The glutathione/glutaredoxin system is essential for arsenate reduction in Synechocystis sp. strain PCC 6803

Abstract

Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by an operon of three genes in which arsC codes for an arsenate reductase with unique characteristics. Here we describe the identification of two additional and nearly identical genes coding for arsenate reductases in Synechocystis sp. strain PCC 6803, which we have designed arsI1 and arsI2, and the biochemical characterization of both ArsC (arsenate reductase) and ArsI. Functional analysis of single, double, and triple mutants shows that both ArsI enzymes are active arsenate reductases but that their roles in arsenate resistance are essential only in the absence of ArsC. Based on its biochemical properties, ArsC belongs to a family that, though related to thioredoxin-dependent arsenate reductases, uses the glutathione/glutaredoxin system for reduction, whereas ArsI belongs to the previously known glutaredoxin-dependent family. We have also analyzed the role in arsenate resistance of the three glutaredoxins present in Synechocystis sp. strain PCC 6803 both in vitro and in vivo. Only the dithiolic glutaredoxins, GrxA (glutaredoxin A) and GrxB (glutaredoxin B), are able to donate electrons to both types of reductases in vitro, while GrxC (glutaredoxin C), a monothiolic glutaredoxin, is unable to donate electrons to either type. Analysis of glutaredoxin mutant strains revealed that only those lacking the grxA gene have impaired arsenic resistance.

FIG. 1.

ORF organization of arsI-containing regions in pSYSM and pSYSX. (A) Schematic representation of the pSYSM and pSYSX regions containing arsI. The repeated sequence is shaded. Arrows indicate the direction of transcription. Filled arrows represent antibiotic resistance cassettes, although for simplicity, only SpΩ is shown. (B) Schematic representation of the pSYSX ΔarsI region present in strains SARSI8, SARSI9, SARSI11, and SARS12. (C) Southern blot analysis of strains SARS4, SARS6, SARS7, SARS8, SARS9, SARS10, SARS11, and SARS12. Genomic DNA was digested with NcoI and hybridized using the same DNA fragment indicated in panel A as a probe. The sizes of the hybridizing bands are given on the right of the gels.

FIG. 2.

Northern blot analysis of the expression of the arsC and arsI genes. Total RNA was isolated from mid-log-phase Synechocystis cells grown in BG11C medium and exposed for 1 h either to 1 mM sodium arsenite [As(III)] or to 10 mM sodium arsenate [As(V)]. Control cells were not exposed to added compounds. Fifteen micrograms of total RNA was denatured, separated by electrophoresis on a 1% agarose gel, blotted, and hybridized with probes for arsC and arsI. The filters were stripped and rehybridized with an rnpB gene probe as a control (see Materials and Methods).

FIG. 3.

Phenotypic characterization of arsI mutants. The tolerance of WT Synechocystis sp. strain PCC 6803 and strains SARS4, SARS6, SARS7, SARS8, SARS9, SARS10, SARS11, and SARS12 to arsenate was examined. Tenfold serial dilutions were spotted onto low-phosphate BG11C plates (− phosphate) supplemented with 100 mM sodium arsenate [As(V)], BG11C plates (+ phosphate) supplemented with 100 mM sodium arsenate [As(V)], or low-phosphate BG11C plates or BG11C plates without additions. Plates were photographed after 10 days of growth.

FIG. 4.

ArsC has a unique catalytic site. (A) Glutaredoxin-dependent arsenate reductase activities of ArsC and ArsI. Portions (25 μg) of purified ArsC and ArsI were assayed with 1 μg of E. coli Grx1 following the couple reaction described in Materials and Methods. A control assay without glutaredoxin addition was carried out. (B) Sequence alignment of ArsC_syn (WT), mutant ArsC_syn (mut), Alr1105 from Anabaena sp. strain PCC 7120 (A.7120), and ArsC from Staphylococcus xylosus plasmid pSX267 or from S. aureus plasmid pI258. Identical amino acids are asterisked; conservative changes are marked with “:” or . as defined by CLUSTAL X. Changes to ArsC_syn in the mutant are underlined. (C) SDS-PAGE of purified mutant ArsC_syn (lane 1), WT ArsC_syn (lane 2), and TrxA(C35S) (lane 4). Lane 3, molecular mass markers. (D) Western blot analysis of interaction experiment between ArsC_syn and TrxA(C35S). Proteins were incubated as described in Materials and Methods. Lanes 1 and 2, reaction mixtures containing TrxA(C35S); lanes 3 and 4, reaction mixtures containing TrxA(C35S) and WT ArsC_syn; lanes 5 and 6, reaction mixtures containing TrxA(C35S) and mutant ArsC_syn. Proteins were incubated in the presence of 100 mM Na₂HAsO₄ (lanes 1, 3, and 5) or in the absence of Na₂HAsO₄ (lanes 2, 4, and 6). The membrane was incubated with anti-TrxA antibodies as described in Materials and Methods.

FIG. 5.

Analysis of the expression of glutaredoxin genes in response to arsenate. (A) Northern blot analysis of the expression of the grxA, grxB, and grxC genes. Total RNA was isolated from mid-log-phase Synechocystis cells grown in BG11C medium and was exposed to 100 mM sodium arsenate for 0.5, 1, and 2 h. Control cells were not exposed. Fifteen micrograms of total RNA was denatured, separated by electrophoresis on a 1% agarose gel, blotted, and hybridized with probes for the grxA, grxB, and grxC genes. The filters were stripped and rehybridized with an rnpB gene probe as the control (see Materials and Methods). (B and C) Northern blot analysis of the expression of grxB in strain SGRXA (B) and of grxA in strain SGRXB (C) after arsenate exposure as described for panel A.

FIG. 6.

Phenotypic characterization of the grx mutants. The tolerance of WT Synechocystis sp. strain PCC 6803 and strains SGRXA, SGRXB, SGRXC, SGRXAB, SGRXAC, SGRXBC, and SGRXABC was tested. Tenfold serial dilutions were spotted onto low-phosphate BG11C plates supplemented with 100 mM sodium arsenate [As(V)], low-phosphate BG11C plates (− phosphate), or BG11C plates. Plates were photographed after 10 days of growth.

FIG. 7.

Phylogenetic analysis of ArsC_syn. Shown is a neighbor-joining tree of full-length amino acid sequences of the following arsenate reductases (sequences are from the ENTREZ protein database): ArsC_Syn (Synechocystis), Alr1105 from Anabaena sp. strain PCC 7120 (A.7120; accession no. NP_485148), Ava 3712 from Anabaena variabilis (ZP_00159236), Glr0004 from Gloeobacter violaceus (NP_922950.1), and ArsC enzymes from Nostoc punctiforme (ZP_00108511), Crocosphaera watsonii WH 8501 (ZP_00349942), Lyngbya sp. strain PCC 8106 (ZP_01620002), Synechococcus sp. strain JA-2-3B′a (Syn_JA-2-3B; YP_478568), Acaryochloris marina MBIC11017 (YP_001514958), Synechococcus sp. strain PCC 7002 (S.PCC7002; YP_001733851), Microcystis aeruginosa NIES-843 (YP_001655268), Cyanothece sp. strain ATCC 51142 (YP_001804412), Bacillus subtilis (BAA12434), Staphylococcus xylosus plasmid pSX267 (Q0125), and S. aureus plasmid pI258 (P30330). Low-molecular-weight phosphatases are Wzb from E. coli (ECU38473), PtpA from Streptomyces coelicolor A3 (NP_628106), Ptp from Acintobacter johnsonii (CAA75430), and YopH from Yersinia pseudotuberculosis (P08538). YopH was used as the outgroup. The scale bar corresponds to 0.1 estimated amino acid substitution per site. Bootstrap values for 1,000 permutations are shown.