The Redox-sensing Transcriptional Regulator RexT ControlsExpression of Thioredoxin A2 in the CyanobacteriumAnabaena sp. Strain PCC 7120*□S

Received for publication, May 22, 2012, and in revised form, September 25, 2012 Published, JBC Papers in Press, October 1, 2012, DOI 10.1074/jbc.M112.384206

Shigeki Ehira‡§ and Masayuki Ohmori‡1

From the ‡Department of Biological Science, Faculty of Science and Engineering, Chuo University, 1-13-27 Kasuga, Bunkyo-ku,Tokyo 112-8551, Japan and §Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and TechnologyAgency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

Background: Thioredoxins (Trxs) play a crucial role in the oxidative stress response.Results:A redox-sensing transcriptional regulator, RexT, controls expression of TrxA2, and TrxA2 regulates the DNA bindingactivity of RexT.Conclusion: The RexT-TrxA2 regulatory system regulates gene expression in response to redox state.Significance: This is the first report on a transcriptional regulator of the trx gene in cyanobacteria.

Thioredoxins are ubiquitous proteins that catalyze thiol-di-sulfide redox reactions. They have a crucial role in the oxidativestress response as well as the redox regulation of metabolicenzymes. In cyanobacteria, little is known about the regulationof trx gene expression despite the importance of thioredoxins incellular functions. In the present study, transcriptional regula-tion of the trx genes under oxidative stress conditionswas inves-tigated in the heterocystous cyanobacterium Anabaena sp.strain PCC 7120. When cells were exposed to H2O2, only thetrxA2 gene (all1866) of seven trx genes was induced. Disruptionof the rexT gene (alr1867), encoding a transcriptional regulatorof the ArsR family, resulted in increased expression of trxA2.RexT bound to the region downstream of the transcription ini-tiation site of trxA2. The DNA binding activity of RexT wasimpaired by H2O2 through the formation of an intramoleculardisulfide bond,which induced expression of the trxA2 gene. Theinactivated DNA binding activity of RexT was restored byreduced TrxA2. Hence, RexT is considered as a redox-sensingtranscriptional repressor of trxA2. These results support theidea that theRexT-TrxA2 regulatory system is important for theoxidative stress response in this cyanobacterium.

Cyanobacteria are a large group of eubacteria characterizedby oxygen-evolving photosynthesis. Anabaena sp. strain PCC7120 (hereafter Anabaena PCC 7120) is a filamentous cyano-bacterium in which certain vegetative cells differentiate intoheterocysts, which are specialized cells for nitrogen fixation (1,2). Heterocysts provide themicrooxic environment for the oxy-gen-labile nitrogenase complex, the enzyme responsible fornitrogen fixation (3). Heterocysts are unable to carry out pho-tosynthesis and depend on vegetative cells for carbohydrate to

generate reductant required for nitrogen fixation. The oxida-tive pentose phosphate pathway is assumed to be the majorroute for sugar catabolism and production of reductant in het-erocysts. Glucose-6-phosphate dehydrogenase, which controlsthe entry of carbon into the oxidative pentose phosphate path-way, is essential for nitrogen fixation (4, 5). Glucose-6-phos-phate dehydrogenase is reductively inactivated and oxidativelyreactivated by thioredoxins (Trxs)2 to prevent a futile cycling,which would occur if the Calvin and oxidative pentose phos-phate pathways operated simultaneously (6, 7). Although Trxshave been shown to be present in heterocysts, whether theyoperate in heterocysts is unclear (8).Trxs are small disulfide-containing redox proteins that

reduce disulfide bonds in other proteins. They can act both as amodulator of enzyme activity by reducing regulatory disulfidebonds in a target protein and as a reducing agent. Trxs regulatea number of Calvin cycle enzymes such as fructose-1,6-bis-phosphatase, glyceraldehyde-3-phosphate dehydrogenase, sedo-heptulose-1,7-bisphosphatase, and phosphoribulokinase aswell as glucose-6-phosphate dehydrogenase in chloroplast (9,10). Moreover, more than 50 proteins involved in various phys-iological functions have been indicated to interact with Trxs inthe cyanobacterium Synechocystis sp. PCC 6803, suggestingtheir regulatory roles in a wide variety of cellular processes(11–13).Transcriptional regulation of trx genes has been well studied

in bacteria. In Escherichia coli, the transcriptional regulatorOxyR, which is activated byH2O2, regulates genes necessary forthe defense against oxidative stress, including trxC (14). Theredox-sensing transcriptional regulator Spx of Bacillus subtilisactivates the transcription of trxA (15). In actinobacteria suchas Streptomyces coelicolor and Corynebacterium glutamicum,the trx gene is under the control of the extracytoplasmic func-tion � factors SigR and SigH, respectively (16, 17). In Syn-echocystis sp. PCC 6803, expression of the trx genes is not* This work was supported by PRESTO of the Japan Science and Technology

Agency (to S. E.) and Grant-in-aid for Scientific Research (C) 23603005 fromthe Japan Society for the Promotion of Science (to S. E.).

□S This article contains supplemental Fig. S1 and Table S1.1 To whom correspondence should be addressed. Tel.: 81-3-3817-7104; Fax:

81-3-3817-7102; E-mail: ohmori@bio.chuo-u.ac.jp.

2 The abbreviations used are: Trx, thioredoxin; AMS, 4-acetamido-4�-maleimi-dylstilbene-2,2�-disulfonic acid; NEM, N-ethylmaleimide; RexT, redox-sens-ing transcriptional regulator of thioredoxin A2.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 48, pp. 40433–40440, November 23, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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induced by oxidative stress (18, 19), whereas transcript levels ofthe trxA and trxB genes are rapidly decreased by transitionsfrom light to dark and restored to the original levels by reillu-mination (13). It has been suggested that the redox state of thephotosynthetic electron transport chain regulates the expres-sion of trxA and trxB, but their molecular mechanisms such asacting as sensor proteins and transcriptional regulators havenot been revealed.In the genome of Anabaena PCC 7120, there are seven trx

genes (20). The trxA2 gene (all1866) has been shown to be up-regulated by nitrogen deprivation (21). In this study, we inves-tigated transcriptional regulation of the trx genes by oxidativestress. It was found that the trxA2 gene was induced by H2O2,and a redox-sensing transcriptional regulator encoded by therexT gene (alr1867) controlled the expression of trxA2. TheDNA binding activity of RexT was regulated through the for-mation of an intramolecular disulfide bond, and TrxA2 wasable to reduce the disulfide bond of RexT. These data suggestthat the RexT-TrxA2 system is important for the oxidativestress response inAnabaena PCC 7120. To our knowledge, thisis the first report on a transcriptional regulator of the trx gene incyanobacteria.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Culture Conditions—Anabaena sp.strain PCC 7120 and a rexT disruptant (DR1867K) were grownin BG-11 medium as described previously (21). Neomycin wasadded to themedium at a final concentration of 30�g/ml whenrequired. Oxidative stress conditions were set up by addition of3 mM H2O2 to cultures in the midlogarithmic phase (OD750 of0.4–0.5).Mutant Construction—All primers used in this study (sup-

plemental Table S1) were designed based on genome data fromCyanoBase (22). The rexT genewas inactivated as follows.DNAfragments upstream and downstream of the rexT gene wereamplified by PCR using the primer pairs 1867-5F and 1867-5Rand 1867-3F and 1867-3R, respectively (supplemental TableS1). The upstream fragment was cloned between the SacI andBamHI sites of pBluescript II KS� (Agilent Technologies), andthen the downstream fragment was cloned between the BamHIandXhoI sites. Aneomycin resistance cassette excised by diges-tion with BamHI from the plasmid pRL161 (23) was insertedinto the BamHI site between the upstream and downstreamfragments. The SacI-XhoI fragment was excised from theresultant plasmid and cloned between the SacI andXhoI sites ofpRL271 (24) to construct pR1867K. pR1867K was transferredby conjugation into Anabaena PCC 7120 according to Elhaiand Wolk (25) to construct the rexTmutant DR1867K.Quantitative Reverse Transcription-PCR—Total RNA was

extracted from whole filaments according to Pinto et al. (26)and treated with DNase I (Takara Bio, Shiga, Japan). cDNAswere synthesized with specific primers using the PrimeScriptfirst strand cDNA synthesis kit (Takara Bio), and quantitativeRT-PCR was performed as described previously (27). Relativetranscript levels were normalized with the value for 16 S rRNAand are represented as means of triplicate experiments.

Mapping of Transcription Initiation Sites by Rapid Amplifi-cation of cDNA Ends-PCR—The transcription initiation site ofthe trxA2 gene was determined using the SMARTer RACEcDNA Amplification kit (Takara Bio) as described previously(27). The resulting PCR product was cloned into the pGEM-TEasy vector (Promega), and seven clones were sequenced.Expression and Purification of His-RexT and His-TrxA2—

For construction of an expression plasmid for the histidine-tagged RexT protein, a DNA fragment containing the alr1867coding region was amplified by PCR using the primer pair1867-F and 1867-R. The amplified DNA fragment was clonedbetween the NdeI and EcoRI sites of the pCold II expressionvector (Takara Bio) to construct pC1867. E. coli BL21(DE3)harboring pC1867 was grown at 37 °C in 200 ml of Luria-Ber-tani medium supplemented with carbenicillin (50 �g/ml). Therecombinant gene was expressed in exponentially growing cells(A600 of 0.4) by rapidly chilling the culturemedium to 15 °C andadding 0.1 mM isopropyl �-D-thiogalactopyranoside. After 24 hof incubation at 15 °C, the cells were harvested by centrifuga-tion. His-RexT was purified with the Ni-NTA Fast Start kit(Qiagen). The elution fraction containing the purified proteinwas loaded onto a PDMidiTrap G-25 column (GE Healthcare)equilibrated with 20 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 10%glycerol, and the protein was eluted with the same buffer. Cys-teine residues at position 40, 41, and 105 of RexTwere replacedwith serine by site-directed mutagenesis using the PrimeSTARmutagenesis basal kit (Takara Bio). Serine-substitutedRexT proteins were expressed and purified as described forHis-RexT.His-TrxA2 was expressed from expression plasmid

pCTrxA2 and purified as described for His-RexT. pCTrxA2was constructed as follows. A DNA fragment amplified by PCRusing the primer pair TrxA2-F and TrxA2-R was clonedbetween the NdeI and SalI sites of the pCold II vector. A PDMidiTrap G-25 column equilibrated with 25 mM Tris-HCl (pH7.9), 150mMNaCl, 10% glycerolwas used for buffer exchange ofpurified His-TrxA2.Gel Mobility Shift Assay—Gel mobility shift assays were

carried out using His-RexT and 0.2 pmol of a DNA probeincluding the intergenic region between trxA2 and rexT in 20�lof binding buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10mMMgCl2, 1mM dithiothreitol (DTT), 10% glycerol). Themix-tures were incubated for 30min at room temperature and thensubjected to electrophoresis on a native 5% polyacrylamidegel. DNA probes were visualized by staining with ethidiumbromide.Thiol Redox State Analyses—His-RexT and its derivatives (10

�g) were reduced by incubation with 1 mM DTT and then oxi-dized with 3 mM H2O2. Proteins were precipitated by additionof TCA at a final concentration of 10% (w/v) and dissolved in200 mM Tris-HCl (pH 8.0) containing 1% SDS and 15 mM

4-acetamido-4�-maleimidylstilbene-2,2�-disulfonic acid (AMS)(Invitrogen). After incubation for 2 h in the dark, 1-�g aliquotsof proteins were separated by non-reducing SDS-PAGE.Mass Spectrometry (MS) Analysis—H2O2-oxidized His-

RexT was incubated with 10 mM N-ethylmaleimide (NEM) for15 min at room temperature. The alkylated His-RexT was sub-jected to non-reducing SDS-PAGE, and Coomassie Brilliant

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Blue-stained bands were excised. The protein was in-geldigested with trypsin, and analyzed by MALDI-TOFMS (Voy-ager-DE STR, Applied Biosystems).

RESULTS

Expression of trxA2 Is Induced by Hydrogen Peroxide andNegatively Regulated by RexT—The complete sequence of theAnabaena PCC 7120 genome revealed seven trx genes (20, 28).According to the phylogenetic analyses of trx genes (28, 29) andthe nomenclature of trx genes of Synechocystis PCC 6803 (13),we designated trx genes of Anabaena PCC 7120 as follows:alr0052 (trxA), all1866 (trxA2), all2367 (trxA3), all2341 (trxB),alr3955 (trxC), all1893 (trxQ), and alr2205 (trxO). The productsof trxA, trxA2, and trxA3 are related to the plant m-type Trx,and trxB and trxQ encode an x- and y-typeTrx, respectively (28,29). The transcript levels of trx genes are known to be up-reg-ulated by oxidative stress in various bacteria such as E. coli, B.subtilis, and S. coelicolor (14, 16, 30). Changes in expression ofthe trx genes in response to oxidative stress were investigated inAnabaena PCC 7120. Filaments in the midlogarithmic phasewere subjected to 3 mM H2O2 for 10 min, and the transcriptlevels of the trx genes were determined by quantitative RT-PCR(Fig. 1). Only expression of trxA2 was induced by H2O2. Thetranscript level of trxA2 was increased about 6-fold after H2O2treatment. A lower concentration of H2O2 (1 mM) did notinduce trxA2 expression (data not shown).The alr1867 gene, encoding a transcriptional regulator of the

ArsR family, is located upstream of trxA2 in the opposite direc-tion (Fig. 2). This genetic organization is also conserved amongother cyanobacteria such asAnabaena variabilisATCC 29413,Nostoc punctiforme PCC 73102, Cyanothece sp. PCC 7425, andAcaryochloris marinaMBIC11017. To ascertain the physiolog-ical role of Alr1867 protein as a transcriptional regulator oftrxA2, the alr1867 gene was deleted from the genome ofAnabaena PCC 7120 to construct an alr1867 disruptant,DR1867K. Expression of trxA2 was drastically increased inDR1867K. The transcript level of trxA2 in DR1867K was morethan 100-fold higher (122.1 � 10.9) than the wild-type levelunder normal growth conditions, whereas expression of theother trx genes was not affected by disruption of alr1867 (datanot shown). These results indicate that Alr1867 protein is anegative regulator of trxA2. We designated the alr1867 gene asrexT (redox-sensing transcriptional regulator of thioredoxinA2).

RexT Protein Binds to the Promoter Region of trxA2—Gelmobility shift assays were carried out with purified His-RexTprotein and DNA probe F1 including the intergenic regionbetween trxA2 and rexT (Figs. 2 and Fig. 3A). His-RexT reducedthe electrophoretic mobility of probe F1, and the amount of theRexT-F1 complex increased in proportion to the concentrationof His-RexT (Fig. 3A). No interaction between His-RexT andDNA probe F2 or F3, which is an internal fragment of the cod-ing region of trxA2 or rexT, respectively (Fig. 2), was observed(Fig. 3A). These observations led to the conclusion that RexTbinds to the intergenic region between trxA2 and rexT in asequence-specific manner.The transcription initiation site of trxA2 was determined by

rapid amplification of cDNA ends-PCR experiments. Thesequence of cloned PCR products was determined by the dyeterminator method. The 5�-end of five clones of seven clonessequenced was situated at position �40 with respect to thetranslation start site of the trxA2 gene (Fig. 2). Putative�10 and�35 promoter regions were found upstream of the transcrip-tion initiation site (Fig. 2). To define the promoter region inter-acting with RexT, four DNAprobes, D1 toD4, in which a 20-bpportion of the intergenic regionwas deletedwere prepared (Fig.2). Although RexT was able to bind to probes D1, D3, and D4,affinity of RexT to probe D2 was markedly reduced (Fig. 3B),suggesting that the region from �18 to �37 with respect to thetranslation start site is important for the binding of RexT to thepromoter region of trxA2.DNA Binding Activity of RexT Is Regulated through the For-

mation of an IntramolecularDisulfide Bond—Expression of thetrxA2was induced by H2O2 (Fig. 1). The effects of H2O2 on theDNA binding activity of RexT were determined by gel mobilityshift assays (Fig. 4). Binding of RexT to probe F1 was preventedby addition of H2O2 (Fig. 4A). Addition of an excess of thereducing agentDTT restored theDNAbinding activity of RexTthat was inactivated by H2O2 (Fig. 4B), indicating that theeffects of oxidation and reduction on the DNA binding activityof RexT are reversible.The RexT protein contains 3 Cys residues, Cys-40, Cys-41,

and Cys-105. These Cys residues are likely to be involved in the

FIGURE 1. Changes in the transcript levels of the trx genes in response toH2O2. The relative transcript levels of the trx genes before (white bars) and 10min after addition of 3 mM H2O2 (black bars) were determined by quantitativeRT-PCR. The transcript level before the H2O2 addition was taken as 1 for eachgene. Means � S.D. (error bars) of three independent experiments are shown. FIGURE 2. The nucleotide sequence of the intergenic region between

trxA2 and rexT. The coding regions of trxA2 and rexT are shaded in gray. Abent arrow and boldface letters indicate the identified transcription initiationsite of trxA2 and the �10 and �35 promoter regions, respectively. The entiresequence corresponds to DNA probe F1 used for gel mobility shift assays inFig. 3. DNA probes F2 and F3 are shown with dotted lines. Boxes D1–D4 areregions deleted from probe F1 in probes D1 to D4, respectively. The invertedrepeat sequence found within the RexT-binding site is indicated with arrows.

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redox-sensitive control of the DNA binding activity. When theHis-RexT protein oxidized with H2O2 was subjected to non-reducing SDS-PAGE, only one band of amolecularmass of�14

kDa, which corresponded to the monomeric form of His-RexT(14.1 kDa), was observed (Fig. 5A), indicating that oxidizedHis-RexT does not undergo dimerization. We next investigatedwhether the thiol groups of Cys residues of His-RexT are mod-ified by treatmentwithH2O2 usingAMS.AMScovalentlymod-ifies free thiol groups, retarding electrophoretic mobility inproportion to the number of free thiol groups in proteins. Theelectrophoretic mobility of His-RexT treated with DTT wasretarded by AMS modification (Fig. 5B, lanes 1 and 2). His-RexT treated with H2O2 was alsomodified by AMS, although itmigrated faster than reduced His-RexT (Fig. 5B, lanes 2–4).The molecular masses of His-RexT modified with one, two, orthree AMSmolecules are 14.6, 15.2, and 15.7 kDa, respectively.Compared with molecular mass markers and RexT derivativesthat contain 1 or 2 Cys residues (Fig. 5B), it was indicated that 1Cys residue of RexT was free when it was oxidized. Theseresults support the conclusion that RexT is inactivated by oxi-dation through the formation of an intramolecular disulfidebond.To determine which Cys residues of RexT are involved in the

formation of a disulfide bond, each Cys residue was replacedwith a Ser residue to generate C40S, C41S, and C105S proteins,which were designated as 2-Cys mutants. 2-Cys mutant pro-teins decreased the electrophoretic mobility of probe F1 (Fig.6A). Binding of 2-Cys mutant proteins to probe F1 was alsoprevented by addition of H2O2, although concentrationsrequired for inactivation were higher than that of the wild-typeprotein (Figs. 4A and 6A). Addition of DTT restored the DNA

FIGURE 3. Gel mobility shift assays with His-RexT and the promoterregion of the trxA2 gene. A, probe F1 (10 nM) was mixed with His-RexT in theamounts indicated above each lane, and then the mixtures were subjected toelectrophoresis. Probe F2 (lanes 1– 4) or probe F3 (lanes 5– 8) was added in afinal concentration of 30 nM as a competitor. B, the binding of His-RexT to DNAprobes D1, D2, D3, and D4 was determined. The sequences of DNA probes areshown in Fig. 2. White, gray, and black arrowheads indicate free probes, com-petitor DNA, and protein-DNA complexes, respectively.

FIGURE 4. Redox-sensitive control of the DNA binding activity of RexT.His-RexT (1 �M) and probe F1 were incubated in the presence of 1 mM DTT (A)or 1 mM H2O2 (B) for 15 min, and then H2O2 (A) or DTT (B) was added in theamounts indicated above each lane. After 15 min, the mixtures were sub-jected to electrophoresis. Lane 1, His-RexT was not added.

FIGURE 5. Oxidation of Cys residues of RexT. A, purified His-RexT proteins (1�g) incubated with 1 mM DTT (lane 1) or 1 mM H2O2 (lane 2) were separated bynon-reducing SDS-PAGE. Lane M, molecular mass markers. B, oxidation of Cysresidues of RexT was determined as follows. Proteins (1 �g) treated with 1 mM

DTT were further incubated with (�) or without (�) 3 mM H2O2, and then freethiol groups were modified with AMS. The modified samples were separatedby non-reducing SDS-PAGE. Open arrowheads indicate the shifted band ofproteins modified with one, two, or three AMS molecules. Red, reduction; Ox,oxidation.

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binding activity of 2-Cys mutant proteins that were inactivatedby 3 mM H2O2, indicating that Cys residues were not overoxi-dized to sulfinic or sulfonic acids (Fig. 6B). The oxidized C40Sand C105S proteins were not modified with AMS, but C41Sproteins were not fully oxidized by 3 mM H2O2 (Fig. 6C), con-sistent with the partial inactivation of C41S by 3mMH2O2 (Fig.6A). These results indicate that 3Cys residues of RexT are capa-ble of forming a disulfide bond between any two of them.To further investigate the correlation between the inactiva-

tion of RexT and the formation of a disulfide bond, we prepared1-Cys mutant proteins of RexT, C41S,C105S, C40S,C105S, andC40S,C41S, which contain only 1 Cys residue. 1-Cys mutantproteins bound to probe F1 in a sequence-specific manner (Fig.7A). However, the DNAbinding activities of 1-Cysmutant pro-teins were not impaired by H2O2 (Fig. 7B). A thiol group ofC40S,C105S and C40S,C41S proteins was free after treatmentwith H2O2, and half of C41S,C105S proteins were oxidized byH2O2 (Fig. 7C). These results confirm that RexT is inactivatedthrough the formation of an intramolecular disulfide bond.Mass Spectrometric Analysis of Oxidized RexT—To ascertain

whether all 3 Cys residues are indeed involved in the disulfidebond formation, oxidized RexT was analyzed by MS. The oxi-dized RexT was alkylated by NEM before trypsin digestion andMALDI-TOF MS analysis. Cys residues that are present in adisulfide bond are not modified with NEM. The results of MSanalysis are summarized in Table 1 and Fig. 8. Peptide 1 con-taining Cys-40 and Cys-41 was identified; its mass was lower by

2.03Da than the predictedmass. The identification of peptide 3containing Cys-105 modified with NEM suggested that someoxidizedRexTproteinswould contain a disulfide bond betweenCys-40 and Cys-41 and free Cys-105. Moreover, peptide 5 withamass corresponding to the sum of two tryptic peptides, 37–50and 98–112, and one NEM modification was identified. Theobserved mass was 2.01 Da lower than that predicted for thesum of the individual peptides, indicating that the 37–50 and98–112 peptides were cross-linked by a disulfide bond.Although it was not possible to distinguish whether a disulfidebond was formed between Cys-105 and Cys-40 or Cys-41 fromthis analysis, it was indicated that Cys-105 was also involved inthe disulfide bond formation. Peptides 2 and 4 contained a freeCys residue, which could result from reduction of a disulfidebond during MS analysis or might be protected from NEMmodification.TrxA2 Regulates the DNABinding Activity of RexT—Because

Trx catalyzes disulfide-dithiol exchange reactions, we deter-mined whether TrxA2 was able to reduce the disulfide bond inthe oxidized form of RexT. The DNA binding activity of RexTthatwas inactivated by 1mMH2O2was not restored by additionof 0.5 mMDTT (Fig. 9). However, incubation with various con-centrations of TrxA2 revealed that as the concentration ofTrxA2 was increased the DNA binding activity of RexT was

FIGURE 6. Redox response of 2-Cys mutant proteins of RexT. The effect ofH2O2 (A) and DTT (B) on the DNA binding activities of 2-Cys mutant proteinswas determined by gel mobility shift assays as described in Fig. 4. C, oxidationof cysteine residues of RexT and 2-Cys mutant proteins was determined asdescribed in Fig. 5B.

FIGURE 7. Redox response of 1-Cys mutant proteins of RexT. A, gel mobilityshift assays with 1-Cys mutant proteins were carried out as described in Fig.3A. B and C, the effect of H2O2 on the DNA binding activities and oxidation ofcysteine residues of 1-Cys mutant proteins were determined as described inFig. 6, A and C.

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gradually restored (Fig. 9). As shown above, RexT is inactivatedthrough the formation of an intramolecular disulfide bond.Thus, TrxA2 is able to reduce the intramolecular disulfide bondof RexT.

DISCUSSION

In the present study, transcriptional regulation of the trxgenes under oxidative stress conditions was investigated inAnabaena PCC 7120. We demonstrated that only the trxA2gene was induced by H2O2 (Fig. 1), and RexT was a redox-sensing transcriptional regulator of the trxA2 gene. Disruptionof the rexT gene resulted in increased expression of trxA2, and

the DNA binding activity of RexT was inhibited by H2O2through the formation of an intramolecular disulfide bond(Figs. 4 and 8), suggesting that expression of trxA2 was dere-pressed by inactivation of the transcriptional repressor RexT.Moreover, the DNA binding activity of oxidized RexT wasrestored by incubation with reduced TrxA2 (Fig. 9). Thus,TrxA2 controls expression of its structural gene by regulat-ing the DNA binding activity of RexT depending on redoxstate.The RexT protein contains 3 Cys residues. An intramolecu-

lar disulfide bond was formed between any 2 of 3 Cys residues,and every disulfide bond inactivated the DNA binding activity

TABLE 1Mass spectrometry results of oxidized RexT

Peptide Sequence NEMa

Mr

IntensityCalb Expc

1 (37–50) GEQCCAEFDFAIAK 0 1,531.66 1,529.63 143,3132 (98–112) SAQPLLTCQQSAIVK 0 1,586.86 1,586.85 20,8283 (98–112) SAQPLLTCQQSAIVK 1 1,711.90 1,711.89 18,9984 (37–50 � 98–112) GEQCCAEFDFAIAK � SAQPLLTCQQSAIVK 0 3,117.50 3,115.48 7,9745 (37–50 � 98–112) GEQCCAEFDFAIAK � SAQPLLTCQQSAIVK 1 3,242.55 3,240.54 68,154

a Number of cysteine residues modified with NEM.b Relative molecular mass based on the matched peptide sequence.c Experimentalm/z.

FIGURE 8. Mass spectrometry analysis of oxidized RexT. Oxidized RexT was analyzed by MALDI-TOF MS after tryptic digestion. Cys residues forming adisulfide bond and modified with NEM are indicated with an asterisk and �, respectively. Because it was not possible to distinguish whether a disulfide bondwas formed between Cys-105 and Cys-40 or Cys-41 from this analysis, both residues are indicated with an asterisk in parentheses.

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of RexT (Figs. 6 and 8). Moreover, the mutant proteins thatcontained only 1 Cys residue were not sensitive to H2O2, indi-cating that the formation of a disulfide bond was essential forthe inactivation of RexT protein (Fig. 7). It is worth noting thatC41S mutant protein was not fully oxidized and inactivatedwith 3mMH2O2, suggesting that Cys-41 is highly susceptible tooxidation (Fig. 6). Cys-41 is conserved among RexT ortho-logues of cyanobacteria and is predicted to be positioned in thehelix-turn-helix domain (supplemental Fig. S1). Orthologousproteins of RexT in A. variabilis and N. punctiforme also con-tain Cys-40, and Cys-105 is characteristic of Anabaena spp.Orthologous proteins in Cyanothece PCC 7425 and A. marinahave another Cys residue at position 68 and 46, respectively.Thus, the formation of a disulfide bond between Cys-41 andanother Cys residue is likely to be the fundamental mechanisminactivating RexT by oxidation. It has been shown that forma-tion of an intramolecular disulfide bond induces conforma-tional changes that remarkably alter the topology of the DNA-binding interface in an ArsR family transcriptional regulator(31).The 20-bp region from�18 to�37with respect to the trans-

lation start site was shown to be important for the binding ofRexT to the promoter region of trxA2 (Fig. 3B). Because thetranscription initiation site of trxA2 was located at position�40 (Fig. 2), the binding of RexT could hinder elongation of thetrxA2 transcript, which is coincident with the function of RexTas a transcriptional repressor. Transcriptional regulators of theArsR family are known to bind DNA as a dimer, and dimerizedDNA-binding proteins often bind to inverted repeat sequences(32, 33). The inverted repeat sequence 5�-ATTCGN15-CGAAT-3� was found within the RexT-binding region (Fig. 2).This sequence is a candidate for the RexT recognitionsequence.Transcriptome analyses of Synechocystis PCC 6803 show

that the trx genes are not induced under oxidative stress con-ditions in contrast to other bacteria such as E. coli and B. sub-tilis (18, 19). Moreover, cyanobacteria do not have homologuesof the redox-sensing transcriptional regulators OxyR and Spx(34). However, we found that the trxA2 gene was induced byH2O2 in Anabaena PCC 7120 (Fig. 1). The antioxidant systemof Synechocystis PCC 6803 relies mainly on a high catalaseactivity (35, 36). In contrast, in Anabaena PCC 7120, the anti-

oxidant mechanism does not rely on catalase, but the 2-Cysperoxiredoxin plays a more prominent role, which is remark-ably similar to the chloroplast system (35). Peroxiredoxins arethiol-based peroxidases that catalyze the reduction of H2O2 bythe concerted action of 2 Cys residues, which form the inter-molecular disulfide bondduring catalysis (37). Recycling of per-oxiredoxin is achieved by reduced Trx. Thus, increased expres-sion of TrxA2 after treatment with H2O2 could contribute toscavenging H2O2, although it remains to be shown whether thelevel of TrxA2 protein is increased concomitantly with up-reg-ulation of the trxA2 transcription.Despite the microoxic environment in heterocysts, H2O2 is

actively generated in heterocysts through the reactions cata-lyzed by superoxide dismutase and nitrogenase reductase andinactivates nitrogenase (38, 39). It has been demonstrated thatrubrerythrin, which is preferentially expressed in heterocysts,decomposesH2O2 generated within heterocysts (39). The tran-script level of the rbrA gene, encoding rubrerythrin, was notaffected by the rexT disruption (data not shown). In addition,DNA microarray analyses showed that no trx genes were up-regulated in heterocysts.3 These observations suggest that theRexT-TrxA2 system might not be active in heterocysts undernormal growth conditions, but under oxidative stress condi-tions such as high light or low temperature, this system couldregulate expression of redox-responsive genes not only in veg-etative cells but also in heterocysts.

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Transcriptional Regulation of the trx Gene in Anabaena

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Shigeki Ehira and Masayuki Ohmori sp. Strain PCC 7120AnabaenaThioredoxin A2 in the Cyanobacterium

The Redox-sensing Transcriptional Regulator RexT Controls Expression of

doi: 10.1074/jbc.M112.384206 originally published online October 1, 20122012, 287:40433-40440.J. Biol. Chem. 

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