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Journal of Bacteriology, September 2003, p. 5363-5371, Vol. 185, No. 18
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.18.5363-5371.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Arsenic Sensing and Resistance System in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Luis López-Maury, Francisco J. Florencio, and José C. Reyes*

Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas, Universidad de Sevilla, E-41092 Seville, Spain

Received 28 March 2003/ Accepted 23 June 2003


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ABSTRACT
 
Arsenic is one of the most important global environmental pollutants. Here we show that the cyanobacterium Synechocystis sp. strain PCC 6803 contains an arsenic and antimony resistance operon consisting of three genes: arsB, encoding a putative arsenite and antimonite carrier, arsH, encoding a protein of unknown function, and arsC, encoding a putative arsenate reductase. While arsB mutant strains were sensitive to arsenite, arsenate, and antimonite, arsC mutants were sensitive only to arsenate. The arsH mutant strain showed no obvious phenotype under the conditions tested. In vivo the arsBHC operon was derepressed by oxyanions of arsenic and antimony (oxidation state, +3) and, to a lesser extent, by bismuth (oxidation state, +3) and arsenate (oxidation state, +5). In the absence of these effectors, the operon was repressed by a transcription repressor of the ArsR/SmtB family, encoded by an unlinked gene termed arsR. Thus, arsR null mutants showed constitutive derepression of the arsBHC operon. Expression of the arsR gene was not altered by the presence of arsenic or antimony compounds. Purified recombinant ArsR protein binds to the arsBHC promoter-operator region in the absence of metals and dissociates from the DNA in the presence of Sb(III) or As(III) but not in the presence of As(V), suggesting that trivalent metalloids are the true inducers of the system. DNase I footprinting experiments indicate that ArsR binds to two 17-bp direct repeats, with each one consisting of two inverted repeats, in the region from nucleotides -34 to + 17 of the arsBHC promoter-operator.


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INTRODUCTION
 
Arsenicals generated from natural and man-made sources are widely distributed contaminants of freshwater, ground water, and seawater (2, 11, 24). Probably because of this, many organisms contain either chromosomal or plasmid-encoded genes involved in arsenical resistance (ars genes). Most of the knowledge about ars genes and their regulation in prokaryotes comes from studies of ars operons in Escherichia coli and Staphylococcus species (recently reviewed in references 21, 26, and 27). The chromosomal ars operon of E. coli and the ars operons from staphylococcal plasmids pI258 and pSX267 are constituted by three genes, arsR, -B, and -C. arsR encodes a trans-acting repressor of the ArsR/SmtB family involved in transcriptional regulation, arsB encodes a membrane-bound arsenite carrier that exports arsenite but not arsenate, and arsC encodes a reductase that converts arsenate to arsenite. In contrast, the ars operons of E. coli plasmids R773 and R46 encode two additional proteins: ArsA, an arsenite-stimulated ATPase, and ArsD, another metalloid-responsive transcriptional repressor.

Three different families of arsenate reductases have been described (for revisions, see references 18 and 26). The first family to be described was the product of the arsC gene from the E. coli plasmid R773 (6). This enzyme uses glutaredoxin as a source of reducing equivalents, and it is present in several gram-negative bacteria. The Staphylococcus aureus pI258 and the Bacillus subtilis ArsC products exhibit no significant similarity with the R773-encoded enzyme (1, 13). This second type of arsenate reductase is related to low-molecular-weight protein tyrosine phosphatases and uses thioredoxin as the source of reducing equivalents. Finally, a third family of arsenate reductases, represented by the Acr2p enzyme from Saccharomyces cerevisiae, is also homologous to a different subfamily of protein phosphatases that includes CDC25a (20, 22).

Arsenate has to be reduced by ArsC to the more toxic form arsenite before being exported by the carrier protein ArsB (for a review, see reference 27). Two independently evolving families of arsenite carriers in bacteria have been described. The arsB gene, present in the E. coli plasmid R773 and in Staphylococcus aureus pI258, encodes an integral membrane protein with 12 membrane-spanning segments that can use the membrane potential to extrude arsenite. Interestingly, ArsB proteins can also function as primary ATP-driven arsenite pumps by interacting with ArsA, an arsenite-stimulated ATPase. The metalloid oxyanion antimonite is also a substrate of this family of ArsB proteins and can stimulate ATPase activity of ArsA (28). The second family of arsenite carriers has been much less characterized and includes the ArsB gene of the B. subtilis ars operon and the ARR3 protein from S. cerevisiae (formerly ACR3) (41). These arsenite transporters (ArsB/ARR3 family) are membrane proteins with 10 predicted membrane-spanning segments. ARR3-disrupted cells accumulate arsenite and are sensitive to arsenite and arsenate but not to antimonite, demonstrating that ARR3p specifically exports As(III) (12).

In addition to these factors, another gene, arsH, has been found close to or cotranscribed with arsenic resistance genes in Yersinia enterocolitica and in Acidothiobacillus ferroxidans (3, 23). The function of the ArsH protein is unknown, but it has been shown to be required for resistance to arsenite and arsenate in Y. enterocolitica.

Cyanobacteria are photosynthetic prokaryotes that carry out oxygenic photosynthesis. Several cyanobacterial species are able to grow in the presence of high concentrations of arsenate (up to 100 mM) and in low-millimolar concentrations of arsenite (36; our unpublished observations). It is worth noting that the maximum contaminant level for arsenic allowed in public water supplies has recently been lowered to 10 µg/liter (approximately 0.13 µM) by the U.S. Environmental Protection Agency (34). The existence of ars genes in cyanobacteria has been suggested by gene homology searches of genome databases, but the function and means of regulation of these genes are unknown. In the present work we have characterized the arsenic resistance system of the cyanobacterium Synechocystis sp. strain PCC 6803.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions. Synechocystis sp. strain PCC 6803 was grown photoautotrophically at 30°C in BG11 medium (25) supplemented with 1 g of HCO3Na per liter (BG11C medium) and bubbled with a continuous stream of 1% (vol/vol) CO2 in air under continuous fluorescent illumination (50 µmol of photons per m2 per s; white light from fluorescent lamps). In the BG11C low-phosphate medium, the concentration of K2HPO4 was reduced to 10 µM. For plate cultures, BG11C liquid medium was supplemented with 1% (wt/vol) agar. Kanamycin and chloramphenicol were added to achieve final concentrations of 50 to 200 and 10 to 40 µg/ml, respectively, when required.

E. coli DH5{alpha} (Bethesda Research Laboratories) grown in Luria-Bertani (LB) broth medium as described previously (29) was used for plasmid construction and replication. E. coli BL21(DE3) grown in LB broth medium was used for expression of the MalE-ArsR protein. E. coli was supplemented with 100 µg of ampicillin/ml, 50 µg of kanamycin/ml, or 40 µg of chloramphenicol/ml and glucose 0.2% (wt/vol) when required.

Insertional mutagenesis of Synechocystis genes. DNA fragments containing loci slr0944, slr0945, slr0946, and sll1945 were amplified by PCR from the cosmids cs0223 and cs0205 (provided by Kazusa DNA Research Institute) using the following oligonucleotide pairs: ARSB2-ARSB3, ARSH1-ARSH2, ARSC1-ARSC2, and ARSR1-ARSR2. The oligonucleotide sequences are available on request. DNA fragments were cloned into pGEM-T (Promega) to generate pARSB1, pARSH1, pARSC1, and pARSR1, respectively. The targeting vector pARSB2(+) was generated by replacing a 79-bp BstEII-BstEII fragment by the C.K1 cassette (8) in the same orientation as that of the arsB gene. The targeting vectors pARSH2(+) and pARSH2(-) were generated by inserting the C.K1 cassette into the EcoRI sites of the arsH gene in the same orientation as that of the ars operon [pARSH2(+)] or in the inverse orientation [pARSH2(-)]. The targeting vector pARSC2(-) was generated by inserting the C.K1 cassette into the SmaI site of the arsC gene in the orientation opposite to that of the ars operon. The targeting vector pARSR2(+) was generated by inserting the C.C1 cassette into the HindIII site of the arsR gene in the same orientation as that of the arsR gene. All targeting vectors were used to transform Synechocystis strain 6803 as previously described (9).

The correct integration and complete segregation of the mutant strain were tested by Southern blotting using standard procedures (29). Total DNA from the cyanobacteria was isolated as previously described (5).

RNA isolation and Northern blot analysis. Total RNA was isolated from 25-ml samples of Synechocystis cultures at the mid-exponential growth phase (3 to 5 µg chlorophyll/ml). RNA extractions were performed as previously described (10).

For Northern blotting, 15 µg of total RNA was loaded per lane and electrophoresed in 1.0% (wt/vol) agarose denaturing formaldehyde gels and transferred to nylon membranes (Hybond N-plus; Amersham). Prehybridization, hybridization, and washes were performed as described in the Amersham instruction manual. Probes were synthesized by PCR with the same oligonucleotide pairs used for cloning of the genes except that the arsR probe was synthesized by using oligonucleotides ARSR2 and ARSR3. DNA probes were 32P labeled with a random-primer kit (Amersham) with [{alpha}-32P]dCTP (3,000 Ci/mmol). All of the filters were stripped and reprobed with a 580-bp HindIII-BamHI probe from plasmid pAV1100 that contains the constitutively expressed RNase P RNA gene (rnpB) from Synechocystis strain PCC 6803 (38). To quantify the radioactive signals in Northern blot hybridizations, an InstantImager electronic autoradiography apparatus (Packard Instrument Company, Meriden, Conn.) was used.

Primer extension analysis. Oligonucleotide ARSB1 end labeled with T4 polynucleotide kinase and [{gamma}-32P]ATP (3,000 Ci/mmol) according to standard procedures (29) was used for primer extension analysis of the arsBHC operon transcript. Primer extension reactions were carried out as previously described (15). One-half of the reaction mixture was electrophoresed on a 6% polyacrylamide sequencing gel, and the arsB promoter region was subjected to a sequencing reaction using the ARSB1 oligonucleotide.

Cloning and purification of MalE-ArsR. The complete arsR open reading frame (ORF) was PCR amplified with oligonucleotides ARSR2 (which introduces an EcoRI site) and ARSR3 (which introduces a PstI site) and cloned in pGEM-T (Promega) to generate pARSR3. This plasmid was sequenced to verify the fidelity of the PCR product. The 452-bp EcoRI-PstI fragment containing the arsR gene was cloned into EcoRI-PstI-digested pMALc-2x (New England Biolabs) to generate pARSR4. The MalE-ArsR recombinant protein was expressed in E. coli BL21(DE3) from the plasmid pARSR4. For that process, 500 ml of culture was grown in LB broth supplemented with 0.2% (wt/vol) glucose to an optical density at 580 nm of 0.6, induced with 1 mM isopropyl-ß-D-thiogalactopyranoside for 2.5 h, harvested by centrifugation, and resuspended in 8 ml of 50 mM Tris-HCl (pH 8.0)-100 mM KCl (buffer A) supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM EGTA, and 1 mM {varepsilon}-aminocaproic acid. Cells were broken by sonication, and insoluble debris was pelleted by centrifugation at 18,000 x g for 15 min. The supernatant was then diluted to 40 ml with buffer A, and 1 ml of bed volume amylose resin (New England Biolabs) was added and allowed to bind for 1 h a 4°C with gentle agitation. The resin was then transferred to a plastic column, washed with buffer A until no protein was detectable in the flowthrough, and eluted with the same buffer containing 10 mM maltose and 10% glycerol.

MalE-ArsR was digested with factor Xa protease (New England Biolabs) to separate the MalE domain from ArsR as described by the manufacturer. Briefly, 400 µg of purified MalE-ArsR was incubated for 12 h at 25°C in buffer A containing 2 mM Cl2Ca with 5 µl of factor Xa in a final volume of 500 µl to yield two protein fragments corresponding to MalE (42 kDa) and ArsR (12.5 kDa).

Gel retardation assays. The probe was synthesized by PCR using oligonucleotides ARSB7 and ARSB8, containing NarI and NcoI restriction sites, respectively, from cosmid cs0205 (provided by Kazusa DNA Research Institute). The resulting DNA was digested with NcoI and end labeled with [{alpha}-32P]dCTP (3,000 Ci/mmol) by using the Klenow fragment. The size of the resulting probe was 95 bp from nucleotides -59 to +36 with respect to the transcription start point. The binding reaction was carried out in a final volume of 25 µl containing 4 ng of labeled DNA and 5 µg of salmon sperm DNA in 50 mM Tris-HCl (pH 8.0)-100 mM KCl-5 mM dithioreitol, 10% glycerol, and different amounts of purified ArsR. The mixtures were incubated for 25 min at room temperature and loaded on a nondenaturing 6% polyacrylamide gel. Electrophoresis was carried out at 4°C and 180 V in 0.25x Tris-borate-EDTA. Gels were transferred to Whatman 3MM filter paper, dried, and autoradiographed.

DNase I footprinting. The probe was synthesized by PCR using oligonucleotides ARSB9 and ARSB10, containing NarI and NcoI restriction sites, respectively. The size of the resulting probe was 237 bp from nucleotides -132 to +105 with respect to the transcription start point. For footprinting the coding strand, the DNA was digested with NcoI and end labeled with [{alpha}-32P]dCTP (3,000 Ci/mmol) by using the Klenow fragment. For footprinting the noncoding strand, the DNA was digested with NarI and end labeled in the same way. The binding reaction was carried out as described for gel retardation assays but without glycerol. DNA-protein complexes were incubated for 30 s or 1 min with 1 U of DNase I (Roche) in the presence of MgCl2. Reactions were terminated by the addition of 90 µl of DNase I stop solution (0.4 M sodium acetate, 0.8% sodium dodecyl sulfate, 12.5 mM EDTA, 1 mg of salmon DNA/ml). The samples were extracted with an equal volume of phenol-chloroform (1:1), precipitated with 2 volumes of ethanol, washed with 70% ethanol, and resuspended in formamide loading buffer. After being heated at 80°C for 20 min, the samples were loaded in a denaturing 6% polyacrylamide sequencing gel together with G+A size standards of the end-labeled fragments by the Maxam and Gilbert methods (described in reference 29). After electrophoresis, the gel was transferred to Whatman 3MM filter paper, dried, and autoradiographed.


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RESULTS AND DISCUSSION
 
A metalloid-regulated operon in Synechocystis. Within the fully sequenced genome of Synechocystis strain PCC 6803 is a cluster of three genes that shows sequence similarity to arsenic resistance genes (Fig. 1A). The first gene of the cluster (the ORF slr0944) encodes a putative arsenite carrier homologous to the S. cerevisiae protein ARR3 (35% amino acid identity) and to the B. subtilis ArsB protein (26% amino acid identity) (41). The second gene of the group (slr0945), separated by 152 bp of slr0944, is related to the Y. enterocolitica arsH gene (67% amino acid identity) (23). Ninety base pairs downstream of slr0945 is the third ORF (slr0946) of the cluster, encoding a predicted protein with sequence similarity to the thioredoxin-dependent arsenate reductases of the Staphylococcus (42% amino acid identity) and Bacillus (37% amino acid identity) strains (1, 13). Based on their respective homologies, these three genes were named arsB, arsH, and arsC. To find evidence of the role of these three genes in metalloid resistance, we analyzed their levels of expression in the presence of different forms of elements of group 15 of the periodic table. Three probes, one for each of the ars genes, were used to hybridize total RNA obtained from mid-log-phase Synechocystis cells grown in BG11C medium and exposed for 1 h to a 1 mM concentration of either sodium arsenite (NaAsO2)[As(III)], sodium arsenate (Na2HAsO4) [As(V)], potassium antimonyl tartrate (C4H4KO7Sb) [Sb(III)], bismuth nitrate [(NO3)3Bi] [Bi(III)], and phosphorous acid (H3PO3) [P(III)]. Control cells were not exposed to added compounds. As shown in Fig. 1B, the three probes hybridized strongly with RNA obtained from As(III)- and Sb(III)-exposed Synechocystis cells and weakly with RNA from As(V)- and Bi(III)-exposed cells. The transcripts were undetected in lanes containing RNA from P(III)-treated or untreated cells. The three probes detected an mRNA fragment of the same size, approximately 2,700 nucleotides, suggesting that arsB, arsH, and arsC form an operon. As(III) was the best inducer of the arsBHC operon, followed by Sb(III) and, finally, As(V). As shown in Fig. 2, different levels of induction were determined by using either an arsB or an arsC probe and suggested a strong polar effect of the gene expression in the arsBHC operon. Time course analysis indicated that arsBHC mRNA was already induced at the maximal level 15 min after the metalloid addition and that this level was maintained at least during the first 4 h of treatment (data not shown).



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  		FIG. 1. Metal-dependent regulation and disruption of Synechocystis ars genes. (A) Schematic representation of ars genomic region in the WT strain and site of insertion of the C.K1 cassette in the arsB::C.K1, arsH::C.K1(+), arsH::C.K1(-), and arsC::C.K1 mutants. B, BstEII; E, EcoRI; S, SmaI. (B) Northern blot analysis of the expression of the ars gene cluster. Total RNA was isolated from mid-log-phase Synechocystis cells grown in BG11C medium and exposed for 1 h to a 1 mM concentration of either sodium arsenite [As(III)], sodium arsenate [As(V)], potassium antimonyl tartrate [Sb(III)], bismuth nitrate [Bi(III)], and phosphorous acid [P(III)]. Control cells were not exposed to added compounds. Fifteen micrograms of total RNA was denatured, separated by electrophoresis in a 1.2% agarose gel, blotted, and hybridized with probes a, b, and c, indicated in panel A (see Materials and Methods). The filters were stripped and rehybridized with an rnpB gene probe as the control. The estimated size of the transcripts (in nucleotides) is indicated. (C) Tolerance of Synechocystis WT and Synechocystis ars mutants to metalloids. Tenfold serial dilutions were spotted on low-phosphate BG11C plates; supplemented with the indicated concentration of sodium arsenite [As(III)], sodium arsenate [As(V)], or potassium antimonyl tartrate [Sb(III)]; and photographed after 10 days of growth.



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  		FIG. 2. Effect of metalloid concentration on the amount of transcript. The indicated concentration of sodium arsenite [As(III)], sodium arsenate [As(V)], or potassium antimonyl tartrate [Sb(III)] was added to mid-log-phase Synechocystis cells grown in BG11C medium. After 1 h, cells were harvested and total RNA was isolated, processed, and hybridized as described in the legend to Fig. 1 by using an arsB gene probe (probe a from Fig. 1A) or an arsC gene probe (probe c from Fig. 1A). Radioactive signals of two independent experiments were quantified with an InstantImager electronic autoradiography apparatus. Levels of mRNA were normalized with the rnpB signal, and plots of increases in induction (n-fold) with increasing doses were drawn. Please note differences in the plotted scales.

arsB and arsC are involved in arsenic resistance. The homologies displayed by the ArsB, ArsH, and ArsC proteins together with the pattern of expression of their genes suggested that the arsBHC operon is involved in arsenic resistance in Synechocystis. In order to verify this hypothesis, mutants were generated by insertion of kanamycin resistance cassettes (C.K1) (8) into each one of the genes of the operon (Fig. 1A). The arsB::C.K1, arsH::C.K1(+), arsH::C.K1(-), and arsC::C.K1 Synechocystis strains were viable, and their growth rates in BG11C medium were similar to those of the wild-type (WT) strain (data not shown). ArsC-disrupted cells were more sensitive to high concentrations of As(V) than the WT cells (Fig. 1C). This phenotype was more apparent in a medium with a small amount of phosphate, probably due to the fact that arsenate enters the cell by some of the phosphate transport systems (reviewed in reference 26). Resistance to As(III) and Sb(III) was not affected by the arsC mutation. This result is in agreement with the notion that arsC encodes an arsenate reductase involved in arsenate detoxification. arsB-disrupted cells were hypersensitive to both As(V) and As(III), supporting the putative role of ArsB in arsenite export. The Synechocystis ArsB protein clusters with the ArsB/ARR3 family of arsenite transporters (41). This family is not related to the ArsB proteins found in many bacterial ars operons (including in E. coli and Staphylococcus aureus) and appears to be the result of convergent evolution (21). In addition to As(III), the E. coli-like ArsB proteins are able to carry out Sb(III) export (37). However, arsB and arr3 mutants in B. subtilis and S. cerevisiae, respectively, are not hypersensitive to Sb(III), suggesting that the proteins encoded by these genes are not able to promote Sb(III) extrusion (30, 41). Interestingly, the Synechocystis arsB::C.K1 strain was hypersensitive to Sb(III), indicating that the Synechocystis ArsB protein is the first member of the ArsB/ARR3 family able to export both As(III) and Sb(III) and providing a new example of convergent evolution between both families of arsenite transporters.

Finally, disruption of the arsH gene showed different phenotypes depending on the orientation of the C.K1 cassette. Since the npt gene of the C.K1 cassette lacks a transcription terminator, insertional mutagenesis in the same orientation as that of the ars operon [indicated by "(+)"] does not suppress transcription of the arsC gene (data not shown). While the arsH::C.K1(-) mutants showed a phenotype similar to that of the arsC::C.K1 mutants, the arsH::C.K1(+) strain did not display any phenotype under the conditions tested (Fig. 1C). This result suggests that the phenotype of the arsH::C.K1(-) strain is a consequence of the absence of the expression of the arsC gene, not of the mutation of the arsH gene. Therefore, the ArsH protein is not required for arsenic or antimony resistance under the conditions tested. Genes homologous to arsH have been found close to arsenic resistance genes in Y. enterocolitica and in A. ferroxidans (3, 23). In Y. enterocolitica, the arsH product is required for resistance to As(III) and As(V). However, when the arsH gene of A. ferroxidans was introduced into E. coli, it was not required for arsenic resistance, but whether the gene is required for arsenic resistance in A. ferroxidans is unknown. There is no correlation between the type of ArsC or ArsB protein present in an organism and the existence of the ArsH protein. For example, Y. enterocolitica contains an E. coli-like ArsB and a glutaredoxin-dependent ArsC protein; however, Synechocystis contains an ArsB/ARR3-type ArsB and thioredoxin-dependent ArsC proteins, but both organisms contain arsH genes. A close inspection of the lowest-scored ArsH homologous proteins indicates that the protein shows some similarity with pyridine nucleotide-dependent oxidoreductase. Cysteine residues have been found in the active sites of the three types of arsenate reductases described previously. Furthermore, it is known that these residues participate in the catalysis of the reaction by forming an intermediary arsenate thioester (16, 17, 19, 44). Since Synechocystis ArsH lacks cysteine residues, it is unlikely that the function of ArsH is the reduction of arsenate. One possibility is that ArsH works as an alternative electron carrier protein under some specific conditions.

E. coli-like ArsB transporters are able to function as membrane potential-driven secondary transport systems or as primary ATP-driven transport systems associated with the ATPase ArsA (28). Nothing is known about the energetics of arsenite transport by the ArsB/ARR3 family of proteins. However, ars operons encoding members of this family of transporters do not contain genes encoding putative ATPases. The sll0086 ORF of the Synechocystis genome has been annotated as a putative ArsA-like arsenical pump-driving ATPase in theCyanoBase (http://www.kazusa.or.jp/cyano/cyano.html). The583-residue E. coli ArsA protein has two homologous halves, A1 and A2 (45); however, the 396-residue Synechocystis protein contains only one domain that displays around 27% identity with each one of the domains of the E. coli protein. Similarly, one-domain ArsA-like proteins have been found in humans and yeast (14, 31). In order to test the role of this protein in arsenic resistance, we inactivated the sll0086 gene by insertional mutagenesis. While the sll0086-inactivated strain presented some growth defects, its degree of resistance to As(III), As(V), or Sb(III) was indistinguishable from that of the WT strain (data not shown). Therefore, our data indicate that the sll0086-encoded ATPase is not involved in arsenic or antimony resistance.

Primer extension mapping of the arsBHC promoter. In order to determine the location of the arsBHC promoter, the starting point of the arsBHC transcript was mapped by primer extension. One single extension product was obtained by using RNA from cells under induced conditions, while no extension product was yielded with RNA from uninduced cells (Fig. 3A). The transcription start site was found 23 bp upstream of the arsB ATG start codon. Putative -10 and -35 promoter boxes in the form TAAGAT and TTGACA, respectively, were found at the appropriate positions. Two 17-bp direct repeats (ATCAAGTTTTTTTGATG) separated by 13 bp were found in the region from nucleotides -29 to +17 of the arsBHC promoter. Each direct repeat is composed of two inverted repeats in the form ATCAA(N)6TTGAT (Fig. 3B).



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  		FIG. 3. Primer extension analysis of the arsBHC transcripts. (A) Primer extension analysis of the arsBHC transcripts from Synechocystis cells exposed to 1 mM sodium arsenite [As(III)], and 1 mM sodium arsenate [As(V)], for 1 h or nonexposed cells (control). Sequencing ladders generated with the same oligonucleotide used for the primer extension are also shown. (B) Sequence of the arsBHC promoter-operator region. The transcript start point is marked with an arrow. Putative -10 and -35 boxes based on the transcription start site are boxed. Direct repeats are underlined. The shaded sequence indicates the binding site for ArsR as defined by DNase I footprinting. The translation start codon is indicated in boldface type.

The arsBHC operon is repressed by a DNA-binding protein of the ArsR/SmtB family. We proceeded to investigate the existence in Synechocystis strain PCC 6803 of a DNA-binding protein responsible for the metalloid-dependent regulation of the arsBHC operon. As previously mentioned, arsenic resistance operons from E. coli and Staphylococcus are controlled at the transcriptional level by a trans-acting repressor of the ArsR/SmtB family (33). Two ORFs encoding proteins of this family exist in the Synechocystis genome. The ziaR gene encodes a transcriptional repressor of the ZiaA Zn-dependent ATPase (35) and therefore is unlikely to be involved in controlling the arsBHC operon. We tested whether the other ORF (sll1957) encodes a protein involved in the regulation of the arsBHC operon. For that experiment, the sll1957 ORF was interrupted by a chloramphenicol resistance cassette (C.C1) (8). The sll1957::C.C1 Synechocystis strain was viable, and its growth rate in BG11C medium was comparable to that of the WT strain (data not shown). Furthermore, resistance to arsenate, arsenite, and antimony was not altered in the sll1957-disrupted strain (data not shown). Then we tested the metalloid-dependent regulation of the arsBHC operon in the disrupted strain. Interestingly, the sll1957::C.C1 cells showed high constitutive expression of the arsBHC operon independently of the presence of As(V), As(III), or Sb(III). These data suggest that the protein encoded by the sll1957 ORF is a repressor of the arsBHC operon, and therefore the protein was named ArsR. The arsR gene was expressed at very low levels both in the absence and in the presence of metalloids (data not shown).

In order to verify whether ArsR is able to interact with the arsBHC promoter-operator region, we purified a recombinant MalE-ArsR chimeric protein expressed in E. coli (Fig. 4A). Proteolysis of the chimeric protein with factor Xa yielded both the MalE protein and a version of the ArsR protein in which the first two amino acids (MK) have been replaced by four amino acids (ISEF). DNA binding was tested by gel retardation assays using a fragment from nucleotide -59 to +36 with respect to the transcription start point of the arsBHC operon. As shown in Fig. 4B, ArsR interacted with the DNA, generating different types of ArsR-DNA complexes. The ArsR-dependent band shift was diminished in the presence of a 50-fold excess of the same unlabeled fragment, and it was unaffected by the presence of an excess of an unrelated DNA fragment (data not shown). Control experiments to demonstrate that a purified MalE protein was not able to bind DNA were also performed (data not shown).



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  		FIG. 4. ArsR binds to the arsBHC promoter-operator region. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified MalE-ArsR. Lane M, molecular mass markers; lane 1, purified MalE-ArsR; lane 2, purified MalE-ArsR digested with factor Xa protease to separate the MalE domain from ArsR. (B) Mobility shift assay of the arsBHC promoter-operator region with increasing quantities (from 0.095 to 3.8 µM) of ArsR protein. A 95-bp fragment containing the putative arsBHC promoter-operator region was used as the probe. (C) Effect of inducers of the arsBHC operon on ArsR-DNA complex formation. A 95-bp fragment containing the putative arsBHC promoter-operator region was incubated with a 1 µM concentration of the ArsR protein in the presence of 1 mM sodium arsenite [As(III)], sodium arsenate [As(V)], or potassium antimonyl tartrate [Sb(III)] or in the absence of metalloid (-). The binding mixture was analyzed in a mobility shift assay.

The Synechocystis ArsR protein contains the proposed metal binding site sequence CVCDLC identified in the E. coli ArsR protein (32, 33). Shi et al. (32) found that the three cysteine residues of this sequence were able to interact with arsenite. In vivo, the arsBHC operon was derepressed by arsenic(III) and antimony(III) as well as by arsenic(V), albeit to a lesser extent. We investigated the effect of in vivo inducers of the arsBHC operon on the in vitro binding activity of ArsR. As shown in Fig. 4C, the ArsR binding activity decreased in the presence of As(III) and Sb(III) but not in the presence of As(V). Similar results have previously been reported for the R773-borne ars system (40). The simplest interpretation of these data is that As(III) and Sb(III) are true inducers of the system but that As(V) has to be reduced to As(III) in vivo to induce the system. Interestingly, the arsBHC operon was induced by As(V) in vivo in a Synechocystis strain where the arsC gene had been inactivated (data not shown). This suggests that, in the absence of ArsC, a significant amount of arsenate is reduced in vivo to arsenite by another enzymatic or a nonenzymatic mechanism.

An interesting difference between the Synechocystis arsenic resistance system and the well-characterized systems in E. coli and Staphylococcus is that in Synechocystis the arsR gene is not autoregulated. Thus, the Synechocystis arsR gene is expressed constitutively at very low levels. The autoregulation of a repressor is usually related to the ability of the system to return to the repressed state. This autoregulatory mechanism is probably related to the fact that overexpression of the E. coli ArsB protein is toxic for the cell (see, for example, references 4 and 39). Therefore, in order to prevent toxicity, it is important to tightly control the expression of the ars operon. A more accurate control of the level of induction has been achieved by the five-gene operons from the R773 and R46 plasmids, where an additional transcription factor, ArsD, represses the operon in the presence of low levels of arsenic (7). In agreement with these data, E. coli arsD mutants overproduce ArsB in the presence of moderated levels of arsenic that lead to growth inhibition (39). In contrast, the Synechocystis arsR-inactivated cells show normal growth parameters in spite of the constitutive expression of the arsB gene. This absence of ArsB overexpression-dependent toxicity may explain why ArsR autoregulation has not been a selective advantage for Synechocystis.

Identification of the DNA binding site for the ArsR repressor. The site of binding of the ArsR protein to the arsBHC promoter-operator region was determined by DNase I footprinting analysis (Fig. 5A). A protected region of 51 bp (-34 to +17) in the coding strand and 52 bp (-36 to +16) in the noncoding strand was observed (Fig. 4B). The protected region overlaps the putative -10 and -35 promoter elements, suggesting that binding of ArsR under noninducing conditions prevents RNA polymerase binding and therefore transcription. This region contains two direct repeats of the sequence ATCAAGTTTTTTTGATG (coding strand), which contains an imperfect dyad symmetry and which may constitute the ArsR binding site. It has been shown that E. coli ArsR proteins are dimers in solution and that dimerization is required for DNA binding (40, 42, 43). Therefore, it is tempting to speculate that two ArsR dimers bind to the arsBHC promoter-operator fragment. However, more than two DNA-ArsR complexes were obtained in gel retardation assays when large amounts of ArsR were added to the binding reaction mixtures. This result may be due to additional ArsR binding to low-affinity sites or to oligomerization of the ArsR protein by protein-protein interaction. Figure 5B shows an alignment of the putative Synechocystis ArsR binding site with those of E. coli chromosomal and R773 plasmid-encoded ArsR DNA binding sites. The sequence TCAT(N)7TTTG has been proposed to be a consensus binding site for both E. coli ArsR proteins. The Synechocystis ArsR putative DNA binding site can be accommodated to this consensus by introducing two more nucleotides in the spacer region.



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  		FIG. 5. DNase I footprints of the ArsR protein bound to the arsBHC promoter-operator region. (A) DNA fragments of the arsBHC promoter-operator region were labeled at positions -132 (coding strand) and +105 (noncoding strand) and subjected to DNase I footprint analysis using increasing quantities of purified ArsR protein (0.025, 0.063, 0.126, and 0.252 µg), as described in Materials and Methods. The regions of the DNA protected by ArsR are indicated by bars. (B) Alignment of ArsR DNA binding sites in the E. coli plasmid R773-encoded and chromosomal (chr) ars operons and in the Synechocystis arsBHC operon. The DNA sequence that was protected in DNase I footprinting experiments is shown in the case of the E. coli ars operons. The Synechocystis sequence corresponds to one of the two identical direct repeats found in the ArsR-protected region (Fig. 3B). The numbers indicate the position of the sequence with respect to the transcription start point.

In summary, we have characterized for the first time the system for metalloid resistance in a cyanobacterium. The system for resistance in Synechocystis strain PCC 6803 combines elements from gram-negative and gram-positive species, as well as interesting particularities. One of the most intriguing questions is the biochemical role of ArsH, which will be the direction of our future research.


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ACKNOWLEDGMENTS
 
We thank Kazusa DNA Research Institute and S. Tabata for providing cs0223 and cs0205 cosmid DNA. We thank Marika Lindahl for critical reading of the manuscript.

Luis López-Maury is a recipient of a fellowship from the Spanish Ministerio de Educación Cultura y Deporte. This work was supported by grant BMC2001-2635 from MCyT and by the Junta de Andalucía (group CV1-802).


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FOOTNOTES
 
* Corresponding author. Mailing address: Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas, Av. Américo Vespucio s/n, 41092 Seville, Spain. Phone: 34 954489573. Fax: 34 954460065. E-mail: jcreyes{at}cica.es. Back


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Journal of Bacteriology, September 2003, p. 5363-5371, Vol. 185, No. 18
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.18.5363-5371.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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