Influence of Iron and Aeration on Staphylococcus aureus Growth, Metabolism, and Transcription

12 Pages·2014·2.26 MB·English

Influence of Iron and Aeration on Staphylococcus aureus Growth,
Metabolism, and Transcription
NagenderLedala,aBoZhang,bJavierSeravalli,cRobertPowers,bGregA.Somervillea
SchoolofVeterinaryMedicineandBiomedicalSciences,UniversityofNebraska—Lincoln,Lincoln,Nebraska,USAa;DepartmentofChemistry,UniversityofNebraska—
Lincoln,Lincoln,Nebraska,USAb;DepartmentofBiochemistry,UniversityofNebraska—Lincoln,Lincoln,Nebraska,USAc
Staphylococcusaureusisaprominentnosocomialpathogenandamajorcauseofbiomaterial-associatedinfections.Thesuccess
ofS.aureusasapathogenisdueinparttoitsabilitytoadapttostressfulenvironments.Asanexample,thetransitionfromre-
sidinginthenarestoresidinginthebloodordeepertissuesisaccompaniedbychangesintheavailabilityofnutrientsandele-
mentssuchasoxygenandiron.Assuch,nutrients,oxygen,andironareimportantdeterminantsofvirulencefactorsynthesisin
S.aureus.Inadditiontoinfluencingvirulencefactorsynthesis,oxygenandironarecriticalcofactorsinenzymaticandelectron D
o
transferreactions;thus,achangeinironoroxygenavailabilityaltersthebacterialmetabolome.Changesinmetabolismcreate w
intracellularsignalsthataltertheactivityofmetabolite-responsiveregulatorssuchasCodY,RpiRc,andCcpA.Toassesstheex- n
lo
tentofmetabolomicchangesassociatedwithoxygenandironlimitation,S.aureuscellswerecultivatediniron-limitedmedium a
and/orwithdecreasingaeration,andthemetabolomeswereexaminedbynuclearmagneticresonance(NMR)spectroscopy.As d
e
expected,oxygenandironlimitationdramaticallydecreasedtricarboxylicacid(TCA)cycleactivity,creatingametabolicblock d

andsignificantlyalteringthemetabolome.Thesechangesweremostprominentduringpost-exponential-phasegrowth,when fr
o
TCAcycleactivitywasmaximal.Importantly,manyoftheeffectsofironlimitationwereobscuredbyaerationlimitation.Aera- m
tionlimitationnotonlyobscuredthemetaboliceffectsofironlimitationbutalsooverrodethetranscriptionofiron-regulated h
genes.Finally,incontrasttopreviousspeculation,weconfirmedthatacidificationoftheculturemediumoccursindependentof tt
p
theavailabilityofiron. :
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.
a
Staphylococcusaureusisaversatilepathogencapableofinfecting lite-responsive regulators (e.g., CodY, CcpA, and RpiRc) that s
m
almostanynichewithinahumanoranimalhost.Aswithmost maintain metabolic homeostasis when nutritional and environ- .
o
bacterialpathogens,successfulpathogenesisrequiresthatS.au- mentalconditionsarefavorableandthatfacilitatemetabolicad- r
g
reus gain entry into a host (e.g., through a breach in the skin), aptationswhennutritionalandenvironmentalconditionschange /

o
adheretoasuitablesurface,avoidbeingkilledbythehost’sim- (6–8).Specifically,CodYrespondstochangesinbranched-chain n
mune system, acquire nutrients, and proliferate. Notably, every amino acids and GTP concentrations (9, 10), while CcpA indi- M
stepinthispathogenicprocessinvolvesmetabolicchangesand/or rectly responds to glucose-6-phosphate and fructose-1,6-bis- a
r
metabolic regulation. Entry into a host is an environmental phosphate(11).WhiletheRpiRregulatorsallhavesugarisomer- c
h
change that dramatically alters the availability of nutrients and asebindingdomains,themetabolitesthatmodulatetheiractivi-
3
cofactors.Similarly,thetransitionfromsynthesizingadhesinsto tieshaveyettobedetermined(8).Thesemetabolite-responsive 0
,
producing tissue-damaging secreted virulence determinants is regulators, i.e., CodY, CcpA, and RpiRc, coordinate both viru- 2
preceded by changes in the bacterial nutritional status (1). To lencedeterminantbiosynthesisandmetabolisminstaphylococci 0
1
avoidbeingkilledbythehost’simmunesystem,S.aureusaltersits (8,9,12).Ingeneral,theactivityofmetabolite-responsiveregula- 9

metabolism to limit damage by oxidative and nitrosative stress tors is controlled by intracellular concentrations of numerous b
y
(2). The act of acquiring nutrients is regulated by metabolite- compounds,suchasbiosyntheticintermediates(13),aminoacids
g
responsiveregulatorsthatrespondtochangesinthebacterialmet- (14),nucleicacids(15),andcofactors(e.g.,iron)(16).InStaphy- u
e
abolic status (3). Finally, proliferation is a carbon- and energy- lococcusepidermidis,theintracellularconcentrationsofmetabo- s
intensive undertaking. In short, the success of S. aureus as a litescanbealteredbyenvironmentalstressorssuchasantibiotics, t
pathogendependsonitsabilitytomeettherapidlychangingnu- ethanol, metal ion limitation, or nutrient depletion (3, 17, 18).
tritionalandenergyrequirementsnecessaryforsurvivalandpro- This creates a mechanism by which extracellular stress can be
liferationwithinahost.Theabilitytomeetthechangingnutri- transducedintointracellularmetabolicsignalsthatelicitregula-
tionalandenergyrequirementsiscomplicatedbythehost,which
limitsaccesstoenzymaticcofactorssuchasoxygenandironby
sequesteringthesecofactorsinhostproteinsandmolecules(e.g., Received21January2014 Accepted28March2014
hemoglobin, heme, and transferrin). The limited availability of Publishedaheadofprint4April2014
thesecofactorscreatesmetabolicblocksthathindervirulencefac- AddresscorrespondencetoGregA.Somerville,[email protected].
torsynthesis(4,5),whichleavesS.aureusvulnerabletothehost’s N.L.andB.Z.areequalcontributors.
immunesystem. Supplementalmaterialforthisarticlemaybefoundathttp://dx.doi.org/10.1128
Thepotentialconsequencesofametabolicblock(s)(i.e.,de- /JB.01475-14.
creasedfecundityandfitnessand/ordeath)createselectivepres- Copyright©2014,AmericanSocietyforMicrobiology.AllRightsReserved.
sure to maintain a functional metabolic state. Over time, this doi:10.1128/JB.01475-14
selectivepressurehasledtotheevolution/acquisitionofmetabo-
2178 jb.asm.org JournalofBacteriology p.2178–2189 June2014 Volume196 Number12
InfluenceofOxygenandIrononS.aureus
tory changes via metabolite-responsive regulators. While the tionconditionandgrowthphase.Bacteriawereharvestedbyusingaster-
transductionofexternalfactorsintointernalmetabolicsignalshas ile0.45-(cid:3)mMicrofilVfiltrationsystem(EMDMilliporeCorporation)
beendemonstratedinS.epidermidis,ithasnotbeenshowninS. thathadbeenprewashedwith5mlofsterilephosphate-bufferedsaline
aureus. (PBS)(pH7.4).Followingfiltration,bacteriawerewashedtwicewith5ml
Two important enzymatic cofactors that influence S. aureus ofice-coldPBS,andeachmembranewastransferredintoa50-mlconical
tubeandquenchedinliquidnitrogen.Afterquenching,theconicaltubes
fitnessandthatarecontrolledbythehostareoxygenandiron.
wereplacedinice,andbacteriawerecollectedfromthefilterbyusing
Numerousstudieshaveshownthatoxygenandironavailability
ice-cold20mMphosphatebuffer(1ml).Thebacterialsuspensionswere
can influence growth, antibiotic tolerance, and the synthesis of
normalizedtoequalOD units(10for1DNMRand20for2DNMR)in
virulencefactors(19–23);however,littleregardhasbeengivento 600
a1-mltotalvolume,transferredintolysingmatrixBtubes,andlysedtwice
themetabolicchangesthataccompanyironandoxygenlimitation
(speed6for40s)byusingaFastPrepFP120instrument(MPBiomedi-
(24–26).Wherethemetaboliceffectsofironoroxygenlimitation cals).Thesampleswererestedonicefor5minbetweenlysings.Toremove
havebeenexamined,thosestudiesaddressedonlythemetabolic celldebrisandglassbeads,sampleswerecentrifugedat17,000(cid:2)gfor2
effects of a single stress and in a single growth phase/state. The minat(cid:4)9°C,and700(cid:3)lofthesupernatantfromeachtubewastrans-
analysis of a single growth phase/state overlooks the fact that ferredintoanew2-mlmicrocentrifugetube(precooledto(cid:4)20°C).One
staphylococcihavetwoverydistinctmetabolicstatesthatcorrelate milliliterofice-coldphosphatebufferwasaddedtotheresidualsamplein D
with the exponential and post-exponential growth phases (27). thelysingmatrixBtubes,mixed,andcentrifuged.Afterthesecondcen- o
Because pathogenic bacteria rarely encounter a single stress, we trifugation,900(cid:3)lofthesupernatantfromeachtubewaspooledwiththe w
n
samplesfromthefirstcentrifugation.Ofthe1.6-mlvolumeforeachsam-
cghenoselitmoietxaatimonineotnheeexffpeoctnoenftcioaml-bianneddsptroessts-eesxopfoinreonntiaanl-dpohxayse- ple,100(cid:3)lwasremoved,andtheproteinconcentrationwasdetermined loa
byusingamodifiedLowryassaykit(ThermoFisherScientific).There- d
msinegtalebsotlroemsseess.(Ii.ne.a,dirdointioorno,xwyegeanlsolimasisteastsioedn)tthoetchoenotvreibrualtlioefnfescotsf amdadiendinpgri1o.r5tmoalnoaflyesaicsh.Asatomtaplleofw5a0s(cid:3)lyMop3h-(iltirziemde,tahnydlsi6ly0l0)p(cid:3)rlopoifoDn2icOacwida-s ed f
ofcombinedstresses(i.e.,ironandoxygenlimitation).Finally,by 2,2,3,3–d4(TMSP-d4)or500(cid:3)MTMSP-d (Sigma-Aldrich)wasadded ro
analyzingthemetaboliceffectsofsinglestresses,wecanidentify toeachsampleastheinternalstandardfor41D1HNMRor2D1H-13C m

commonmetabolicsignals,similartowhatwasdonewithS.epi- HSQCexperiments,respectively. h
t
dermidis(17,18).Inthecurrentstudy,theeffectsofoxygenand Datacollection.1Ddatacollectionwasperformedasdescribedpre- tp
ironlimitationonthemetabolomeofS.aureusstrainSA564were viously(30),while2Dtimezeroextrapolated1H-13CHSQC(HSQC0) ://
examinedbyusingnuclearmagneticresonance(NMR)metabo- NMRspectrawereprocessedasdescribedpreviously(31).Foreachsam- jb
lomics.Inaddition,aselectsetofgeneswasexaminedfortran- ple,atotalof1,024datapointswithaspectrumwidthof10.00ppminthe .a
scriptional changes that might be associated with the altered 1H dimension and a total of 64 data points with a spectrum width of sm
140.00ppminthe13Cdimensionwerecollected.Atotalof16dummy
growthconditions. .o
scansand64scanswithareceivergainof9,195.2andarelaxationdelayof
r
MATERIALSANDMETHODS 1.5swereappliedforeachsample. g/
Dataanalysis.1D1HNMRspectrawereprocessedasdescribedpre- o
Bacterial strains, media, and cultivation conditions. S. aureus strain viously (18). 2D 1H-13C HSQC spectra were processed by using the n
SA564wasgrownintrypticsoybroth(TSB;BDBiosciences)orontryptic M
NMRPipesoftwarepackage(32).Peakpickingandpeakmatchingwere
soyagar(TSA).Forironlimitationcultivationconditions,deferratedTSB a
donebyusingNMRViewJversion8(33).Atableofpeaksalongwiththe
(DTSB)mediumwasused(17).Inthisstudy,iron-limitedmediumwas r
respectiveintensitieswererecorded,andtheconcentrationsofmetabo- c
preferredover“iron-free”mediumbecauseiron-freemediumrequires h
theadditionofadulterantssuchasdeferoxaminemesylate.Theseadulter- liteswerecalculatedbyusingastandardcurvegeneratedbyusingnine 3
a(2n8ts),awrehiacdhdmedaktoescchoemlatpearirisoonn;shboewtweveeern,cthueltyivaaltsioonchceolnatdeitoiothnesrimcaptiroanc-s mglyectainbeo-l1i3teCs2w,iDthL-kanlaonwinnec-o3n-1c3eCn,trasotidoinusm[D-pgylurucvoasete--1133CC63,,D-sfurucccitnoisce-a13cCid6-, 0, 2
ticalbecausemorethanonevariablewouldbechanged.Topreventpy- 13C4,sodiumacetate-13C2,2-keto-3-(methyl-13C)-butyricacid-4-13C,so- 01
rolysisoflabilemetabolites,DTSBandTSBwerepreparedbyfiltersteril- diumsalt,and2-ketobutyricacid-4-13Csodiumsalthydrate(Sigma-Al- 9
ization. For iron-limiting conditions, bacterial cultures were grown drich)] (31). A Student t test was used to determine the statistical b
overnightin10mlofDTSB,harvestedbycentrifugationat3,795(cid:2)g, significance(P(cid:5)0.05)ofmetaboliteconcentrationchanges. y
washedoncein10mlofDTSB,andsuspendedinfreshDTSB.Thisculture Theprincipalcomponentanalysis(PCA)andorthogonalprojections gu
was used to inoculate the starter cultures at a 1:100 dilution, and the tolatentstructures-discriminantanalysis(OPLS-DA)scoreplotsandthe e
startercultureswerecultivatedin100mlofDTSBorTSBfor2h.After2 shared and unique structure (SUS) plots were generated by using the st
hofgrowth,thesecultureswereusedtoinoculatetheprimaryculturesto SIMCA12.0(cid:6)statisticalpackage(Umetrics).TheOPLS-DAmodelswere
anopticaldensityat600nm(OD )of0.06.Eachprimaryculturewas validatedbyusingamodifiedleave-one-outmethod(34,35)andanalysis
600
cultivatedinprewarmedDTSBorTSBatdefinedflask-to-mediumratios. ofvarianceofcross-validatedresiduals(CV-ANOVA)(36).Themetabo-
Allculturesweregrownat37°Cwithconstantaeration(225rpm)and lomicstreediagrams(dendrograms)andtheellipsescorrespondingtothe
growth(OD ),andpHsweremeasuredathourlyintervalsfor12h.For 95%confidencelimitsfromanormaldistributionforeachclusterwithin
600
thepurposeofchangingcultureaerationduringgrowth,flask-to-medium thePCAscoreplotsweregeneratedbyusingourPCA/PLS-DAutilities
ratios(vol/vol)of10:8,10:4,10:2.5,and10:1wereused.Thisapproach (http://bionmr.unl.edu/pca-utils.php)(37,38).
takesadvantageofthepoorcoefficientofdiffusionofoxygenintowaterto Datainterpretation.Thepeakswereassignedtometabolitesbyusing
altertheamountofoxygenavailabletothebacteria(29). chemical shift references from the Human Metabolomics Database
SamplepreparationforNMRmetabolomicanalysis.Bacterialcul- (HMDB)(39),thePlatformforRIKENMetabolomics(PRIMe)(40),the
tureswereharvestedduringtheexponential(2h)andpost-exponential(6 BiologicalMagneticResonanceDataBank(BMRB)(41),andChenomx
h)growthphases.Forone-dimensional(1D)1HNMRexperiments,eight NMRSuite7.6software(Chenomx,Inc.,Edmonton,Canada).Heatmaps
biologicalreplicates(10OD unitseach)wereharvestedforeachculti- weregeneratedinRwithagplotspackage(42).
600
vationconditionandgrowthphase.Fortwo-dimensional(2D)1H-13C ICP-MSanalysisofDTSB.CultivationofS.aureusstrainSA564was
heteronuclearsinglequantumcoherence(HSQC)experiments,fourbio- performedasdescribedabove.Forthedeterminationofironconcentra-
logicalreplicates(20OD unitseach)wereharvestedforeachcultiva- tionsintheculturesupernatants,1mlofeachculturewasharvesteddur-
600
June2014 Volume196 Number12 jb.asm.org 2179
Ledalaetal.
TABLE1Real-timeRT-PCRprimersusedinthisstudy RESULTS
Primer Mu50ORFa Sequence GrowthofS.aureusunderconditionsofironandoxygenlimi-
tation.Thetransitionfromanoxygen-andiron-repleteenviron-
16SrRNARTF SAVrRNA16 CGTGCTACAATGGACAATACAAA
16SrRNARTF SAVrRNA16 ATCTACGATTACTAGCGATTCCA ment(e.g.,theskinandthenares)toanoxygen-andiron-limited
citBRTF SAV1350 GCGCAACAGCAACTGATTTA environment (e.g., blood) constrains metabolic possibilities be-
citBRTR SAV1350 GTTGTACACCTGGACCAAAGA causemanyenzymaticreactionsrequireironandoxygenascofac-
feoBRTF SAV2584 GGAATGACAGCAACACAGTTAC tors.Theseconstraintshavefitnessconsequences,specificallythe
feoBRTR SAV2584 GTGCTGACTGACCACCTAAA abilitytoplaceprogenyintothenextgeneration(44–46).Toassess
sbnARTF SAV0116 TTCTGTAGGGCAAACACCTATG the extent of the fitness cost of iron and oxygen limitation on
sbnARTR SAV0116 GCTGCCTCCAGGATTCATATAC
growth yield, S. aureus strain SA564 was cultivated under iron-
sstCRTF SAV0735 GACCTAATGGTGCGGGTAAG
and/or oxygen-limited conditions, and growth was monitored
sstCRTR SAV0735 CAGACATGAGCTGTCCATCTATT
(Fig.1AandB).Asexpected,thegrowthrateswerelargelyinde-
aGenedesignationsbasedontheStaphylococcusaureusstrainMu50genomicDNA
pendent of iron- and/or oxygen-limited conditions. Consistent
sequence(68).ORF,openreadingframe.
with the Pasteur effect (47), oxygen-limited growth conditions
dramaticallydecreasedbiomassgeneration,whereasironlimita- D
o
tiondidnotsignificantlyalterthebiomassgeneratedpermmolof w
ingtheexponential(2h)orpost-exponential(6h)growthphasebycen- glucose (Fig. 1C). Importantly, these data demonstrate the ease n
trifugationat3,795(cid:2)gfor5minatroomtemperature.Toremoveany withwhichabatchculturecanbecomemicroaerobic/anaerobicat lo
a
residual bacteria, samples were passed through Nalgene 0.2-(cid:3)m poly- anatmosphericoxygenconcentrationof20.946%(29)andwith d
ethersulfone membrane syringe filters (Thermo Fisher Scientific). All vigorousagitationat225rpm.Thistransitiontoamicroaerobic/ ed
sampleswerediluted1:20withasolutioncontaining52.6ppb71Gain anaerobic status is due to two factors: first, the poor diffusion f
0.1%HNO3,whichresultedina71Gaconcentrationof50ppb,whichwas coefficientforoxygenintowater,andsecond,therelativelysmall ro
usedasaninternalstandard.Inductivelycoupledplasmamassspectrom- m
etry(ICP-MS)analyseswereperformedattheUniversityofNebraska— surfaceareathatisexposedtoatmosphericoxygenasthevolume h
Lincoln Spectroscopy Core Facility by using an Agilent Technologies ofmediuminacultureflaskisincreased.Incontrasttooxygen tt
p
limitation, iron limitation decreased the growth yield of strain
ICP-MS7500csinstrumentwithanESISC-4high-throughputautosam- :
pler(ElementalScientific,Inc.).Theconcentrationofmetalswascalcu- SA564 by only (cid:8)25% relative to growth in iron-replete culture //jb
latedbyusingChemStationforICPsoftwarewithacalibrationcurvefor medium(Fig.1A).InS.aureus,thedemandforironisgreatestin .a
18elementsfromserialdilutionsofastandardstockfromInorganicVen- the post-exponential growth phase (5), when the tricarboxylic s
m
tures.Theinstrumentwasoperatedwithanoctopolecollisionreaction acid(TCA)cycleandtheelectrontransportchainaremostactive;
.
cellfilledwithHeataflowrateof5.0ml/minandthefollowingArplasma hence,thesmallamountofironintheculturemedium(Fig.1B)is o
r
conditions:plasmapowerof1,500W,plasmagasat17liters/min,auxil- sufficient to maintain the exponential growth rate but not the g/
iarygasat1liter/min,samplegasat0.9to1.1liters/min,makeupgasat0.1 growthyield(Fig.1A). o
to0.2liters/min,andasampleflowrateof0.1ml/min.Foreachsample, n
S. aureus is a facultative anaerobe and is well adapted for
threereplicatesfromthreeindependentcultureswereanalyzed.Statistical M
significancewasdeterminedbyone-wayANOVAusingSigmaPlot11.2 growth in low-oxygen environments when nutrients are abun- a
software(SystatSoftware,Inc.). dant. As nutrients become scarce, S. aureus catabolizes incom- rc
h
Aconitaseactivityassay.Aconitaseenzymaticactivitywasmeasured pletelyoxidizedcarboncompounds(e.g.,lacticacidoraceticacid)
3
asdescribedpreviously(17).Proteinconcentrationsweredeterminedby that accumulate in the medium during the exponential growth 0
usingamodifiedLowryproteinassaykit(ThermoFisherScientific). phase(27);hence,thepHoftheculturemediumwillalkalinize.As ,
2
Real-timequantitativeRT-PCR(qRT-PCR).TotalRNAwasisolated expected, strain SA564 grown under oxygen- and iron-replete 0
1
from10OD600unitsofbacteriaharvestedintheexponential(2h)and conditions began to alkalinize the culture medium during the 9
post-exponential(6h)growthphasesbyusingtheFastRNAProBluekit transitionfromtheexponentialgrowthphasetothepost-expo- b
(MPBiomedical)andtheRNeasykit(Qiagen),asdescribedpreviously y
nentialgrowthphase(Fig.1D).Iron-limitedgrowthslightlyde-
(43).FormRNAanalysis,atwo-stepreal-timereversetranscription-PCR g
layed and decreased the alkalinization of the culture medium, u
(RT-PCR)wasperformedwithcDNAsynthesisfromtotalRNAfollowed while oxygen limitation severely restricted alkalinization of the e
byPCR.TenmicrogramsofTurboDNase(Bio-Rad)-treatedtotalRNA s
medium. t
for each sample was used to prepare cDNA by a reverse transcriptase
Incontrasttopreviousspeculation,ironlimitationdoesnot
reactionusingiScriptmastermix(Bio-Rad)accordingtothemanufac-
turer’sprotocol.Thereactionmixturesforreal-timePCRcontained10(cid:3)l lead to the acidification of the culture medium (26). As stated
of 2(cid:2) SsoAdvanced SybrGreen Supermix (Bio-Rad), 7 pmol of each above,theacidificationoftheculturemediumistheresultofthe
primer(Table1),and5(cid:3)lof10-fold-dilutedcDNA,inatotalvolumeof inefficient use of carbohydrates during growth under nutrient-
20(cid:3)l.Asaninternalreference,primersfor16SrRNAwereusedinthe rich conditions, which occurs irrespective of the availability of
samereactionvolumecontaining1,000-fold-dilutedcDNAasatemplate. iron or oxygen (Fig. 1C to F). The decreased ability of oxygen-
Thereal-timePCRcyclingconditionswereaninitialdenaturationstepat limited cultures and, to a lesser extent, iron-limited cultures to
94°Cfor3min,followedby40cyclesat94°Cfor15s,60°Cfor25s,and
alkalinizethemediumwasmostlikelyduetoadiminishedcapac-
72°Cfor20s.Relativetranscriptlevelsweredeterminedbythecompara-
itytocatabolizeorganicacidsandaminoacids.Toaddressthis
tivethreshold((cid:7)(cid:7)C )method(Bio-Rad).Experimentalsetupanddata
T possibility,theconcentrationsofaceticacidandlacticacidinthe
analysiswerecarriedoutbyusingaCFX96real-timePCRdetectionsys-
culturemediaweremeasuredthroughoutthegrowthcycle(Fig.
temandBio-RadCFXManagersoftwareversion3.0(Bio-Rad).
Determinationofacetate,glucose,andlactatelevelsinmediumsu- 1EandF).Duringmicroaerobic/anaerobicgrowth(i.e.,whenthe
pernatant.Metaboliteconcentrationsintheculturemediaweredeter- flask-to-mediumratiois10:8),pyruvateislargelyreducedtolactic
minedbyusingkitspurchasedfromR-biopharm,Inc. acid (48, 49) (Fig. 1F). In contrast, when the flask-to-medium
2180 jb.asm.org JournalofBacteriology
InfluenceofOxygenandIrononS.aureus
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FIG1CultivationofS.aureusstrainSA564underaeration-and/oriron-limitingconditions.(A)Growthcurves.(B)Ironconcentrationsofuninoculatedmedia
andcultivationmediaharvestedintheexponentialandpost-exponentialgrowthphases.(C)Glucoseutilizationasafunctionofgrowth.(D)pHprofiles.(E)
Acetateaccumulationanddepletioninculturemedia. (F) Lactateaccumulationanddepletioninculturemedia.Dataarerepresentativeofexperiments
performedatleasttwice.ThedatainpanelBarepresentedasthemeansof3independentexperimentsdoneintriplicate,witherrorbarsrepresentingstandard
deviations.TSB,trypticsoybroth;DTSB,deferratedTSB.Flask-to-mediumratioswere10:1(aerobic)and10:8(microaerobic/anaerobic).
June2014 Volume196 Number12 jb.asm.org 2181
Ledalaetal.
ratiowasalteredto10:1,pyruvatewasoxidizedtoacetylcoenzyme
A(acetyl-CoA)andCO bythepyruvatedehydrogenasecomplex
2
(50)(Fig.1E).Intheexponentialgrowthphase,acetyl-CoAisused
to generate the small phospho-donor acetyl-phosphate, which
servesasasubstrateforacetatekinaseinsubstrate-levelphosphor-
ylationtogenerateATPandaceticacid(Fig.1E).Duringthepost-
exponential growth phase, this acetic acid can be used for the
synthesis of acetyl-CoA to fuel the TCA cycle. When iron was
limiting,thecatabolismofacetateandthebiomassgeneratedfrom
thatcatabolismwerereduced(Fig.1E).Asexpected,oxygenlim-
itationinhibitedthecatabolismofacetate;however,ithadonlya
slighteffectonlactatecatabolism(Fig.1EandF).Thelatterdata
weresurprisingbecauseacetatecatabolismthroughtheTCAcycle
(27) and lactate catabolism through L-lactate-quinone oxi-
doreductase(Lqo)(51)bothrequireelectronacceptorstobalance D
theredoxstatus. o
w
Ironandoxygenlimitationscreatemetabolicblocks.Staph-
n
ylococcilackaglyoxylateshuntandcatabolizeacetatethroughthe lo
TCAcycle(27,52);hence,adiminishedabilitytocatabolizeace- a
d
tate (Fig. 1E) suggests that the TCA cycle had reduced activity e
d
underiron-andoxygen-limitedgrowthconditions.Totestthis
f
suggestion, the TCA cycle enzyme aconitase was assayed for ro
activity under iron- and oxygen-limited growth conditions m

(Fig.2AandB).Sinceaconitaseisaniron-sulfurcluster-con- h
t
tainingenzyme,ironlimitationwouldbeexpectedtodecrease tp
its activity. As expected, growth of S. aureus strain SA564 in :/
/
iron-limitedculturemediumdramaticallydecreasedpost-expo- jb
.
nential-growth-phaseaconitaseactivity(Fig.2A).Asstatedabove, a
s
onefunctionofironistofacilitateelectrontransferreactions,a m
process that requires an electron donor (e.g., NADH and .o
NADPH)andanelectronacceptor(e.g.,oxygen).Inthepresence rg
of iron but in the absence of an electron acceptor, reduced di- /
o
nucleotideswillaccumulate,whichshouldinhibitenzymeactivity n

throughafeedbackmechanism.Totestthishypothesis,welimited M
oxygen availability by changing the flask-to-medium ratio and a
r
assayedforaconitaseactivity(Fig.2B)(29).Asthemediumvol- c
h
umewasincreased,thesurfaceareaavailableforoxygendiffusion
3
decreased,resultinginapost-exponential-growth-phasedecrease 0
,
in aconitase activity. While aconitase is an excellent metabolic 2
0
sentinel,ironandoxygenlimitationwillaffecttheactivityofmany
1
enzymesinbacteria. FIG2Temporalandstress-relatedaconitaseactivitydifferencesinS.au- 9
Ironand/oroxygenlimitationalterstheS.aureusmetabo- reusstrainSA564intheexponential(2h)andpost-exponential(6h)growth b
phases.(A)Aconitaseactivityduringiron-limitedgrowth.(B)Aconitaseac- y
lome.Iron-andoxygen-limitedgrowthcreatedametabolicblock
tivitywithdecreasingaerationflask-to-mediumratiosof10:8,10:5,10:2.5,and g
at aconitase in the TCA cycle (Fig. 2A and B); however, these 10:1.(C)AdditionofirontoDTSBmediumrestoresaconitaseactivity. u
e
stressesalsoinhibittheactivitiesofmanydifferentmetabolicen- s
t
zymesandpathways.Itisimpracticaltoassayforallenzymesthat
mightbeaffectedbyiron-and/oroxygen-limitedgrowth,somet- growniniron-limitedmediumandwithreducedaerationclus-
abolicchangeswereassessedbyusingNMRmetabolomics(Fig. tered more closely to the aeration-limited cultures than to the
3).Asexpected,principalcomponentanalysis(PCA)ofexponen- iron-limitedculturesinaPCAscoreplot(Fig.3B).Inotherwords,
tial-growth-phase metabolomes revealed that they were mini- theeffectofironlimitationwasmaskedbygrowthundercondi-
mally affected by iron limitation, whereas oxygen limitation tionsthatlimitthediffusionofoxygenintotheculturemedium.
causedmodestchangesinthemetabolomes(Fig.3AandC).The Thiscanalsobeeasilyseeninthemetabolicheatmap,wherethe
heterogeneity of the exponential-growth-phase metabolomes is metabolic changes in post-exponential-growth-phase (6 h) cul-
easilydemonstratedinaPCAsharedanduniquestructure(SUS) turesgrownunderconditionsofaerationlimitationareverysim-
plot,whereveryfewsharedorinverselysharedmetaboliteswere ilar,irrespectiveofironlimitation(Fig.4).Takentogether,these
identified(Fig.3E).Incontrasttotheexponentialgrowthphase, datademonstratethatthesignificanceofironlimitationinS.au-
ironlimitationsignificantlyalteredthepost-exponential-growth- reusmetabolismisdeterminedlargelybythegrowthphaseandthe
phasemetabolomePCAscoreplot(Fig.3B,D,andF).Similarly, availabilityofoxygen.
oxygenlimitationhadaverypronouncedeffectonthemetabo- Toidentifymetabolitesthatcontributedtotheseparationof
lome. Interestingly, strain SA564 metabolomes from cultures thesampleclusters,OPLS-DAwasemployed(seeFig.S1andS2in
2182 jb.asm.org JournalofBacteriology
InfluenceofOxygenandIrononS.aureus
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FIG3Exponential-andpost-exponential-growth-phasemetabolomicchangesassociatedwithiron-and/oroxygen-limitedcultivation.(AandB)PCA rc
scoreplotsoftheexponential(A)andpost-exponential(B)growthphasesofiron-and/oroxygen-limitedculturesofS.aureusstrainSA564.Theellipses h

inthePCAscoreplotscorrespondtothe95%confidencelimitsfromanormaldistributionforeachcluster.(CandD)MetabolictreegeneratedbyusingthePCA 3
scoreplotdatademonstratingtherelationshipofiron-and/oroxygen-limitedculturesintheexponentialgrowthphase(C)andpost-exponentialgrowthphase 0
,
(D).ThestatisticalsignificanceofeachnodeinthemetabolictreeisindicatedbyaPvalue.(EandF)Sharedanduniquestructure(SUS)plotsgeneratedfromthe 2
PCAmodeloftheexponential(E)andpost-exponential(F)growthphasescomparingtheclassesofDTSBata10:1ratioandTSBat10:8toacommonreference 0
(TSB,10:1).Theredboxeshighlightthechemicalshiftbinswithintensitychangesthataresharedbybothmodels.Theblueboxeshighlightthechemicalshiftbins 1
9
withintensitychangesthatarenegativelysharedbybothmodels(relativeintensitychangesareintheoppositedirection).Thebrownboxeshighlightthechemical
b
shiftbinswithintensitychangesuniquetomodel1(TSBat10:8versusTSBat10:1).Thegreenboxeshighlightthechemicalshiftbinswithintensitychanges y
uniquetomodel2(DTSBat10:1versusTSBat10:1).Inthepost-exponentialgrowthphase,73binswerefoundtobepresentintheshared/inverselysharedregion
g
of the plot, compared to 29 bins found in the same regions in the exponential phase. The flask-to-medium ratios were 10:1 (aerobic) and 10:8 u
(microaerobic/anaerobic). e
s
t
the supplemental material). In contrast to PCA, OPLS-DA is a tionscausedbyironlimitationwerelargelysuppressedwhenaer-
supervisedclassificationanalysiswhereallthevariationleadingto ation was also limiting. As expected, the majority of the
separationbetweenthetwogroupsisalignedinthexaxis(Pp)and metabolicdifferencesbetweenaerobicgrowthandoxygen-lim-
all other variation is aligned in the y axis (Po) (53). To achieve ited growth include fermentation products, metabolites of
separationbasedonaerationinanOPLS-DAscoringplot,clusters pyruvatehomeostasis,andaminoacids(seeFig.S1inthesup-
ofTSBata10:1dilutionandDTSBat10:1wereassignedavalueof plementalmaterial).Toachieveseparationbasedonironavail-
0,andclustersofTSBat10:8andDTSBat10:8wereassigneda abilityinanOPLS-DAscoreplot,clustersofTSBat10:1and
valueof1(seeFig.S1inthesupplementalmaterial).Thismodel TSBat10:8wereassignedavalueof0,andclustersofDTSBat
resulted in one predictive and two orthogonal (1 (cid:6) 2) compo- 10:1andDTSBat10:8,wereassignedavalueof1(seeFig.S2in
nentswithacross-validatedpredictiveabilityofQ2(Y)(cid:9)0.883, thesupplementalmaterial).Thismodelalsoproducedanex-
whichindicatedthatthedatafitwellwithinthemodel.Themodel cellentfitthatresultedinonepredictiveandthreeorthogonal
wasvalidatedwithCV-ANOVA,producingastatisticallysignifi- (1(cid:6)3)componentswithacross-validatedpredictiveabilityof
cantPvalueof8.27(cid:2)10(cid:4)12.Basedonthismodel,theperturba- Q2(Y)(cid:9)0.969(seeFig.S2inthesupplementalmaterial).This
June2014 Volume196 Number12 jb.asm.org 2183
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FIG4Heatmapsshowingthechangesinmetabolitesasafunctionofcultivationconditionsandgrowthphase.CulturemediawereTSB(trypticsoybroth)and jb
DTSB(deferratedTSB).Flask-to-mediumratioswere10:1(aerobic)and10:8(microaerobic/anaerobic).Statisticalsignificance(P(cid:5)0.05)isindicatedby** .a
(bothoxygenandironlimitation),*(onlyoxygenlimitation),and(cid:6)(onlyironlimitation). s
m
.
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modelwasalsovalidatedbyCV-ANOVA,producingastatisti- growthconditions.Thesedatademonstratethatiron-dependent /
o
callysignificantPvalueof1.33(cid:2)10(cid:4)17.Inthismodel,thefour regulationoftranscriptioninS.aureuscanbesuppressedunder n

metabolomic clusters are largely separated from each other, conditionsoflowoxygenavailability.Inaddition,thesedatahigh- M
suggestingthatoxygenlimitationasastressorcannotbeover- lightthenecessityofcarefullyconsidering,andreporting,bacte- a
r
whelmed by iron limitation. Altogether, the clustering of rialcultivationconditions(29). c
h
metabolomesinbothmodelssuggestedthattheeffectsofiron
3
limitationweresuppressedbyoxygenlimitation. DISCUSSION 0
,
Iron-regulatedgenetranscriptionvarieswithoxygenavail- WhenculturesofStaphylococcusepidermidisarechallengedwith 2
0
ability.Ifoxygenlimitationcanmaskmetabolicchangesassoci- ethanol, antibiotics, iron limitation, or high glucose concentra-
1
atedwithironlimitation,itraisesthequestionastowhetheriron- tions,thesestressescausecommonphenotypicchanges;namely, 9

dependent transcriptional changes can be obscured by oxygen theyfavorbiofilmformationand/orbiosynthesisofpolysaccha- b
y
limitation.Toaddressthisquestion,bacteriaweregrownunder rideintercellularadhesin(56–59).Thesesamestressescausethe
g
iron- and/or oxygen-limited culture conditions, and transcrip- post-exponential-growth-phase metabolomic profiles to resem- u
e
tionalchangesingenesregulatedbyironwereassessedbyusing blethatofanS.epidermidisTCAcyclemutant(17,18).Thefact s
t
qRT-PCR(Fig.5).Asapositivecontrol,transcriptionoftheaco- that divergent environmental challenges produce similar meta-
nitasegeneacnA(alsoknownascitB)wasassessedandcompared bolicandphenotypicalterationssuggeststhatcommonregulatory
to the enzyme activity data (Fig. 2A). Overall, the acnA mRNA changes are occurring to produce the similar phenotypic out-
levelscorrelatedwell(r(cid:9)0.83)withtheenzymaticactivitydata. comes(3).Thissuggestionwasconfirmedwhenitwasobserved
Similartothemetabolomicsdata,oxygenlimitationwasableto thatanS.epidermidisccpAmutantfailedtorespondtometabolic
masktheeffectofiron-limitedgrowthonsomegenes.Specifically, changesassociatedwithTCAcyclestress(7).SimilartoS.epider-
oxygen limitation dramatically decreased the exponential- midis,wehypothesizethatdivergentenvironmentalstressescause
growth-phase (2 h) and post-exponential-growth-phase (6 h) commonmetabolicchangesinS.aureusthataltertheactivityof
transcriptionoftheferrousirontransporterfeoBandthefirstgene metabolite-responsiveregulators.Thefirststepinidentifyingme-
(i.e.,sbnA)ofthestaphyloferrinBbiosyntheticlocus(54).This tabolite-responsiveregulatorsistodefinethecommonmetabolic
reduction in transcript levels occurred despite the fact that the changesassociatedwithenvironmentalstresses(Fig.4and6).The
culturemediumwasironlimited(Fig.1B).IncontrasttofeoBand metabolic effects of many of the environmental stresses that S.
sbnA, some genes (e.g., sstC, which codes for an ATP binding aureusencountershavebeenstudiedindetailbutwithlittle,orno,
cassette in a siderophore transporter [55]) had only minimal deferencetothetemporalnatureandtheinterconnectednessof
changes in mRNA levels under iron- and/or oxygen-limited metabolicchanges(26,60–62).Theintentofthecurrentstudywas
2184 jb.asm.org JournalofBacteriology
InfluenceofOxygenandIrononS.aureus
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FIG5Oxygenlimitationinfluencestranscriptionofiron-regulatedgenesintheexponential(2h)andpost-exponential(6h)growthphases.RelativemRNA
3
levelsforacnA,sbnA,feoB,andsstCweredeterminedbyqRT-PCR.Thedataarethemeansandstandarderrorsofthemeansofatleast2biologicalreplicates,each 0
determinedinduplicate.CulturemediawereTSB(trypticsoybroth)andDTSB(deferratedTSB).Flask-to-mediumratioswere10:1(aerobic)and10:8 ,
2
(microaerobic/anaerobic). 0
1
9

b
y
to identify common metabolic changes associated with iron TCAcyclestressinducestheformationofanN-acetylglucosamine
g
and/oroxygenlimitationthatcouldaltertheactivityofmetabo- polymerknownaspolysaccharideintercellularadhesin(43,64). u
e
lite-responsiveregulators;theidentificationoftheseregulatorsis Together,thesedatasuggestthatTCAcycle-dependentregulation s
t
anactiveareaofresearchinourlaboratories.Asasidebenefit,this ofpolysaccharideintercellularadhesinsynthesisiscommontothe
studyreinforcesthenecessityofcarefullyconsideringandreport- staphylococciandnotjustS.epidermidis.Whileaminosugarbio-
ingcultivationconditions(63). synthesiswasincreasedduringoxygen-andiron-limitedgrowth,
Whilethemostprominentmetabolomicdifferencesareinthe S.aureusacidifiedtheculturemediumirrespectiveofironorox-
post-exponential growth phase, the exponential-growth-phase ygen availability (Fig. 1D). The latter observation is consistent
changesaregreatestunderconditionsofcombinediron-andox- withpreviousobservationsofstaphylococcalcarbohydratecatab-
ygen-limitedgrowth(Fig.3,4,and6).Forexample,bothironand olism(27,65,66)yetisinconsistentwithmorerecentspeculation
oxygenlimitationredirectcarbonintoaminosugarbiosynthesis (26).
(e.g.,UDP-N-acetylglucosamine,glucosamine-6-phosphate,and As stated above, the metabolic perturbations during iron-
acetyl-glucosamine).Thisredirectionofcarbonappearstobeat and/oroxygen-limitedcultivationaregreatestinthepost-expo-
theexpenseoftheexponential-growth-phasebasal-levelTCAcy- nentialgrowthphase(Fig.3AandB).Thesedifferencesarepri-
cle carbon flow because the concentration of succinate is de- marilyaconsequenceofthefollowingthreefactors:(i)theTCA
creased relative to that under iron- and oxygen-replete growth cycleiscataboliterepressedduringtheexponentialgrowthphase
conditions (Fig. 4 and 6). We observed a similar redirection of (27),(ii)derepressionoftheTCAcyclecreatesalargedemandfor
carbon into amino sugar biosynthesis in S. epidermidis, where ironinthepost-exponentialgrowthphase(Fig.1F)(5),and(iii)
June2014 Volume196 Number12 jb.asm.org 2185
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FIG 6Schematic representation of the central metabolic changes associated with iron or oxygen limitation. The black and blue arrows indicate /

exponential/post-exponential-growth-phase(EXP/PE)increasesordecreasesinintracellularconcentrationsofmetabolitesinstrainSA564cultivatedin o
TSBwitha10:8flask-to-mediumratiorelativetoacontrolculturegrowninTSBwithaflask-to-mediumratioof10:1.Theredandorangearrowsindicate n

exponential/post-exponential-growth-phaseincreasesordecreasesinintracellularconcentrationsofmetabolitesinstrainSA564growninDTSBwitha M
flask-to-mediumratioof10:1relativetoacontrolcultureofS.aureusstrainSA564growninTSBwithaflask-to-mediumratioof10:1.Statisticalsignificance a
atthe95%confidencelevel(P(cid:5)0.05)isdenotedbyasterisksabovethearrows.Glucose-1-P,glucose-1-phosphate;FBP,fructose-1,6-bisphosphate;DHAP, rc
dihydroxyacetonephosphate;GAP,glyceraldehyde-3-phosphate;3PG,3-phosphoglycerate;OAA,oxaloacetate;2-KG,(cid:10)-ketoglutarate. h

3
0
,

2
0
TCAcycleactivitygeneratesreducingpotential,whichcreatesa coneogenesis,whichisessentialforgrowthwhenglucosehasbeen
1
needforanelectronacceptor,mostcommonlyoxygen.Onceglu- depletedfromthemedium.Combined,theaminoacidauxotro- 9

coseisdepletedfromtheculturemediumduringgrowthunder phies,alackofgluconeogenesis,andtheinhibitionoftheelectron b
y
iron-andoxygen-repleteconditions(Fig.1B),cataboliterepres- transportchain(i.e.,duetothelackofanelectronacceptor)result
g
sionoftheTCAcyclegenesisrelieved,andenzymaticactivities inadecreasedgrowthyield(Fig.1A). u
e
increase(Fig.2AandBand5).Incontrast,whenS.aureuscellsare Inhibitionofelectrontransportforcesbacteriatousealterna- s
t
cultivatedunderiron-and/oroxygen-limitedconditions,levelsof tivepathwaystooxidizedinucleotides;however,thesealternatives
transcriptionofTCAcyclegenesandenzymaticactivityremain are strongly dependent upon carbon availability. When carbon
lowdespitethedepletionofglucose(Fig.1,2A,and5).Although sourcesthatcangeneratepyruvate(e.g.,glucoseandserine)are
oxygenandironarebothimportantforpost-exponential-phase abundantinthemedium,bacteriacanmaintainredoxbalanceby
growth(Fig.1A),theavailabilityofoxygenisthemajordetermi- usingenzymeslikelactatedehydrogenase,whichleadstotheac-
nantofthemetabolome(Fig.3Band4).Theabsenceofoxygen cumulationoflacticacidinthemedium(Fig.1Fand4).Inaddi-
preventscarbonflowthroughtheTCAcycle,decreasingthecon- tiontolactatedehydrogenase,S.aureuscanoxidizedinucleotides
centrationsofbiosyntheticintermediatesandtheaminoacidsde- throughalaninedehydrogenase,aresultconsistentwiththepost-
rivedfromtheseintermediates(Fig.2AandBand3);specifically, exponential-growth-phase accumulation of D-alanyl–D-alanine
levels of citric acid, succinate, (cid:10)-ketoglutarate, glutamate, glu- andalanine.Thesedatasuggestthatasreadilycatabolizablecar-
tamine,aspartate,andasparaginearedecreased(Fig.4and6).In bonsourcesarebeingdepletedfromthemedium,severalpath-
other words, the decreased carbon flow through the TCA cycle waysareusedtooxidizedinucleotides,resultinginanaccumula-
resultsinmultipleaminoacidauxotrophies.Inaddition,thede- tion of NAD(cid:6) (Fig. 4). While oxidation of dinucleotides is
creasedcarbonflowthroughtheTCAcyclelikelydecreasedthe important, it is only one part of redox homeostasis, the other
availabilityofoxaloacetate/phosphoenolpyruvate(PEP)forglu- beingreduction.
2186 jb.asm.org JournalofBacteriology
InfluenceofOxygenandIrononS.aureus
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tentwiththeoxygen-limitedaccumulationofdihydroxyacetone 10. PohlK,FrancoisP,StenzL,SchlinkF,GeigerT,HerbertS,GoerkeC,
(thephosphateislabileandcanbelostduringharvestandsample SchrenzelJ,WolzC.2009.CodYinStaphylococcusaureus:aregulatory
linkbetweenmetabolismandvirulencegeneexpression.J.Bacteriol.191:
preparationtogeneratethemorestabledihydroxyacetone)(Fig.
2953–2963.http://dx.doi.org/10.1128/JB.01492-08.
4).WhencarbonflowthroughtheTCAcycleandpentosephos-
11. DeutscherJ,KusterE,BergstedtU,CharrierV,HillenW.1995.Protein
phatepathwayisdecreased,theabilitytogeneratereducingpo- kinase-dependentHPr/CcpAinteractionlinksglycolyticactivitytocar-
tentialviatheNAD(P)H-dependentglycerol-3-phosphatedehy- boncataboliterepressioningram-positivebacteria.Mol.Microbiol.15: D
drogenase (Fig. 4) can be important for redox homeostasis and 1049–1053.http://dx.doi.org/10.1111/j.1365-2958.1995.tb02280.x. o
12. SeidlK,StuckiM,RueggM,GoerkeC,WolzC,HarrisL,Berger-Bächi w
biosynthesis.Thatbeingsaid,thismetabolicrearrangementisin-
B,BischoffM.2006.StaphylococcusaureusCcpAaffectsvirulencedeter- n
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metabolome than does the availability of iron (Fig. 3B). In the acocltii.vJa.tBoracptreortioeiln.1r9eg0u:6l5at9e8N–-6a6c0e8ty.lhmttupr:a//mdxic.daocii.docrga/t1ab0o.1l1is2m8/iJnBE.0s0ch64er2i-c0h8ia. fro
absenceofanelectronacceptor,ironisnotneededforelectron 14. ShiversRP,SonensheinAL.2004.ActivationoftheBacillussubtilisglobal m

transport;hence,thedemandforironislower.Thisalsoexplains regulatorCodYbydirectinteractionwithbranched-chainaminoacids. h
whyoxygenavailabilityaltersthetranscriptionofgenesinvolved Mol.Microbiol.53:599–611.http://dx.doi.org/10.1111/j.1365-2958.2004 ttp
inironacquisition(Fig.5).Inessence,decreasedoxygenavailabil- .04135.x. :/
itysuppressestheiron-sparingresponse,aphenomenonsimilar 15. RomlingU,GalperinMY,GomelskyM.2013.Cyclicdi-GMP:thefirst25 /jb
yearsofauniversalbacterialsecondmessenger.Microbiol.Mol.Biol.Rev.
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tothatseeninSaccharomycescerevisiae(67). 77:1–52.http://dx.doi.org/10.1128/MMBR.00043-12. a
s
16. ErnstJF,BennettRL,RothfieldLI.1978.Constitutiveexpressionofthe m
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G.A.S.wassupportedbyfundsprovidedthroughtheHatchActtothe 17. Sadykov MR, Zhang B, Halouska S, Nelson JL, Kreimer LW, Zhu Y, g
University of Nebraska Institute of Agriculture and Natural Resources PowersR,SomervilleGA.2010.UsingNMRmetabolomicstoinvestigate o/
andbyfundsprovidedthroughtheNationalInstitutesofHealth(grant tricarboxylicacidcycledependentsignaltransductioninStaphylococcus n
AI087668) to G.A.S. and R.P. R.P. was supported by funds provided epidermidis.J.Biol.Chem.285:36616–36624.http://dx.doi.org/10.1074 M
throughtheNIH(grantsP20RR-17675andP30GM103335)andfrom /jbc.M110.152843. a
theAmericanHeartAssociation(grant0860033Z).Portionsofthisre- 18. ZhangB,HalouskaS,SchiaffoCE,SadykovMR,SomervilleGA,Powers rc
searchwereperformedinfacilitiesrenovatedwithsupportfromtheNIH R.2011.NMRanalysisofastressresponsemetabolicsignalingnetwork.J. h
(grantRR015468-01). ProteomeRes.10:3743–3754.http://dx.doi.org/10.1021/pr200360w. 3
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