The RclR Protein Is a Reactive Chlorine-specific Transcription Factor in Escherichia coli

12 Pages·2013·2.41 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.288,NO.45,pp.32574–32584,November8,2013
©2013byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
The RclR Protein Is a Reactive Chlorine-specific Transcription
Factor in Escherichia coli*
Receivedforpublication,July18,2013,andinrevisedform,September23,2013Published,JBCPapersinPress,September27,2013,DOI10.1074/jbc.M113.503516
BenjaminW.Parker‡,EmilyA.Schwessinger‡,UrsulaJakob‡§1,andMichaelJ.Gray‡2
Fromthe‡DepartmentofMolecular,Cellular,andDevelopmentalBiologyandthe§CellularandMolecularBiologyProgram,
UniversityofMichigan,AnnArbor,Michigan48109
Background:Reactivechlorinecompoundsareimportantnaturalantimicrobialsproducedbytheimmunesystem.
Results: Reactive chlorine treatment leads to RclR cysteine oxidation, activation of DNA binding, and expression of RclR-
controlledgenes.
Conclusion:RclRisatranscriptionalactivatorthatrespondsspecificallytoreactivechlorine.
Significance:Understandingreactivechlorineresponsesisimportantforunderstandinginteractionsbetweenbacteriaandthe
hostimmunesystem.
Reactivechlorinespecies(RCS)suchashypochlorousacidare householdsettings(4,5).Moreover,theyaremajorbactericidal
powerfulantimicrobialoxidants.Usedextensivelyfordisinfec- componentsoftheoxidativeburstofneutrophils(3,6,7)and
tioninhouseholdandindustrialsettings(i.e.asbleach),RCSare areimplicatedincontrollingbacterialcolonizationofepithelial
also naturally generated in high quantities during the innate surfaces(8–10).Thereisnowalsoincreasingevidencethatpro-
immuneresponse.BacterialresponsestoRCSarecomplexand ductionofRCSisacommonstrategyamongdiverseeukaryotes
differ substantially from the well characterized responses to for controlling bacterial populations (11, 12). Understanding
otherphysiologicallyrelevantoxidants,likeperoxideorsuper- howbacteriasenseandrespondtoRCSisthereforeimportant
oxide. Several RCS-sensitive transcription factors have been forunderstandingtheinteractionsbetweenbacteriaandtheir
identified in bacteria, but most of them respond to multiple eukaryotic hosts, with obvious implications for the study of
stressors whose damaging effects overlap with those of RCS, humanhealthanddisease.
including reactive oxygen species and electrophiles. We have Bacterialresponsestoreactiveoxygenspecies(ROS),suchas
nowusedinvivogeneticandinvitrobiochemicalmethodsto hydrogenperoxide(H O )orsuperoxide(O.),havebeenstud-
2 2 2
identify and demonstrate that Escherichia coli RclR (formerly iedextensively(reviewedrecentlyinRefs.13–16).Itisknown
YkgD)isaredox-regulatedtranscriptionalactivatoroftheAraC thattheseresponsesdependonselecttranscriptionfactors(e.g.
family,whosehighlyconservedcysteineresiduesarespecifically OxyR,SoxR,PerR),whichareabletospecificallysensepartic-
sensitivetooxidationbyRCS.Oxidationofthesecysteinesleads ular oxidants, often through the oxidation state of conserved
to strong, highly specific activation of expression of genes cysteine residues. These regulators control the expression of
requiredforsurvivalofRCSstress.Theseresultsdemonstrate genesthatcontributedirectlytoROSdetoxificationortothe
the existence of a widely conserved bacterial regulon devoted repairofROS-mediateddamage.Theabilityofredox-sensitive
specificallytoRCSresistance.
regulatorstodistinguishamongdifferentoxidantsisakeyfac-
torinredoxsignaling(17).
Recentstudieshaveidentifiedseveralbacterialtranscription
Reactive chlorine species (RCS),3 including hypochlorous factorsthatrespondtoRCStreatment.TheseincludetheEsch-
acid(HOCl)andchloramines,arepowerfulantimicrobialoxi- erichia coli transcription factors HypT and NemR and the
dants capable of chlorinating and oxidizing a wide range of BacillussubtilistranscriptionfactorsOhrRandHypR(18–21).
biomolecules(1–3).RCS,suchasthosepresentinbleach,are Interestingly,whereasHypTissofaronlyknowntorespondto
important disinfectants in human medical, industrial, and HOCl (19, 22), the other RCS-sensing transcription factors
respond to a variety of other stress signals, including cysteine-
modifyingelectrophiles(20,21,23)andorganichydroperoxides
*Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth
GrantsR01-GM065318andR21-AI097893(toU.J.). (18,24).IncontrasttoROSandtoxicelectrophiles,whichhavea
1Towhomcorrespondencemaybeaddressed:Dept.ofMolecular,Cellular, morelimitedsetofcellulartargets(25–27),RCSarecapableof
andDevelopmentalBiology,UniversityofMichigan,830N.UniversityAve.,
damagingproteins,DNA,lipids,andmostothercellularcompo-
Ann Arbor, MI 48109. Tel.: [phone]; Fax: [phone]; E-mail:
nents(1–3).ItthereforeappearsthattosuccessfullycombatRCS,
[email protected].
2SupportedbyNationalResearchServiceAwardF32GM096613.Towhom organismsrequireamultifacetedstressresponsesystem,regulated
correspondence may be addressed: Dept. of Molecular, Cellular, and byadiversesetofredox-sensitiveregulators.
DevelopmentalBiology,UniversityofMichigan,830N.UniversityAve.,
Here we report the discovery of RclR (formerly known as
Ann Arbor, MI 48109. Tel.: [phone]; Fax: [phone]; E-mail:
[email protected]. YkgD),ahighlyRCS-specifictranscriptionalactivatorinE.coli.
3Theabbreviationsusedare:RCS,reactivechlorinespecies;HOCl,hypochlo- InvivoandinvitrostudiesdemonstratethatRclRreliesonthe
rous acid; qRT-PCR, quantitative RT-PCR; ROS, reactive oxygen species;
reversibleoxidationofconservedredox-sensitivecysteineres-
TPEN,tetrakis-(2-pyridylmethyl)ethylenediamine;NBD-chloride,4-chloro-7-
nitro-1,2,3-benzoxadiazole. iduestocontroltheexpressionofthreegenesessentialforbac-
32574 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER45•NOVEMBER8,2013
This is an Open Access article under the CC BY license.
RclRIsaReactiveChlorine-specificActivator
TABLE1
Primersusedinthisstudy
Primer Sequence
[1] 5(cid:2)-TAC TAA GAT TGT TGG TGT TTG TAA TCA AAA ACC ACT CAG GAG TCT GAT ATG GTG TAG GCT GGA GCT GCT TC-3(cid:2)
[2] 5(cid:2)-AAC TCC CGG AAT GCG GAT TAT CTT TGC CCT GAT TTC TGG CGG TTT TAT TCA CAT ATG AAT ATC CTC CTT AG-3(cid:2)
[3] 5(cid:2)-CGT CTA TAG TCA TGA TGT CAA ATG AAC GCG TTT CGA CAG GAA ATC ATC ATG GTG TAG GCT GGA GCT GCT TC-3(cid:2)
[4] 5(cid:2)-CTT TTC TCT GAG ACG CCA GAA TAT TTG TTC TGG CGT CTG ATT TTG AGT TTA CAT ATG AAT ATC CTC CTT AG-3(cid:2)
[5] 5(cid:2)-TCT CTG TTA CTC TAT AAC TTC TTA ATC ACT TCA TTG ATG GTA TTT TAT ATG GTG TAG GCT GGA GCT GCT TC-3(cid:2)
[6] 5(cid:2)-TAT CTC CCC GAC TTA ACA GGT GCA GGT ATT TTT CCA TTG TGA ACA TCC TTA CAT ATG AAT ATC CTC CTT AG-3(cid:2)
[7] 5(cid:2)-TAA TAA TTC AAC CGC AAC AGC AGT ATT GTA TAA ATA AGG ATG TTC ACA ATG GTG TAG GCT GGA GCT GCT TC-3(cid:2)
[8] 5(cid:2)-AGG GGG CCT GAC GCC TGA AAA AGT GAA CAA CAG ACA GTG TTC GGA TTA TCA CAT ATG AAT ATC CTC CTT AG-3(cid:2)
[9] 5(cid:2)-TTC GAA TTC AGG AGT CTG ATA TGG ATG CCC-3(cid:2)
[10] 5(cid:2)-CTT AAG CTT TCA GGG TGC AAG CTG TCT GA-3(cid:2)
[11] 5(cid:2)-GAA CGA TCG ATA AGA ATG CGG TGT TAG GAA GTG ACT GG-3(cid:2)
[12] 5(cid:2)-CGA CCT GTA TTG TCG CGG GCA CTC TTC GGT TG-3(cid:2)
[13] 5(cid:2)-GAA TTA TCG GTT ATT CGT TGG GCG GCG TTA ACG CAA GGA GCG GC-3(cid:2)
[14] 5(cid:2)-CCA CAA AAT TCA GCT GCG CGC CTG AGT CAT GTC G-3(cid:2)
[15] 5(cid:2)-CAC GAT TGA TAA AAA CGC GGT CCT GGG TTC TGA CTG-3(cid:2)
[16] 5(cid:2)-GAA TCC ACC TGC ATT GTT GCG GGT ACG CTG CGT CTG CAA C-3(cid:2)
[17] 5(cid:2)-ACT GAG CGT TAT TCG TTG GGC GGC ACT GAC CCA GGG TGC A-3(cid:2)
[18] 5(cid:2)-AGA ATA GTG CGG CGC GCC TGT CCC ACG TGG ATA A-3(cid:2)
[19] 5(cid:2)-AAG AAC TTA CCT GGT CTT GAC ATC-3(cid:2)
[20] 5(cid:2)-CAG TTT ATC ACT GGC AGT CTC CTT-3(cid:2)
[21] 5(cid:2)-AAA AAC ATT CTC AGT TTG CTT CTG C-3(cid:2)
[22] 5(cid:2)-CGC TAT TTG TAG ACG CAA CTT TGT T-3(cid:2)
[23] 5(cid:2)-AGG GTT GGT ATG ACA GAA GAA CAA G-3(cid:2)
[24] 5(cid:2)-TAT ATT GAT CAT CTC GTG GGA GTC A-3(cid:2)
[25] 5(cid:2)-CAC TAT TAT CTG GCG TTA TGG CAT T-3(cid:2)
[26] 5(cid:2)-TAT ACA ATA CTG CTG TTG CGG TTG-3(cid:2)
[27] 5(cid:2)-TCT TTT ATG AAC ACC CGG AAG ACT A-3(cid:2)
[28] 5(cid:2)-ATC AGT CCT CCC AAT AAA CCT AAC C-3(cid:2)
[29] 5(cid:2)-GGT CCT TCT ATC ACT ACT TTC GCT CT-3(cid:2)
[30] 5(cid:2)-GTT TTA CTG TCA GGC AAC CAC TGA T-3(cid:2)
[31] 5(cid:2)-GAT GAT TTC CTG TCG AAA CGC-3(cid:2)
[32] 5(cid:2)-ATC AGA CTC CTG AGT GGT TTT TG-3(cid:2)
terial survival of reactive chlorine stress (rclA, rclB, and rclC; chloramphenicol resistance cassettes and subsequent resolu-
formerly ykgC, ykgI, and ykgB, respectively). Deletion of any tion to yield in-frame deletions, as described previously (35),
oneofthesegenes,whoseRCS-mediatedtranscriptionalup- usingthefollowingprimers(Table1):rclR,[1]and[2];rclA,[3]
regulationisamongthemostpronouncedinE.coli,dramat- and[4];rclB,[5]and[6];rclC,[7]and[8].
icallyincreasesRCSstresssensitivity.Theseresultssuggest PlasmidConstruction—TherclRcodingsequenceplus11bp
that we have identified an important member of the RCS of5(cid:2)-sequencewasamplifiedfromE.coliMG1655genomic
stressresponsesysteminbacteria. DNAwithprimers[9]and[10](Table1)andclonedintothe
EcoRI and HindIII sites of plasmid pBAD30 (36) to yield
EXPERIMENTALPROCEDURES
plasmidpRCLR1.TheQuikChangesite-directedmutagene-
Sequence Analysis and Primer Design—Gene and protein
siskit(Stratagene),modifiedtouseonlyasingleprimerand
sequenceswereobtainedfromtheIntegratedMicrobialGenomes
35cyclesofamplification,wasusedtomutatepRCLR1with
database(28).CustomPythonscriptsemployingtheBiopython
primers [11], [12], [13], or [14] to yield plasmids pRCLR8,
1.57toolkit(29)wereusedtosearchforandsortRclRhomologs
pRCLR9, pRCLR14, and pRCLR15, containing rclR alleles
fromtheNationalCenterforBiotechnologyInformationdata-
encoding RclRC21A, RclRC89A, RclRH42A, and RclRH75A,
bases. Sequence alignments were performed using MUSCLE
respectively.ThesamemethodwasusedtomutatepRCLR8
3.8 (30), and sequence logos were generated using WebLogo
withprimer[12],yieldingplasmidpRCLR12,containingan
3.1.MutagenicprimersweredesignedwithPrimerX,qRT-PCR
rclRalleleencodingRclRC21A,C89A.
primers were designed with Primer3 0.4.0, and PCR and
ForpurificationofRclRwild-typeandmutantproteins,the
sequencingprimersweredesignedwithWebPrimer.Allprim-
rclR sequence was codon-optimized for expression in E.coli
ersusedinthisstudyarelistedinTable1.
(GenScript,Inc.),yieldinganrclRopt.alleleencodingthewild-
Bacterial Strains and Growth Conditions—All strains and
type RclR amino acid sequence. The rclRopt. allele was then
plasmidsusedinthisstudyarelistedinTable2.DNAmanipu-
cloned into the NdeI and BamHI sites of plasmid pET-15b
lationswerecarriedoutbystandardmethods(31)inE.coliXL1
Blue (Stratagene), and the identity of all constructs was con- (Novagen)toyieldplasmidpRCLR7.TheQuikChangesite-
firmed by sequencing (GENEWIZ, Inc.). E.coli was grown in directedmutagenesiskit(Stratagene),modifiedtouseonlya
lysogenic broth (LB; Fisher) (32) or MOPS minimal medium single primer and 35 cycles of amplification, was used to
(Teknova,Inc.)containing0.2%glucose,1.32mMK HPO ,and mutatepRCLR7withprimer[15],[16],[17],or[18]toyield
2 4
10(cid:1)Mthiaminewithampicillin(100(cid:1)gml(cid:1)1)orchloramphen- plasmidpRCLR10,pRCLR11,pRCLR16,orpRCLR17.These
icol(12.5(cid:1)gml(cid:1)1)asindicated.ChemicalswerefromFisheror plasmids contained rclRopt. alleles encoding RclRC21A,
Sigma-Aldrich. N-Chlorotaurine was synthesized before each RclRC89A,RclRH42A,andRclRH75A,respectively.
use(33).NullmutationsinE.coliMG1655(F(cid:1),(cid:2)(cid:1),rph-1ilvG(cid:1) HOCl Survival Assays—HOCl survival assays were per-
rfb-50) (34) were constructed by replacement of genes with formedasdescribedpreviously(20).
NOVEMBER8,2013•VOLUME288•NUMBER45 JOURNALOFBIOLOGICALCHEMISTRY 32575
RclRIsaReactiveChlorine-specificActivator
TABLE2
Strainsandplasmidsusedinthisstudy
Unlessotherwiseindicated,allstrainsandplasmidsweregeneratedinthecourseofthiswork.TcR,tetracyclineresistance;NxR,nalidixicacidresistance;CmR,chloram-
phenicolresistance;ApR,ampicillinresistance.
Strainname Marker(s) Relevantgenotype Source
E.colistrains
XL1-Blue TcRNxR endA1gyrA96(nalR)thi-1recA1relA1lacglnV44F’[::Tn10(tet(cid:5))proAB(cid:5)lacI (cid:6)(lacZ)M15]hsdR17(r (cid:1)m (cid:5)) Stratagene
BL21(DE3) F(cid:1),ompTgaldcmlonhsdSB(r (cid:1)m (cid:1))(cid:2)(DE3[lacIlacUV5-T7gene1ind1sqam7nin5]) K K Novagen
MG1655 F(cid:1),(cid:2)(cid:1),rph-1ilvG(cid:1)rfb-50 B B (34)
MJG013 CmR F(cid:1),(cid:2)(cid:1),rph-1ilvG(cid:1)rfb-50rclC::cat(cid:5)
MJG014 CmR F(cid:1),(cid:2)(cid:1),rph-1ilvG(cid:1)rfb-50rclA::cat(cid:5)
MJG015 CmR F(cid:1),(cid:2)(cid:1),rph-1ilvG(cid:1)rfb-50rclR::cat(cid:5)
MJG016 CmR F(cid:1),(cid:2)(cid:1),rph-1ilvG(cid:1)rfb-50rclB::cat(cid:5)
MJG043 F(cid:1),(cid:2)(cid:1),rph-1ilvG(cid:1)rfb-50(cid:6)nemR (20)
MJG045 F(cid:1),(cid:2)(cid:1),rph-1ilvG(cid:1)rfb-50(cid:6)rclC
MJG046 F(cid:1),(cid:2)(cid:1),rph-1ilvG(cid:1)rfb-50(cid:6)rclA
MJG047 F(cid:1),(cid:2)(cid:1),rph-1ilvG(cid:1)rfb-50(cid:6)rclB
MJG059 F(cid:1),(cid:2)(cid:1),rph-1ilvG(cid:1)rfb-50(cid:6)rclR
Plasmids
pET-15b ApR N-terminalHis tag Novagen
6
pBAD30 ApR P arabinose-induciblepromoter (36)
pRCLR1 ApR rcBlARD(cid:5)
pRCLR7 ApR rclRopt.
pRCLR8 ApR rclRT61G,G62C,C63G(encodingRclRC21A)
pRCLR9 ApR rclRT265G,G266C,C267G(encodingRclRC89A)
pRCLR10 ApR rclRopt.(T61G,G62C,T63G)(encodingRclRC21A)
pRCLR11 ApR rclRopt.(T265G,G266C,T267G)(encodingRclRC89A)
pRCLR12 ApR rclRT61G,G62C,C63G,T265G,G266C,C267G(encodingRclRC21A,C89A)
pRCLR16 ApR rclRopt.(C124G,A125C,C126G)(encodingRclRH42A)
pRCLR17 ApR rclRopt.(C223G,A224C,T225G)(encodingRclRH75A)
GeneExpressionAnalysisbyqRT-PCR—E.coliwasgrownin modifications. P , the 225-bp intergenic region between
rclRA
MOPS medium with shaking at 37°C to an A (cid:3) 0.4–0.5. rclAandrclR,wasamplifiedusingprimers[31]and[32].P
600 nemR
Then, potential RclR inducers were added as indicated. For was prepared as described previously (20). RclR protein vari-
plasmid-containingstrains,mediacontainedampicillinand1 antswereexchangedinto150mMNaCl,50mMsodiumphos-
mMarabinose.Samples(0.5ml)werecollectedbycentrifuga- phate(pH6.8)usingBio-SpinP-30(Bio-Rad)gelfiltrationcol-
tion(1minat16,100(cid:4)g)immediatelybeforeandatindicated umnsandcentrifugedfor30minat16,000(cid:4)gat4°Ctoremove
timepointsafteradditionoftherespectivestressors.RNAiso- aggregates. RclR proteins were then incubated with the indi-
lation and quantitative RT-PCR analyses were performed as cated oxidants at 37°C for 15 min. To remove the oxidants,
describedpreviously(20).PrimersusedinRT-PCRswere:rrsD, RclRwasappliedtoaBio-SpinP-30column,equilibratedwith
[19]and[20];rclR,[21]and[22];rclA,[23]and[24];rclB,[25] 150mMNaCl,50mMsodiumphosphate,10%glycerol(pH6.8)
and[26];rclC,[27]and[28];nemR,[29]and[30].Student’sttest andcentrifugedfor30minat16,000(cid:4)gat4°C.P DNA(0.1
rclRA
wasusedtodeterminewhetherthemeansoflog-transformed pmol)wasincubatedwith0–10.0pmolofRclRproteinin10-(cid:1)l
expressionvaluesdifferedsignificantlyfromeachother. reactionsfor30minat37°C,separatedon10%polyacrylamide
PurificationofRclR—N-terminallyHis -taggedRclRvariants gelsinTrisborate/EDTAbufferfor90minat100V,andstained
6
were overexpressed in E.coli BL21(DE3) (Novagen). Cultures with ethidium bromide; band intensity was quantified using
weregrowninLBwithshakingat37°CtoanA (cid:3)0.6–0.8 ImageJ 1.44o (37). Student’s t test was used to determine
600
andinducedwith0.5mMisopropyl1-thio-(cid:3)-D-galactopyrano- whethermeanbandintensityvaluesdifferedsignificantlyfrom
side,thenincubatedat20°Cfor20h.Cultureswerecentrifuged eachother.Forreductionandre-reduction,sampleswereincu-
at6000rpmfor20min,resuspendedin0.5MNaCl,5mMimid- bated in 1 mM DTT at 37°C for 15 min immediately before
azole,20mMsodiumphosphate(pH7.4)plusEDTA-freeCom- incubation with P . Metal-free reagents were prepared by
rclRA
plete Mini protease inhibitor (Roche Applied Science). Cells incubationwith50mgl(cid:1)1Chelex100(Sigma)accordingtothe
werelysedbythreepassagesthroughaFrenchpressat1200psi. manufacturer’sinstructions.
Lysates were spun down for 60 min at 20,000 rpm at 4°C, Analysis of Cysteine Thiol Status—Cysteine thiols in 8 (cid:1)M
passedthrougha0.8-(cid:1)mfilter,thenappliedtoanickel-loaded reduced N-chlorotaurine-oxidized or H O -oxidized RclR
2 2
HiTrap Chelating HP affinity column (GE Healthcare). RclR weredeterminedunderdenaturingconditions(50mMsodium
waspurifiedusinganÄKTAfastproteinliquidchromatogra- phosphate,6MguanidiniumHCl,1mMEDTA(pH8.5))using
physystem(AmershamBiosciences)witha17-columnvolume Ellman’sreagent(5,5(cid:2)-dithiobis-(2-nitrobenzoicacid)(DTNB))
lineargradientto0.5MNaCl,0.5Mimidazole,20mMsodium (38).ForH O -oxidizedsamples,excessH O wasremovedby
2 2 2 2
phosphate (pH 7.4). Fractions containing pure RclR were additionof1unitofbovinecatalase(Sigma)andincubationat
pooledanddialyzedagainst50mMsodiumphosphate(pH8.5), 37°Cfor15minbeforeadditionofDTNB.Additionofcatalase
0.5MNaCl,2mMdithiothreitol(DTT),10%glycerolandstored itself resulted in no detectable thiols under these conditions
at(cid:1)80°C. (datanotshown).
Electrophoretic Mobility Shift Assay (EMSA)—EMSAs were The sulfenic acid-specific probes NBD-chloride and dime-
performed as described previously (20), with the following donewereusedasdescribedpreviouslytoexaminereducedand
32576 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER45•NOVEMBER8,2013
RclRIsaReactiveChlorine-specificActivator
FIGURE1.TheRclR-regulatedrcllocusisrequiredforHOClsurvival.A,operonstructureofthercllocusshowstheformernamesoftherclgenesin
parentheses.B,E.coliMG1655(wildtype)andisogenicmutantstrainswereincubatedinMOPSglucosemediumcontaining2.5mMHOClfor30min,then
dilutedandspot-titeredontoLBagar.Plateswereincubatedat37°Covernight.C,E.coliMG1655(wildtype)andthe(cid:6)rclRmutantweregrowntomid-logphase
inMOPSglucosemediumandtreatedwith0.4mMHOCl.ExpressionratiosofrclR(opencircles),rclA(bluetriangles),rclB(redsquares),orrclC(blackcircles)were
determinedbyqRT-PCR(mean(cid:8)S.D.(errorbars)).
N-chlorotaurine-oxidized RclR, RclRC21A, and RclRC89A (39,
40).
TestingtheRoleofMetalsinRclRActivation—Metalcontent
ofpurifiedwild-typeRclRwasanalyzedusinginductivelycou-
pled plasma-high resolution mass spectrometry at the Keck
ElementalGeochemistryLaboratory,DepartmentofGeologi-
cal Sciences (University of Michigan). To prepare metal-free
proteinsamples,RclR(40(cid:1)M)wasincubatedwith1mMEDTA,
TPEN, 2,2(cid:2)-dipyridyl, or EGTA at 37°C for 1 h inmetal-free
buffer.
RESULTS
TheRclR-regulatedrclLocusIsRequiredforHOClSurvival—
MicroarrayanalysesinanumberofRCS-stressedE.colistrains
revealedthemassiveup-regulationofthreegenesofunknown FIGURE2.RclRisspecificallyactivatedbyreactivechlorinespeciesinvivo.
functionykgB,ykgI,andykgC(20,41,42).Allthreegenesare E.coliMG1655wasgrowntomid-logphaseinMOPSglucosemediumand
divergently transcribed from ykgD (Fig. 1A), encoding an treatedwithchlorinatedcompounds(0.4mMHOCl,0.1mMN-chlorotaurine,
uncharacterized AraC family transcriptional activator (43) 1b%utyclhhloyrdorfooprmero(CxiHdCel(3T),BoHr)0,2.1m%MtrHic2hOlo2,ro0.a4cmetMicmaceidth(yTlCvAio))l,oRgOeSn,(0o.r225(cid:1)mgMmtel(cid:1)rt1-
whose expression was also significantly up-regulated after pamotiansesinuimtricteollxuirditee((DKE2ATeNOO3))o),rre0a.1ctmivMenpietrroogxyennitsrpiteec)i,eosr(eRlNeSc)tr(o0p.2hmileMsd(0ie.5th(cid:1)yMl-
HOCl treatment. We therefore hypothesized that this locus
diamide,0.2mMmethylglyoxal(MGO),or0.1mMN-ethylmaleimide(NEM)).
mightbeinvolvedinbacterialsurvivalofHOClstressandthat -FoldinductionofrclBafter10minwasdeterminedbyqRT-PCR(mean(cid:8)S.D.
(errorbars)).Expressionvaluesindicatedwithasterisksaresignificantlydiffer-
ykgD might encode a HOCl-responsive transcription factor.
entfromexpressionintheabsenceoftreatment(Student’sttest;**,p(cid:9)0.01;
AnalysisoftheHOClsensitivityofin-framedeletionmutants ***,p(cid:9)0.001).
lacking any one of the four ykg genes fully agreed with this
assumption.Allmutantstrainswerefoundtobemoresensitive scriptionalactivatorthatcontrolstheexpressionofrclA,rclB,
to HOCl treatment than wild-type cells (Fig. 1B), indicating and rclC and contributes significantly to bacterial HOCl
thatthesegenesareindeedinvolvedinsurvivingHOClstress. survival.
WethereforerenamedtheykgD,ykgC,ykgI,andykgBgenesto RclR-dependentGeneExpressionIsSpecificallyActivatedby
rclR, rclA, rclB, and rclC, respectively, to reflect their role in Reactive Chlorine Species—Of the few HOCl-sensitive tran-
reactive chlorine resistance (Fig. 1A). Quantitative RT-PCR scriptionfactorsthathavebeenidentifiedtodate,onlyHypT
analysis confirmed the microarray analysis and revealed that, appears to be specific for RCS whereas all others respond to
uponsublethalHOClstress,expressionofrclA,rclB,andrclC multiple stresses, including organic peroxides and electro-
wasrapidlyinduced(cid:7)100-fold,whereasexpressionoftheputa- philes.TodeterminethespecificityofRclRactivationinvivo,
tive regulator rclR increased 10-fold (Fig. 1C, upper panel). wethereforetreatedwild-typeE.coliwithavarietyofchlori-
Expressionlevelsremainedhighforatleast30minafterHOCl nated compounds, reactive oxygen species, reactive nitrogen
treatment.InductionofrclA,rclB,andrclCwaslargelyelimi- species,andelectrophiles(Fig.2).Thechosenconcentrationsof
nated in a (cid:6)rclR mutant (Fig. 1C, lower panel). These results eachcompoundledtoashortgrowthdelayunderourcondi-
servetodemonstratethatrclRencodesaHOCl-sensitivetran- tions,butdidnotresultincelldeath(datanotshown).Addition
NOVEMBER8,2013•VOLUME288•NUMBER45 JOURNALOFBIOLOGICALCHEMISTRY 32577
RclRIsaReactiveChlorine-specificActivator
FIGURE3.Reactivechlorine-specificoxidationactivatesRclRDNAbindingactivityinvitro.A,EMSAsofthebindingofRclR(0,0.25,0.5,0.75,1(cid:1)M)torclRA
promoterDNA(PrclRA,10nM)beforeandaftertreatmentwitha1:1molarratioofN-chlorotaurine.Totestthereversibilityofoxidativeactivation,N-chlorotau-
rine-oxidizedRclRwasincubatedfor30minwiththethiol-reducingagentDTT(1mM).Representativegelsareshown,alongwithquantificationresults
(mean(cid:8)S.D.(errorbars)).B,EMSAofN-chlorotaurine-oxidizedRclRbindingtonemRpromoterDNA(P ).C,EMSAsofRclRbindingtoP aftertreatment
nemR rclRA
with5:1,10:1,or20:1molarratiosofH O .
2 2
ofHOClorN-chlorotaurine,areactivechloraminegenerated (GenScript,Inc.).Thisstrategyallowedhighoverexpressionof
bythereactionofHOClwithtaurineandfoundathighconcen- RclRandthepurificationoftheproteinto(cid:7)99%purity.EMSAs
trationsinneutrophils(7),resultedin100–500-foldinduction revealedthat,whereaspurifiedRclRdidnotbindtothisDNA
ofrclBgeneexpression.Noactivationwasseenwithother,non- fragmentunderreducingconditions,itboundstronglytoDNA
reactivechlorinatedcompounds,includingchloroformandtri- afterincubationwitha1:1molarratioofN-chlorotaurine(Fig.
chloroaceticacid.RclRalsodidnotrespondtooxidativestress 3A).Thisactivationwaslargelyreversiblebyincubationofoxi-
causedbytheorganichydroperoxidetert-butylhydroperoxide, dizedRclRwith2mMofthethiol-specificreducingagentDTT,
H O , the superoxide-generating redox cycling agent methyl stronglysuggestingthatactivationofRclRinvolvesformation
2 2
viologen (44), or by potassium tellurite (45). Similarly, no of reversible oxidative cysteine modifications. N-Chlorotau-
induction of rclB was observed after treatment with reactive rine-activatedRclRdidnotbindtoaDNAfragmentcontaining
nitrogenspecies,includingthenitricoxidedonordiethylamine the nemR promoter region (20) (Fig. 3B), indicating that the
nitric oxide and peroxynitrite, or with reactive electrophiles, DNAbindingofRclRissequence-specificandthatRclRdoes
including diamide, methylglyoxal, and N-ethylmaleimide. not affect expression of the HOCl-responsive nemR operon.
TheseresultsdemonstratethatwithRclR,wehaveidentifieda Confirmingthisresult,qRT-PCRshowednochangesinnemR
memberoftheAraCfamilythatrespondsspecificallytoreac- expressionina(cid:6)rclRmutantstrainorinrclBexpressionina
tivechlorinespecies. (cid:6)nemR mutant (data not shown), indicating that NemR and
Reactive Chlorine-specific Oxidation Activates RclR DNA RclRcontrolindependentHOCl-responsiveregulonsinE.coli.
BindingActivity—ToinvestigatetheabilityofRclRtobindand Incubation of purified RclR with increasing molar ratios of
shiftDNAinapotentiallyredox-dependentmanner,wecon- H O resultedinsomeactivationofDNAbinding(Fig.3C);but
2 2
ductedEMSAsusingpurifiedRclRandtherclR-rclAintergenic even at a ratio of 20:1, the activity of H O -treated RclR was
2 2
region(P )asaDNAsubstrate.Duetotheremarkablyhigh significantlylower(Student’sttest,p(cid:9)0.05)thantheactivityof
rclRA
number of rare codons in the rclR coding sequence (i.e. one RclR treated with a 1:1 ratio of N-chlorotaurine to RclR (Fig.
AGG,twoAGA,andfourCGGargininecodons,twoATAiso- 3A).TheinvitrosensitivityofRclRtoH O wasmuchlower
2 2
leucine codons, and one CCC proline codon) (46), which than that reported for the bona fide H O -sensing activator
2 2
resultedinpooroverexpressionfromthenativegenesequence OxyR,whichispresentintheoxidized,activeformwithoutthe
(datanotshown),RclRwasoverexpressedforpurificationfrom additionofH O invitro(47)andisfullyactivatedbyaslittleas
2 2
anrclRgenethatwascodon-optimizedforexpressioninE.coli 10(cid:1)MH O invivo(48).Incontrast,2mMH O hadnoeffect
2 2 2 2
32578 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER45•NOVEMBER8,2013
RclRIsaReactiveChlorine-specificActivator
domainshomologoustotheRclRN-terminaldomain(BLAST
e-value(cid:9)0.0001)from70bacterialspecies.Alignmentofthese
sequences(Fig.5A)revealedthepresenceoftwoconservedcys-
teineresidues(Cys-21andCys-89ofE.coliRclR)andtwocon-
served histidine residues (His-42 and His-75 of E.coli RclR).
WealsonotedthattheRclRN-terminaldomainappearstobea
memberofthepoorlycharacterizedCupin_6(pfam12852)pro-
FIGURE4.CharacteristicsofoxidativelyactivatedRclR.A,cysteinethiol teinfamily(50).Cupinsareafunctionallydiverseproteinsuper-
statusofRclR,asdeterminedbyEllman’sassay,beforeandaftertreatment familysharingacommonthermostablesmall(cid:3)-barrelfold(51).
witha1:1molarratioofN-chlorotaurine(NCT)ora10:1molarratioofH O .
2 2 However, very little is known about the functions or detailed
Asterisks indicate significant differences from the untreated sample (Stu-
dent’sttest;**,p(cid:9)0.01;****,p(cid:9)10(cid:1)8).B,nonreducingSDS-PAGEofRclR(2 structureoftheCupin_6subfamily.Fig.5Bshowsanalignment
(cid:1)g)beforeandaftertreatmentwitha1:1molarratioofN-chlorotaurineora
of the E.coli RclR sequence with the Cupin_6 consensus
10:1molarratioofH O .
2 2 sequence, showing that Cys-89, His-42, and His-75 are con-
on RclR-dependent gene expression in vivo (Fig. 2). These served in both RclR and the larger Cupin_6 family, but that
resultsdemonstratethatactivationofRclRDNAbindingactiv- Cys-21appearstobespecifictoRclR.
ityishighlysensitiveandspecifictoRCSandsuggestthatH O RclRDoesNotAppeartoRequireMetalsforActivation—The
2 2
isnotaphysiologicallyrelevantactivatorofRclR.Thefactthat presenceoftwoconservedcysteinesandtwoconservedhisti-
RclR activation is largely reversible in vitro by DTT strongly dines in RclR, along with the known presence of biologically
suggeststhatitisalsoreversibleinvivo. important metal ions in many cupin proteins (51), led us to
Thiol Properties and Structural Characteristics of Oxida- hypothesizethatRclRmightbeametalloprotein.Boundmetal
tivelyActivatedRclR—Tofurthercharacterizethepropertiesof ionscanhavedramaticeffectsontheredoxpropertiesofcys-
oxidativelyactivatedRclR,wenextmeasuredthecysteinethiol teine residues (52), and 2Cys-2His motifs are very common
statusinreduced,N-chlorotaurine-oxidized,orH O -oxidized among zinc-binding proteins (53). However, inductively cou-
2 2
RclRusingEllman’sassay(Fig.4A).Ofthe6cysteineresidues pledplasmamassspectrometryfailedtoidentifyanystoichio-
thatarepresentinRclR,wedetectedanaverageof5.5reduced metricmetalsboundtopurified,redox-activeRclR.Totestfur-
cysteinesinreducedRclRcomparedwith3.6and5.0reduced therwhetherthepresenceofmetalshadanyeffectonactivation
cysteinesinRclRtreatedwitha1:1molarratioofN-chlorotau- of RclR, we treated purified RclR in metal-free buffers with a
rineora10:1molarratioofH O ,respectively.Incombination varietyofmetal-bindingchelators.AsshowninFig.6,extended
2 2
with the findings presented in Fig. 3, this result suggests that treatmentofRclRwithhighconcentrationsofeitherthestrong
activationofRclRbyN-chlorotaurinemayinvolveformationof zinc-bindingchelatorTPEN(K forZn2(cid:5)(cid:3)10(cid:1)16M(cid:1)1)(54)or
d
onedisulfidebondandexplainsthemuchreducedcapacityof withthegeneralchelatorEDTAhadnosignificanteffectonthe
H O toactivateRclR.NonreducingSDS-PAGEofreducedand activation of RclR in vitro (Student’s t test, p (cid:7) 0.3). Similar
2 2
oxidized RclR samples did not reveal the formation of higher results were observed with other chelators, including EGTA
molecularmassspeciesuponoxidation(Fig.4B),excludingthe and2,2-dipyridyl(datanotshown).Theseresultsledustocon-
formation of intermolecularly disulfide-bonded oligomers. cludethat,atleastinvitro,RclRdoesnotrequiremetalionsfor
N-Chlorotaurinetreatmentofcysteineisexpectedtoresultin activationbyN-chlorotaurine.
theoxidationofthethiol(-SH)grouptosulfenicacid(-SOH) RoleofConservedCysteineandHistidineResiduesintheActi-
(49).Reducedthiolsandsulfenicacidscanbedetectedusingthe vationofRclR—Tofirstdeterminewhatroletheconservedcys-
probe NBD-chloride, which forms adducts with absorption teines Cys-21 and Cys-89 play in redox sensing by RclR, we
maximaof420nmforthiolsand350nmforsulfenicacid(39). constructedplasmidsencodingRclRvariantsinwhichoneor
However, no sulfenic acid adducts of NBD-chloride were bothconservedcysteinesweremutatedtoalanine.Theability
detected in N-chlorotaurine-oxidized RclR (data not shown). ofthesemutantstorespondtoRCSwasthentestedbothinvivo
To confirm this result, N-chlorotaurine-oxidized RclR was and in vitro. In vivo, qRT-PCR analysis (Fig. 7) showed that
treated with the sulfenic acid-specific nucleophile dimedone whereasE.colistrainsencodingthewild-typeRclRonaplas-
and examined using antibodies specific to the resulting mid had higher base-line levels of rclB expression than did a
thioether product (40). Whereas sulfenic acid formation was strain encoding RclR on the chromosome (probably due to
clearlydetectedinperoxide-treatedGAPDH(40),nosulfenic increasedrclRcopynumber),rclBexpressionwasstronglyfur-
acid was detected in RclR by this method either (data not ther induced by the addition of 0.4 mM HOCl. In contrast,
shown).Theseresultssuggestthatanysulfenicacidintermedi- strainsencodingRclRvariantslackingeitherCys-21,Cys-89,or
atesformedduringtheactivationofRclRmustexistonlyvery bothCys-21andCys-89wereunabletoinducerclBexpression
transiently.Theprecisenatureoftheoxidativemodification(s) inresponsetoHOCl,indicatingthatbothCys-21andCys-89
inactivatedRclRremainstobeelucidated. playimportantrolesintheactivationofRclRinvivo.Invitro
MechanismofRclRActivation—BasedonRclRhomologyto EMSA using the purified mutant RclR variants revealed that
regulators of the AraC family (43), RclR is predicted to be a whereastheRclRC21Avariantwasnolongerabletorespondto
two-domainproteinwithanN-terminaldomainpresumedto N-chlorotaurinetreatmentbyactivationofDNAbinding(Fig.
beinvolvedinsensing(residues1–162)andaC-terminalDNA- 8,middlepanels),theRclRC89Amutantvariantmaintainedits
bindingdomain.Toidentifyconservedresiduesthatmightbe redox response (Fig. 8, bottom panels). These results showed
important for RclR function, we identified proteins with that,whereasbothCys-21andCys-89residuesarerequiredfor
NOVEMBER8,2013•VOLUME288•NUMBER45 JOURNALOFBIOLOGICALCHEMISTRY 32579
RclRIsaReactiveChlorine-specificActivator
FIGURE5.ConservedresiduesinRclR.A,alignmentofRclRhomologsfrom70bacterialspecies,withcysteineresiduesindicatedinredandhistidineresidues
inblue.ColorednumbersinbracketsindicatetheE.colinumberingofconservedcysteineandhistidineresidues.B,alignmentofE.coliRclRwiththeCupin_6
domainconsensussequence(50),withconservedcysteineandhistidineresiduesindicatedwithblackhighlighting.Asteriskindicatescysteineconserved
amongRclRhomologsbutnotintheCupin_6consensus.ColorednumbersinbracketsindicatetheE.colinumberingofconservedcysteineandhistidine
residues.
FIGURE6.RclRdoesnotrequiremetalsforactivationinvitro.RclRwaspreparedbytreatmentundermetal-freeconditionsfor1hat37°Cwiththeindicated
cahloenlagtowristh(1qmuMan),ttihfiecantaiosnsaryeesdulbtsy(EmMeSaAnf(cid:8)orSb.iDn.d(ienrgrotrobParcrlsR)A).beforeandaftertreatmentwitha1:1molarratioofN-chlorotaurine.Representativegelsareshown,
32580 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER45•NOVEMBER8,2013
RclRIsaReactiveChlorine-specificActivator
FIGURE7.RoleofconservedcysteineandhistidineresiduesintheactivationofRclRinvivo.E.coli(cid:6)rclRstrainscontainingpBAD30-derivedplasmids
encodingtheindicatedRclRvariantsweregrowntomid-logphaseinMOPSglucosemediumandtreatedwith0.4mMHOCl.-FoldinductionofrclBafter10min,
relativetoexpressioninuntreatedwild-typeE.coliMG1655,wasdeterminedbyqRT-PCR(mean(cid:8)S.D.(errorbars)).Expressionvaluesindicatedwithasterisks
directlyabovethebarsaresignificantlydifferentfromexpressionunderthesameconditioninthewild-type,whereasasterisksabovebracketsindicateRclR
variantsleadingtosignificantlydifferentexpressionwithandwithoutHOCltreatment(Student’sttest;*,p(cid:9)0.05;**,p(cid:9)0.01).
andshowedincreasedDNAbindingbyRclRH75Aunderreduc-
ingconditionscomparedwiththewildtype(Fig.9).Bindingof
the RclRH42A and RclRH75A variants after N-chlorotaurine
treatment(Fig.9)wassimilartothatofthewildtype,indicating
thatthelackofthesehistidinesdidnotimpairtheactivationof
RclRbyN-chlorotaurine.Interestingly,however,invitroDNA
binding of both RclRH42A and RclRH75A was activated more
stronglybya10:1excessofH O thanwasthewildtype(Fig.9).
2 2
Indeed,theRclRH42Avariantwassignificantlymoresensitiveto
oxidationbybothN-chlorotaurineandH O thanthewildtype
2 2
(Student’sttest,p(cid:9)0.05).Incombination,theseresultssug-
gestedthatHis-42andHis-75playrolesinmaintainingRclRin
aninactivestateunderreducingconditionsandcontribute,bya
yettobedefinedmechanism,totheRCSspecificityandoxida-
tionsensitivityofRclR.
DISCUSSION
We have now identified RclR as a transcriptional activator
that relies on conserved redox-sensitive cysteine residues to
specificallysenseRCSandcontroltheexpressionofgenesthat
contributetotheabilityofE.colitosurviveHOClstress.The
stronginvivoRCSspecificityofRclRdistinguishesitfrommost
otherHOCl-responsivetranscriptionfactorssofardescribed.
Forexample,E.coliNemRandB.subtilisHypRrespondtoboth
HOCl and cysteine-modifying electrophiles (e.g. N-ethylma-
leimideordiamide)(20,21),andB.subtilisOhrRrespondsto
bothHOClandorganichydroperoxides(18,55,56).Ourresults
FIGURE8.RoleofconservedcysteineresiduesintheactivationofRclRin
vitro.EMSAsshowbindingofRclRwild-type,RclRC21A,andRclRC89Avariant suggest a model in which initial oxidation of Cys-21 leads to
proteinstoP beforeandaftertreatmentwitha1:1molarratioofN-chlo- partialactivationofRclR,followedbytheformationofanintra-
rclRA
rotaurine.Representativegelsareshown,alongwithquantificationresults
molecular disulfide bond between Cys-21 and Cys-89, which
(mean(cid:8)S.D.(errorbars)).
stabilizesthefullyactiveformoftheproteininvivo.Cys-21is
RCS-responseinvivo,theRclR-specificCys-21appearstoplay necessary for activation and is likely to be the critical redox-
themorecriticalroleintheRclRredoxresponse. sensitivecysteineofRclR.TheabilityoftheRclRC89Avariantto
Next,weusedthesamestrategytodeterminetherolesofthe beactivatedinvitrosupportsthisconclusionanddemonstrates
conservedhistidinesHis-42andHis-75inRCSsensingbothin thatdisulfidebondformationisindeednotrequiredforRclR
vivoandinvitro.Invivo,mutationofeitherhistidineresidueto activationinvitro.AsshowninFig.8,theC89Avariantcanin
alanine resulted in substantially higherrclB expression in the factbefullyactivatedinvitro.FormationofaCys-21–Cys-89
absence of HOCl, along with a slight further increase after disulfidebondmightservetostabilizetheactiveformofRclR
HOCltreatment(Fig.7).Theseresultssuggestthatbothcon- underphysiologicalredoxconditionsinvivoandmightprevent
served His residues are involved in preventing DNA binding premature reduction and inactivation by cellular reductases
whenRclRisreduced.InvitroEMSAsagreedwiththeseresults (e.g.thioredoxinorglutaredoxin).Regardless,invivo,bothcon-
NOVEMBER8,2013•VOLUME288•NUMBER45 JOURNALOFBIOLOGICALCHEMISTRY 32581
RclRIsaReactiveChlorine-specificActivator
FIGURE9.RoleofconservedhistidineresiduesintheactivationofRclRinvitro.EMSAsshowbindingofRclRwild-type,RclRH42A,andRclRH75Avariant
proteinstoP beforeandaftertreatmentwitha1:1molarratioofN-chlorotaurineora10:1molarratioofH O .Representativegelsareshown,alongwith
quantificatiornclRreAsults(mean(cid:8)S.D.(errorbars)). 2 2
served cysteines are required for sensing of RCS. This model Hsp33 relies on the presence of bound zinc to modulate the
resemblesthemechanismofredoxactivationofOxyR,inwhich redoxsensitivityofitscysteineresidues(52,62),whereasHypT,
initialoxidationofCys-199precedesformationofaCys-199– theonlyotherRCS-specifictranscriptionfactorsofardescribed,
Cys-208 disulfide bond in the fully activated protein (57). relies on HOCl-dependent methionine oxidation (19, 22). The
Although no structural information for RclR or any other mechanismofRclRactivationalsoappearstobedistinctfromthat
Cupin_6 family member is available, modeling of the RclR ofotherknownHOCl-responsivetranscriptionfactors,includ-
N-terminaldomainwiththeI-TASSER(58)andSwiss-Model ingNemRandHypR,whoseDNAbindingactivityisinactivated
(59) structure prediction algorithms supports this model.
by the formation of disulfide-bonded oligomers (20, 21) or
Despite substantial differences in the structures predicted by
OhrR,whoseinactivationdependsonS-bacillithiolationorsul-
thesetwoalgorithms,theyagreeinplacingCys-21andCys-89
fenamide formation at a conserved cysteine residue (18, 55).
adjacenttoeachotheronadjoining(cid:3)strands(datanotshown),
WearecurrentlypursuingstructuralanalysisofRclRtoidentify
wellpositionedtoformadisulfidebond.
both the specific nature of the structural changes associated
RclRisunusualnotonlyforitshighspecificityandsensitivity
with oxidative activation and properties of Cys-21 or Cys-89
toRCS,butalsoforbeingaredox-sensitivetranscriptionalacti-
which may contribute to the exquisite sensitivity of RclR to
vator. Most known redox-sensing transcription factors func-
RCS.
tionasrepressorsandareinactivatedbyoxidation(e.g.NemR,
Anintriguingunansweredquestioniswhybacteriaappearto
OhrR,PerR,andHypR)(20,21,24,60).Inactivationthenleads
have evolved multiple overlapping RCS-sensitive regulons as
to their release from DNA and to the de-repression of gene
opposed to the simpler regulatory architecture devoted to
expression.RclRnowjoinstherelativelysmallgroupofregula-
resistingotheroxidativestressconditionssuchasperoxideor
torswhosefunctionsarespecificallyactivatedbyoxidation(e.g.
superoxide.E.colicontainsonetranscriptionfactordevotedto
OxyRandSoxR)(13,14).IncontrasttotheactivationofDNA
binding observed in RclR, however, OxyR and SoxR bind to sensing H2O2 (OxyR) and one devoted to sensing superoxide
DNAinboththeirreducedandoxidizedforms,withoxidation (SoxR)(13).ThereisnowevidencethatE.colipossessesatleast
resultinginstructuralchangesthatleadtotranscriptionalacti- threedifferentregulatorsthatrespondverysensitivelytoHOCl
vation(47,61). (RclR,HypT,andNemR)andwhichcontroldistinct,nonover-
ThemechanismunderlyingRclRspecificityforRCSremains lappingstressresponses(19,20).Itremainstobedetermined
to be determined, but our results demonstrate that it differs whatpropertiesofRCSstressrequiresuchacomplextranscrip-
fromthatofotherknownRCS-specificproteins:thechaperone tionalresponse.
32582 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER45•NOVEMBER8,2013
RclRIsaReactiveChlorine-specificActivator
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unknown.RclAisagroupIflavindisulfidereductase(63),pre-
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dictedtobeverysimilartothioredoxinreductaseandmercuric
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reductase. RclB is an 80-amino acid periplasmic protein, and
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However,inothercladestherclRhomologsarefoundassoci- lussubtilisasrevealedbytranscriptomicsandredoxproteomics.Mol.Cell
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Winter,J.(2013)Methionineoxidationactivatesatranscriptionfactorin
AhpDalkylhydroperoxidase(64),anoxidativestressresistance
responsetooxidativestress.Proc.Natl.Acad.Sci.U.S.A.110,9493–9498
enzymewhichwehypothesizemayplayasofaruncharacter- 20. Gray,M.J.,Wholey,W.Y.,Parker,B.W.,Kim,M.,andJakob,U.(2013)
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servedtranscriptionalactivatorthatdependsontheoxidation 21. Palm,G.J.,KhanhChi,B.,Waack,P.,Gronau,K.,Becher,D.,Albrecht,D.,
Hinrichs,W.,Read,R.J.,andAntelmann,H.(2012)Structuralinsights
ofconservedcysteineresiduestorespondverysensitivelyand
into the redox-switch mechanism of the MarR/DUF24-type regulator
specifically to RCS. In E.coli, RclR regulates the high level
HypR.NucleicAcidsRes.40,4178–4192
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rolesinsurvivingRCStreatment. tenmüller,A.,Ganzinger,K.A.,Braun,N.,Franzmann,T.M.,andWinter,
J.(2012)Identificationofahypochlorite-specifictranscriptionfactorfrom
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Acknowledgment—We thank Kate Carroll (Scripps Research Insti-
23. Lee,C.,Shin,J.,andPark,C.(2013)NovelregulatorysystemnemRA-gloA
tute)forthesulfenicacid-detectingantibodies.
forelectrophilereductioninEscherichiacoliK-12.Mol.Microbiol.88,
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