Reduced Aeration Affects the Expression of the NorB Efflux Pump of Staphylococcus aureus by ...

12 Pages·2012·2.58 MB·English

Reduced Aeration Affects the Expression of the NorB Efflux Pump of
Staphylococcus aureus by Posttranslational Modification of MgrA
QueChiTruong-Bolduc,aLiaoChunHsing,a*RegisVillet,aGillesR.Bolduc,bZoeEstabrooks,bGFlorentTaguezem,band
DavidC.Hoopera
DivisionofInfectiousDiseasesandMedicalServices,MassachusettsGeneralHospital,andHarvardMedicalSchool,Boston,Massachusetts,USA,aandMassasoit
CommunityCollege,Brockton,Massachusetts,USAb
WepreviouslyshowedthatatacidpH,thetranscriptionofnorB,encodingtheNorBeffluxpump,increasesduetoareductionin
thephosphorylationlevelofMgrA,whichinturnleadstoareductioninbacterialkillingbymoxifloxacin,asubstrateofthe
NorBeffluxpump.Inthisstudy,wedemonstratedthatreducedoxygenlevelsdidnotaffectthetranscriptlevelsofmgrAbut
D
modifiedthedimerizationoftheMgrAprotein,whichremainedmostlyinitsmonomericform.Underreducedaeration,wealso o
observeda21.7-foldincreaseinthenorBtranscriptlevelsafter60minofgrowththatcontributedtoa4-foldincreaseinthe w
n
MICsofmoxifloxacinandsparfloxacinforStaphylococcusaureusRN6390.TherelativeproportionsofMgrAinmonomericand lo
dimericformswerealteredbytreatmentwithH O ,butincubationofpurifiedMgrAwithextractsofcellsgrownunderreduced a
2 2 d
butnotnormalaerationpreventedMgrAfrombeingconvertedtoitsdimericDNA-bindingform.Thismodificationwasassoci- e
d
atedwithcleavageofafragmentofthedimerizationdomainofMgrAwithoutchangeinMgrAphosphorylationandanincrease
f
intranscriptlevelsofgenesencodingserineproteasesincellsincubatedatreducedaeration.Takentogether,thesedatasuggest ro
thatmodificationofMgrAbyproteasesunderliesthereversalofitsrepressionofnorBandincreasedresistancetoNorBsub- m

stratesinresponsetoreduced-aerationconditions,illustratingathirdmechanismofposttranslationalmodification,inaddition h
t
tooxidationandphosphorylation,thatmodulatestheregulatoryactivitiesofMgrA. tp
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Staphylococcusaureusisamajorhumanpathogenresponsible expressionofawiderangeofproteins,includingvirulencefactors a
s
fornosocomialinfectionsandseriousdiseases,includingtoxic andproteasessuchastheSspAserineprotease(8). m
shocksyndrome,endocarditis,pneumonia,andsepticemia.S.au- Duringthecourseofinfection,S.aureussurvivesbydevelop- .o
r
reusrecognizesseveralenvironmentalsignalsthathelpittosense ingaversatiledefensesystemthatincludesenzymessuchascata- g
/
thehostenvironmentandtocoordinatetheexpressionofspecific laseorsuperoxidedismutasetoprotectitselffromreactiveoxi- o
regulators, which in turn modulate the expression of virulence dants, such as hydrogen peroxide or superoxides produced by n

factors for adaptation and host invasion. Various studies have macrophages (11). Furthermore, S. aureus must compete with J
a
demonstratedthatenvironmentalstresses,suchasalowoxygen other pathogens in certain habitats, such as the nasopharynx, n
u
levelorahighcarbondioxidelevelataneutralpH,arenecessary whereoneofitsknowncompetitorsisStreptococcuspneumoniae. a
r
fortheproductionofexotoxins,includingthesynthesisoftoxic S.aureusproducescatalasetodefenditselfagainsthydrogenper- y

shocksyndrometoxin1(TSST-1)(18,30).Recently,wedemon- oxideproducedbyS.pneumoniaeinthenasopharyngealmucosa 1
2
stratedthatacidpHinfluencestheexpressionoftheMgrAtran- (15). ,

2
scriptional regulator, modulating its state of phosphorylation OxidativestresscausedbyreactiveoxidantscanbesensedbyS. 0
through changes in the Ser/Thr kinase PknB (22). S. aureus is aureus proteins, MgrA and SarZ, which belong to the MarR 1
9
facultatively anaerobic and able to grow under reduced-oxygen (multiple-antibioticresistanceregulator)familyandregulatethe
b
conditionsbyswitchingitsaerobicrespiratorymechanismtofer- expressionofgenesinvolvedinresistancetoantibiotics,resistance y
mentationornitraterespiration,withlactateornitrate(NO (cid:1)) to oxidative stress, and bacterial virulence (2, 21, 24–26). The g
3 u
andnitrite(NO (cid:1))asterminalelectronacceptorsunderanaero- abilityofMgrAandSarZtobindspecificDNAsitesismodified e
2 s
bicconditions(9,19).TheregulatorymechanismsthatenableS. after oxidation of the unique cysteine in the monomer (5, 16). t
aureustomodifyitsgeneexpressionpatternsinadaptingtovari- Cys ,thereactivecysteineinMgrA,islocatedinthehydrophobic
12
ations in oxygen level are still incompletely understood. Recent pocketneartheN-terminalportionofthe(cid:1)1-helixoftheMgrA
studiesdemonstratedthattheSrrAB(staphylococcalrespiratory monomer,whichisinthedimerizationdomain.Cys isrecog-
12
responseAB)two-componentsystemregulatesfermentationen- nizedbyresiduesSer andTyr oftheoppositemonomervia
113 38
zymesandalsoenzymesinthetricarboxylicacidcycleinresponse
tooxygendepletion(20).Theoxygen-responsiveNreABCregu-
lonofS.aureuswasrecentlycharacterizedasessentialfortran- Received10November2011 Accepted18January2012
scriptionalactivationofgenesparticipatinginnitraterespiration Publishedaheadofprint27January2012
(19).TheRexfamilyrepressorwasalsoinvolvedintheregulation AddresscorrespondencetoDavidC.Hooper,[email protected].
ofgeneexpressioninS.aureusunderanaerobicconditions(14). *Presentaddress:DivisionofInfectiousDiseases,Far-EasternMemorialHospital,
Moreimportantly,thisredox-sensingrepressorregulatedtheex- Taipei,Taiwan.
pressionoftheClpproteasesinmultiplestresssituations,leading Copyright©2012,AmericanSocietyforMicrobiology.AllRightsReserved.
toactivedegradationofvariousproteins,includingtheRottran- doi:10.1128/JB.06503-11
scriptionalregulator(12).Rotisaglobalregulatorthataffectsthe
0021-9193/12/$12.00 JournalofBacteriology p.1823–1834 jb.asm.org 1823
Truong-Bolducetal.
hydrogenbondstoformthehomodimer.InthepresenceofH O , grownat37°Cunderaerobicconditionswithshaking(200rpm)untilan
2 2
the hydrogen bonds around Cys are disrupted by oxidation, OD of0.5wasreached.Fifteen-milliliteraliquotsofthebacterialcul-
12 600
leadingtothedissociationofMgrAfromDNA,aswasobserved turewerethendistributedquicklyintoaseriesof15-mltubes,whichwere
withthesarVpromoter(5).TheoxidizedformsofMgrAandSarZ tightlyclosedtocreateareduced-aerationenvironment(9).Thetubes
canundergoconformationalchangestobeconvertedbacktothe werelabeledastimezero,10,30,60,and120minandwereincubatedat
DNA-binding reduced form when oxidative stress is removed. 37°Cwithshakinginthesameshaker/incubatorastheaeratedculture.
Theremainingculturewasincubatedundershakingat37°Calongsidethe
ThisphenomenonemphasizestheadaptabilityofS.aureustoad-
othertubes.Foreachtimepoint,culturesundernormalaeration(AE)and
justtooxidativestressbyrapidreversiblemodificationofexisting
reducedaeration(RAE)werecollected,andthedissolvedoxygenamounts
regulatoryproteins(16).
oftheculturesweremeasuredusingaVernierdissolvedoxygenprobe
S.aureusisthemostcommonbacteriumassociatedwithskin
(VernierSoftwareandTechnology,Beaverton,OR).Theprobewascon-
abscessformation.MicroarraydataobtainedfromS.aureusex-
nectedtoacomputerwithdatacollectionsoftwareasrecommendedby
tractedfromsubcutaneousabscessesinamousemodelindicated
themanufacturer.Theprobewasfirstcalibratedwiththezero-oxygen
asubstantialincreaseinnorBtranscripts,whichencodetheNorB solutionprovidedbythemanufacturer.Theunitformeasurementwas
effluxpump,whichcontributestobacterialfitnessintheabscess milligramsofoxygendissolvedin1literofculture(mg/liter).Theprobe
D
environment(7).Sinceabscessesconstituteamildlyacidicenvi- wasplaceddirectlyintothetube,andthereadingwasrecordedafter45sof
o
ronment(pH5.5)andalsohaveareducedoxygenlevel(28),we probe-culturecontact. w
investigatedtheinfluenceofthesetwoenvironmentalstimulion TreatmentofcelllysateswithH O andproteaseinhibitors.H O n
theexpressionoftheNorBeffluxpumpviaitsregulatorMgrA.We waspurchasedfromSigmaChemica2lC2o.(St.Louis,MO).ThermoS2ci2- lo
a
previously showed that under acid pH, the transcription of the entificHaltproteaseandphosphataseinhibitorcocktailcontainedinhib- d
norBeffluxpumpincreasesduetoareductioninthephosphory- itorsagainstthemajorclassesofproteasesandphosphatases,particularly ed
lationstateofMgrA.TheincreaseinnorBtranscriptsleadstoa aminopeptidases,cysteineandserineproteases,andserine/threonineand f
r
tyrosinephosphatases(ThermoFisherScientific,Rockford,IL). o
reductioninbacterialkillingbymoxifloxacin,asubstrateofthe
ForWesternblotassaysusingcellextracts,cultureswereincubated m
NorBeffluxpump(22). undernormalorreducedaerationandthencollectedattimepoint60 h
Inthisstudy,wedemonstratedthatreducedoxygenlevelsdid t
min.TheS.aureuspelletswerewashedtwicewithTris-HCl,pH7.5,buf- t
notaffectthetranscriptlevelsofmgrA,whichisresponsibleforthe p
fer,followedbytreatmentwithlysostaphin.Celllysateswererecovered :
/
repressionofnorBexpression,butincreasedthetranscriptionof aftercentrifugationat10,000(cid:3)gfor20min.ProteaseinhibitorsorH2O2 /jb
severalserineproteases,whichwasassociatedwithchangesinthe wasaddedimmediatelytothecelllysatespriortoWesternblotassays.We .a
abilityofMgrAtodimerizeandtobindthenorBpromoterwith- usedTris-glycinenativegels(4%to20%)andSDS-15%PAGE(Invitro- s
m
outhavinganeffectonMgrA’sphosphorylationstate.Takento- gen,Carlsbad,CA)fortheseassays.
.
gether,thesephenomenaledtoanincreaseinnorBtranscriptsand ForWesternblotsusingpurifiedMgrA-His,extractsoftheS.aureus o
r
areductioninsusceptibilitytomoxifloxacin. cellsgrownasdescribedaboveundernormalorreducedaerationwere g
/

preparedforincubationofpurifiedMgrA-His,asdescribedbelow. o
n
TreatmentofpurifiedMgrA-Hisafterincubationwithcellextracts.
MATERIALSANDMETHODS PurifiedMgrA-His(8(cid:2)g)(27)wasdividedintotwoseries(4(cid:2)geach)and Ja
Bacterial strain and growth conditions. S. aureus strain RN6390 was incubatedwithcelllysates(100(cid:2)g)preparedfromculturesgrownunder n
grown in Luria-Bertani broth (LB) unless otherwise stated (25). For normalorreducedaeration.Thecelllysatescontainedthefollowingcom- ua
growthcurvesandRNAisolation,overnightculturesofS.aureuswere ponents:(i)celllysateswithproteaseinhibitorcocktail(1(cid:3),finalconcen- ry
dilutedtoanopticaldensityat600nm(OD600)of0.01inLBandincu- tration),(ii)celllysateswithoutproteaseinhibitorcocktail,and(iii)cell 1
batedunderaerobicconditionsat37°Cwithorbitalshakingat200rpm. lysateswithH O (100(cid:2)M).Theseconcentrationswerebasedonstudies 2
WhenthecultureOD600reached0.5,15mlofculturewastransferredto carriedoutby2Ch2enetal.(5).Afterincubation,MgrA-Hiswasrepurified , 2
15-mlscrew-toptubestocreatelow-aerationconditions,aswasdoneby usinganickelaffinitycolumnandusedforWesternblotting. 0
Fuchsetal.(9).Sampleswerecollectedattimepoints0,10,30,60,and120 1
MgrA-Hiswaselutedby200mMimidazoleanddialyzedovernightin 9
mininparallelwithaculturegrownunderfull-aerationconditions.
the same buffer without imidazole. For all subsequent protein sample b
pHwasmonitoredusingcolorpHindicatorstrips(pH0to14;EM y
Science,Gibbstown,NJ)andapHmeter(FisherScientific,Pittsburgh, preparations,weusedsamplebufferwithSDS(2%)withoutdithiothreitol g
PA)aspreviouslydescribed(22). (DTT). u
Real-timeRT-PCRassays.Real-timereversetranscription-PCR(RT- e
Drugsusceptibilitydeterminations.Moxifloxacinandsparfloxacin s
PCR)assayswerecarriedoutaspreviouslydescribed(23,25).TotalS. t
werepurchasedfromSigmaChemicalCo.(St.Louis,MO).TheMICwas
aureus RNA was prepared by extraction from lysostaphin-treated cells
thelowestconcentrationofantibioticthatyieldednovisiblegrowthafter
collectedatdifferenttimepointsundernormal-andreduced-aeration
incubationat37°Cfor24h.TheMICsofsparfloxacinandmoxifloxacin
conditions,usingtheRNeasymidikit(Qiagen,Valencia,CA).cDNAs
weredeterminedbybrothmicrodilution,aspreviouslydescribed(24),
withmodificationstocreatealow-aerationenvironmentbasedonatech- weresynthesizedusingtheVersocDNAsynthesiskit(ThermoScientific,
nique developed by Cramton et al. (6). A mid-log-phase culture of S. ABgene,Epsom,Surrey,UnitedKingdom),followedbyreal-timequan-
aureus(OD (cid:2)0.5)growninLBmediawasdiluted100-foldandincu- titativeRT-PCR(qRT-PCR)assaysusingEvaGreendyeandtheCFX96
600
batedinmicrotiterplates(FisherScientific,Pittsburgh,PA)inthepres- real-timesystem(Bio-Rad,Hercules,CA).
enceofdrugsforatleast16h.Wedivideda96-wellmicrotiterplateinto AllprimersusedinthisstudyweresynthesizedattheTuftsUniversity
twoparts:halfwassubmittedtoareduced-aerationcondition,andthe CoreFacility,Boston,MA,andarelistedinTable1.Thehousekeeping
otherhalfwassubmittedtoanormal-growthcondition.Tocreatealow- genegmkwasusedasaninternalcontrol.Allsampleswereanalyzedin
aerationcondition,thewellsonamicrotiterplatewerefilledtothemax- triplicateandnormalizedagainstgmktranscriptlevels.
imumlevel(300(cid:2)l)andsealedwithadhesivetape.Theotherhalfwas Western blots. Western blotting was carried out as previously de-
filledto200(cid:2)l(two-thirds)andleftopentotheatmosphere. scribedusingcellextractsorrepurifiedMgrA-Hisafterincubationwith
Measurementofthedissolvedoxygeninbacterialcultures.Anover- celllysates(1).MgrA-Hiswaselutedin1mlbufferA(20mMTris-HCl,
nightcultureofS.aureusRN6390wasdiluted(100-fold)inLBbrothand pH7.5,150mMNaCl,5%glycerol,containing200mMimidazole),fol-
1824 jb.asm.org JournalofBacteriology
norBExpressionIsDependentonOxygen
TABLE1Primersusedinthisstudy
S.aureusNCTC8325genome
orDNA-proteingelmobility LengthofPCR
shiftbindingassaytarget Gene Primernames Primersequences product(bp)
SA01176 gmk gmk-F 5=-TCAGGACCATCTGGAGTAGGTAAAG-3= 108
gmk-R 5=-TCACGGATTTGACGTGTTG-3=
SA00703 norA norA-F 5=-GACATTTCACCAAGCCATCAA-3= 102
norA-R 5=-TGCCATAAATCCACCAATCC-3=
SA01448 norB norB-F 5=-GCTACACCATCAACAGATACAGCAA-3= 117
norB-R 5=-ACTCAATGCGACGCCAAA-3=
SA00058 norC norC-F 5=-GTTGTTGGAGCAGGTGGTCT-3= 114
norC-R 5=-TGCCGGTAAATTCGTAATAA-3=
SA02762 norD norD-F 5=-ATGAAAGGTGCAATGGCT-3= 101
norD-R 5=-CCTCGTAACGGTATAAA-3=
SA00099 tet38 tet38-F 5=-ATGAATGTTGAATATTCTAA-3= 106
D
tet38-R 5=-TGGCTACAGAAATCAAT-3= o
SA00647 abcA abcA-F 5=-GGTGGTATCTTTGTCATCAATGC-3= 103 w
abcA-R 5=-CCCATAAAACTGAGCGTATCG-3= n
lo
SA00694 mgrA mgrA-F 5=-AGCTGAAGCGACTTTGTCAGATGC-3= 110
a
mgrA-R 5=-AGCGTGAACGTTCCGAAGTCGA-3= d
e
SA00958 htrA htrA-F 5=-AAATTATATAGA-3= 100
d
htrA-R 5=-ATGTTTTTTACC-3=
f
r
SA01935 splF splF-F 5=-TTGTGCTTTCAC-3= 119 o
splF-R 5=-TATAGTGTTCATC-3= m
SA01936 splE splE-F 5=-TTAATTTCAGGA-3= 111 h
splE-R 5=-TTCAGCTGTATT-3= tt
p
SA01938 splD splD-F 5=-TATTTATCTAAA-3= 106 :
/
splD-R 5=-GTGTTATGTAT-3= /jb
SA01939 splC splC-F 5=-CCATTATATTCA-3= 114 .
a
splC-R 5=-TTTTGATGCATA-3= s
m
SA01941 splB splB-F 5=-ATATGCATTTCT-3= 125
.
splB-R 5=-GTATTGTATATT-3= o
r
SA01942 splA splA-F 5=-AATAAACACCGA-3= 124 g
/
splA-R 5=-TTTGATGCGTAT-3=
o
n
norBpromoter Sense ATAAGGTAAGATAACTAGCA 0.15 J
Antisense ATCTCTATTTGCCTCCCTATA a
n
u
a
r
y

1
lowedbydialysisinbufferA.ThemixturesweresubjectedtoSDS-PAGE formed,andthemembraneswereexposedtoX-rayfilmaccordingtothe 2
(15%gel),followedbyWesternblotting. manufacturer’srecommendations. ,
2
EqualamountsofpurifiedMgrA-Hisproteins(200ng)orcellextracts Massspectrometry.Theimmunoreactiveproteinbandsofinterest 0
(100ng)wereseparatedbySDS-15%PAGE(intheabsenceof(cid:3)-mercap- identifiedfromWesternblotswereexcisedfromthegelafterelectropho- 1
9
toethanolorDTTunlessotherwisestated)andtransferredontonitrocel- resisthroughanSDS-15%PAGEgel.Massspectrometryanalyseswere
b
lulosemembranesaspreviouslydescribed(25).Allwashingstepswere performedattheTuftsUniversityCoreFacility,Boston,MA. y

performedatroomtemperature.Themembranewaswashedtwicefor10 DNAmobilityshiftassays.Primersdesignedtoamplifytheputative g
u
minwithTBSbuffer(10mMTris-HCl,150mMNaCl,pH7.5),followed promoterregionofnorBarelistedinTable1.Oneoftheprimerswasbiotin-
e
by incubation fo r 1 h inblocking buffer (5% bovine serum albumin ylatedbytheTuftsUniversityCoreFacility(Tufts,Boston,MA).Thegel s
t
[BSA],0.1%Tween20inTBSbuffer).Membraneswerewashedtwicefor mobilityshiftassaywascarriedoutusingtheLightShiftchemiluminescent
10mininTBS-Tween-Tritonbuffer(20mMTris-HCl,500mMNaCl, electrophoreticmobilityshiftassay(EMSA)kit(Pierce,Rockford,IL),asrec-
0.05% Tween 20, 0.2% Triton X-100, pH 7.5) and once with TBS ommendedbythemanufacturer.ThepurifiedMgrAproteins(50ng)were
buffer.Membraneswerethenincubatedwitheitherantiphosphoser- mixedwithbiotin-labeledDNAin20(cid:2)lofbindingbuffer(10mMHEPES,
ine(Qiagen,Valencia,CA)oranti-MgrA(GenScript,Piscataway,NJ) pH8,60mMKCl,4mMMgCl,0.1mMEDTA,0.1mg/mlofbovineserum
2
antibodysolutions(1/100dilutionofantibodyinTBS-Tween20buf- albumin,25mMdithiothreitol)containing1(cid:2)gofpoly(dI-dC),200ngof
fer)at4°Covernightandthenwashedtwicefor10mininTBS-Tween- shearedherringspermDNA,and10%glycerolfor20minatroomtempera-
Triton buffer and once in TBS buffer. The anti-MgrA antibody was ture.Afterincubation,thebindingmixturewasanalyzedby5%nondenatur-
producedusinga14-amino-acidpeptidesequence(QRQVNRYYSNK ingpolyacrylamideelectrophoresis(25).
VFK)oftheMgrAproteinasdescribedpreviously(22).Membraneswere
incubatedwithsecondaryantibodies(goatanti-rabbitIgG–horseradish
RESULTS
peroxidase[HRP]forMgrAantibodyandrabbitanti-mouseIgG–IgM–
S.aureusRN6390growthratesunderdifferingaerationcondi-
HRPforphosphoserineantibody)(GenScript,Piscataway,NJ,andJack-
sonImmunoResearch,WestGrove,PA,respectively)(1:10,000dilution) tions.TostudytheeffectofaerationonthegrowthofS.aureus
for1hatroomtemperatureandwashed4timesfor10mineachtimein RN6390,abacterialcultureatanOD600of0.5wasdividedinto
TBS-Tween-Triton.Thechemiluminescencedetectionreactionwasper- twoseriesandallowedtogrowundernormal-andreduced-aera-
April2012 Volume194 Number7 jb.asm.org 1825
Truong-Bolducetal.
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FIG1S.aureusRN6390andgrowthunderreducedaeration.(A)GrowthcurvesofS.aureusRN6390underAEandRAE.ThebacteriaweregrowninLBmedium
at37°Cundernormal-aerationconditionstoanOD of0.5.Atthattime,theculturewasdividedintotwoseries.Oneculturecontinuedtogrowundernormal
600
aeration,andtheotherwascultivatedunderreducedaeration.Thetimepointswere0,10,30,60,and120min.(B)Measurementsoftheamountofdissolved
oxygeninmg/literundernormalandreducedaeration.TheamountofdissolvedoxygenwasmeasuredusingaVernierdissolved-oxygenprobe.Thereadings
weretakenafter45sofcontactbetweentheprobeandtheculture.(C)RelativelevelsofexpressionofnorBtranscriptsundernormalandreducedaeration.The
valuesarethemeansofresultsofthreeseparatebiologicalsamplesandarenormalizedagainstthegmkgeneasaninternalcontrol.RrepresentstheratioofnorB
transcriptionunderreducedaerationcomparedtothatundernormalaeration.
tionconditions.Timezerowasthetimeatwhichtheculturewas thanwhenthesameculturewasgrownatnormalaeration,butat
divided for the different growth conditions. The OD s of the 120min,neitherculturehadreachedstationaryphase(Fig.1A).
600
culturesweremeasuredoveraperiodof120min.Weobserved,as The pH values of the cultures under both conditions remained
expected,alowergrowthrateforthecultureatreducedaeration unchanged(pH7.6)forupto120min.
1826 jb.asm.org JournalofBacteriology
norBExpressionIsDependentonOxygen
TABLE2Real-timeRT-PCRresults tant, QT5 (norB::cat), exhibited no change in moxifloxacin and
sparfloxacinsusceptibility(MIC(cid:2)0.06(cid:2)g/ml)atreducedaera-
S.aureusRN6390 Foldchangein
genomea Gene Description expressionlevelb tion, indicating that the differences in susceptibility seen in the
SA00703 norA MDReffluxpump (cid:5)1.1 parentalstrainwithdifferingaerationconditionsrequiredintact
SA01448 norB MDReffluxpump (cid:5)20.0 NorB.
SA00058 norC MDReffluxpump (cid:5)1.5 MgrA in cell extracts at normal and reduced aeration and
SA02762 norD PutativeMDRefflux (cid:5)1.5 effects of H2O2 on the dimerization of MgrA monomers. We
pump carried out Western blotting to examine the homodimer MgrA
SA00099 tet38 MDReffluxpump (cid:5)1.5 proteinanditsphosphorylationstatusundernormalandreduced
SA00647 abcA ABCtransporter (cid:1)4.0 aeration.
SA00694 mgrA Globalregulator (cid:5)1.3 Proteins in cell extracts were separated by electrophoresis
SA00958 htrA Serineprotease (cid:5)1.0
through a Tris-glycine native gel or an SDS-PAGE 15% gel
SA01935 splF Serineprotease (cid:5)2.5
(Fig.2A).Regardlessofthegeltype,weobservedbothdimericand
SA01936 splE Serineprotease (cid:5)2.7
SA01938 splD Serineprotease (cid:5)2.1 monomericformsofMgrAundertheAEcondition,whileonly
SA01939 splC Serineprotease (cid:5)2.0 the monomeric form was present under the RAE condition. In Do
SA01941 splB Serineprotease (cid:5)2.5 subsequentWesternblotassays,wecarriedouttheexperiments w
SA01942 splA Serineprotease (cid:5)2.2 usingSDS-PAGEgels. n
After60minofincubationwithH O andproteaseinhibitors, lo
aAnnotationsarefromS.aureusNCTC8325. 2 2 a
bCellswerecollectedat30minfollowinggrowthundernormalandreducedaeration. MgrA in cell extracts from a culture at AE appeared mostly di- d
e
ChangesinvaluesoftheS.aureusRN6390genetranscriptswerenormalizedbetween meric.Incontrast,incellextractsfromculturesatRAE,allMgrA d
valuesatnormalaerationovervaluesatreducedaeration.(cid:5)indicatesanincreasein remainedinthemonomericformafterH O exposure(Fig.2B). f
transcriptlevel,and(cid:1)indicatesadecreaseintranscriptlevel.Thehousekeepinggene Westernblottingwasalsoperformed2usi2ngthesamecellex- ro
gmkwasusedastheinternalcontrol.Allassaysweredoneintriplicate. m
tracts and the anti-phosphorylated-serine (anti-Ser-P) antibody

h
to assess the phosphorylation status of monomer and dimer t
t
Changesindissolvedoxygenunderdifferingaerationcondi- MgrA. We observed that phosphorylation occurred with both p
:
/
tions. Under normal-aeration conditions, the dissolved oxygen monomericanddimericformsofMgrAinAEcellextract.Under /
jb
levelofthebacterialcultureincreasedfrom1.075mg/literattime RAE conditions, we found an additional small immunoreactive .
a
zeroto2.1mg/literat10minandthenstabilizedat1.5mg/liter proteinof(cid:4)6kDawiththeanti-Ser-Pantibody(Fig.2C). s
m
from30minupto120min.Underreduced-aerationconditions, TheseobservationssuggestedthatthemonomericMgrAincell
.
weobservedareductionintheoxygenlevelofthebacterialculture extractundernormalaerationwasdifferentfromthatunderre- o
r
from1.075mg/literattimezeroto0.725mg/literat10min,fol- duced aeration. This hypothesis was supported by the fact that g
/

lowedbystabilizationat0.7mg/literupto120min(Fig.1B). H O couldnotdrivethedimerizationofMgrAunderreduced o
2 2 n
Changesineffluxpumpgenetranscriptsunderdifferentcul- aerationasitdidundernormalaeration(Fig.2B).
J
ture conditions. Total RNAs were extracted from S. aureus To explain this difference, we incubated purified MgrA-His a
n
RN6390grownatnormalandreducedaerationattimepoints0, proteinwithcellextractstoassesstheireffectsonthepropertiesof
u
10,30,60,and120min,andrelativetranscriptlevelsweremea- thepurifiedprotein. a
r
suredbyquantitativereal-timeRT-PCR.Undernormalaeration, PurifiedMgrAproteinandtheeffectsofH O andprotease y
2 2
1
weobservedaninitialdecreaseofthenorBtranscripts,whichre- inhibitors on the dimerization of MgrA. Purified His-tagged
2
mainedlowrelativetotheirlevelattimezero(0.54-foldat10min MgrAproducedheterologouslyinEscherichiacoliis(cid:4)21kDaand ,
2
and0.37-foldat120min)(Fig.1C).Underreducedaeration,we appearsdominantlymonomeric(SDS-PAGE)(Fig.3A).Afterin- 0
observed an initial increase of 12.5-fold in the norB transcripts cubation of the purified protein with H O (100 (cid:2)M), we pre- 1
2 2 9
after 10 min. A further increase continued up to 60 min (21.7- paredtheproteinsamplesusingdifferentcompositionsofsample b
fold) followed by a decrease at 120 min, which still remained buffertoassesswhetherH O treatmentgeneratedanintermolec- y
2 2
g
abovetranscriptlevelsattimezero(3.3-fold)(Fig.1CandTable ulardisulfidebondbetweentheCys ofthetwomonomersofthe
12 u
2).Incontrast,thetranscriptlevelsofnorA,norC,norD(encoding MgrAprotein(Fig.3A).TheMgrAproteinswereanalyzedby15% e
s
aputativeeffluxpump[unpublished]),andtet38remainedun- SDS-PAGE,withorwithout100mMDTTinthesamplebuffer. t
changed(1.1-foldfornorAand1.5-foldfortheothereffluxpump AfterincubationwithH O ,MgrAappearedmostlydimericand
2 2
genes).ThetranscriptlevelofabcA,whichencodesanAbcAtrans- becamemonomericinthepresenceofDTTinthesamplebuffer
porter, was reduced 4-fold. The transcript level of mgrA, which (Fig.3A).ThesedatasuggestedthattwoMgrAmonomersforman
encodestheMgrAglobalregulator,waslittlechanged(1.3-fold) intermoleculardisulfidewithH O treatment.Chenetal.previ-
2 2
(Table2). ouslydemonstratedtheformationofsuchanintermoleculardi-
Susceptibilitytomoxifloxacinandsparfloxacinunderdiffer- sulfidebondbetweenthetwoCysresiduesCys andCys ofthe
30 62
ingaerationconditions.TocorrelatechangesinnorBtranscript redoxproteinMexRofPseudomonasaeruginosa(4,5).
levelswithsusceptibilitytomoxifloxacinandsparfloxacin,wede- MgrA monomer is differentially modified by bacterial cell
terminedMICsunderthedifferingconditionsofaeration,usinga lysates grown under normal and reduced aeration. We incu-
previouslydescribedmethodforthereduced-aerationcondition batedpurifiedMgrA-Hiswithcelllysatesfrombacterialcultures
(6).Weobservedanincreaseof4-foldintheMICsofmoxifloxa- grownundernormalandreduced-aerationconditionsinthepres-
cinandsparfloxacinforS.aureusatreducedaeration(0.25(cid:2)g/ml) enceorabsenceofproteaseinhibitors.WealsoincubatedMgrA-
compared to the MICs for the same culture at normal aeration HiswiththetwocelllysatesinthepresenceofH O withoutpro-
2 2
(0.06 (cid:2)g/ml). Importantly, under these conditions, a norB mu- tease inhibitors. Following the incubation step, MgrA-His was
April2012 Volume194 Number7 jb.asm.org 1827
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9

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FIG2MgrAincrudecellextractscollectedfromculturesgrownundernormalaeration(AE)andreducedaeration(RAE).(A)Westernblottingwasperformed
usinganti-MgrAantibody.DimerandmonomerformsofMgrAareindicated.MM,molecularmass(SeeBluePlus2markerfornativeandSDS-PAGEgels).(B)
EffectsofHO onMgrAincrudecellextracts.Westernblotwithanti-MgrAantibody.Cellextractswerecollectedat60min.Celllysateswereincubatedwith
2 2
HO for60mininthepresenceofproteaseinhibitors.Undernormalconditions,thelevelofthedimericformofMgrAincreasesafterincubationwithHO.
2 2 2 2
Withcellextractsfromreduced-aerationconditions,MgrAisnotconvertedtothedimericformbytreatmentwithHO.(C)PhosphorylationstatusofMgrA
2 2
forms.Westernblotwithanti-Ser-Pantibody.Cellswerecollectedat60min.TheWesternblotswerecarriedoutusingcrudecellextractstreatedwithprotease
inhibitors.ThedimerandmonomerMgrAsandcleavageproductsareindicated.Boththemonomericanddimericformsarephosphorylated.
repurified using a nickel column. The MgrA-His (monomeric) anda21-kDaprotein(Fig.3B).Wealsoobserveda40-kDapro-
protein was retained by the column, while other proteins were teinafterincubationwithAEcelllysatesupplementedwithH O
2 2
eliminatedviatheflowthroughandwashingsteps. andintheabsenceofproteaseinhibitors.
IntheMgrAsampleincubatedwiththeAEcelllysate,inthe Incontrast,incubationwiththeRAEcelllysaterevealedone
presenceorabsenceofproteaseinhibitors,weobserveda40-kDa lowerbandat(cid:4)15kDaintheMgrAsamplewithoutprotease
1828 jb.asm.org JournalofBacteriology
norBExpressionIsDependentonOxygen
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FIG3 SDS-PAGEgelsandWesternblotsofMgrA-His.MgrA-Hisproteinwaspurifiedbynickelaffinitychromatography.Lysateswerepreparedfromcells a
r
collectedat60minundernormalandreducedaeration.(A)SDS-PAGE(15%gel)ofpurifiedMgrA-His.Thesizeofthetotalprotein,includingthehistidine- y
taggedportion,is(cid:4)21kDa.TheproteinMgrA-HiswasincubatedwithHO for60minpriortoelectrophoresis.TheSDSsamplebufferwithorwithoutDTT 1
isindicatedinthetableforeachsample.Theproteinsampleswereboiledfo2r22minat100°C.MM,molecularmass(broad-rangemarkerforSDS-PAGEgels).(B) 2
,
MgrA-Histreatedwithcellextractsfromculturesgrownundernormalaeration(AE)andreducedaeration(RAE).MgrAwasrepurifiedafterincubationwithcell 2
extracts,andWesternblottingwascarriedoutusinganti-MgrAandanti-Ser-Pantibodies.TheMgrAproteinwasincubatedwithcrudecellextractsfromcells 0
grownunderAEandRAEconditionsinthepresenceorabsenceofserineproteaseinhibitorsandinthepresenceofHO for60min.Allproteinsampleswere 1
2 2 9
preparedusingsamplebufferwithoutDTT,boiledfor2minat100°C,andthenelectrophoresedthroughSDS-PAGE15%gels,followedbyWesternblotting.
b
MonomerMgrAs(arrowsA,B,andC)areproteinbandsthatwereselectedformassspectrometryanalyses.MgrA-His(monomeranddimer)reactedtothe y
anti-Ser-Pantibody,indicatingthattheywerephosphorylatedbytheAEcelllysate.MgrA-His(15kDa)fromtheRAEcelllysatedidnotreactwiththeanti-Ser-P g
antibody.(cid:5),presenceofproteaseinhibitors;(cid:1),absenceofproteaseinhibitors.A6-kDapeptidethatwaspresentintheSDS-PAGEgelaftertheincubationstep u
withcelllysatesunderRAEreactedwiththeanti-Ser-Pantibodybutnotwiththeanti-MgrAantibody. e
s
t
inhibitors.Furthermore,incubationwithH O didnotaffect anti-MgrAantibodywasnotreactivewiththeanti-Ser-Pantibody
2 2
this15-kDaprotein,andnodimerizationoccurredunderthis (Fig.3B).
condition (Fig. 3B). Thus, incubation with an RAE cell lysate MassspectrometryofrepurifiedMgrAaftertreatmentwith
alteredtheabilityofMgrAtobemodifiedbydifferentoxidative celllysatesfromculturesgrownundernormalandreducedaer-
conditions. ation.Toassessthenatureofthe21-kDaandthe15-kDabands,
Western blots were performed using anti-Ser-P antibody on weisolatedthesebands(Fig.3B,arrowsA[21kDa],B[21kDa],
MgrA-Hisincubatedwithcellextractstoassessthephosphoryla- and C [15 kDa]) from SDS-PAGE gels for mass spectrometry.
tionstatusofmonomeranddimerformsofMgrA.Weobserved ProteinbandAwasextractedfromasampletreatedwiththeAE
thephosphorylationofboththemonomericanddimericformsof celllysateinthepresenceofproteaseinhibitors.Massspectrome-
MgrAuponincubationwithextractsofcellsgrownundernormal- try revealed that this protein matched (130/147 aa) the MgrA
aerationconditions.Incontrast,onlyan(cid:4)6-kDabandwasim- aminoacidsequence.Thus,basedonmassspectrometryandmo-
munoreactiveaftertreatmentofMgrA-Hiswithextractsofcells lecularweight,thisbandwasconfirmedtobeanMgrAmonomer
grownunderreducedaeration.The15-kDabanddetectedwith (Fig.4A).
April2012 Volume194 Number7 jb.asm.org 1829
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FIG4MassspectrometryofMgrAfragmentsA,B,andC.ThepeptideswerematchedwiththeaminoacidsequenceofMgrA.Thematchedportions(bluecolor) :/
/
areinbold.Thepeptideusedtogeneratetheanti-MgrAantibodyisinboldandunderlined.Importantaminoacidsareindicatedbytheirlocationnumbersand jb
underlined.(A)MgrA(21kDa)incubatedwithcelllysate(AE)inthepresenceofproteaseinhibitors;(B)MgrA(21kDa)incubatedwithcelllysate(AE)inthe .a
absenceofproteaseinhibitors;(C)MgrAfragment(15kDa)incubatedwithcelllysate(RAE)intheabsenceofproteaseinhibitors. s
m
.
o
r
ProteinbandBwasextractedfromthesampletreatedwiththe mericform(Fig.5B).SuchamodificationofMgrAwouldnotbe g
/

AEcelllysateintheabsenceofproteaseinhibitors.Bymassspec- predictedtoaffectitsphosphorylationstatusbutwouldprevent o
n
trometry,peptidesofthisproteinalsoproducedamatchwiththe dimerizationandtheabilityofMgrA-PtobindtothenorBpro-
J
MgrAaminoacidsequence.ThecrucialaminoacidSer ,neces- moter.Thus,posttranslationalmodificationofMgrAbyproteases a
113 n
saryforthedimerizationofMgrA,wasidentifiedinonepeptide inadditiontoitsmodificationbykinasescanaffectitsresponsesto
u
(inboldfaceinLS NAS DKVASASSLSQDEV)ofthedimer- changing environmental conditions and can modulate the re- a
110 113 r
izationdomainthatoriginatedfrombothproteinbandsAandB; sponsesofthegenesthatitregulates(25). y

1
thus,thisfindingisconsistentwiththeabilityofthismonomeric Serine protease expression under reduced aeration. Given
2
formtodimerizewithchangesinoxidationconditions(Fig.4B). thepresenceofa15-kDafragmentaftertheincubationofMgrA- ,
2
ProteinbandC(15kDa)wasextractedfromthesampletreated HiswithRAEcelllysatesbutnotAEcelllysates,wehypothesized 0
withtheRAEcelllysateintheabsenceofproteaseinhibitors.Mass thatproteaseswereinvolvedinthecleavageofMgrAatasite(s) 1
9
spectrometry revealed that this protein matched 63 out of 147 crucialforMgrAdimerizationbasedonthemodelproposedby b
aminoacidswiththeMgrAaminoacidsequence.Thus,proteinC Chen et al. (5) and that these proteases would be increased in y

g
wasafragmentoftheMgrAmonomer.Furthermore,aminoacids responsetoreducedaeration.
u
Ser andSer foundinbothbandsAandBwerenotidentified Todetermineifchangesinserineproteaseexpressioncouldbe e
110 113 s
inthisMgrAfragment(Fig.4C).Sincetheseaminoacidsarees- correlatedwithchangesinaeration,wemeasuredbyRT-PCRthe t
sentialfordimerizationofMgrA,theirabsenceisconsistentwith transcriptlevelsofsevengenesencodingserineproteases,namely,
theinabilityofthisMgrAfragmenttobedimerizedbytreatment htrA,splA,splB,splC,splD,splE,andsplF(17).Theseserinepro-
withH O (Fig.3B). tease genes, with the exception of htrA, formed an operon and
2 2
ThestructureoftheMgrAproteinhasbeensolvedbyChenet demonstratedanincreaseofatleast2-foldunderreduced-aera-
al.(5).UsingthepublishedMgrAdataandthepublicprogram tionconditions(Table2).
Jmol(http://www.rcsb.org),weattemptedtovisualizetheinter- MgrA-P binding to the norB promoter under normal and
actionsbetweenkeyaminoacids,Cys ,Tyr ,Tyr ,andSer . reducedaeration.Gelshiftbindingexperimentswereperformed
12 26 38 113
We hypothesized that under reduced aeration, a peptide ((cid:4)6.0 toevaluatetheabilityofMgrAtobindtothenorBpromoter.After
kDa)containingSer wascleavedfromthemonomericMgrAby incubation with the cell lysates (AE or RAE), without protease
113
a protease(s) such as a serine protease (Fig. 5A). This cleavage inhibitors,werepurifiedtheMgrA-Hisproteinsusingnickelcol-
wouldleadtoadisruptionofthehydrogeninteractionsbetween umns. Only repurified MgrA-His incubated with cell extracts
Cys ofonemonomerandSer -Tyr -Tyr oftheothermono- (CE-AE) reacted with anti-Ser-P antibody, indicating that the
12 113 38 26
mer,aswasshowninthemodelofChenetal.(5).Themonomeric proteinswerephosphorylatedaftertheincubationstep(Fig.3B).
MgrAfragmentwithoutSer wouldthenbelockedinitsmono- MgrA-P(CE-AE)createdthesamenorBpromoter-shiftingpat-
113
1830 jb.asm.org JournalofBacteriology
norBExpressionIsDependentonOxygen
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FIG6 DNAgelmobilityshiftassaysusingthenorBpromoterandthepurified
f
MgrA-Hisproteinsincubatedwithcelllysates.MgrAandMgrA-P,purified ro
MgrAwithandwithoutphosphorylationbyPknBinvitro;MgrA(CE-AE), m
purifiedMgrAincubatedwithcellextractspreparedfromculturesgrownun-
h
dernormalaerationandrepurified;MgrA(CE-RAE),purifiedMgrAincu- t
t
batedwithcellextractspreparedfromculturesgrownunderreducedaeration p
andrepurified. :/
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aureusNorBeffluxpumpisaprotonantiporteroftheMFSfamily,
.
whichwasfoundtobeinvolvedinbacterialfitnessinasubcuta- o
r
neous abscess mouse model as well as in bacterial resistance to g
FIG5 MgrAproteinandinteractionsamongCys ,Ser ,Tyr ,andTyr /
aminoacids.(A)Aminoacidsinvolvedintheint1e2ractio1n13sbetw26eenthetw3o8 quinolones(7,24).Underreducedaeration,weobservedasignif- o
n
MgrAmonomersareindicatedinboldandunderlined.Thepeptideusedfor icantincreaseinnorBtranscriptlevelsassociatedwithadecrease
J
thegenerationoftheanti-MgrAantibodyisunderlined.Putativecleavagesites in the amount of dissolved oxygen in the medium. Thus, low- a
byproteasesareindicatedbyarrows.(B)Schematicrepresentationofinterac- n
oxygenconditionsareatriggerforexpressionofnorB,encoding
tionsamongkeyaminoacidsofthetwoMgrAmonomers.Wehypothesize u
theNorBeffluxpump.NorBwasrecentlyshowntobeacompo- a
tthioanttahnedctlheauvsacgoenofifnmesoMnogmrAertoMigtsrAmaotnroemsideruiecSfoerrm113.Vpriseuvaelniztsattihoendoifmaemriiznao- nentofthebacterialadaptationtoanabscessenvironment,with ry
acidinteractionswasdoneusingtheWeb-basedpublicprogramJmol,http: acidicconditionstriggeringincreasedexpression(22).Reductions 1
2
//www.rcsb.org,andMgrAdatafromtheworkofChenetal.(5). inoxygenlevelswereassociatedwithareducedgrowthrate,butit ,
2
isunlikelythatachangeingrowthphaseresultedinachangein 0
norB expression, since the induction of expression occurred 1
9
ternasthatofMgrA-P/norB(phosphorylatedbyPknB)(25).In within 10 min of a shift in conditions, and cells did not reach b
contrast,MgrA(CE-RAE)didnotgenerateanyshiftingpattern stationary phase under either condition over the course of the y

whenincontactwiththenorBpromoter(Fig.6).Thesedatasug- experiment. g
u
gestedthattheabsenceofDNAbindingbetweenMgrA(RAE)and ToassessiftheincreaseinnorBtranscriptswasassociatedwith e
s
thenorBpromoterwasduetoalackofdimerizationandanex- increased resistance to moxifloxacin and sparfloxacin, two sub- t
pectedlackofphosphorylationoftheresultingMgrAfragment, stratesoftheNorBeffluxpump(24),wecarriedoutMICassays
whichlackedserinephosphorylation(Ser andSer )(25). undernormal-andreduced-aerationconditions.Anincreaseof
110 113
4-foldintheMICsofmoxifloxacinandsparfloxacinwasassoci-
DISCUSSION atedwithgrowthatreducedaeration.ForthenorBmutantQT5at
S.aureusisafacultativeanaerobethatcanswitchfromaerobicto reducedaeration,nochangeintheMICsofbothquinoloneswas
anaerobicgrowthusingeitherrespiratoryorfermentativemetab- observed,thusindicatingtheinvolvementoftheintactNorBpro-
olismtosynthesizeATP(10).Protonmotiveforcegeneratedby teinintheincreaseinresistancetomoxifloxacinunderreduced
theelectrochemicalgradientofhydrogenionsdiffersdepending aeration.
on growth conditions. Under aerobic conditions, proton trans- PhosphorylatedMgrA(MgrA-P)bindstothenorBpromoter
portiscarriedoutbytherespiratorychain.Themembranepro- andrepressesnorBexpression(25).Tounderstandtheregulation
ton-translocatingATPaseoperatesinthedirectionofprotonin- of norB in the presence of reduced oxygen levels, we evaluated
flux and ATP synthesis. Under anaerobic conditions, ATP is MgrA protein and its phosphorylation status. MgrA is a ho-
generatedbyfermentation,andthemembraneATPaseoperatesin modimer and is involved in the regulation of numerous genes
the direction of proton efflux and ATP hydrolysis (10). The S. responsible for a wide variety of functions, including virulence
April2012 Volume194 Number7 jb.asm.org 1831
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FIG7 ModelofposttranslationalregulationofnorBexpressionbyMgrA.EffectsofreducedaerationonMgrAarederivedfromthisstudy.Effectsofacidic ro
conditionsweretakenfromreference22,andeffectsofoxidizingconditionsweretakenfromreference5. m

h
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p
:
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factors, autolysis, capsule synthesis, and transporters, as well as Chenetal.demonstratedthatCys ,locatedattheN-terminal /
other regulators, such as the SarZ (2). MgrA is involved in the (cid:1)1-helix in the dimerization domain12of MgrA is recognized by jb.
a
oxidative-stressresponseinS.aureusandissensitivetotheeffects Ser and Tyr of the other monomer via hydrogen bonding. s
113 38 m
ofreactiveoxidants(H O )andreductants(DTT)(5).Oxidized InteractionsamongthethreeresiduesCys ,Tyr ,andSer are
2 2 12 38 113 .
MgrAlostitsabilitytobindtothesarVpromoterandrecovered necessary for dimerization (5) (Fig. 4B). The proposed cleaved o
r
thisabilityaftertreatmentwithDTT.Thus,theoxidativestateof peptide(Phe -Lys )containsSer andSer ,whichaccounts g
94 147 110 113 /

MgrAaswellasitsphosphorylationstatecanaffectitsfunction, fortheinabilityoftheMgrAfragmentfromcellsunderreduced o
n
addingadditionalpotentiallyrapidcapabilitiesofregulatorycon- aerationtobedimerized,thuslockingMgrAinamonomericform
J
trolbeyondmodulationoflevelsofproteinexpression. andpreventingitsbindingtothenorBpromoterandrepressionof a
n
The reduced-aeration condition did not affect the transcript norBexpression.
u
levelofmgrA,butWesternblotdataindicatedthatitaffectedthe Asyet,itisnotknownwhichspecificprotease(s)actsonMgrA a
r
MgrA protein. Under normal aeration, addition of H O in- under reduced-aeration conditions, but protease expression is y
2 2
1
creasedthequantityofMgrAhomodimers.Underreducedaera- knowntobemodulatedunderanaerobicconditionsinS.aureus.
2
tion,MgrAproteinremainedmostlyinthemonomericformre- Underanaerobicconditions,theRexfamilyrepressorisactivated ,
2
gardlessoftheadditionofH O .Thisfindingsuggestedthatthe andthereisincreasedexpressionoftheClpproteases,whichin 0
2 2
monomericformsofMgrAobtainedfromcellsundernormaland turnactivelydegradeotherproteins,includingthetoxinrepressor 1
9
reducedaerationhadstructuraldifferences.Intheabsenceofpro- Rot.Rotregulatesawiderangeofvirulencefactorsandproteases, b
teaseinhibitorsandwithlysatefromAEcells,weobtainedMgrA- suchasserineproteases(SspAfamily)(8).Withreducedorabsent y

g
His(21kDa)initsmonomericform,whichcouldbeconvertedto amountsofRot,serineproteasesmayactivelydegradeothertarget
u
a dimeric form upon treatment with H O . In contrast, we ob- proteins,andMgrAmayalsobeatargetofthisfamilyofproteases. e
2 2 s
tained a 15-kDa fragment of MgrA-His after treatment with a We demonstrated that genes encoding six serine proteases t
lysatefromRAEcells.Massspectrometryanalysesindicatedthat (splA,-B,-C,-D,-E,and-F)exhibitedincreasedtranscriptlevels
thisMgrAfragmentlackedtheaminoacidportion((cid:4)6kDa)car- ofatleast2-foldinbacteriagrownunderreducedaeration.These
ryingtheSer necessaryforthedimerizationofMgrA(5,16). serineproteasegenesareinanoperonstructure,aswasdemon-
113
Thus,weproposeamodelinwhichproteasecleavageofthe strated by Reed et al. (17). These data were consistent with the
monomeric form of MgrA prevented its dimerization (Fig. 7). findings of Fuchs et al., who demonstrated that splC and splD
Basedonthepreferenceofserineproteasestocleaveatbulkyhy- increasedapproximately2-foldinatranscriptionalmicroarrayof
drophobicresidues,wesuggestthattheMgrAmonomercanbe S.aureusunderanaerobicconditions(9).Fuchsetal.alsoshowed
cleavedatresiduePhe (F ),generatingapeptideof50amino thatthetranscriptionofrotincreased2-fold.Thesefindingssug-
94 94
acids((cid:4)6kDa)whichencompassesthetwoSer andSer res- gestthatrottranscriptionincreasedbutthattheRotproteinwas
110 113
iduesbutlacksthepeptideusedtogeneratetheanti-MgrAanti- degraded by Clp proteases, thus explaining the increase of the
body (Fig. 5A) (3, 13). Consistent with this model, the 15-kDa transcription of the splC and splD genes, encoding serine pro-
fragment reacted only with the anti-MgrA antibody, and the teases, as shown by Frees et al. (8). Thus, MgrA, like Rot, is a
6-kDafragment,generatedfromtheMgrAcelllysate(RAE),re- regulatoryprotein,thefunctionofwhichmaybemodulatedby
actedonlywiththeanti-Ser-Pantibody. proteasesunderanaerobicgrowth.
1832 jb.asm.org JournalofBacteriology