Regulation of quinone detoxification by the thiol stresssensing DUF24/MarR-like repressor, YodB in Bacillus subtilis

Montira Leelakriangsak,1 Nguyen Thi Thu Huyen,2

Stefanie Töwe,2 Nguyen van Duy,2 Dörte Becher,2

Michael Hecker,2 Haike Antelmann2* andPeter Zuber1**1Department of Environmental and BiomolecularSystems, OGI School of Science and Engineering,Oregon Health and Science University, Beaverton, OR,USA.2Institute of Microbiology, Ernst-Moritz-Arndt-Universityof Greifswald, F.-L.-Jahn-Str. 15, D-17487 Greifswald,Germany.

Summary

Recently, we showed that the MarR-type repressorYkvE (MhqR) regulates multiple dioxygenases/glyoxalases, oxidoreductases and the azoreductaseencoding yvaB (azoR2) gene in response to thiol-specific stress conditions, such as diamide, catecholand 2-methylhydroquinone (MHQ). Here we report onthe regulation of the yocJ (azoR1) gene encodinganother azoreductase by the novel DUF24/MarR-typerepressor, YodB after exposure to thiol-reactivecompounds. DNA binding activity of YodB is directlyinhibited by thiol-reactive compounds in vitro. Massspectrometry identified YodB-Cys-S-adducts that areformed upon exposure of YodB to MHQ and catecholin vitro. This confirms that catechol and MHQ areauto-oxidized to toxic ortho- and para-benzoquinoneswhich act like diamide as thiol-reactive electrophiles.Mutational analyses further showed that the con-served Cys6 residue of YodB is required for optimalrepression in vivo and in vitro while substitution of allthree Cys residues of YodB affects induction of azoR1transcription. Finally, phenotype analyses revealedthat both azoreductases, AzoR1 and AzoR2 conferresistance to catechol, MHQ, 1,4-benzoquinone anddiamide. Thus, both azoreductases that are controlledby different regulatory mechanisms have commonfunctions in quinone and azo-compound reduction

to protect cells against the thiol reactivity ofelectrophiles.

Introduction

The Gram-positive bacterium, Bacillus subtilis is exposedto a variety of toxic and antimicrobial compounds in thesoil, which induce general and specific stress responsesin growing cells. As part of the stress response, specificdetoxification systems confer resistance to reactive aro-matic compounds. Catecholic and hydroquinone-likeintermediates are formed within the catabolic pathways ofmany aromatic compounds (Vaillancourt et al., 2006).They are metabolized by specific ring-cleavage dioxy-genases. Two classes of ring-cleavage dioxygenaseshave been identified based on the mode of ring cleavage:intradiol and extradiol dioxygenases, which cleave thearomatic ring ortho and meta to the hydroxyl substituentsrespectively. Catechol and hydroquinones are auto-oxidized to toxic ortho- and para-benzoquinones in thepresence of oxygen. Quinones can exert their toxicityas oxidants and/or electrophiles (O’Brian, 1991; Monkset al., 1992; Kumagai et al., 2002). As oxidants, quinonesact as catalysts in the generation of reactive oxygenspecies (ROS). As substrates for cellular reductases,quinones can undergo one-electron reduction to yieldsemi-quinone radical anions that reduce molecularoxygen, producing ROS such as superoxide anion, andlead to redox cycling reactions. As electrophiles, quinonescan form S-adducts with cellular thiols through reactionsinvolving a 1,4-reductive Michael-type addition. As ortho-and para-benzoquinones have a one-electron reductionpotential that far exceeds the range for redox-cyclingreactions, these are primarily electrophilic (Rodriguezet al., 2005). Quinones are also biologically activecompounds that function as lipid electron carriers in theelectron-transport chain (e.g. ubiquinone, menadione)(Unden and Bongaerts, 1997).

Previous proteome and transcriptome analysesshowed a strong overlap in the response of B. subtilis tocatechol, 2-methylhydroquinone (MHQ) and diamide.Diamide is an electrophilic azo-compound that reacts withprotein thiolates leading to the formation of disulphidesor S-thiolated proteins (Leichert et al., 2003; Hochgräfeet al., 2007). Thus, it seems that diamide, catechol and

Accepted 1 January, 2008. For correspondence. *E-mail antelman@uni-greifswald.de; Tel. (+49) 3834 864237; Fax (+49) 3834 864202.**E-mail pzuber@ebs.ogi.edu; Tel. (+1) 503 748 7335; Fax(+1) 503 748 1464

Molecular Microbiology (2008) 67(5), 1108–1124 � doi:10.1111/j.1365-2958.2008.06110.xFirst published online 16 January 2008

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd

MHQ are common thiol-reactive electrophiles that cangenerate mixed disulphides and Cys-S-adducts that leadto depletion of cellular protein thiolates. Several regula-tory factors participate in controlling this complex thiol-specific stress response to electrophilic compounds,such as Spx, CtsR, PerR, CymR and the novel MarR-type repressors YodB and MhqR (YkvE) (Leichert et al.,2003; Nakano et al., 2003a; Zuber, 2004; Leelakriang-sak et al., 2007; Töwe et al., 2007). The Spx protein ofB. subtilis is a global transcriptional regulator that con-trols genes whose products function in maintenance ofthiol homeostasis under disulphide stress conditions(Nakano et al., 2003a; 2005; Zuber, 2004). Regulationby Spx requires interaction with the C-terminal domain ofthe RNA polymerase a subunit (Nakano et al., 2003a,b).Recent studies showed that spx transcription initiation atthe P3 promoter located upstream of spx is induced inresponse to diamide treatment (Leelakriangsak andZuber, 2007; Leelakriangsak et al., 2007). Two negativetranscriptional regulators, PerR and YodB, were discov-ered to interact directly with and repress transcriptioninitiation from the spx P3 promoter (Leelakriangsak andZuber, 2007). PerR was previously characterized as arepressor of the peroxide stress regulon (Mongkolsukand Helmann, 2002). The novel DUF24/MarR-typefamily transcriptional regulator YodB also controls thedivergently transcribed yodC gene which encodes aputative nitroreductase and is induced in response todisulphide stress conditions (Duy et al., 2007; Leelakri-angsak et al., 2007). Moreover, three cysteine residuesare present in the YodB protein, which could be thetargets for the redox-sensitive control of repressor activ-ity that responds to thiol-reactive compounds. Thus,YodB might function as a thiol-responsive regulator ofB. subtilis. Recently, we have shown that another MarR-type repressor MhqR (YkvE) controls three putativedioxygenases/glyoxalases (MhqA, MhqO and MhqE),the nitroreductase MhqN and the azoreductase YvaB(AzoR2) in response to thiol-reactive electrophiles.These confer resistance to MHQ and catechol inB. subtilis (Töwe et al., 2007).

In this study, we have identified a paralogue of azoR2,the azoreductase-encoding azoR1 (yocJ ) gene, which isthe most strongly derepressed member of the YodBregulon under thiol-specific stress conditions. Mutationalanalyses indicate that the Cys residues of YodB play arole in repressor activity and the repressor’s response toinducing phenolic compounds. The thiol-specific quinonereactivity of oxidized MHQ and catechol was confirmed invitro using mass spectrometry. Finally, we show that theparalogous enzymes, AzoR1 and AzoR2 that are regu-lated by YodB and MhqR both contribute to electrophileresistance in B. subtilis. Hence, these azoreductases,which are conserved among Gram-positive bacteria could

play major roles as quinone reductases, that prevent toxicquinones from undergoing redox-cycling and causingdepletion of cellular thiolates.

Results

Microarray and proteomic analyses identified theazoreductase-encoding yocJ (azoR1) gene as moststrongly derepressed in DyodB mutant cells

The novel MarR-type repressor, YodB was shown tocontrol the expression of spx and that of thenitroreductase-encoding yodC gene in response to thiolstress conditions in B. subtilis (Duy et al., 2007; Leelakri-angsak et al., 2007). To identify the genes of the YodBregulon, B. subtilis wild-type and DyodB mutant strainswere subjected to proteome and transcriptome com-parison. Table 1 lists genes that are at least threefoldderepressed in the DyodB mutant according to themicroarray hybridization analysis. The most stronglyderepressed, YodB-controlled gene was identified asyocJ, which encodes a putative FMN-dependent NADH-azoreductase and is also referred to as AzoR1 accord-ing to Swiss-Prot database annotations (AccessionNo. O35022). The very high derepression ratio of azoR1transcription in the yodB mutant could be due to the factthat azoR1 is subject to regulation by YodB only, in con-trast to spx. The azoR1 (yocJ) transcriptional start sitewas mapped previously and the concentration of azoR1transcript was observed to increase after treatmentwith diamide (Nakano et al., 2003a). In addition, AzoR1protein synthesis is strongly increased by diamide, cat-echol, MHQ and nitrofurantoin treatment as shown byproteomic analysis (Fig. 1, Table 1). However, AzoR1protein synthesis was not induced in response to oxida-tive stress provoked by the addition of H2O2 (Fig. 1). Theproteomic analyses verified the derepression of azoR1and yodC in the DyodB mutant (Fig. 1). The proteomicdata also confirmed that AzoR1 production is several-fold more elevated than YodC in the DyodB mutant(Fig. 1, Table 1). Recently, we showed that production ofthe paralogous azoreductase, AzoR2 (YvaB), is inducedunder thiol stress conditions and azoR2 expression isregulated the MarR-type repressor, MhqR (YkvE) (Töweet al., 2007) (Fig. 1). However, in contrast to AzoR1,AzoR2 protein synthesis was not induced after diamideor nitrofurantoin stress (Fig. 1). Interestingly, synthesis ofMhqR-regulated azoR2 expression was reduced in theDyodB mutant and, vice versa, the level of the YodB-controlled azoR1 and yodC expression was lower in theDmhqR mutant under control conditions (Fig. 1). In con-clusion, the microarray and proteome results show thatYodB negatively controls the expression of azoR1, yodCand spx.

Regulation and function of the YodB repressor 1109

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 67, 1108–1124

Expression of azoR1 and azoR1-bsrB is induced bydiamide, catechol and MHQ and derepressed in theDyodB mutant

Northern blot analyses were performed to examine thelevel and size of azoR1 transcript in wild-type and DyodBmutant cells after exposure to MHQ, catechol, diamideand H2O2. The results verified the very strong transcriptionof azoR1 in response to catechol, MHQ and diamidestress in wild-type cells and derepression in the DyodBmutant (Fig. 2). Consistent with the proteome results,there was no increase in azoR1 transcription in responseto oxidative stress caused by H2O2. In addition, the basallevel of azoR1 mRNA was reduced in the DmhqR mutant,which is also consistent with the proteomic data.

Two different azoR1 transcripts were detected using anazoR1-specific RNA probe (Fig. 2A and B). The smaller0.7 kb transcript is most strongly elevated after catechol,MHQ and diamide stress and corresponds to an azoR1-specific monocistronic transcript. The larger 1.1 kb RNAspecies corresponds to the azoR1-bsrB-specific transcriptwhich is only visible in yodB mutant cells and in wild-typecells after catechol, MHQ and diamide treatment. The bsrBgene encodes a highly abundant small 6S RNA that istranscribed during exponential growth and decreases inconcentration during stationary phase (Ando et al., 2002;Barrick et al., 2005). The conserved features of this 6SRNA suggest that it binds RNA polymerase by mimickingthe structure of DNA in an open promoter complex, therebyregulating RNA polymerase activity (Wassarman andSaecker, 2006; Wassarman, 2007). Synthesis of azoR1-bsrB mRNA in treated wild-type cells was also verifiedusing a bsrB-specific mRNA probe (Fig. 2C). These differ-ent azoR1-specific transcripts observed in the Northernblot suggest that the azoR1-bsrB mRNA might be sub-jected to RNA processing. However, Northern analysisusing the bsrB-specific probe indicates that expression ofbsrB can be attributed largely to constitutive transcriptionfrom its own promoter that resides downstream of theazoR1 coding sequence. Thus, the azoR1-bsrB transcriptmakes only a minor contribution to bsrB transcription underthiol stress conditions. Transcription of bsrB as part of theazoR1-bsrB mRNA under thiol stress conditions mightresult from readthrough past the azoR1 terminator owing tothe very high induction of azoR1 transcription.

The DyodB mutant is resistant to catechol and MHQstress, which is attributable to derepression of theazoR1 gene

The growth phenotype of the DyodB mutant cells afteraddition of the thiol-reactive compounds diamide, cat-echol and MHQ to exponentially growing cells wasanalysed. The growth of the DyodB mutant was similar tothat of the wild-type after exposure to diamide (data notTa

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1110 M. Leelakriangsak et al. �

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 67, 1108–1124

shown). As reported earlier, the growth of wild-type cellswas inhibited in the presence of 4.8 mM catechol and0.66 mM MHQ (Tam et al., 2006; Duy et al., 2007). Inter-estingly, the DyodB mutant could grow in the presence of9.6 mM catechol and 0.83 mM MHQ, suggesting thatYodB-controlled genes, derepressed in the yodB mutant,confer resistance to these phenolic compounds (Fig. 3A).Likewise, the DmhqR mutant displayed a catechol andMHQ resistance phenotype as it was able to grow with12 mM catechol and 1 mM MHQ (Töwe et al., 2007).Moreover, DyodBDmhqR double mutant cells showed anadditive hyper-resistance to catechol and MHQ as growthwas still possible in the presence of 19.2 mM catechol and2 mM MHQ (Fig. 3B).

The next step was to determine if the paralogous azore-ductases AzoR2 and AzoR1, production of which is con-trolled by MhqR and YodB, respectively, contribute to

catechol and MHQ resistance. As reported previously, thegrowth of the DazoR2 mutant was impaired in the pres-ence of 3.6 mM catechol and 0.5 mM MHQ (Töwe et al.,2007). Surprisingly, no growth difference was detected forthe azoR1 mutant compared with the wild-type parentafter exposure to these compounds (data not shown).However, deletion of both azoreductases in theDazoR2DazoR1 double mutant resulted in a cessation ofgrowth after exposure to 2.4 mM catechol and cellularlysis after treatment with 0.5 mM MHQ (Fig. 3C). Thus,the DazoR2DazoR1 double mutant was more sensitive tocatechol and MHQ stress compared with the DazoR2single mutant. Moreover, DazoR2DazoR1 mutant cellsshowed a growth defect in the presence of 1 mM diamidecompared with wild-type cells whereas the DyodB DmhqRdouble mutant grew faster in the presence of 1 mMdiamide than the wild-type parent (Fig. 3D). These

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Fig. 1. Proteome analysis of the YodB regulon in B. subtilis. Induction of AzoR1 and YodC synthesis by catechol, MHQ, diamide,nitrofurantoin and H2O2 treatment of B. subtilis wild-type cells; elevated AzoR1 and YodC production in the DyodB mutant and reduced AzoR1and YodC levels in the DmhqR(ykvE) mutant. B. subtilis wild-type cells grown in minimal medium to an OD500 of 0.4 were pulse-labelled for5 min each with L-[35S]methionine at control conditions (green image) and in the wild type 10 min after exposure to 2.4 mM catechol (cat),0.33 mM MHQ, 1 mM diamide (dia), 0.1 mM nitrofurantoin (NT) and 100 mM H2O2 or in the DyodB and DmhqR mutant at control conditions(red image).

Fig. 2. Trancriptional organization (A) andtranscript analyses of azoR1 (B) and bsrB (C)in the wild type in response to MHQ, catechol,diamide and H2O2 stress and in the DyodBand DmhqR mutants. For Northern blotexperiments 5 mg RNA isolated from theB. subtilis strains before (co) and 10 min afterexposure to 2.4 mM catechol, 0.33 mM MHQ,1 mM diamide and 100 mM H2O2 were appliedto each well of the denaturing agarose gel.The Northern blots were hybridized withazoR1- (B) and bsrB-specific (C) mRNAprobes. The arrows point toward the sizes ofthe azoR1-, azoR1-bsrB- and bsrB-specifictranscripts. The percentage amounts ofazoR1- and azoR1-bsrB-specific mRNAs arecalculated with the ImageJ software and areshown on the right.

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Regulation and function of the YodB repressor 1111

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 67, 1108–1124

data confirmed that paralogous azoreductases encodedby azoR2 and azoR1 are major MhqR- and YodB-controlled determinants contributing to catechol andMHQ resistance. The increased sensitivity of theDazoR2DazoR1 double mutant towards thiol-reactivecompounds compared with the DazoR2 single mutantsuggests that AzoR2 could complement AzoR1 deficiencywith respect to detoxification.

To verify that the resistance of the DyodB mutant tocatechol and MHQ was attributable to azoR1 derepres-sion, the DazoR1 mutation was introduced into the DyodBmutant. Notably, the DyodB DazoR1 double mutant was

similarly sensitive to catechol and MHQ as the wild-typestrain (data not shown). These results confirmed that theresistance of the DyodB mutant was caused by the over-production of AzoR1.

Diamide, hydrogen peroxide and MHQ impair binding ofYodB protein to azoR1 promoter DNA

To identify the region of the azoR1 promoter DNA requiredfor transcriptional repression by YodB interaction, DNaseI footprinting was performed. The DNA that was end-labelled on the top strand of the azoR1 promoter was

Fig. 3. The DyodB mutant is resistant tocatechol and MHQ (A), the DyodBDmhqRdouble mutant is hyper-resistant (B) andthe DazoR2DazoR1 double mutant ishyper-sensitive to catechol, MHQ (C) anddiamide stress (D). B. subtilis wild type (wt),DyodB, DyodBDmhqR, DazoR2 andDazoR2DazoR1 mutant strains were grownin minimal medium to an OD500 of 0.4 andtreated with catechol, MHQ or diamide at thetime points that were set to zero. OD500 of thecultures was then measured at the timeintervals indicated.

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mixed with YodB protein (Leelakriangsak et al., 2007),followed by digestion with DNase I. YodB protected aregion from approximately -20 to +10 relative to the tran-scription start site (Fig. 4A, lanes 3 and 8). Promoteralignments identified putative sites of YodB recognition(YodB boxes) that were uncovered by DNase I footprintingexperiments of YodB with the spx (Leelakriangsak et al.,2007) and azoR1 promoters. YodB boxes contained acommon 15 bp consensus sequence of TACTWTTTgWWAGTA (Fig. 4C). In previous work, mutations in the spxpromoter at positions corresponding to the nucleotides 6(T), 7 (T) and 12 (A) of the consensus sequence impairedYodB binding (Leelakriangsak et al., 2007).

As shown previously, YodB binding activity on spx P3

promoter DNA was impaired in the presence of diamideand H2O2 (Leelakriangsak et al., 2007). A fourfold higherconcentration of YodB was used to demonstrate specificbinding to the spx P3 promoter then was used for the yocJpromoter owing to the lower affinity of YodB for the spxtarget sequence. Notably, sensitivity of YodB activity to the

toxic electrophiles was still observed despite the highconcentration of YodB used in these experiments. Wefurther determined whether the binding of YodB to theazoR1 promoter is reversed by the addition of diamide,H2O2 or MHQ in varying concentrations to the footprintingreactions. As shown in lanes 4, 5, 9–11 in Fig. 4A andlanes 4–6 in Fig. 4B, YodB bound poorly to the operatorsequence when diamide, H2O2 or MHQ at levels causingtoxicity in wild-type cells was present compared with thereactions that contained YodB alone (lanes 3, 8 in Fig. 4Aand lane 3 in Fig. 4B). However, the presence of diamide,MHQ and H2O2 at concentrations that inhibit YodB–targetDNA interaction did not effect DNA binding of CymRrepressor to the yrrT promoter (Leelakriangsak et al.,2007; M. Leelakriangsak and P. Zuber, unpublished),showing that these compounds do not have a generaleffect on protein–DNA interaction. In addition, neitherdiamide, H2O2 nor MHQ at the higher concentrationstested in the DNA-protein-binding experiments affectedDNase I activity (Fig. 4A, lanes 2 and 7 and Fig. 4B, lane2). These results confirmed that YodB acts as a repressorby interaction with azoR1 promoter DNA and impairmentof YodB–DNA interaction occurs when thiol-reactive com-pounds are present.

Role of Cys residues in repression of azoR1transcription by YodB in vivo

The YodB protein contains three cysteine (Cys or C) resi-dues, one at the N-terminal position 6 (C6), and two at theC-terminal positions 101 (C101) and 108 (C108). TheN-terminal Cys residue (C6) is conserved among yodBhomologues in low GC Gram-positive bacteria, unlike thetwo C-terminal Cys residues. These Cys residues may beinvolved in the redox-sensitive control of YodB activity, ashas been shown for Cys residues of other regulatoryfactors involved in the oxidative stress response, such asOxyR, Hsp33 and OhrR (Barbirz et al., 2000; Graumannet al., 2001; Fuangthong et al., 2002; Hong et al., 2005;Nakano et al., 2005; Lee et al., 2007). To investigate therole of Cys residues in YodB-mediated regulation, eachwas replaced with alanine, thus generating the yodBC6A,yodBC101A and yodBC108A alleles, along with thedouble mutant yodBC101,108A and triple mutantyodBC6,101,108A. All mutant proteins were produced atsimilar levels as shown by Western blot analysis (Fig. S1).The expression of azoR1–lacZ fusions were examinedin partial diploid strains bearing the DyodB allele andecotopically expressed versions of the C-to-A alleles inte-grated at the thrC locus (Fig. 5A and B). In yodB mutantcells (ORB7106), azoR1–lacZ activity was ~60-fold highercompared with wild-type cells (ORB7094). The azoR1–lacZ activity in strains ORB7109 (DyodB/yodBC101A),ORB7110 (DyodB/yodBC108A) and ORB7111 (DyodB/

Fig. 4. Effect of diamide, hydrogen peroxide (A) and MHQ (B) onDNA binding activity of YodB to the azoR1 promoter and YodB boxalignments (C). DNaseI footprinting was used to assess the effectof diamide, hydrogen peroxide and MHQ on YodB binding to thetop strand of the azoR1 promoter. The region protected by YodB isindicated by the solid line. The positions relative tothe transcriptional start site are shown on the left. The 15 bpYodB consensus includes a 4-7-4 bp inverted repeat which isprotected by YodB in the azoR1 and spx promoters. BSA, bovineserum albumin.

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yodBC101,108A) was similar to the wild-type strain(ORB7094). However, azoR1–lacZ activity was approxi-mately eightfold derepressed in the yodBC6A mutantunder control conditions compared with the untreated wildtype. In addition, derepression of azoR1–lacZ activity wassimilar in the yodBC6A and triple yodBC6,101,108Amutants, suggesting that Cys6 is essential for YodB-dependent repression of azoR1 transcription undernon-inducing conditions. However, there is still YodB-dependent repression in the yodBC6A and tripleyodBC6,101,108A mutants suggesting that other resi-dues are required for optimal YodB repressor activity.

Northern blot analysis and primer extension were per-formed to determine the level of azoR1 transcription aftertreatment of yodB Cys-to-Ala mutants with MHQ, catechol

(Fig. 5C and D) and diamide (Fig. 5E). As already shownin Fig. 2, the level of azoR1 transcript increased afterdiamide, MHQ or catechol treatment in the wild-type cells(Fig. 5C and D, lanes 2–3; Fig. 5E, lane 2). The transcriptconcentration of azoR1 was high in the untreated DyodBmutant (Fig. 5C and D, lane 4; Fig. 5E, lane 3) comparedwith untreated wild-type cells (Fig. 5C and D, lane 1;Fig. 5E, lane 1). Induction of azoR1 transcription in theyodBC101, yodBC108 and yodBC101,108A mutants inresponse to catechol, MHQ and diamide was similar tothat of the wild type. However, significantly higher basallevels of azoR1 transcripts were observed in theuntreated yodBC6A and triple yodBC6,101,108A mutants(Fig. 5C and D, lanes 7 and 10; Fig. 5E, lanes 7 and 15)compared with untreated wild-type cells (Fig. 5C and D,

Fig. 5. Role of YodB Cys mutants in regulation of azoR1–lacZ activity (A, B) and azoR1 transcription (C, D, E) in vivo. The yodBC6A mutantshows a catechol resistance phenotype (F).A. Expression of azoR1–lacZ fusion cells containing wild-type yodB (�, ORB7094), DyodB mutant (�, ORB7106), yodB, DyodB mutant(�, ORB7107), yodBC6A (�, ORB7108), yodBC101A (¥, ORB7109), yodBC108A (�, ORB7110), yodBC101,108A (+, ORB7111) andyodBC6,101,108A (�, ORB7112) were determined. The cells were grown in DSM, and their b-galactosidase activities were determined. Timezero indicates the mid-log phase. Duplicate experiments from two independently isolated strains of yodB Cys mutants were performed. (B)Expression of azoR1–lacZ fusion cells containing wild-type and yodB Cys mutants but not the DyodB mutant (�, ORB7106) were shown.(C, D) Quantitative Northern blot analysis and (E) primer extension analysis of azoR1 transcription using RNA extracted from strains ORB6284(wild-type), ORB6288 (DyodB mutant), ORB6599 (yodBC6A), ORB6715 (yodBC101A), ORB6716 (yodBC108A), ORB6777 (yodBC101,108A)and ORB7083 (yodBC6,101,108A) (co and 10 min) and after treatment with 2.4 mM catechol (C), 0.33 mM MHQ (M) or 1 mM diamide (10D)for 10 min. For primer extension analysis, labelled primer oSN03-63 (Table S2) was used to examine the level of azoR1 transcript. For thedideoxynucleotide sequencing shown on the left, the nucleotide complementary to the dideoxynucleotide added in each reaction mixtureis indicated above the corresponding lane (T′, A′, C′ and G′). (F) For the growth phenotypes, B. subtilis wild type, DyodB, yodBC6A andyodBC6,101,108A strains were grown in minimal medium to an OD500 of 0.4 and treated with 4.8 mM catechol at the time points that were setto zero. OD500 of the cultures was then measured at the time intervals indicated.

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lane 1; Fig. 5E, lane 1), which is consistent with theb-galactosidase activity data (Fig. 5A and B). Quantifica-tion of the Northern blots revealed ~25-fold derepressionof azoR1 transcription in the DyodB mutant (Fig. 5D). Inthe untreated yodBC6A and triple yodBC6,101,108Amutants, the amounts of azoR1 mRNAs were approxi-mately eightfold increased, compared with the untreatedwild type (Fig. 5C, lanes 7 and 10). There was a two- tothreefold induction of azoR1 transcription in the yodBC6Aand yodBC6,101,108A triple mutants after exposure tocatechol and MHQ. But the level of expression in the caseof the triple mutant was lower than the induced level ofwild-type cells (Fig. 5D and E). Furthermore, primerextension analysis of the yodBC6,101,108A triple mutantshowed little if any induction following diamide treatment(Fig. 5E, lanes 15 and 16), with a lower level of transcriptthan that observed in reactions of RNA from wild-typecells treated with diamide. Together, the Northern andprimer extension analysis suggests that the Cys residuesplay a sensory function required for the repressor’sresponsiveness to the inducing compounds.

The DyodB mutant acquires an increased resistanceto catechol and MHQ owing to the upregulation ofAzoR1. To confirm that the yodBC6A mutant retains thisresistance phenotype owing to weakened repressoractivity, growth experiments were performed in the pres-ence of different concentrations of catechol. The resultsshowed that the yodBC6A single or yodBC6,101,108Atriple mutants could grow with 4.8 mM catechol (Fig. 5F).In contrast, growth of the yodBC101A, yodBC108A andyodBC101,108A mutants was similar to the wild typeand inhibited in the presence of 4.8 mM catechol (datanot shown). However, resistance of the yodBC6A singleor yodBC6,101,108A triple mutants was lower than thatof DyodB mutant cells. These results support the hypoth-

esis that the Cys residues are at least in part involvedYodB repressor activity.

To examine YodBC6A protein binding activity to spx andazoR1 promoter DNA, the YodBC6A protein was purified(see Experimental procedures) and used in DNase I foot-printing experiments to compare mutant DNA-bindingprotein activity with that of wild-type YodB. YodBC6Aprotein exhibited reduced binding activity to the targetDNA when compared with the wild-type YodB protein(Fig. 6A and B, lanes 3 and 6). To further clarify whetherthe binding of YodBC6A protein was sensitive to a toxicoxidant, diamide was added to the footprinting reactions.As had been shown previously, diamide reduced YodBbinding to target DNA (Leelakriangsak et al., 2007).Diamide also caused a loss of YodBC6A binding activity tospx and azoR1 promoter DNA in vitro (Fig. 6A and B,lanes 7–8). These data indicated that YodBC6A could stillfunction and respond somewhat to thiol-reactive com-pounds despite its reduced DNA-binding activity.

Mass spectrometry analysis of YodB modified in vitro bycatechol and MHQ

We next examined whether treatment of YodB with thethiol-reactive compounds catechol and MHQ leads to spe-cific Cys6 modifications. Purified His6-YodB was cleavedwith Tev (tobacco-etch virus) protease to leave only thetripeptide GAH N-terminal extension of the YodB protein.This modified YodB protein was incubated for 10 min witheach compound and then digested with trypsin for 2 h. Thetryptic digest was analysed using FT-ICR-MS (Fouriertransform ion cyclotron resonance MS) in the positive ionmode. In the untreated YodB sample, the 573.75 Da masspeak corresponding to the double charged T1 peptide(m/z = 1145.49 + 2H+) was found as the most abundantpeptide, suggesting that Cys6 is in the reduced form

Fig. 6. Reduction of YodBC6A proteinbinding activity to the spx P3 promoter (A)and azoR1 promoter (B) in vitro. DNase Ifootprinting was used to assess the bindingactivity of YodB and YobBC6A protein and theeffect of diamide on DNA binding. Theconcentrations of proteins used were 4 mM(A, lanes 3–8) and 0.5 mM (B, lanes 3–8).Theprotected region by YodB is indicated by thesolid line. The positions relative to thetranscriptional start site are shown. Theconcentrations of diamide used were 1 and1.5 mM for spx, 125 and 250 mM for azoR1.BSA, bovine serum albumin.

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(Fig. 7A). However, small amounts of T1 peptides withmethionine sulphoxides and the fully oxidized cysteinesulphonic acids were also detected, which could begenerated upon protein purification or during samplepreparation.

The treatment of YodB with 1 mM MHQ resulted in theappearance of a new mass peak of 634.76 Da correspond-ing to the double charged T1 peptide with a 122 Da modi-fication (m/z = 1267.52 + 2H +) (Fig. 7B). This mass peakis representative of the sulphhydryl-bound form of MHQ(m/z = T1 + 122 +2H+) as there is the loss of 2 Da owing tothe phenolic ring-conjugation with the thiolate (-2H+). Thecollision induced dissociation MS/MS analysis of thedouble charged m/z = 1267.52 peak confirmed that MHQis covalently linked to the Cys6 residue (Fig. S2).

The treatment of YodB with 10 mM catechol caused theformation of a new mass peak of 627.76 Da correspond-ing to the double charged T1 peptide with a 108 Damodification (m/z = 1253.52 + 2H+) (Fig. 7C). Thismass peak represents the Cys6-S-adduct of catechol(m/z = T1 + 108 + 2H+), which was confirmed by collisioninduced dissociation MS/MS analysis of this peptide(Fig. S3). The reduced Cys101 and Cys108 containing1680.73 Da mass peak was observed in the untreatedYodB sample corresponding to the double chargedT9-peptide (m/z = 3359.45 + 2H+) (data not shown). Inaddition, the disulphide-bound form of the T9 peptide wasdetected in the untreated YodB sample as judged fromthe loss of 2 Da of this peptide (m/z = 3357.45 + 2H+)(data not shown). Sulphhydryl-bound forms of MHQ andcatechol were also detected for this T9 peptide, suggest-ing that both MHQ and catechol auto-oxidize to quinones,which react as electrophiles with protein thiolates.

Discussion

YodB and MhqR affect quinone and azo-compounddetoxification by controlling production ofparalogous azoreductases

In this study, we uncovered the azoreductase-encodingazoR1 gene as the most strongly derepressed member ofthe YodB regulon. The yodB mutant showed increasedresistance to catechol and MHQ, which could be attrib-uted to the function of AzoR1. The YodB-controlled azoR1gene encodes an azoreductase, AzoR1, which is a para-logue of AzoR2, specified by the MhqR-regulated azoR2gene. Production of both enzymes responds to

thiol-specific electrophiles and confers resistance to cat-echol and MHQ in B. subtilis (Töwe et al., 2007). Thus,both enzymes have similar functions related to the detoxi-fication of catechol and MHQ. Catechol and hydroquino-nes can be auto-oxidized to the corresponding ortho-andpara-benzoquinones that can act as toxic oxidants andelectrophiles (Fig. 8). The formation of the benzoquinonescould be detected by the appearance of a red-colouredmedium if cells are exposed to growth-inhibitory concen-tration of catechol and MHQ.

Quinones are enzymatically reduced via two differentpathways: (i) formation of semi-quinone radicals and reac-tive superoxide anions via one-electron reduction (e.g.NADPH-cytochrome P450 reductase, z-crystallin) and (ii)formation of hydroquinones in the two-electron reductionby NAD(P)H quinone oxidoreductases (Ernster, 1987).The functions of the azoreductases AzoR1 and AzoR2are related to NADH:quinone reductases, also called dia-phorases, which catalyse the NAD(P)H-dependent two-electron reductions of quinones, quinoneimines, azodyesand nitrogroups to protect cells against the toxic effects offree radicals and ROS arising from the one-electronreductions (Ernster, 1987; Li et al., 1995; Cavelier andAmzel, 2001). The azoreductases could function todetoxify the quinones back to redox-stable catechol andMHQ to protect cells against the redox-cycling actionand reaction as electrophiles targeting protein thiolates(Fig. 8). This thesis is further supported by the fact that theDmhqR and DyodB mutants show a four- to fivefoldincreased resistance to para-benzoquinone (our unpub-lished data). The toxicity of para-benzoquinone is morethan 50-fold higher than that of hydroquinone inB. subtilis, which might explain the thiol-reactive electro-philic nature. It has been further shown, that AzoR2 pos-sesses NADH:DCIP oxidoreductase and azoreductaseactivity (Nishiya and Yamamoto, 2007). Azoreductasesare flavoenzymes that have the unique ability to catalysea wide range of biochemical reactions and have broadsubstrate specificity (Fraaije and Mattevi, 2000). Thus,both azoreductases, AzoR1 and AzoR2 could alsofunction in the reductive cleavage of the electrophileazo-compound diamide to 1,1′-dimethylurea. Another fla-voenzyme, the nitroreductase YodC is also regulated byYodB, and is a paralogue of MhqN, the gene for which iscontrolled by MhqR. Thus, paralogous nitroreductasesare co-regulated with paralogous azoreductases inresponse to thiol-reactive electrophiles. Nitroreductasescatalyse the two-electron reduction of nitrogroups in

Fig. 7. Modification of YodB in vitro by MHQ and catechol. Purified YodB (5 mg) was incubated for 10 min with 1 mM MHQ and 10 mMcatechol and analysed by FT-ICR-MS in the positive ion mode after tryptic digestion. In the untreated YodB sample, the double charged T1peptide was mostly detected in the Cys6-reduced form (m/z = 573.75) (A). Methionine sulphoxide modifications of T1 are indicated with anasterisk and cysteine sulphonic acid modification of T1 is indicated by #. After treatment of YodB protein with MHQ, a new peak correspondingto the double charged T1 with Cys6-S-conjugated MHQ was detected (m/z = 634.76) (B). The treatment with catechol resulted in a new masspeak corresponding to the double charged T1 with Cys6-S-conjugated catechol (m/z = 627.76) (C).

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nitroaromatic compounds to nitroso, hydroxylamine inter-mediates and finally primary amines (Bryant and DeLuca,1991). As quinones also represent substrates for nitrore-ductases (Nivinskas et al., 2002), YodC and MhqN couldfunction in quinone reduction in B. subtilis. There is thequestion: Why have paralogous azo- and nitroreductasesevolved that act upon the same class of substrates, butare separately regulated by YodB and MhqR? Twooxygen-insensitive FMN-containing nitroreductases, NfsAand NfsB, have been also characterized in Eschericia coli.Of these, NfsA uses NADPH as electron donor whereasNfsB can use either NADH or NADPH as source ofreduction equivalents (Zenno et al., 1996a,b). Thus, theparalogous nitro- and azoreductases of B. subtilis thatcontribute to quinone resistance could use different elec-tron donors for quinone reduction. The detailed mecha-nism of quinone reduction remains to be elucidated,however.

Interestingly, neither of these azo- or nitroreductaseswas induced by oxidative stress provoked by H2O2 or theredox-cycling agent paraquat (our unpublished data)supporting the thesis that these enzymes are specificallyinvolved in quinone and electrophile reduction and not inthe oxidative stress response. As ROS are producedupon auto-oxidation of MHQ and catechol, this couldaccount for the induction of the PerR-controlled oxidativestress response by electrophiles. Other quinone oxi-doreductases such as QorA of Staphylococcus aureusare produced in response to oxidative stress and thesecatalyse the one-electron reduction of quinones, thusproducing superoxide anions (Maruyama et al., 2003).Interestingly, QorA is homologous to the quinone oxi-doreductase YfmJ of B. subtilis. YfmJ protein was alsoinduced by MHQ treatment (Duy et al., 2007) and indeedfunctions in quinone reduction in vitro (A. Maruyama,pers. comm.). In addition, production of many other

Proposed mechanism for electrophile detoxification in B. subtilis

O

O

+ R-SHOH

OH

Catechol

ortho-

benzoquinone

ring cleavage

productOH

OH

S-R

AzoR1

AzoR2

NADH+H+NAD+

MhqA

MhqO

MhqE

+ R-SH

NN

O

NN

O

diamide

NH2

O

H3C

H3CCH3

CH3

2HN N

O

CH3

CH3

H3C

H3CN

NN

O

NN

O

H

Hydrazine

OH

CH3

OH O

O

CH3MHQ

Methyl-para-

benzoquinone

ring cleavage

product

OH

OH

CH3

S-R

+ R-SH

AzoR1

AzoR2

NADH+H+NAD+

+ R-SH

+ 2 R-SH

1,1'-Dimethylurea

AzoR1

AzoR2

2 NADH+H+

2 NAD+

MhqA

MhqO

MhqE

H

+ R-S-S-R

H3C

H3C

CH3

CH3

½ O2 H2O

½ O2 H2O

Fig. 8. Thiol-specific reactions of electrophilic compounds and model for the functions of MhqR- und YodB-controlled azoreductases anddioxygenases/glyoxalases in B. subtilis. Catechol and MHQ are oxidized to the corresponding ortho- and para-benzoquinones that causeS-adduct formation via 1,4-reductive addition of thiols to quinones (Michael-type addition) (Rodriguez et al., 2004). The paralogousazoreductases AzoR1 and AzoR2 are major quinone resistance determinants and function in quinone reduction to catechol and MHQ(Cavelier and Amzel, 2001). Diamide is an electrophilic azo-compound that reacts with protein thiolates and causes disulphide bond formationor mixed disulphides with LMW thiols (Leichert et al., 2003; Hochgräfe et al., 2007). AzoR1 and AzoR2 also confer resistance to diamide andfunctionin detoxification of diamide via the reductive cleavage of the azo group, resulting in two molecules 1,1′-Dimethylurea. The paralogousdioxygenases/glyoxalases which are co-regulated with the azoreductases (Töwe et al., 2007) might be involved in the thiol-specificdetoxification of the LMW-thiol-S-adducts via dioxygenolytic cleavage of the compounds. Thus, this mechanism of electrophile resistance iscoupled to the thiol-specific stress responsive regulatory network.

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NAD(P)H-oxidoreductases responds to MHQ or catecholand might contribute to either the one-or two-electron reduction of quinones in B. subtilis(Duy et al., 2007).

The mechanism of quinone reduction by nitro- andazoreductases is coupled to the thiol-specific stressresponse in B. subtilis

The question arises why regulation of quinone reductionby the azoreductases is coupled to the thiol-specificstress response in B. subtilis. Quinones exert their tox-icity mainly as electrophiles via the 1,4-reductive nucleo-philic addition to protein thiolates (Michael-type addition)leading to thiol-S-adduct formation (Fig. 8). Thus,quinone and diamide exposure results in depletion ofprotein thiolates and non-protein-low molecular weight(LMW) thiolates. B. subtilis does not possess glutathioneand instead, the major LMW thiols of B. subtilis are likelycysteine, CoASH, or the recently reported 398 Da bio-thiol (Newton et al., 1996; Gusarov and Nudler, 2005;Lee et al., 2007; Hochgräfe et al., 2007). Recently, it hasbeen shown that diamide triggers S-cysteinylation in vivoin a number of B. subtilis cytoplasmic proteins that havecatalytic cysteine residues (e.g. GuaB, MetE and PpaC)(Hochgräfe et al., 2007). The formation of mixed disul-phides with the LMW thiol cysteine is proposed as amechanism to protect catalytic cysteine residues againstoxidation to irreversible sulphonic acid. Thus, the LMWthiol cysteine could function as a GSH equivalent indetoxification of electrophiles via cysteine-S-adduct for-mation. The CymR regulon which regulates cysteine bio-synthesis is also derepressed under the conditions ofdiamide, catechol and MHQ stress which supports theidea of cysteine depletion owing to S-adduct formationwith electrophiles.

The MarR-type repressor MhqR regulates expressionof azoR2 and genes encoding multiple dioxygenase/glyoxalase family enzymes in response to thiol stressconditions (Töwe et al., 2007). These dioxygenases/glyoxalases could be involved in the thiol-dependent ringcleavage of the catechol- and MHQ-S-adducts (Fig. 8).This proposed mechanism of electrophile detoxification issimilar to the GSH-dependent detoxification of methylgly-oxal and other electrophiles (e.g. N-ethylmaleimide) inE. coli which involves glutathione-S-adduct formation withthe electrophiles and detoxification via the glyoxalasepathway or release of these GSH-adducts from the cell(Ferguson et al., 1998, 1999; McLaggan et al., 2000;Booth et al., 2003). The results reported herein suggestthat azo- and nitroreductases function in the detoxificationof electrophiles whereas dioxygenases/glyoxalase couldbe involved mainly in detoxification of the redox-stableS-hydroquinone adducts.

The Cys residues of YodB function in sensingthiol-reactive electrophiles

In this study, the regulation of the MarR/DUF24-familyrepressor YodB in response to thiol-specific electrophileswas uncovered in B. subtilis. YodB is highly conservedamong low-GC Gram-positive bacteria and several para-logues are encoded within the B. subtilis genome. YodB-like regulators share the conserved N-terminal cysteine inposition 5 or 6 followed by a conserved proline residue.As reported herein, this conserved Cys6 is essential forrepression of YodB-controlled target genes in vivo and invitro. Inactivation of Cys6 in a yodBC6A mutant resulted ina decreased repression of azoR1 in vivo. Furthermore,the conserved Cys6 was found to play a role in regulationof YodB activity as observed by examining the phenotypeof the yodBC6A mutant, which was more resistant tocatechol than the wild-type cells. Thus, it could be pos-sible that the Cys6 residue has a higher reactivity to formthe nucleophilic thiolate than the Cys101 and Cys108residues. There was still some repression of YodB-dependent genes in the yodBC6A mutant in vivo as thebasal level of azoR1 transcription was lower in theyodBC6A mutant compared with the yodB null mutant.Transcription of azoR1 was still two- to threefold induciblein the yodBC6A mutant after exposure to catechol andMHQ, suggesting that there might be other sites in theYodB protein that are involved in regulation of YodBactivity. Electrophilic quinone-like compounds could reactwith other nucleophilic amino acids present in the YodBprotein, thereby influencing YodB DNA-binding activity.Interestingly, there are reports that quinone-like electro-philes can also form chemical adducts on protein nitrogennucleophiles at the N-terminus and at the basic aminoacid side-chains of lysine, arginine and histidine residues(Person et al., 2003; 2005). As several basic lysine andarginine residues are present in the N-terminal part of theYodB protein, further mutagenesis studies are required toresolve this issue. Notably, the C-terminal C101 and C108residues are not required for YodB repression, but werefound also in disulphide bond form in vitro as revealedby mass spectrometry. The triple mutant, C6,101,108A,shows reduced repression, but also lower responsivenessto inducers, suggesting the Cys residues of YodB couldserve a sensory function by reacting directly with thiol-reactive compounds.

Using mass spectrometry we uncovered the formationof Cys-S-adducts upon treatment of YodB with MHQ andcatechol in vitro confirming the thiol reactivity of the quino-nes produced upon catechol and MHQ auto-oxidation.Thus, the derepression of YodB in response to diversearomatic compounds might be caused by their commonelectrophilic nature, which leads to covalent cysteinemodifications. Cysteine residues are strong nucleophiles

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and form redox centres in an emerging group of oxidativestress transcription factors such as the E. coli oxidativestress regulator OxyR and the B. subtilis organic hydrop-eroxide repressor OhrR (Fuangthong et al., 2002; Pagetand Buttner, 2003; Hong et al., 2005; Lee et al., 2007). Allattempts to detect YodB modifications upon treatmentwith diamide failed using mass spectrometry. We onlyobserved that the peptide containing Cys6 was absentin the MS spectrum after diamide treatment (data notshown). Thus, the mechanism of YodB inactivation upondiamide treatment is currently unknown. As S-thiolationhas been shown as one mechanism of OhrR and OxyRregulation (Kim et al., 2002; Lee et al., 2007), modificationof YodB could occur in vivo in response to diamide andother electrophiles via the formation of mixed disulphidescontaining cysteine, CoASH or other LMW thiols. Onegoal of our future proteomic studies will be to determinewhether the YodB repressor is regulated by S-thiolation orS-adduct formation by electrophiles in vivo. However,detection of S-conjugates with quinone-like electrophiles,which are formed in vivo will be difficult using mass spec-trometry as these are rather unstable modifications andare often lost upon prefractionation procedures.

In summary, our studies uncovered the function andregulation of the YodB repressor that controls the expres-sion of spx, and the genes that encode the azoreductaseAzoR1 and the nitroreductase YodC in response to thiol-specific electrophiles. We show that the conserved Cys6residue of YodB is required for optimal YodB-mediatedrepression in vivo and in vitro and that the three Cysresidues function in inducer sensing. In addition, wereport that paralogous azoreductases AzoR1 and AzoR2both confer resistance to catechol and MHQ in B. subtilis.Hence, we presume that these azoreductases ofB. subtilis function as quinone reductases and constitutemajor detoxification pathways to protect bacteria fromquinone toxicity. As these azo- and nitroreductases arehighly conserved among pathogenic Gram-positive bac-teria, it will be interesting to find the naturally ocurringquinone-like substrates for these enzymes.

Experimental procedures

Bacterial strains and growth conditions

Bacillus subtilis strains used in this study are derivatives ofJH642 and 168 and are listed in Table S1. Cells were culti-vated in a shaking water bath at 37°C in Difco sporulationmedium (DSM) for b-galactoisidase assays or TSS minimalmedium and Belitsky minimal medium (Stülke et al., 1993) fortreatments with thiol-reactive compounds. Diamide andhydrogen peroxide were purchased from Sigma-Aldrich.MHQ was purchased from Acros and catechol was pur-chased from Fluka.

The isogenic azoR1 mutant was constructed by transfor-mation of JH642 with chromosomal DNA of the azoR1D

(yocJ::pMutin4) strain, which carries an azoR1–lacZ fusion,resulting in a strain designated ORB6734. The yodB mutantbearing an azoR1–lacZ fusion was constructed by transfor-mation of ORB6734 with chromosomal DNA of ORB6208(yodB mutant) (Leelakriangsak et al., 2007) to generateORB6735.

The azoR1–lacZ fusion was constructed by transformationof JH642 competent cells with plasmid pML78, which carriesan azoR1–lacZ fusion, resulting in strain ORB7093. pML78was generated by PCR using primers oSN03-64 and oSN03-65. The PCR fragment (-155 to +110 with respect to theazoR1 transcription start site) was digested with enzymesEcoRI and BamHI, and then was inserted into plasmidpDH32 which was digested with the same enzymes. TheazoR1 sequence was verified by DNA sequencing. PlasmidpCm::tet was used to transform ORB7093, replacing the Cmr

cassette with a Tetr marker to generate strain ORB7094.The yodB disruption strain bearing azoR1–lacZ fusion wasconstructed by transformation of ORB7094 with chromo-somal DNA of ORB6208 (Leelakriangsak et al., 2007).The resulting strain was designated ORB7106 (azoR1–lacZ,yodB). Diploid yodB strains bearing azoR1–lacZ fusion wereconstructed as follows. The chromosomal DNA of ORB6606(thrC::yodB) (Leelakriangsak et al., 2007), ORB6592(thrC::yodBC6A), ORB6713 (thrC::yodBC101A), ORB6714(thrC::yodBC108A), ORB6775 (thrC::yodBC101,108A) andORB7081 (thrC::yodBC6,101,108A) were used to transformORB7106 (azoR1–lacZ, yodB) to generate ORB7107,ORB7108, ORB7109, ORB7110, ORB7111 and ORB7112respectively. The yodB::pMutin4 and azoR2::pMutin4mutants were constructed during the course of the Europeanand Japanese B. subtilis functional analysis project.Chromosomal DNA of the azoR2::pMutin4 (ST8) andyodB::pMutin4 (ST5) mutants were introduced by trans-formation into the DazoR1 mutant (ST7) to generate theDazoR1DazoR2 (ST9) and DazoR1DyodB (ST10) doublemutants respectively. The DmhqR mutant (TF176) was trans-formed with chromosomal DNA of the yodB::pMutin4 (ST5)mutant to generate the DyodBDmhqR double mutant (ST6).The lesions harboured by the mutants were verified by PCR.

When necessary, the following antibiotics were added (at aconcentration shown in parenthesis): ampicillin (25 mg ml-1),chloramphenicol (5 mg ml-1), erythromycin plus lincomycin (1and 25 mg ml-1 respectively), and tetracycline (12.5 mg ml-1).

Construction of yodBC6A, C101A, C108A, C101,108Aand C6,101,108A mutants

Plasmids pML60(C6A), pML65(C101A), pML66(C108A),pML69(C101,108A) and pML76(C6,101,108A) were pro-duced by using PCR mutagenesis. First-round PCR wasperformed in two separate reactions with primers oyodB-EcoRI with oyodB-C6AR and primers oyodB-C6AF withoyodB-HisB (Table S2) using JH642 chromosomal DNA astemplate. The PCR products were hybridized and subse-quently amplified by a second round of PCR using primersoyodB-EcoRI and oyodB-HisB. The products from the secondround PCR were then digested with EcoRI and BamHIrestriction enzymes and inserted into plasmid pUC19 thatwas digested with the same enzymes to generate pML60.pML76 was generated by the same procedure as that used

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for construction of pML60, but using pML69 as template. ThePCR product (ª 60 bp) that was synthesized using primeroyodB-C101A and oyod-HisB was used as the primertogether with oyodB-EcoRI for second round PCR to gener-ate pML65. pML66 was generated by using primers oyodB-EcoRI and oyodB-C108A. Primers oyodB-EcoRI and oyodB-C108A (Table S2) were used to produce pML69 using pML65as the template. The yodB sequences were verified by DNAsequencing. The plasmids pML60, pML65, pML66, pML69and pML76 were digested with EcoRI and BamHI and thereleased fragments bearing the mutations were then insertedinto pDG795 that was digested with the same enzymesto generate pML61, pML67, pML68, pML72 and pML77respectively. pML61, pML67, pML68, pML72 and pML77were introduced by transformation with selection forerythromycin-lincomycin resistance, into JH642, whereyodBC6A, C101A, C108A, C101,108A and C6101,108A frag-ments integrated into the thrC locus. The resulting strainswere designated ORB6592 (thrC::yodBC6A), ORB6713(thrC::yodBC101A), ORB6714 (thrC::yodBC108A),ORB6775 (thrC::yodBC101,108A) and ORB7081(thrC::yodBC6,101,108A). Chromosomal DNA of ORB6592,ORB6713, ORB6714, ORB6775 and ORB7081 were usedto transform ORB6288 to generate ORB6599, ORB6715,ORB6716, ORB6777 and ORB7083 respectively.

Transcriptome analysis

For microarray analysis, the B. subtilis 168 wild-type andisogenic DyodB (TF277) mutants were grown in minimalmedium and harvested at OD500 of 0.4. Total RNA was iso-lated by the acid phenol method as described (Majumdaret al., 1991). The generation of fluorescence-labelled cDNAand hybridization with B. subtilis whole-genome microarrays(Eurogentec) was performed as described previously (Jürgenet al., 2005). Two independent hybridization experimentswere performed using RNAs from two independent sets ofcultures. Genes showing induction ratios of at least threefoldin two independent experiments were considered as signifi-cantly induced. An independent microarray experiment wasconducted using strains ORB6208 (DyodB::cat) and theJH642 parent. Procedures for RNA purification and micro-array analysis were described previously (Nakano et al.,2003a; Choi et al., 2006).

Assay of b-galactosidase activity

Toluene treatment and assays of b-galactosidase activityusing o-nitrophenyl-b-D-galactopyranoside were performedas previously described (Nakano et al., 1988; Schrogel andAllmansberger, 1997). b-Galactosidase activity is expressedas Miller Units (Miller, 1972).

Protein purification

The yodB coding sequence was amplified by PCR usingprimers oyodB-HisNC6A and yodB-HisB. The PCR productswere digested with NdeI and BamHI restriction enzymes andinserted into pPROEX-1(Life Technologies) digested with thesame enzymes to generate pML74. Cells were cultured and

the protein was purified as described previously (Leelakriang-sak et al., 2007).

Primer extension analysis

Strains ORB6284, ORB6288, ORB6599, ORB6715,ORB6716 and ORB6777 were grown at 37°C in TSSmedium. Primer extension analysis was performed asdescribed previously (Leelakriangsak and Zuber, 2007).Primers oML02-15 and oSN03-63 were used to synthesizethe primer extension products to examine the transcripts ofspx and azoR1 respectively.

DNase I footprinting

A DNA probe for azoR1 (position corresponding -160 to +110with respect to transcription start site) was synthesized byPCR amplification using primers oSN6-03–64 and oSN03-65.Dideoxy sequencing ladders were obtained using the ThermoSequenase cycle sequencing kit (USB) and the PCR productspecifying azoR1 as template. The same primers used for thefootprinting reactions were also used for sequencing. DNaseI footprinting reactions were performed as described previ-ously (Leelakriangsak et al., 2007).

Proteome analysis

Cells grown in minimal medium to an OD500 of 0.4 werepulse-labelled for 5 min each with 5 mCi of L-[35S]methionineper ml before (control) and 10 min after exposure to 0.33 mMMHQ, 2.4 mM catechol, 1 mM diamide, 0.1 mM nitrofuran-toin and 100 mM H2O2. Preparation of cytoplasmicL-[35S]methionine-labelled proteins and separation by two-dimensional gel electrophoresis using the immobilized pHgradients in the pH range 4–7 was performed as described(Tam et al., 2006). The quantitative image analysis was per-formed with the DECODON Delta 2D software (http://www.decodon.com).

Northern blot analyses

Northern blots were performed as described (Wetzstein et al.,1992) using RNA isolated from B. subtilis wild-type andDyodB (TF277) mutant cells before (control) as well as 10and 20 min after treatment with 2.4 mM catechol or 0.33 mMMHQ. Hybridizations specific for azoR1 and bsrB wereperformed with the digoxigenin-labelled RNA probes synthe-sized in vitro using T7 RNA polymerase from T7 promoter-containing PCR products amplified with azoR1-specificprimers (yocJ_for and yocJ_rev) and bsrB-specific primers(bsrB-for and bsrB-rev).

FT-ICR-MS analysis

LC-ESI MS experiments were performed using an LTQ (linearion trap) FTICR mass spectrometer (Thermo Electron, SanJose, CA). The SEQUEST algorithm Version 27.12 (ThermoElectron) was used to search for modifications of the YodBtryptic peptides. A mass deviation of 0.01 Da for precursor

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ions as well as for fragment ions and one missed cleavagesite of trypsin were allowed. The search result was filteredusing BioWorks 3.2 (Thermo Electron). A multiple thresholdfilter applied at the peptide level consisted of the followingcriteria: (i) peptide sequence length: 7–30 amino acids; (ii)RSp � 4; (iii) percentage of ions: 70 (70% of all theoretical b-and y-ions of a peptide are experimentally found); and (iv)Xcorr versus charge state: 1.90 for singly charged ions, 2.20for doubly charged ions and 3.75 for triply charged ions.

Acknowledgements

The authors wish to thank C. Lee and A. D. Grossman fortechnical assistance with microarray experiments; and K.Kobayashi and H. Yoshikawa for gifts of B. subtilis strainsused in the experiments described herein. We thank M. M.Nakano for advice and valuable discussions. Researchreported herein was supported by Grant GM45898 from theNational Institutes of Health, USA; and by grants from theDeutsche Forschungsgemeinschaft, the Bundesministeriumfür Bildung und Forschung (BACELL-SysMo 031397A), theFonds der Chemischen Industrie, the Bildungsministeriumof the country Mecklenburg-Vorpommern and EuropeanUnion grants BACELL-Health (LSHG-CT-2004-503468) andBACELL-BaSysBio (LSHG-CT-2006-037469) to M.H., by ascholarship of the ‘Ministry of Education and Training ofVietnam’ (MOET) to N.v.D.

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