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Originally published In Press as doi:10.1074/jbc.M310560200 on February 11, 2004

J. Biol. Chem., Vol. 279, Issue 17, 17810-17818, April 23, 2004
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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)*

Tong Liu{ddagger}§, Susumu Nakashima{ddagger}, Kazunobu Hirose{ddagger}, Mineo Shibasaka{ddagger}, Maki Katsuhara{ddagger}, Bunichi Ezaki{ddagger}, David P. Giedroc§, and Kunihiro Kasamo{ddagger}||

From the {ddagger}Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan and the §Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128

Received for publication, September 24, 2003 , and in revised form, February 10, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1-3). However, they are required only in trace amounts and, when present in excess in the environment, even essential metals can be cytotoxic, like heavy metal pollutants (4-6). All organisms have evolved a range of mechanisms that govern metal homeostasis, defined as maintaining the intracellular bioavailable concentrations of essential metal ions within a range compatible with cell viability (7-9). Multiple lines of evidence from the past decade reveal that heavy metal homeostasis is maintained in all organisms by a small number of critical processes that include metal sensing, chelation, and transport (10-18).

Two distinct mechanisms play prominent roles in governing metal ion homeostasis and resistance in many organisms. One involves the uptake or efflux of specific heavy metal ions across biomembranes, mediated by ATP-coupled high affinity metal ion transporters such as those derived from the CPx-ATPase family (19-21). Another mechanism involves the specific chelation of metal ions by intracellular chelators, e.g. metallothioneins (MTs),1 now known to be widely distributed in nature (13, 22, 23). To meet the diverse biological requirements of specific metal ions, various strategies have evolved to regulate the transcription of genes encoding these heavy metal homeostasis proteins. In prokaryotes, the expression of these genes is tightly controlled by specific metalloregulators or "metal-sensing" transcriptional regulators (12, 24, 25). One such family of homologous metal sensor proteins is the SmtB/ArsR family, named for Synechococcus PCC7942 SmtB, a Zn(II)-responsive transcriptional repressor (26, 27) that negatively regulates the transcription of SmtA, a cyanobacterial metallothionein (13, 23), and Escherichia coli R773-encoded ArsR, an As(III)/Sb(III) regulator of the ars operon (28, 29). Other members include Staphylococcus aureus pI258 CadC (30, 31) and S. aureus CzrA (32, 33) that regulate the expression of a metal-transporting CPx-ATPase (CadA) and a cation-facilitated diffusion anti-porter (CzrB), respectively.

Despite the progress made in understanding the structure, mechanism, and metal selectivity of diverse metal sensor proteins (17, 33-36), many questions remain as to how specific heavy metal ions are specifically sensed in a common cytosol, particularly when challenged with multiple metal ions (37, 38). Recent studies reveal that the metal ion actually sensed in vivo is at least partly dictated by the cytoplasmic milieu; this suggests that metal uptake/efflux, compartmentalization, and/or trafficking may play an important role in functional metal selectivity in vivo (37, 38). Very little is known about how organisms integrate these complex metal-specific systems to achieve heavy metal ion homeostasis, particularly under conditions of vanishingly small concentrations of "free" metal ions thought to exist in cells (39, 40). Although metal transport and metal chelation systems are likely to be closely interdependent (26, 35, 41), a molecular understanding of this interdependence in a single living organism remains poorly defined because these two resistance systems are usually found in separate bacteria.

Oscillatoria brevis is the first bacterium identified that harbors both a zinc-related metallothionein, BmtA, and a CPx-ATPase, Bxa1, proposed to efflux metals outside of the cytoplasm, in order to achieve multiple heavy metal co-tolerance (41, 42). We proposed previously that bmtA and bxa1 are components of the same metal homeostasis system (42). The studies presented here reveal that a newly identified SmtB/ArsR metal-sensing repressor, BxmR, represses the expression of two distinct transcription units, one encoding Bxa1 and the other a divergently transcribed operon encoding the metallothionein, BmtA, and the repressor itself, BxmR. Strikingly, the derepression of this complex metal homeostasis system is strongly regulated by both monovalent (Cu(I) and Ag(I)) and divalent (Zn(II) and Cd(II)) heavy metal ions. This is achieved by the direct binding of each of these metals to BxmR, which in turn, reduces the affinity of the regulator for the operator/promoter region, thereby inducing transcription. These findings suggest that the homeostasis governing storage and trafficking of multiple heavy metal ions in living cells could be mediated by a system that minimally employs a metallothionein, a metal transporter, and a metalloregulatory protein, analogous to zinc homeostasis in mammalian cells (43).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Growth Conditions—The filamentous cyanobacterium O. brevis was grown at 25 °C in 200 ml of modified CT medium as described (41). Liquid cultures grown to an optical density of 0.5 (660 nm) after {approx}60 days were collected for genomic DNA isolation or metal-inducing transcriptional analysis after adding heavy metals to the cultures. Plasmid TOPO PCR 2.1-TOPO was obtained from Invitrogen. Plasmids were constructed and introduced to E. coli strain TOPO10 (Invitrogen) for amplification following the manufacturer's directions.

DNA Manipulation—Genomic DNA and total RNA for PCR were isolated as described previously (41).

Identification of bxmR—Several cycles of thermal asymmetric interlaced PCR were employed as described previously (41) to amplify the fragment upstream or downstream of bxa1 or bmtA gene. The amplified products were separated and subcloned into the TOPO PCR 2.1-TOPO vectors for DNA sequencing as described (41). GENETYX (Software Development Co., Ltd.) was used for DNA sequence and amino acid analysis.

Preparation of the Cell Extracts from O. brevis—Cultures (1 liter) with an OD660 of about 0.5 (containing {approx}0.1 g wet weight cells) were harvested and resuspended in 200 ml of medium containing ZnSO4 (60 µM), CdCl2 (16 µM), CuSO4 (16 µM), and AgNO3 (5 µM), respectively. After incubation for 24 h, cells were harvested and washed three times with buffer A (buffer A: 10 mM Tris-HCl, pH 7.3, 1 mM phenylmethylsulfonyl fluoride, and 2 mM EDTA). The cells were frozen by liquid nitrogen and resuspended in buffer A without EDTA and sonicated. Cellular debris was pelleted by ultracentrifugation (30,000 x g, 30 min) after incubation with DNase I. The resulting supernatants were further treated with 100,000 Mr cut-off membrane (Millipore), and the filtrates were immediately concentrated and desalted in buffer A without EDTA using an Ultrafree-4 centrifugal filter unit (10,000 Mr cut-off, Millipore) before storage at -80 °C. The amount of final product was determined using a Bio-Rad protein assay kit (Bio-Rad) with bovine serum albumin as a standard.

Overexpression and Purification of BxmR—The primers 5'-GGGGCTCGAGTGTCACCGAAGTCTGCTGTTAAC and 5'-GACAAGAATTCCTAATGACTACTGACTAC were used to amplify bxmR using O. brevis genomic DNA as a template for PCR. Two restriction sites were incorporated into these primers, a forward site (XhoI) and a reverse site (EcoRI). Competent E. coli TOPO10 cells were transformed with the plasmid pBAD/BxmR constructed by subcloning the amplified product into the plasmid expression vector pBAD/MycB restricted with XhoI and EcoRI; the integrity of the plasmid construction was confirmed by dsDNA sequencing. LB medium (2 liters) supplemented with 50 mg/ml ampicillin was inoculated with the pBAD/BxmR-transformed E. coli cells (20 ml) incubated overnight. After incubation for 3 h, 0.02% L-arabinose was added, and cultures were further incubated at 37 °C with vigorous shaking/aeration for 4 h. The cells were harvested by centrifugation and washed with 10 mM Tris-HCl, pH 7.3, buffer containing 1 mM phenylmethylsulfonyl fluoride. The resuspended cells were disrupted by ultrasonication. The low speed supernatant was collected after incubation with 0.02 mg/ml DNase I (Fig. 1, lanes 2 and 5). Recombinant BxmR fusion protein containing an N-terminal polyhistidine tag (N-His6-BxmR) was purified from the supernatant by Ni(II)-chelate chromatography using a MagExtractor fusion protein purification kit (Toyobo, Japan) (Fig. 1, lanes 3 and 6). N-His6-BxmR was then digested with enterokinase overnight at 25 °C to remove the N-terminal polyhistidine tag and further purified according to the manufacturer's protocol (Invitrogen) (Fig. 1, lanes 4 and 7). Recombinant BxmR thus purified was estimated to be 96% homogeneous as assessed by scanning a photograph of the Coomassie-stained gel (Fig. 1, lane 4). The monomer concentration of BxmR was determined using the Bradford method standardized to bovine serum albumin.



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  		FIG. 1.
SDS-PAGE (lanes 1-4) and Western blotting (lanes 5-7) analysis of recombinant O. brevis BxmR. N-terminally His-tagged BxmR was overexpressed in E. coli and purified as described under "Experimental Procedures." Lanes 1-4, proteins were stained with Coomassie Blue. Lane 1, standard molecular weight markers (kDa); lane 2, soluble protein fraction following sonication and low speed centrifugation (20 µg of total protein); lane 3, 2 µg of His-tagged BxmR following nickel-chelate chromatography; lane 4, 2 µg of His-tagged BxmR following enterokinase digestion to remove the N-terminal His-tag. Lanes 5-7, Western blot of lanes 2-4 using an anti-His antibody.

 
Electrophoretic Mobility Shift Analysis (EMSA)—The DNA probes for EMSA designated P5 (Fig. 2A) and P11 (Fig. 2B) were prepared by PCR using the primers shown (underlined sequences in Fig. 2, A and B). The 3'-end of each DNA fragment was labeled with digoxigenin (DIG) using terminal transferase and DIG-ddUTP and EMSA experiments carried out using a DIG-Gel shift kit (Roche Applied Science) according to the manufacturer's instructions or standard methods. Purified recombinant BxmR at the concentration indicated or a crude O. brevis cellular extract was incubated with 80 fmol of DIG-P5 or DIG-P11 in a buffer containing 25 mM Tris-HCl, pH 7.3, 10% glycerol, 1 µg of poly(dI-dC) (or 5 µg for cell extracts), and/or poly(dA-dT)·poly(dA-dT), 1 mM dithiothreitol, in a total volume of 20 µl at ambient temperature. The effect of exogenous metals on protein-DNA complex formation was examined by addition of either ZnSO4, CdCl2, CuSO4, or CuCl2 plus 4 mM dithiothreitol, and AgNO3 to the indicated total concentration. Reaction mixtures were incubated at ambient temperature for 30 min, and DNA-protein complexes were separated at 4 °C on an 8% nondenaturing PAGE and transferred to a positively charged nylon membrane (Roche Applied Science). The DIG-derived chemiluminescent signal was detected using the Lumi-Imager work station (Fuji Film), with the migration of protein-DIG-P5 or DIG-P11 complexes directly compared with DIG-labeled dsDNA markers of known molecular weight (Roche Applied Science). The intensity of the individual gel bands (Ii) was quantitated using an Image Analysis System/Molecular Analyst software (Bio-Rad) and converted to mol fractions of the ith DNA species, {Theta}i, using {Theta}i = Ii/{sum}Ii.



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  		FIG. 2.
A, DNA fragments derived from the promoter regions of bxmR/bmtA, and B, bxa1 used for the EMSA with BxmR. The open rectangles and associated arrows denote the genes and their relative transcription orientation. The DNA sequences of P5 and P11 used for the EMSAs (see Fig. 4) are shown with the primers used for PCR amplification underlined. C, alignment of the 12-2-12 inverted repeat elements that give rise to metal-regulated transcription of genes encoding BxmR/BmtA and Bxa1 in O. brevis compared with other SmtB/ArsR metalloregulatory repressors in cyanobacteria. The genes are described in Fig. 3.

 
In Vivo Analysis of Expression of bmxR, bmtA, and bxa1 Induced by Heavy Metal Ions Using Real Time Quantification RT-PCR (rQRTPCR)—For the experiments designed to examine the metal concentration dependence of expression, O. brevis cells (about 0.1 g wet weight) were suspended in 200 ml of a medium containing ZnSO4 (4, 8, 16, 32, 60, 92, or 122 µM), CdCl2 (2, 4, 8, 16, or 32 µM), CuSO4 (4, 8, 12, 16, 24, or 32 µM), and AgNO3 (2, 4, 7, 10, 15, or 18 µM) salts, respectively. Samples were harvested for total RNA isolation after incubation for 6 h. For the experiments designed to examine the kinetics of metal induction, ZnSO4 (60 µM), CdCl2 (16 µM), CuSO4 (16 µM), or AgNO3 (5 µM) was added to the same wet weight of O. brevis cultures as above. The cultures were then incubated at 25 °C for 0.5, 1, 2, 4, 8, 24, 48, 72, and 168 h and harvested for RNA isolation as described above. The quantification of absolute mRNA concentrations induced by heavy metal ions was performed by rQRT-PCR using external standards as described previously (41, 42). The integrity of the amplification of specific fragments was confirmed by melting-curve analysis and DNA sequencing (42).

Transcription/Translation of bxa1 in E. coli Using an in Vitro bxa1 Promoter-driven Expression System—Three DNA fragments (designated P9bxa1, P10bxa1, and P11bxa1; see Fig. 2B) were amplified from O. brevis genomic DNA using PCR and the following primers: 5'-GGAATTAGGATGAGTGGCGT (sense primer for P10bxa1), 5'-GAAGCTAAAAGAAGATTCAGCCTTG (sense primer for P9bxa1), 5'-GAGTTAAAAATCTAGTATAT-3' (sense primer for P11bxa1), and 5'-GGGGCATGCCTACTTTAAAACTCTAGTTGC-3' (universal antisense primer). These PCR products were gel-purified and used directly as DNA templates in an in vitro transcription/translation assay carried out with a commercial E. coli S30 lysate (Extract System for Linear Templates from Promega). Reaction mixtures containing DNA template, purified BxmR, and metal ions (when added) were preincubated at 4 °C and then added to the E. coli 30 S lysate to a total volume of 50 µl, as recommended by the manufacturer. In vitro transcription/translation was allowed to proceed at 37 °C for 2 h, with the translation products subsequently precipitated with acetone. The resulting pellet was resuspended with 20 µl of SDS-PAGE loading buffer and loaded onto a denaturing 8% SDS-PAGE gel for electrophoresis. The expression level of Bxa1 was analyzed and quantified by Western blotting by using an antisera raised against Bxa1 as described previously (41).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
BxmR Is an SmtB/ArsR Family Metalloregulatory Protein—Our previous work in heavy metal tolerance in O. brevis identified the genes encoding Bxa1, a putative heavy metal-transporting CPx-ATPase, and BmtA, a metal-sequestering MT (41, 42). However, those studies failed to identify a transcriptional regulator of the expression of these genes. In many bacterial heavy metal resistance systems, the genes encoding the transcriptional regulator and the resistance protein, i.e. an MT or a metal transporter, are organized either as two consecutive open reading frames (ORFs) or as a divergently transcribed operon (12). We therefore sequenced DNA fragments carrying the bxa1 and bmtA genes derived from O. brevis. This led to the identification of a 411-bp ORF, found 506 bp upstream of the start codon of bmtA and in the opposite coding orientation relative to bmtA. We denote this gene bxmR (Fig. 2A).

Blast searches using the deduced amino acid sequence of bxmR as query reveal that BxmR is a member of the SmtB/ ArsR family of metalloregulatory proteins (12, 28) and is most closely related to Synechocystis PCC 6803 ZiaR, with 65% identity (44) (Fig. 3). BxmR also shares 56% identity with Synechococcus PCC 7942 SmtB (26) and {approx}25-30% identity with other SmtB/ArsR family members in other bacteria (12). The highest degree of sequence identity was found to reside in the putative helix-turn-helix DNA-binding domain, as is typical for other SmtB/ArsR family members (12).



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  		FIG. 3.
Multiple sequence alignment of BxmR with other ArsR family regulatory proteins. Sequences shown are as follows: BxmR from O. brevis; ZiaR from Synechocystis PCC 6803; SmtB from Synechococcus PCC 7942; all7621, an open reading frame divergent from an ORF encoding a putative CPx-ATPase gene (ZiaA homolog) from Anabaena PCC 7120; tlr1018, a divergent open reading frame from an ORF encoding a putative MT (SmtA homolog) from Synechococcus elongatus; CadC from S. aureus (chromosomally encoded); ArsR from E. coli (chromosomally encoded). The conserved and semi-conserved residues are gray, with cysteines colored red and histidines colored blue. The {alpha}3N (red circles) and {alpha}5 (green circles) metal-binding residues previously identified in S. aureus pI258 CadC (31) and Synechococcus SmtB (34), respectively, are also highlighted (see text for details) (12).

 
DNA sequence analysis of the region between bxmR and bmtA reveal the presence of an imperfect 12-2-12 inverted repeat element (IRE) with a conserved central core 5'-TGAAXXXXXXTTCA (Fig. 2, A and C). Previous work has shown that similar inverted repeats function as DNA-binding sites for other SmtB/ArsR homologs in a wide range of bacteria (Fig. 2C) (44, 45), where mutagenesis typically leads to constitutive expression of the target gene (26, 44, 46, 47). Analysis of the putative promoter regions of the all7621 and tlr1018 genes from the cyanobacteria genome data base (www.kazusa.or.jp/cyanobase/) also reveals the existence of a 12-2-12 inverted repeat showing high identity to the smt O/P (Fig. 2C). These data support the hypothesis that the highly conserved 12-2-12 inverted repeat elements in all cyanobacteria function as specific recognition sites for SmtB/ArsR-mediated regulation of target genes in response to specific metal ions.

Most interesting, a 12-2-12 IRE with significant nucleotide sequence identity to that of bxmR/bmtA IRE was also found in the promoter region of bxa1, which encodes a putative CPx-ATPase (Fig. 2B). The presence of this IRE in the promoter region of bxa1 initially suggested that another SmtB homolog might be encoded close to bxa1. However, extensive DNA sequencing on both sides of bxa1 failed to locate a gene encoding an SmtB/ArsR homolog, nor was there any other identifiable candidate regulatory gene close by. Because previous studies indicated that bxa1 is strongly induced by metal ions (41, 42), this sequence analysis makes the prediction that BxmR metalloregulates bxa1 transcription, like bmtA expression (see below).

BxmR Specifically Binds to the 12-2-12 Inverted Repeat Elements in bmtA/bxmR and bxa1 Operator/Promoters with Similar Apparent Affinities—EMSAs were next carried out to determine whether recombinant BxmR binds specifically to the operator/promoter regions of both bmtA/bxmR or bxa1 in vitro. In order to roughly map the boundaries of the BxmR-binding site in both regions, the 506-bp region between the bmtA and bxmR ORFs was divided into four overlapping DNA segments of {approx}200 bp designated P1, P2, P3, and P4, with P1 overlapping the start codon for bxmR and P4 overlapping that for bmtA (Fig. 2A). A similar fragmentation was also performed for a 683-bp region upstream of bxa1, to create EMSA probes P7, P8, P9, and P10, with P7 overlapping the initiation codon for bxa1 (Fig. 2B). When EMSA was performed with these DNA fragments, an electrophoretic mobility shift with saturating BxmR was observed only with P1 and P7, each of which contain the imperfect 12-2-12 inverted repeat as described above (data not shown). To confirm that BxmR specifically binds to the 12-2-12 IREs, we further truncated P1 and P7 into {approx}80-100-bp fragments designated P5 and P11, respectively, each of which contain the intact 12-2-12 inverted repeat region (see Fig. 2, A and B, for nucleotide sequences). These results are shown in Fig. 4.



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  		FIG. 4.
In vitro EMSA analysis of the binding of recombinant BxmR to the putative promoter region in bxmR/bmtA (A) and bxa1 (B). A, DNA fragment P5 (see Fig. 2A) was incubated with increasing concentrations of BxmR monomer as indicated (lanes 3-9) or no protein (lane 2). In lane 10, the binding reaction was carried out under the same conditions as in lane 7 except that unlabeled P5 was added in 100-fold excess to Dig-labeled P5. Lane 1, DNA marker. FP, C1, C2, and C3 correspond to free DNA and BxmR-DNA complexes 1, 2 and 3, respectively. B, DNA fragment P11 (see Fig. 2B) was used for this experiment, carried out exactly as described in A. C, quantitation of the relative intensities of the ith gel band (Ii) from the BxmR-P5 DNA EMSA experiment shown in A. Relative intensities were directly converted to mol fractions of the ith P5 species ({Theta}i) from {Theta}i = Ii/{sum}Ii. D, quantitation of the relative intensities of the ith gel band (Ii) from the BxmR-P11 DNA EMSA experiment shown in B, with {Theta}i calculated as described for BxmR-P5 mixtures.

 
As can be seen, both oligonucleotide P5, derived from the bmtA/bxmR operator/promoter (Fig. 4, A and C), and oligonucleotide P11, from the bxa1 operator/promoter (Fig. 4, B and D), give rise to multiple resolvable complexes when titrated with recombinant BxmR in an EMSA experiment in the presence of 1-5 µg of poly(dI-dC) and/or poly(dA-dT)·poly(dA-dT). With respect to the bmtA/bxmR operator/promoter fragment P5, two slowly migrating bands designated C1 and C2 are visible at a total BxmR monomer concentration of 0.07 µM, in addition to free P5 (FP). As the total BxmR concentration is increased to >=0.70 µM, a third migration complex, designated C3, is observed. Even at the highest concentration of BxmR tested, both C2 and C3 persist, with C2 {approx}3-fold more abundant than C3 at 7.0 µM (Fig. 4C). The BxmR concentration dependence of binding to the bxa1 operator/promoter fragment P11 was found to be similar to that of P5 (Fig. 4, B and D); this suggests that BxmR binds specifically to the P11 fragment in vitro with an apparent binding affinity similar to that of P5. A 100-fold molar excess of unlabeled P5 or P11 probe was sufficient to render all three slowly migrating complexes undetectable (Fig. 4, A and B, lane 10), revealing that the binding is indeed sequence-specific. Although the BxmR-DNA binding stoichiometry in C1, C2, and C3 is unknown and any linkage to the BxmR monomerdimer equilibrium (33) was not investigated in these experiments, these gel mobility shift patterns bear a qualitative resemblance to those published previously for apoSmtB from Synechococcus PCC7942 where up to four dimers are known to bind to a single 12-2-12 inverted repeat, two with high affinity (27, 45, 48).

Similar EMSA results were obtained with DNA probes P5 (Fig. 5A) and P11 (Fig. 5B) when cell extracts from O. brevis, incubated for 24 h in a medium containing either Zn(II), Cd(II), Cu(I), or Ag(I), were used in place of purified BxmR. Two slowly migrating complexes that coincide with the mobilities of the C2 and C3 complexes (see Fig. 4) were detected in these cells treated with heavy metals but not in untreated cells. This suggests that the expression of BxmR is induced in vivo by both monovalent and divalent metal ions and, as a result, is capable of binding to both P5 (bmtA/bxmR) and P11 (bxa1) fragments, findings compatible with the EMSA results obtained with recombinant BxmR (Fig. 4).



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  		FIG. 5.
EMSA analysis of the binding of endogenous BxmR from crude O. brevis cell extracts to the putative promoter regions in bxmR/bmtA (A) and bxa1 (B). Cell extracts were isolated from O. brevis cultures subjected to a 24-h exposure to either 60 µM ZnSO4 (lane 2), 16 µM CdCl2 (lane 3), 16 µM CuCl2 (lane 4), or 10 µM AgNO3 (lane 5) or not exposed to heavy metals (lane 1). Each 20-µl reaction mixture contained 5 nM P5 (bxmR/bmtA) fragment (A) or P11 (bxa1) fragment (B) and 5 µg of partially purified cell extract from O. brevis. dsDNA molecular weight markers are shown in lane 6 in both panels.

 
BxmR Is Dissociated from Both bmtA/bxmR and bxa1 O/Ps Upon the Addition of Both Divalent and Monovalent Metal Ions—A common characteristic of SmtB/ArsR metal-regulated repressors is that the operator/promoter binding affinity is strongly negatively regulated by inducing metal ions in vitro, i.e. preincubation with metal salts inhibits protein-DNA complex formation (31, 33, 45). This is also found to be the case for BxmR (Fig. 6). Addition of increasing total concentrations of Zn(II), Cd(II), Cu(II) plus 4 mM dithiothreitol or Ag(I) reduced the formation of all BxmR-DNA complexes formed with either the P5 (bmtA/bxmR) (Fig. 6A) or P11 (bxa1) (Fig. 6B) DNA fragments. The metal-mediated inhibition of BxmR-P5 or BxmR-P11 complexes by monovalent Ag(I) is particularly surprising and has not been observed for any other previously characterized SmtB/ArsR regulator (12, 38). A similar result was obtained by using crude cell extracts from O. brevis in place of purified BxmR (data not shown). In all cases, inhibition of complex formation requires approximately twice as much Zn(II) (50-100 µM) relative to the other metal ions, which require 20-50 µM. In addition, with all metals tested, the concentration of metal salts required to achieve similar levels of metal-mediated inhibition was generally found to be {approx}2x smaller for P11 (bxa1)-BxmR complexes (Fig. 6B) relative to P5 (bmtA/bxmR)-BxmR complexes (Fig. 6A). Although potentially interesting (see "Discussion"), the origin of this effect is unknown and requires further investigation. In any case, the formation of BxmR-P5 and BxmR-P11 complexes is clearly regulated by multiple monovalent and divalent heavy metal ions, consistent with the hypothesis that the repression and derepression of transcription of both bxmR/bmtA and bxa1 is mediated by the metal-sensing repressor BxmR in O. brevis in vivo.



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  		FIG. 6.
The effect of multiple heavy metal ions on the binding of recombinant BxmR to the putative promoter region in bxmR/bmtA (A) and bxa1 (B). Each 20 µl of reaction mixture contained 5 nM P5 (A) or P11 (B) DNA fragments, 0.70 µM total BxmR monomer, and the indicated total concentration of each metal salt.

 
Recombinant BxmR Functions as a Repressor of the Expression of bmtA, bxa1, and bxmR in a Coupled in Vitro E. coli Transcription/Translation System—Because the EMSA results reveal that multiple metal ions reversibly regulate the binding of BxmR to promoter regions of both bxa1 and bmtA/bxmR, direct evidence for transcriptional regulation was sought. A fragment containing the entire bxmR/bmtA operator/promoter region was subcloned into the vector pBAD/HisA and transformed into E. coli. rQRT-PCR analysis indicated that the transcription of both bxmR and bmtA was induced in E. coli transformed with the bxmR/bmtA O/P upon addition of both divalent and monovalent metal ions in the media. An in vitro transcription/translation experiment was next carried out to examine the ability of recombinant BxmR to regulate bxa1 expression using an E. coli in vitro transcription/translation system. This experiment was facilitated by the high similarity of the putative -10 (TATATT) and -35 (TAGACT) regions of the bxa1 gene relative to consensus E. coli promoters (cf. Ref. 49).

Because the complete promoter region of bxa1 was not yet defined, three DNA fragments, designated P10bxa1 (including 683 bp upstream of the start codon for bxa1 plus the entire bxa1 gene), P9bxa1 (505 bp upstream), and P11bxa1 (89 bp upstream) (see Fig. 2B), were employed as DNA templates in a coupled transcription/translation using E. coli extracts. All of these fragments contain the imperfect 12-2-12 inverted repeat region. As expected, transcription/translation of bxa1 was achieved in the E. coli system (Fig. 7A), with P10bxa1 or P9bxa1 templates effective to nearly the same degree. In contrast, the Bxa1 synthesis driven by P11bxa1 was poorly effective, indicating that a DNA fragment containing only an inverted repeat motif is not sufficient for bxa1 transcription/translation.



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  		FIG. 7.
Recombinant BxmR regulates bxa1 transcription/translation in vitro in a heavy metal-dependent fashion. A, suppression of Bxa1 protein synthesis by recombinant BxmR in an in vitro E. coli transcription/translation system using DNA fragments encoding the bxa1 promoter as template. 2 µg of P10bxa1 (lane 2), P9bxa1 (lane 3), or P11bxa1 (lane 4) DNA fragments (see Fig. 2B) was added to each 50 µl of reaction mixture as transcription/translation template. Lanes 5-9, 2 µg of P10bxa1 in the presence of BxmR at the indicated total concentration. Lane 1, molecular marker (kDa). B, effect of BxmR and heavy metal ions on the transcription/translation of Bxa1. Each 50 µlof reaction mixture contained 5 nM P10bxa1 DNA fragment, 12 µM BxmR monomer, and metal salts at the indicated concentrations (µM). Lane 1, molecular marker. The in vitro transcription/translation reaction was carried out at 37 °C for 2 h. The translation products were purified, subjected to SDS-PAGE, and the gel Western blotted as described under "Experimental Procedures." The intensity analysis was performed as described and normalized to the lane containing no added metal ions (lane 5, A), with fractional intensities given below the lane numbers. The inhibition of coupled transcription/translation in the presence of 100 µM ZnSO4 or 50 µM AgNO3 is likely due to template degradation.

 
As expected for a repressor, addition of recombinant BxmR to these reaction mixtures resulted in a dose-dependent decrease in Bxa1 production, with 92% inhibition obtained with 12 µM BxmR monomer (Fig. 7A, lane 9). These data directly show that BxmR alone is capable of efficient repression of Bxa1 synthesis. Furthermore, this BxmR-mediated repression is quantitatively reversed upon addition of 50 µM zinc, cadmium, or copper salts to the coupled transcription/translation reaction mixture, with 20 µM silver mediating the same level of derepression (Fig. 7B). When higher concentrations of either Zn(II) (100 µM) or Ag(I) (50 µM) were added, significant inhibition of Bxa1 synthesis was observed (Fig. 7B, lanes 3 and 9); this behavior might be due to degradation of template DNA, which was also observed in the EMSA experiments (data not shown).

Metal-mediated Regulation of bxa1, bmtA, and bxmR mRNA Levels in Vivo by BxmR—Given the genetic organization of the putative bxmR/bmtA divergon (Fig. 2A), the metal-regulated transcription of the bxmR gene in O. brevis might parallel the expression of bmtA, which may or may not parallel the metal induction profile of bxa1. To investigate this, rQRT-PCR was employed to measure both the metal concentration dependence and the kinetics of the expression of bxmR, bmtA, and bxa1 mRNA induced by multiple heavy metals in O. brevis cultures. As a control, the mRNA expression profile in O. brevis cells grown under the same culture conditions without exposure to heavy metal ions was also examined.

Fig. 8 shows how the expression of bxmR, bmtA, and bxa1 is influenced by a 6-h incubation with the indicated total concentrations of metal salts added to the media. Similar metal dose-response curves were obtained for all three genes with Cd(II) (Fig. 8B), Cu(I) (Fig. 8C), and Ag(I) (Fig. 8D), with maximal inductions obtained with 4 µM Cd(II), 8 µM Cu(I), and 4 µM Ag(I). Such an induction profile is consistent with the idea that the same factor mediates the expression of bxmR, bmtA, and bxa1 in response to multiple metal ions. Most interesting, a lower concentration of Zn(II) was required to induce bxmR and bmtA transcription ({approx}8-16 µM), relative to bxa1 transcription, the latter of which only occurred at a much higher total metal concentrations ({approx}60 µM).



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  		FIG. 8.
Quantification of the expression of bxmR, bmtA, and bxa1 by rQRT-PCR from O. brevis incubated with the indicated concentration of metal salts for 6 h prior to harvest and total RNA isolation. O. brevis cultures were treated with ZnSO4 (A), CdCl2 (B), CuSO4 (C), and AgNO3 (D) with RNA isolation, generation of external standards, and rQRT-PCR conducted as described under "Experimental Procedures." Note that the values indicated on the abscissas are x105 bmtA mRNA copies/ng total RNA versus 102 bxmR and bxa1 mRNA copies/ng total RNA. Data were obtained from at least three independent analyses, and S.D. of the mean is indicated.

 
The temporal pattern of bxmR, bmtA, and bxa1 expression was next examined at a fixed concentration of heavy metal salts: 60 µM Zn(II) (Fig. 9A), 16 µM Cd(II) (Fig. 9B), 16 µM Cu(I) (Fig. 9C), and 5 µM Ag(I) (Fig. 9D). Strikingly, the kinetics of metal-mediated induction of mRNA synthesis is identical for all four metal salts. The expression of bxa1, which encodes a putative CPx-ATPase metal transporter, is maximal ~1 h after addition of metal salts to the media, with parallel induction of bxmR and bmtA, which both becomes maximal only much later in the 8-24-h time window. These data make two important points. The expression kinetics of bxmR completely parallel that of bmtA. Because EMSAs have established bxmR and bmtA share the same protein-binding site (Fig. 4), it is this site that is likely responsible for the co-transcriptional regulation of these two genes. The second is that metal-mediated derepression of the transporter might function as the first or acute phase of defense against metal toxicity in O. brevis, with the repressor and the putative metallothionein, BmtA, providing a secondary system designed to counter chronic exposure to the same metal ions.



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  		FIG. 9.
Analysis of the kinetics of expression of bxmR, bmtA, and bxa1 mRNAs by rQRT-PCR in the presence of a fixed concentration of the indicated metal salt. O. brevis cultures were treated with either 60 µM ZnSO4 (A), 16 µM CdCl2 (B), 16 µM CuSO4 (C), or 5 µM AgNO3 (D) for the indicated time between 0.5 and 48 h prior to harvest and RNA isolation. Sample treatment, RNA isolation, generation of external standards, and rQRT-PCR were conducted as described under "Experimental Procedures." Data were obtained from at least three analyses, and the S.D. is indicated.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A Novel SmtB/ArsR Metal Sensor Protein from O. brevis, BxmR, Mediates the Metalloregulated Transcription of bxmR, bmtA, and bxa1—Previous studies (41, 42) revealed that heavy metal tolerance in the fresh water cyanobacterium, O. brevis, involved the Zn(II)- and Cd(II)-dependent up-regulation of both a putative heavy metal sequestering metallothionein, encoded by the bmtA gene, and a putative CPx-ATPase metal transporter, encoded by bxa1. Consistent with this physiological role in O. brevis, overexpression of either bmtA or bxa1 in E. coli increases metal resistance in this system as well (42). In this paper, we establish two additional important points. First, a single metal-regulated repressor, designated BxmR, is necessary and sufficient to mediate metal-dependent transcriptional regulation of the expression of both bmtA and bxa1 genes. Amino acid sequence analysis clearly establishes that BxmR is a member of the SmtB/ArsR family of metal-sensing repressors (12, 28). Although other SmtB/ArsR metal sensor proteins regulate the expression of either an MT or a metal transporter (12), to our knowledge this is the first example of a single repressor mediating the metalloregulation of both a metallothionein and a putative metal efflux pump in the same organism. The second significant conclusion reached here is that O. brevis BxmR metalloregulates the expression of all three genes in response to both the coinage metals Ag(I) and Cu(I), as well as Zn(II) and Cd(II), a novel characteristic not only in the SmtB/ArsR family of regulators (12) but also in other metal-loregulatory protein families, e.g. MerR family (25, 35). Although the mechanism of metal-dependent regulation requires further investigation, the studies reported here are consistent with the hypothesis that BxmR harbors metal-binding site(s) for both Cu(I) and Zn(II) that allosterically regulate bxa1 and bxmR/bmtA O/P binding (Fig. 6). The other possibility, of course, is that induction by Cu(I) is indirect and occurs upon redistribution of Zn(II) pools in O. brevis by copper salts.

On the Mode of Metal Binding by BxmR—Here we show that the temporal pattern of Zn(II)/Cd(II) induction of bxa1 versus bmtA genes observed previously (42) is paralleled precisely by the induction by both monovalent Ag(I) and Cu(I). Ag(I) and Cu(I) often form isostructural coordination complexes distinct from that of Zn(II) and Cd(II). Ag(I) and Cu(I) typically adopt either low coordination number (n = 2 or 3) linear bis-thiolato (35) or trigonal tris-thiolato (50) mononuclear metal complexes, or polynuclear clusters in which multiple Ag(I)/Cu(I) ions are assembled into a Cu4-Cys6 cage structures, e.g. in the metal-regulatory sites of S. cerevisiae Mac1 and AceI, and in metal-lothioneins (51, 52). Formation of a polynuclear cluster with a limited number of donor ligands would provide exquisite selectivity for Cu(I) and Ag(I). Although it is not yet known how BxmR binds metal ions of any type, BxmR contains many candidate ligands in the N-terminal region (including His15, Cys23, His27, and Cys31; Fig. 3) as well as a Cys pair (Cys75-Val76-Cys77) near the middle of the protein. Each of these two regions harbor known metal ligands to the Cd(II)/Pb(II) ion in S. aureus pI258 CadC (31). BxmR also contains all four {alpha}5 metal site ligands known to comprise the Zn(II)-sensing site in zinc sensors Synechococcus PCC7942 SmtB and S. aureus CzrA (34, 45) (Fig. 3). Direct metal-binding experiments carried out with metal salts in an anaerobic atmosphere will provide further insight on the metal stoichiometry, coordination chemistry, metal selectivity, and allosteric regulation of DNA binding in BxmR.

Temporal Patterns of Metal Resistance Gene Expression in O. brevis—The finding that the bxmR, bmtA, and bxa1 genes are reversibly regulated by BxmR is consistent with the scenario that all three genes belong to a same metal homeostasis system in O. brevis. Previous kinetic studies of the expression of bxa1 and bmtA have already established that Zn(II) and Cd(II) strongly induce bxa1 expression within 1 h of metal treatment, with bmtA expression maximally induced 8 or more hours later (42). These results are consistent with the idea that the metal transporter Bxa1 is a rapid response determinant required to handle the trafficking of intracellular metal ions and function as first line of defense against acute metal toxicity. In contrast, the metallothionein BmtA is a relatively slowly emerging factor that may protect O. brevis from chronic metal toxicity that results from a more longer term change in the environment.

The molecular mechanism of the rapid induction of bxa1 relative to bmtA is currently unknown. One way to achieve rapid induction or derepression of bxa1 specifically is to favor intracellular conditions under which constitutively expressed BmxR is bound primarily to the bxa1 O/P, with the bxmR/bmtA O/P free of the regulatory factor. However, unless other factors influence the occupancy of the O/Ps in vivo, both sites are likely to be bound by BxmR because they appear to have comparable affinities for BxmR (Fig. 4); furthermore, the expression of all three genes is strongly induced by metal ions (Figs. 8 and 9). Another possible mechanism is that metal ion-mediated disassembly of the BxmR-bxa1 promoter complexes takes place preferentially relative to the BxmR-bxmR/bmtA promoter complexes in vivo. This could occur either via a greater sensitivity of the BxmR-bxa1 complexes to the effective concentrations of inducing metal ion that would exist in an acute (early) metal toxicity phase, relative to chronic (longer term), metal toxicity conditions, or on purely kinetic grounds, i.e. the rate of disassembly is more rapid for BxmR-bxa1 relative to the BxmR-bxmR/bmtA promoter complexes. Initial experiments suggest that the BxmR-bxa1 promoter complex may be dissociable by lower total concentrations of the metal salts relative to BxmR-bxmR/bmtA promoter complexes (Fig. 6), potentially consistent with the former scenario. The bmtA mRNA (Fig. 8) and BmtA itself (47) are both abundant in O. brevis, even in the absence of metal inducer (Fig. 8), relative to both bxa1 and bxmR mRNAs and proteins. Perhaps constitutively expressed BmtA sufficiently buffers metal ion concentrations in the acute phase, which is nonetheless sufficient to derepress bxa1 expression. Bxa1 would then rapidly move to the membrane and efficiently efflux metal ion outside of the cytosol. This process of metal ion efflux may be enabled by the ability of BmtA-metal complexes to deliver metal ion directly to the efflux pump, i.e. function formally as metallochaperones for the transporter (53). Only when the available metal-binding sites on BmtA in the cytosol are filled would the up-regulation of the expression of the bmxR/bmtA regulon by metal ions occur. Finally, as the intracellular concentrations of both BmtA and Bxa1 rise, the effective or free metal ion concentration would then fall dramatically and a new homeostasis that balances efflux and intracellular chelation be established thereby allowing O. brevis to survive under conditions of chronic metal toxicity.

Like that shown here for O. brevis, the genome of another filamentous cyanobacterium Anabaena PCC 7120 (www.kazusa.or.jp/cyanobase) also encodes homologs of both a CPx-ATPase metal transporter and a metallothionein with high similarities to Synechocystis PCC6803 ZiaA (44) and Synechococcus PCC7942 SmtA (13), respectively. However, in a situation opposite to O. brevis, the BxmR-like regulatory gene is upstream of the ORF encoding the putative ZiaA-like protein, with no SmtB/ArsR homolog close to the gene encoding the putative metallothionein. The significance of this difference is unknown. In any case, the metal homeostasis systems in Oscillatoria and Anabaena closely parallel that in mammalian cells, where the zinc finger transcription factor MTF-1 is known to mitigate the effects of Zn(II)/Cd(II) toxicity by transcriptionally activating the genes encoding both a metal efflux pump, ZnT1, as well as a metallothionein, MT-I (54-56). The physiological relationship between the MT (BmtA) and the metal transporter (Bxa1) in O. brevis is unknown; however, it is tempting to speculate that BmtA and Bxa1 may be formally analogous to Atx1 and CCC2 that function in copper trafficking in yeast (53, 57). Although no zinc-specific metallochaperones have been reported, MTs are known to perform a variety of functions, one of which is to kinetically facilitate the loading of Zn(II) into apoenzymes (58). Indeed, recent studies2 reveal that the addition of BmtA and cysteine increases the ATPase activity of purified, recombinant Bxa1 reconstituted in lipid vesicles. Further investigations of the exchange of heavy metal ions among BxmR, BmtA, and Bxa1 will help further elucidate the mechanisms of metal resistance and trafficking of metal ions in living cells.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB085750 [GenBank] , AB085749 [GenBank] , and AB073990 [GenBank] .

* This work was supported in part by grants from the Sumitomo Foundation (to S. N.), the Oohara Foundation for Agricultural Sciences, Grant-in-aid for the Future Program from the Japanese Society for the Promotion of Science 9616001 (to S. N.), Grants for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan 11440237 (to K K.) and 10878087 (to S. N.), and National Institutes of Health Grant GM042569 (to D. P. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence may be addressed. Tel.: 81-86-434-1211; Fax: 81-86-434-1249; E-mail: snakashi{at}rib.okayama-u.ac.jp.

|| To whom correspondence may be addressed. Tel.: 979-845-4231; Fax: 979-845-4946; E-mail: giedroc{at}tamu.edu.

1 The 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. Back

2 T. Liu, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Professors Paul M. Hasegawa (Purdue University) and Hideaki Matsumoto (Okayama University) for their comments on the manuscript and continued interest in this project and the Norwegian Institute for Water Research for providing O. brevis. The critical suggestions of Professor Nigel J. Robinson (University of Newcastle, United Kingdom) are also gratefully acknowledged.



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 RESULTS
 DISCUSSION
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