The Zinc-responsive Regulator Zur and Its Control of theznu Gene Cluster Encoding the ZnuABC Zinc Uptake System in Escherichia coli

Abstract

The synthesis of the Escherichia colizinc transporter, encoded by the znuACB gene cluster, is regulated in response to the intracellular zinc concentration by thezur gene product. Inactivation of the zur gene demonstrated that Zur acts as a repressor when binding Zn2+. Eight chromosomal mutant zur alleles were sequenced to correlate the loss of Zur function with individual mutations. Wild-type Zur and ZurΔ46–91 formed homo- and heterodimers. Dimerization was independent of metal ions since it also occurred in the presence of metal chelators. Using an in vivo titration assay, the znu operator was narrowed down to a 31-base pair region overlapping the translational start site of znuA. This location was confirmed by footprinting assays. Zur directly binds to a single region comprising a nearly perfect palindrome. Zinc chelators completely inhibited and Zn2+ in low concentrations enhanced DNA binding of Zur. No evidence for autoregulation of Zur was found. Zur binds at least 2 zinc ions/dimer specifically. Although most of the mutant Zur proteins bound to the znu operator in vitro, no protection was observed in in vivo footprinting experiments. Analysis of the mutant Zur proteins suggested an amino-terminal DNA contact domain around residue 65 and a dimerization and Zn2+-binding domain toward the carboxyl-terminal end. The synthesis of the Escherichia colizinc transporter, encoded by the znuACB gene cluster, is regulated in response to the intracellular zinc concentration by thezur gene product. Inactivation of the zur gene demonstrated that Zur acts as a repressor when binding Zn2+. Eight chromosomal mutant zur alleles were sequenced to correlate the loss of Zur function with individual mutations. Wild-type Zur and ZurΔ46–91 formed homo- and heterodimers. Dimerization was independent of metal ions since it also occurred in the presence of metal chelators. Using an in vivo titration assay, the znu operator was narrowed down to a 31-base pair region overlapping the translational start site of znuA. This location was confirmed by footprinting assays. Zur directly binds to a single region comprising a nearly perfect palindrome. Zinc chelators completely inhibited and Zn2+ in low concentrations enhanced DNA binding of Zur. No evidence for autoregulation of Zur was found. Zur binds at least 2 zinc ions/dimer specifically. Although most of the mutant Zur proteins bound to the znu operator in vitro, no protection was observed in in vivo footprinting experiments. Analysis of the mutant Zur proteins suggested an amino-terminal DNA contact domain around residue 65 and a dimerization and Zn2+-binding domain toward the carboxyl-terminal end. polymerase chain reaction kilobase pair nucleotide(s) polyacrylamide gel electrophoresis dithiothreitol dimethyl sulfate N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (S,S)-ethylenediaminedisuccinic acid Zinc is essential for all organisms and plays a catalytic as well as a structural role in many proteins. However, zinc can also be toxic. It interferes with vital functions by competing with other metal ions for biologically important ligands, such as active sites of enzymes and transporter proteins, especially when a critical concentration is exceeded. Therefore, the intracellular zinc level must be precisely regulated. Compared with eukaryotes, little is known about zinc homeostasis in bacteria. In Escherichia coli, a zinc efflux system (ZntA) and a zinc import system (ZnuABC) have been described recently. ZntA (Zn2+transport ortolerance) is an ion-motive P-type ATPase that exports Zn2+, Cd2+, and Pb2+ (1Beard S.J. Hashim R. Membrillo-Hernández J. Hughes M.N. Poole R.K. Mol. Microbiol. 1997; 25: 883-891Crossref PubMed Scopus (158) Google Scholar, 2Rensing C. Sun Y. Mitra B. Rosen B.P. J. Biol. Chem. 1998; 273: 32614-32617Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). The Zn2+-specific uptake system Znu (Zn2+uptake) belongs to the ABC transporter family and is composed of the periplasmic binding protein ZnuA, the ATPase ZnuC, and the integral membrane protein ZnuB (3Patzer S.I. Hantke K. Mol. Microbiol. 1998; 28: 1199-1210Crossref PubMed Scopus (379) Google Scholar). In the znu gene cluster, the transcription of theznuA gene is divergent to that of the znuCBgenes, and the genes are separated by an unusually short intergenic region of 24 base pairs (4Blattner F.R. Plunkett G.I. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (5973) Google Scholar).As we have reported earlier, the zinc uptake system ZnuABC is regulated by Zur (Zn2+uptakeregulator) (3Patzer S.I. Hantke K. Mol. Microbiol. 1998; 28: 1199-1210Crossref PubMed Scopus (379) Google Scholar), and the corresponding genes lie ≈50 min apart on the E. coli chromosome. Since then, Zur proteins from Bacillus subtilis (5Gaballa A. Helmann J.D. J. Bacteriol. 1998; 180: 5815-5821Crossref PubMed Google Scholar) and Listeria monocytogenes (6Dalet K. Gouin E. Cenatiempo Y. Cossart P. Héchard Y. FEMS Microbiol. Lett. 1999; 174: 111-116Crossref PubMed Google Scholar) have been described. Also ZntR, theZn2+-responsive transcriptionalregulator of zntA that belongs to the family of MerR-like prokaryotic transcriptional regulators, has been identified (7Brocklehurst K.R. Hobman J.L. Lawley B. Blank L. Marshall S.J. Brown N.L. Morby A.P. Mol. Microbiol. 1999; 31: 893-902Crossref PubMed Scopus (201) Google Scholar). Other zinc-specific metalloregulatory proteins from bacteria known to date are SmtB and ZiaR. SmtB in cyanobacteria represses the expression of the gene encoding the metallothionein SmtA, which confers resistance to heavy metal ions. SmtB binds zinc and other heavy metals and belongs to the ArsR family of metallorepressors, which control mainly prokaryotic metal resistance operons (8Shi J. Lindsay W.P. Huckle J.W. Morby A.P. Robinson N.J. FEBS Lett. 1992; 303: 159-163Crossref PubMed Scopus (54) Google Scholar). ZiaR is an SmtB-like repressor of the expression of the gene encoding the zinc efflux pump ZiaA (zinc ATPase) in Synechocystissp. (9Thelwell C. Robinson N.J. Turner-Cavet J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10728-10733Crossref PubMed Scopus (152) Google Scholar). According to sequence similarities, Zur is considered to be a member of the Fur family of metalloregulatory proteins, which include the Fur (ferric iron uptakeregulator) proteins of E. coli and other bacteria involved in iron regulation (10Hantke K. Braun V. Walden W. Silver S. Metal Ions in Gene Regulation. Chapman and Hall, Inc., New York1997: 11-44Google Scholar) as well as PerR of B. subtilis, which controls the peroxide stress response (11Bsat N. Herbig A. Casillas-Martinez L. Setlow P. Helmann J.D. Mol. Microbiol. 1998; 29: PubMed Scopus Google we the role of Zur in of the znu gene Wild-type and mutant Zur proteins were and and the with metal ions and with operator DNA in and in vivo was Zur is a in the of zinc other metal as by gel in the presence of chelators. sequence of was the znu sequence is for and Zur protein was to directly and to DNA with sequence independent of DNA in vivo and in binding site in the presence of zinc other metal at low as by the footprinting Zur a on of the znu operator with a of This that of such as proteins, the repressor from Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The observed is of of the at the no about the at concentrations of no and of the region as has been observed with Fur at in footprinting V. S. M. J. Bacteriol. PubMed Google and M.A. J. Bacteriol. PubMed Google Scholar) and by and E. M. A. E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google is active in the a it has to the in the Mol. Biol. Google Scholar). In vitro, the of Zur are to as by the on with Zur DNA of to be in bacteria and as by sequence In B. subtilis, apart from Fur and PerR stress response (11Bsat N. Herbig A. Casillas-Martinez L. Setlow P. Helmann J.D. Mol. Microbiol. 1998; 29: PubMed Scopus Google Scholar), the can as Zur (5Gaballa A. Helmann J.D. J. Bacteriol. 1998; 180: 5815-5821Crossref PubMed Google Scholar). The sequence coli Zur is to the and bacteria. zur gene has also been in L. monocytogenes (6Dalet K. Gouin E. Cenatiempo Y. Cossart P. Héchard Y. FEMS Microbiol. Lett. 1999; 174: 111-116Crossref PubMed Google Scholar). on sequence we that the Fur protein C. Hantke K. FEMS Microbiol. Lett. PubMed Google Scholar) is Zur Fur It is that other proteins as Fur to be Zur proteins. S. A. E. Y. N. M. M. S. A. A. N. K. S. S. C. A. M. M. S. DNA PubMed Scopus Google Scholar) also have a Zur the and are to Zur to Fur from E. It is that is directly and is of the genes encoding the many of the sequenced of a Zur is in for for of for and Since organisms also a system to we that proteins are of zinc of proteins are in all mutant Zur proteins be in it can be that the Zur is the of low protein expression of an Zur protein that is by This that the in the mutant Zur proteins are important for the function of Zur. The of the acid of the with Zur proteins from other organisms that all of the in well which has an in the amino-terminal of bound as Zn2+ as Zur and formed and with DNA from region is for binding of is essential for dimerization Zn2+ by an of the mutant Zur protein can be by for the Zn2+ by of active with Zur. and were in vivo as were to to Zn2+ to In all mutant proteins, in the amino-terminal region are region at least the individual are for binding of Zn2+ In to inhibited DNA binding of Zur in footprinting and of which can be for by the of the mutant Zur proteins and ZurΔ46–91 were in to DNA since DNA from in vitro, in This that DNA by the Zur protein are in the region around residue region is in all Zur and to the in the in Fur that has been suggested to with DNA L. C. M. R. R. R. PubMed Scopus Google Scholar). However, the in to the the is to the zinc for Zur was at least 2 which that at least site of the Zur is by Zn2+. the of the protein concentration was in the in Zur the of around acid that is in Fur and that is to be involved in binding of Fur Mol. Microbiol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). the in E. and are involved in Zn2+ binding are in at least all Zur proteins from other E. coli Fur structural zinc in to the site that the intracellular iron concentration L. A. 1998; PubMed Scopus Google Scholar). The zinc is by at least L. A. 1998; PubMed Scopus Google Scholar) and and A. C. A. L. E. 1999; PubMed Scopus Google Scholar). are in all Zur proteins and as Zn2+ also in observed in the footprinting Zn2+ at low concentrations acts as a in Zur and can be in by other metal ions such as for in is by Fur with Cd2+, Zn2+ and by and with Cd2+, Zn2+ as in footprinting (8Shi J. Lindsay W.P. Huckle J.W. Morby A.P. Robinson N.J. FEBS Lett. 1992; 303: 159-163Crossref PubMed Scopus (54) Google Scholar, V. S. M. J. Bacteriol. PubMed Google Scholar, R.K. Mol. Microbiol. PubMed Scopus Google Scholar, M. L. R.K. PubMed Google Scholar), the response of genes is in It is which binding of the individual metal to the in the the from the footprinting can the in vivo to a This is also by the binding of most of the mutant Zur proteins to the in in footprinting as was by of in the zur and the of protection of the znu operator by at least in in vivo footprinting znu gene was by metal ions such as and This can be by binding of to the an that no represses by metal ions by the intracellular zinc The be by the metal ions competing with Zn2+ for an transporter and uptake of Zn2+. in regulation with the znu system a it is the Zur binding in footprinting with the as the the for footprinting and the in Zur bound Zn2+, and binding of the Zn2+ to at least and concentrations no for a of iron with Zur. Zinc is essential for all organisms and plays a catalytic as well as a structural role in many proteins. However, zinc can also be toxic. It interferes with vital functions by competing with other metal ions for biologically important ligands, such as active sites of enzymes and transporter proteins, especially when a critical concentration is exceeded. Therefore, the intracellular zinc level must be precisely regulated. Compared with eukaryotes, little is known about zinc homeostasis in bacteria. In Escherichia coli, a zinc efflux system (ZntA) and a zinc import system (ZnuABC) have been described recently. ZntA (Zn2+transport ortolerance) is an ion-motive P-type ATPase that exports Zn2+, Cd2+, and Pb2+ (1Beard S.J. Hashim R. Membrillo-Hernández J. Hughes M.N. Poole R.K. Mol. Microbiol. 1997; 25: 883-891Crossref PubMed Scopus (158) Google Scholar, 2Rensing C. Sun Y. Mitra B. Rosen B.P. J. Biol. Chem. 1998; 273: 32614-32617Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). The Zn2+-specific uptake system Znu (Zn2+uptake) belongs to the ABC transporter family and is composed of the periplasmic binding protein ZnuA, the ATPase ZnuC, and the integral membrane protein ZnuB (3Patzer S.I. Hantke K. Mol. Microbiol. 1998; 28: 1199-1210Crossref PubMed Scopus (379) Google Scholar). In the znu gene cluster, the transcription of theznuA gene is divergent to that of the znuCBgenes, and the genes are separated by an unusually short intergenic region of 24 base pairs (4Blattner F.R. Plunkett G.I. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (5973) Google Scholar). we have reported earlier, the zinc uptake system ZnuABC is regulated by Zur (Zn2+uptakeregulator) (3Patzer S.I. Hantke K. Mol. Microbiol. 1998; 28: 1199-1210Crossref PubMed Scopus (379) Google Scholar), and the corresponding genes lie ≈50 min apart on the E. coli chromosome. Since then, Zur proteins from Bacillus subtilis (5Gaballa A. Helmann J.D. J. Bacteriol. 1998; 180: 5815-5821Crossref PubMed Google Scholar) and Listeria monocytogenes (6Dalet K. Gouin E. Cenatiempo Y. Cossart P. Héchard Y. FEMS Microbiol. Lett. 1999; 174: 111-116Crossref PubMed Google Scholar) have been described. Also ZntR, theZn2+-responsive transcriptionalregulator of zntA that belongs to the family of MerR-like prokaryotic transcriptional regulators, has been identified (7Brocklehurst K.R. Hobman J.L. Lawley B. Blank L. Marshall S.J. Brown N.L. Morby A.P. Mol. Microbiol. 1999; 31: 893-902Crossref PubMed Scopus (201) Google Scholar). Other zinc-specific metalloregulatory proteins from bacteria known to date are SmtB and ZiaR. SmtB in cyanobacteria represses the expression of the gene encoding the metallothionein SmtA, which confers resistance to heavy metal ions. SmtB binds zinc and other heavy metals and belongs to the ArsR family of metallorepressors, which control mainly prokaryotic metal resistance operons (8Shi J. Lindsay W.P. Huckle J.W. Morby A.P. Robinson N.J. FEBS Lett. 1992; 303: 159-163Crossref PubMed Scopus (54) Google Scholar). ZiaR is an SmtB-like repressor of the expression of the gene encoding the zinc efflux pump ZiaA (zinc ATPase) in Synechocystissp. (9Thelwell C. Robinson N.J. Turner-Cavet J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10728-10733Crossref PubMed Scopus (152) Google Scholar). According to sequence similarities, Zur is considered to be a member of the Fur family of metalloregulatory proteins, which include the Fur (ferric iron uptakeregulator) proteins of E. coli and other bacteria involved in iron regulation (10Hantke K. Braun V. Walden W. Silver S. Metal Ions in Gene Regulation. Chapman and Hall, Inc., New York1997: 11-44Google Scholar) as well as PerR of B. subtilis, which controls the peroxide stress response (11Bsat N. Herbig A. Casillas-Martinez L. Setlow P. Helmann J.D. Mol. Microbiol. 1998; 29: PubMed Scopus Google Scholar). In we the role of Zur in of the znu gene Wild-type and mutant Zur proteins were and and the with metal ions and with operator DNA in and in vivo was Zur is a in the of zinc other metal as by gel in the presence of chelators. sequence of was the znu sequence is for and Zur protein was to directly and to DNA with sequence independent of DNA in vivo and in binding site in the presence of zinc other metal at low as by the footprinting Zur a on of the znu operator with a of This that of such as proteins, the repressor from Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The observed is of of the at the no about the at concentrations of no and of the region as has been observed with Fur at in footprinting V. S. M. J. Bacteriol. PubMed Google and M.A. J. Bacteriol. PubMed Google Scholar) and by and E. M. A. E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google is active in the a it has to the in the Mol. Biol. Google Scholar). In vitro, the of Zur are to as by the on with Zur DNA of to be in bacteria and as by sequence In B. subtilis, apart from Fur and PerR stress response (11Bsat N. Herbig A. Casillas-Martinez L. Setlow P. Helmann J.D. Mol. Microbiol. 1998; 29: PubMed Scopus Google Scholar), the can as Zur (5Gaballa A. Helmann J.D. J. Bacteriol. 1998; 180: 5815-5821Crossref PubMed Google Scholar). The sequence coli Zur is to the and bacteria. zur gene has also been in L. monocytogenes (6Dalet K. Gouin E. Cenatiempo Y. Cossart P. Héchard Y. FEMS Microbiol. Lett. 1999; 174: 111-116Crossref PubMed Google Scholar). on sequence we that the Fur protein C. Hantke K. FEMS Microbiol. Lett. PubMed Google Scholar) is Zur Fur It is that other proteins as Fur to be Zur proteins. S. A. E. Y. N. M. M. S. A. A. N. K. S. S. C. A. M. M. S. DNA PubMed Scopus Google Scholar) also have a Zur the and are to Zur to Fur from E. It is that is directly and is of the genes encoding the many of the sequenced of a Zur is in for for of for and Since organisms also a system to we that proteins are of zinc of proteins are in all mutant Zur proteins be in it can be that the Zur is the of low protein expression of an Zur protein that is by This that the in the mutant Zur proteins are important for the function of Zur. The of the acid of the with Zur proteins from other organisms that all of the in well which has an in the amino-terminal of bound as Zn2+ as Zur and formed and with DNA from region is for binding of is essential for dimerization Zn2+ by an of the mutant Zur protein can be by for the Zn2+ by of active with Zur. and were in vivo as were to to Zn2+ to In all mutant proteins, in the amino-terminal region are region at least the individual are for binding of Zn2+ In to inhibited DNA binding of Zur in footprinting and of which can be for by the of the mutant Zur proteins and ZurΔ46–91 were in to DNA since DNA from in vitro, in This that DNA by the Zur protein are in the region around residue region is in all Zur and to the in the in Fur that has been suggested to with DNA L. C. M. R. R. R. PubMed Scopus Google Scholar). However, the in to the the is to the zinc for Zur was at least 2 which that at least site of the Zur is by Zn2+. the of the protein concentration was in the in Zur the of around acid that is in Fur and that is to be involved in binding of Fur Mol. Microbiol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). the in E. and are involved in Zn2+ binding are in at least all Zur proteins from other E. coli Fur structural zinc in to the site that the intracellular iron concentration L. A. 1998; PubMed Scopus Google Scholar). The zinc is by at least L. A. 1998; PubMed Scopus Google Scholar) and and A. C. A. L. E. 1999; PubMed Scopus Google Scholar). are in all Zur proteins and as Zn2+ also in observed in the footprinting Zn2+ at low concentrations acts as a in Zur and can be in by other metal ions such as for in is by Fur with Cd2+, Zn2+ and by and with Cd2+, Zn2+ as in footprinting (8Shi J. Lindsay W.P. Huckle J.W. Morby A.P. Robinson N.J. FEBS Lett. 1992; 303: 159-163Crossref PubMed Scopus (54) Google Scholar, V. S. M. J. Bacteriol. PubMed Google Scholar, R.K. Mol. Microbiol. PubMed Scopus Google Scholar, M. L. R.K. PubMed Google Scholar), the response of genes is in It is which binding of the individual metal to the in the the from the footprinting can the in vivo to a This is also by the binding of most of the mutant Zur proteins to the in in footprinting as was by of in the zur and the of protection of the znu operator by at least in in vivo footprinting znu gene was by metal ions such as and This can be by binding of to the an that no represses by metal ions by the intracellular zinc The be by the metal ions competing with Zn2+ for an transporter and uptake of Zn2+. in regulation with the znu system a it is the Zur binding in footprinting with the as the the for footprinting and the in Zur bound Zn2+, and binding of the Zn2+ to at least and concentrations no for a of iron with Zur. Zur is a in the of zinc other metal as by gel in the presence of chelators. sequence of was the znu sequence is for and Zur protein was to directly and to DNA with sequence independent of DNA in vivo and in Zur binding site in the presence of zinc other metal at low as by the footprinting Zur a on of the znu operator with a of This that of such as proteins, the repressor from Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The observed is of of the at the no about the at concentrations of no and of the region as has been observed with Fur at in footprinting V. S. M. J. Bacteriol. PubMed Google and M.A. J. Bacteriol. PubMed Google Scholar) and by and E. M. A. E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Zur is active in the a it has to the in the Mol. Biol. Google Scholar). In vitro, the of Zur are to as by the on with Zur DNA of Zn2+. Zur to be in bacteria and as by sequence In B. subtilis, apart from Fur and PerR stress response (11Bsat N. Herbig A. Casillas-Martinez L. Setlow P. Helmann J.D. Mol. Microbiol. 1998; 29: PubMed Scopus Google Scholar), the can as Zur (5Gaballa A. Helmann J.D. J. Bacteriol. 1998; 180: 5815-5821Crossref PubMed Google Scholar). The sequence coli Zur is to the and bacteria. zur gene has also been in L. monocytogenes (6Dalet K. Gouin E. Cenatiempo Y. Cossart P. Héchard Y. FEMS Microbiol. Lett. 1999; 174: 111-116Crossref PubMed Google Scholar). on sequence we that the Fur protein C. Hantke K. FEMS Microbiol. Lett. PubMed Google Scholar) is Zur Fur It is that other proteins as Fur to be Zur proteins. S. A. E. Y. N. M. M. S. A. A. N. K. S. S. C. A. M. M. S. DNA PubMed Scopus Google Scholar) also have a Zur the and are to Zur to Fur from E. It is that is directly and is of the genes encoding the In many of the sequenced of a Zur is in for for of for and Since organisms also a system to we that proteins are of zinc of proteins are in Since all mutant Zur proteins be in it can be that the Zur is the of low protein expression of an Zur protein that is by This that the in the mutant Zur proteins are important for the function of Zur. The of the acid of the with Zur proteins from other organisms that all of the in well which has an in the amino-terminal of bound as Zn2+ as Zur and formed and with DNA from region is for binding of is essential for dimerization Zn2+ by an of the mutant Zur protein can be by for the Zn2+ by of active with Zur. and were in vivo as were to to Zn2+ to In all mutant proteins, in the amino-terminal region are region at least the individual are for binding of Zn2+ In to inhibited DNA binding of Zur in footprinting and of which can be for by the of the mutant Zur proteins and ZurΔ46–91 were in to DNA since DNA from in vitro, in This that DNA by the Zur protein are in the region around residue region is in all Zur and to the in the in Fur that has been suggested to with DNA L. C. M. R. R. R. PubMed Scopus Google Scholar). However, the in to the the is to the The zinc for Zur was at least 2 which that at least site of the Zur is by Zn2+. the of the protein concentration was in the in Zur the of around acid that is in Fur and that is to be involved in binding of Fur Mol. Microbiol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). the in E. and are involved in Zn2+ binding are in at least all Zur proteins from other E. coli Fur structural zinc in to the site that the intracellular iron concentration L. A. 1998; PubMed Scopus Google Scholar). The zinc is by at least L. A. 1998; PubMed Scopus Google Scholar) and and A. C. A. L. E. 1999; PubMed Scopus Google Scholar). are in all Zur proteins and as Zn2+ also in Zur. observed in the footprinting Zn2+ at low concentrations acts as a in Zur and can be in by other metal ions such as for in is by Fur with Cd2+, Zn2+ and by and with Cd2+, Zn2+ as in footprinting (8Shi J. Lindsay W.P. Huckle J.W. Morby A.P. Robinson N.J. FEBS Lett. 1992; 303: 159-163Crossref PubMed Scopus (54) Google Scholar, V. S. M. J. Bacteriol. PubMed Google Scholar, R.K. Mol. Microbiol. PubMed Scopus Google Scholar, M. L. R.K. PubMed Google Scholar), the response of genes is in It is which binding of the individual metal to the in the the from the footprinting can the in vivo to a This is also by the binding of most of the mutant Zur proteins to the in in footprinting as was by of in the zur and the of protection of the znu operator by at least in in vivo footprinting experiments. The znu gene was by metal ions such as and This can be by binding of to the an that no represses by metal ions by the intracellular zinc The be by the metal ions competing with Zn2+ for an transporter and uptake of Zn2+. in regulation with the znu system a it is the Zur binding in footprinting with the as the the for footprinting and the in Zur bound Zn2+, and binding of the Zn2+ to at least and concentrations no for a of iron with Zur. the for for amino-terminal protein Braun for and A. for critical of the