. in bxa1 and from O. brevis and the for for for and and used as in in with a E. for from containing BxmR, and metal ions and to the E. to a of as by the In to for with the with with of and a gel for expression of and by by as T. S. M. M. J. 2002; PubMed Scopus Google Scholar). BxmR SmtB/ArsR in heavy metal in O. brevis identified the genes encoding Bxa1, a heavy and BmtA, a T. S. M. M. J. 2002; PubMed Scopus Google Scholar, T. S. M. M. 2003; PubMed Scopus Google Scholar). However, those to a transcriptional of the expression of these In many heavy metal resistance the genes encoding the transcriptional and the resistance or a metal transporter, are either as two open reading or as a divergently transcribed operon (12Busenlehner L.S. Pennella M.A. Giedroc D.P. FEMS Microbiol. 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Rev. 2003; 27: 131-144Crossref PubMed Scopus (314) Google Scholar). of the bxmR and bmtA reveal the of 12-2-12 inverted repeat with a A and has that similar inverted as for SmtB/ArsR in a range of C. Robinson N.J. J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus (155) Google Scholar, Giedroc D.P. 2002; PubMed Scopus Google to expression of the J.S. Robinson N.J. Nucleic Acids Res. 21: PubMed Scopus Google Scholar, C. Robinson N.J. J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus (155) Google Scholar, W. J. Rosen B.P. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J.S. Robinson N.J. Mol. Microbiol. 7: PubMed Scopus Google Scholar). of the regions of the and genes from the reveals the of a 12-2-12 inverted repeat high to the the that the 12-2-12 inverted repeat elements in all as specific for regulation of genes in to specific metal ions. a 12-2-12 with to that of in the of bxa1, a CPx-ATPase of in the of bxa1 that be to However, both of bxa1 to a encoding SmtB/ArsR that bxa1 is induced by metal ions T. S. M. M. J. 2002; PubMed Scopus Google Scholar, T. S. M. M. 2003; PubMed Scopus Google the that BxmR bxa1 like bmtA expression BxmR to the 12-2-12 in and bxa1 with to BxmR to the operator/promoter regions of both or bxa1 in In to the of the in both the the bmtA and bxmR of and with the for bxmR and that for bmtA A similar for a of bxa1, to and with the for bxa1 with these electrophoretic mobility shift with BxmR only with and of the 12-2-12 inverted repeat as To that BxmR to the 12-2-12 and and of the 12-2-12 inverted repeat A and for are in can be both derived from the operator/promoter A and and from the bxa1 operator/promoter and to multiple resolvable complexes when with BxmR in in the of of to the operator/promoter two and are a BxmR of in to the BxmR is to a is the of BxmR both and with BxmR of to the bxa1 operator/promoter to be similar to that of and that BxmR to the in with apparent affinity similar to that of A excess of or to all three complexes A and that the is the in and is and to the BxmR M.A. N.J. Giedroc D.P. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google in these these gel mobility shift a to those for from Synechococcus PCC7942 to are known to to a single 12-2-12 inverted two with high affinity J.L. Nucleic Acids Res. 23: PubMed Scopus Google Scholar, Giedroc D.P. 2002; PubMed Scopus Google Scholar, J. S. 2001; PubMed Scopus Google Scholar). with and when cell from O. for in a containing either or used in of Two complexes that with the of the and complexes in these with heavy metals in that the expression of BxmR is induced in by both monovalent and divalent metal ions and, as a is of to both and compatible with the with BxmR BxmR from and bxa1 the of and of SmtB/ArsR is that the operator/promoter affinity is by metal ions in with metal L.S. Giedroc D.P. J. Mol. Biol. 2002; PubMed Scopus Google Scholar, M.A. N.J. Giedroc D.P. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, Giedroc D.P. 2002; PubMed Scopus Google Scholar). is to be the for BxmR of concentrations of or the of all complexes with either the or of or complexes by monovalent is and has been for characterized SmtB/ArsR (12Busenlehner L.S. Pennella M.A. Giedroc D.P. FEMS Microbiol. Rev. 2003; 27: 131-144Crossref PubMed Scopus (314) Google Scholar, J.S. W. Robinson N.J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). A similar by cell from O. brevis in of BxmR In all of as relative to the metal ions, In with all metals the of metal required to similar of to be for complexes relative to complexes the of is and In the of and complexes is by multiple monovalent and divalent heavy metal ions, with the that the and of transcription of both and bxa1 is mediated by the BxmR in O. brevis in BxmR as a of the of bmtA, bxa1, and bxmR in a in E. the reveal that multiple metal ions regulate the of BxmR to regions of both bxa1 and evidence for transcriptional regulation A containing the operator/promoter the and E. that the transcription of both bxmR and bmtA induced in E. with the upon of both divalent and monovalent metal ions in the in to the of BxmR to regulate bxa1 expression E. in by the high of the and regions of the bxa1 relative to E. J. P. C. Scholar). the of bxa1 three of the for bxa1 the bxa1 and as in a E. All of these the 12-2-12 inverted repeat of bxa1 in the E. with or to the In the by that a containing only inverted repeat is for bxa1 for a of BxmR to these in a in with with BxmR that BxmR is of of is upon of zinc, or to the with the of concentrations of either or of and be to of in the experiments of bxa1, bmtA, and bxmR in by the of the the transcription of the bxmR in O. brevis the expression of bmtA, or the metal of To to both the metal and the of the expression of bmtA, and bxa1 induced by multiple heavy metals in O. brevis a the expression in O. brevis the to heavy metal ions the expression of bmtA, and bxa1 is by a with the concentrations of metal to the metal for all three genes with and with with and is with the that the the expression of bmtA, and bxa1 in to multiple metal ions. a of required to bxmR and bmtA transcription relative to bxa1 the of only a metal concentrations temporal of bmtA, and bxa1 expression a of heavy metal and the of of is for all metal expression of bxa1, a CPx-ATPase metal transporter, is of metal to the with of bxmR and bmtA, both only in the time two expression of bxmR that of have bxmR and bmtA the is that is for the regulation of these two is that of the as the first or of metal in O. with the and the BmtA, a to to the metal ions. A SmtB/ArsR from O. BxmR, the of bmtA, and T. S. M. M. J. 2002; PubMed Scopus Google Scholar, T. S. M. M. 2003; PubMed Scopus Google that heavy metal in the O. the and of both a heavy metal sequestering by the bmtA and a CPx-ATPase metal transporter, by with in O. of either bmtA or bxa1 in E. metal resistance in as well T. S. M. M. 2003; PubMed Scopus Google Scholar). In two a single BxmR, is and to transcriptional regulation of the expression of both bmtA and bxa1 that BxmR is a of the SmtB/ArsR family of (12Busenlehner L.S. Pennella M.A. Giedroc D.P. FEMS Microbiol. Rev. 2003; 27: 131-144Crossref PubMed Scopus (314) Google Scholar, W. J. Rosen B.P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). SmtB/ArsR metal sensor proteins regulate the expression of either or a metal (12Busenlehner L.S. Pennella M.A. Giedroc D.P. FEMS Microbiol. Rev. 2003; 27: 131-144Crossref PubMed Scopus (314) Google to is the first of a single the of both a and a metal efflux in the is that O. brevis BxmR the expression of all three genes in to both the metals and as well as and a novel only in the SmtB/ArsR family of regulators (12Busenlehner L.S. Pennella M.A. Giedroc D.P. FEMS Microbiol. Rev. 2003; 27: 131-144Crossref PubMed Scopus (314) Google in e.g. family N.L. Stoyanov J.V. Kidd S.P. Hobman J.L. FEMS Microbiol. Rev. 2003; 27: 145-164Crossref PubMed Scopus (553) Google Scholar, A. J. O'Halloran T.V. A. Science. 2003; PubMed Scopus Google Scholar). the mechanism of regulation the are with the that BxmR for both and that regulate bxa1 and of is that by is and upon of in O. brevis by the of by that the temporal of of bxa1 bmtA genes T. S. M. M. 2003; PubMed Scopus Google is by the by both monovalent and and complexes distinct from that of and and either number or A. J. O'Halloran T.V. A. Science. 2003; PubMed Scopus Google or A.K. O'Halloran T.V. Nat. Struct. Biol. 2000; 7: PubMed Scopus Google metal or in multiple ions are a e.g. in the of S. and and in Keller G.L. Winge D.R. 2002; PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). of a with a number of for and is known BxmR metal ions of BxmR many in the and as well as a the of the of these two regions known metal to the ion in S. L.S. Giedroc D.P. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). BxmR all metal known to the in Synechococcus PCC7942 and S. C. Pennella M.A. Giedroc D.P. J. Mol. Biol. 2003; PubMed Scopus Google Scholar, Giedroc D.P. 2002; PubMed Scopus Google experiments with metal in the metal metal and regulation of in of Gene in O. that the bmtA, and bxa1 genes are by BxmR is with the that all three genes to a metal homeostasis in O. brevis. of the expression of bxa1 and bmtA have that and bxa1 expression within of metal treatment, with bmtA expression induced or T. S. M. M. 2003; PubMed Scopus Google Scholar). are with the that the metal is a required to the of intracellular metal ions and as first of metal In the is a that O. brevis from metal that from a in the mechanism of the of bxa1 relative to bmtA is One to or of bxa1 is to intracellular is to the bxa1 O/P, with the of the However, the of the in both are to be by BxmR they to have for BxmR the expression of all three genes is induced by metal ions and Another mechanism is that metal disassembly of the BxmR-bxa1 complexes relative to the BxmR-bxmR/bmtA complexes in either a of the BxmR-bxa1 complexes to the concentrations of metal ion that in metal relative to metal or the of disassembly is for BxmR-bxa1 relative to the BxmR-bxmR/bmtA complexes. experiments that the BxmR-bxa1 be by concentrations of the metal relative to BxmR-bxmR/bmtA complexes with the bmtA and J.S. Robinson N.J. Mol. Microbiol. 7: PubMed Scopus Google are both in O. even in the of metal relative to both bxa1 and bxmR mRNAs and proteins. metal ion concentrations in the is to bxa1 to the and efflux metal ion of the of metal ion efflux be by the of complexes to metal ion to the efflux as for the P.J. Culotta V.C. O'Halloran T.V. Science. 1997; 278: PubMed Scopus Google Scholar). when the in the are the of the expression of the by metal ions as the intracellular concentrations of both and the or metal ion and a homeostasis that efflux and intracellular chelation be O. brevis to of metal that for O. the of cyanobacterium of both a CPx-ATPase metal and a with high to C. Robinson N.J. J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus (155) Google and Synechococcus PCC7942 (13Robinson N.J. Whitehall S.K. Cavet K.S. Adv. Microbiol. Physiol. 2001; 44: 183-213Crossref PubMed Scopus (97) Google However, in a to O. the is of the encoding the with SmtB/ArsR to the encoding the metallothionein. of is In the metal homeostasis in Oscillatoria and that in the transcription is known to the of by the genes encoding both a metal efflux as well as a Georgiev O. M. M. Schaffner W. EMBO J. PubMed Scopus Google Scholar, P.J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). the and the metal in O. brevis is is to that and be to and that in in P.J. Culotta V.C. O'Halloran T.V. Science. 1997; 278: PubMed Scopus Google Scholar, L. S. O'Halloran T.V. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). have been are known to a of one of is to the of W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). reveal that the of and the of in of the of heavy metal ions among BxmR, BmtA, and the mechanisms of metal resistance and of metal ions in M. and for the and in and the for for O. brevis. critical of J. Robinson of are