APPLIEDANDENVIRONMENTALMICROBIOLOGY,Nov.2010,p.7559 Vol.76,No.22 0099-2240/10/$12.00doi:10.1128/AEM.01126-10 Copyright2010,AmericanSocietyforMicrobiology.AllRightsReserved.ExploringtheLow-PressureGrowthLimit:Evolutionof Bacillussubtilis intheLaboratorytoEnhancedGrowthat5KilopascalsWayneL.Nicholson,1*PatriciaFajardo-Cavazos,1JeffreyFedenko,1JoseL.Ortz-Lugo,1AndreaRivas-Castillo,1SamanthaM.Waters,1andAndrewC.Schuerger2DepartmentsofMicrobiologyandCellScience1andPlantPathology,2UniversityofFlorida,KennedySpaceCenter,Florida32899Received10May2010/Accepted15September2010Growthof Bacillussubtilis cells,normallyadaptedatEarth-normalatmosphericpressure( 101.3kPa),was progressivelyinhibitedbyloweringofpressureinliquidLBmediumuntilgrowthessentiallyceasedat2.5kPa. Growthinhibitionwasimmediatelyreversibleuponreturnto101.3kPa,albeitataslowerrate.Apopulation of B.subtilis cellswascultivatedatthenear-inhibitorypressureof5kPafor1,000generations,whereastepwise increaseingrowthwasobserved,asmeasuredbytheturbidityof24-hcultures.Anisolatefromthe1,000generationpopulationwasobtainedthatshowedanincreaseintnessat5kPawhencomparedtotheancestral strainorastrainobtainedfromaparallelpopulationthatevolvedfor1,000generationsat101.3kPa.The resultsfromthispreliminarystudyhaveimplicationsforunderstandingtheabilityofterrestrialmicrobesto growinlow-pressureenvironmentssuchasMars. Microorganismsgrowoptimallywithinacharacteristicrange offundamentalphysicalparameterssuchastemperature,osmolarity,pH,andpressure.Cellsthatcangrowattheextreme limitsoftheseparametersareknowncollectivelyasextremophiles(reviewedinreference8).Thus,inextremeenvironmentsonEarththereexisthalophilesinhypersalineniches, psychrophilesandthermophilesinextremecoldandhotenvironments,andacidophilesoralkaliphilesinenvironmentsof extremeacidicorbasicpH.Regardingextremesofpressure, piezophiles(barophiles)havebeenisolatedfromhigh-pressure (hyperbaric)submarineenvironmentsandhavebeenstudied ratherextensively;inaddition,highpressurehasbeenshownto exertlethalandinhibitoryeffectsonvariousmicrobialsystems notnormallyadaptedtohighpressure(reviewedinreference 19).Ontheoppositeextreme,therehasbeenverylittleinvestigationconcerningthesurvivalandgrowthofmicrobesunder conditionsofextremelylowatmosphericpressure(hypobaria), primarilybecausesuchenvironmentsdonotexistinnatureon theEarthâ€™ssurface.However,someinvestigatorsintheemergingeldofastrobiologyhavebecomeconcernedwithmicrobial survivaland/orgrowthatlowpressuresbecause(i)theclosest potentiallife-bearingplanet,Mars,containsalow-pressure atmosphere,and(ii)thepossibilityexiststhatterrestrialmicrobescouldbetransferredfromEarthtoMarsasaresultof naturalimpacts(23)orhumanspaceightactivities(24).Most oftheexperimentsconductedhaveconcentratedontestingthe abilityofterrestrialmicrobesmerelytosurviveintheMars surfaceenvironment(reviewedinreferences6,23,and28); relativelyfewexperimentshavetestedtheabilityofspecic Earthmicrobestogrowormetabolizeatreducedpressure(12, 14).Wepreviouslyreportedthatgrowthofatleast37different microorganismsonsemisolidagarmediumwasinhibitedat pressuresapproaching 2.5to3.5kPa(3,4,27,30).(Notethat atmosphericpressuresatthesurfacesofEarthandMarsaverage 101.3kPaand 0.7kPa,respectively.)These observationssuggestthatthereexistsalow-pressurebarrierto thegrowthofterrestrialmicrobes.Wereasonedthatoneexperimentalapproachtostudyingthecellulartarget(s)andmolecularmechanism(s)involvedintheprokaryoticresponseto hypobaricstresswouldbetousereducedpressureasaselectiveconditiontoisolateandcharacterizemutantstrainsthat hadevolvedanenhancedcapabilityforgrowthatlowpressure; therstresultsoftheseexperimentsarepresentedinthis communication.MATERIALSANDMETHODS Bacterialstrains,media,andgrowthconditions. Theancestralstrainusedin thisstudywasourcommonlaboratorystockof Bacillussubtilis,strain168. Congenic B.subtilis strainsWN624(trpC2amyE::spc)andWN628(trpC2 amyE::cat)werederivedfromstrain168bytransformation(17).Luria-Bertani (LB)medium(21)wasusedthroughoutandwassupplementedwithspectinomycin(Spc)(100 g/ml)orchloramphenicol(Cm)(5 g/ml)toselectforWN624 orWN628,respectively,andtheirdescendants.ForgrowthunderEarth-normal conditions,liquidcultureswereincubatedinawaterbathwithmoderaterotary shaking(150rpm).Forgrowthwithlimitedoxygen,liquidcultureswereincubatedinlledscrew-captesttubesplacedverticallyinanincubatorwithout shaking.Whereindicated,thealternateelectronacceptorssodiumnitrate(11) andsodiumsulfate(1)wereaddedtoLBtonalconcentrationsof10mMand 1mM,respectively.Forgrowthatlowpressure,culturesinKlettaskswere grownundervacuuminawaterbathwithmoderaterotaryshaking(150rpm). Vacuumwassuppliedbyapumpingsystem(KNFNeuberger,Trenton,NJ)tted with0.2-m-porein-lineairlters.GrowthwasmeasuredatEarth-normalatmosphericpressure(101.3 0.6kPa)and10,7.5,5,and2.5kPa.Optical densities(ODs)weremeasuredusingaKlett-Summersoncolorimeterttedwith theno.66(red,660-nm)lter.(Notethatforpurposesofcomparison,100Klett units 1ODunitat660nm[OD660].) Conditionsforlaboratoryevolutionofbacteriawereessentiallyasdescribed previously(17,18)withslightmodications.Briey,strainsWN624andWN628 werepropagatedin125-mlsidearm(Klett)asksin10mlofliquidLBmedium containingtheappropriateselectiveantibioticinarotaryshakerbathat27C. StrainWN628waspropagatedat101.3 0.6kPa,whilestrainWN624was propagatedat5.0 0.2kPa.Atdailyintervals,theODofeachpopulationwas determined,eachculturewasdiluted1:100intofreshselectivemedium,and incubationcontinued.Underthisregimen,eachpopulationprogressedthrough *Correspondingauthor.Mailingaddress:SpaceLifeSciencesLaboratory,BuildingM6-1025,Room201-B,KennedySpaceCenter,FL 32899.Phone:(321)861-3487.Fax:(321)861-2925.E-mail:WLN@u .edu.Publishedaheadofprinton1October2010. 7559
6.6generationsperday.At 50-generationintervals,analiquotofeachculture wasstoredin25%(vol/vol)glycerolat 70C. Competitionexperiments. Strainstobecomparedinpairwisecombination werecultivatedovernightinselectiveLBliquidat27Candnormalatmospheric pressure.Fiftymicrolitersoftwodifferentovernightcultures,containingapproximatelyequalnumbersofcells,wasinoculatedinto10mlofnonselectiveLB mediumina125-mlErlenmeyerask,cultivatedat101.3kPaor5kPa,and diluted1:100intofreshLBeachday,asdescribedabove.Atdailyintervals,an aliquotwasremovedfromeachcultureandserial10-folddilutionsinphosphatebufferedsaline(PBS)buffer(25)wereplatedonbothplatescontainingLB mediumplusCm(LBCm)andLBmediumplusSpc(LBSpc).Theproportionofthetotalpopulationconsistingofchloramphenicol-resistant(Cmr)or spectinomycin-resistant(Spcr)colonieswascalculatedforeachmixedpopulation andplottedversuselapsedgenerations. Statisticalanalyses. Basicstatisticalparametersandanalysesofvariance (ANOVA)wereperformedusingcommercialstatisticalsoftware(Kaleidagraph, version3.6.2;SynergySoftware,Reading,PA).Differenceswith P valuesof 0.05wereconsideredstatisticallysignicant.RESULTSANDDISCUSSION Growthinhibitionof B.subtilis atlowpressure. Previous resultsshowedthatincubationofseveraldifferentmicroorganismsonagarplatesatprogressivelylowerpressuresresultedin adiminutionandeventualcessationofgrowth(4,27,30).To investigatethiseffectfurther, B.subtilis strain168wascultivatedat27CinliquidLBmediumat101.3,10,7.5,5,and2.5 kPa.ODmeasurementsweretakentodetermine(i)the growthrateand(ii)thenal24-hcultureyieldateachpressure.Itwasobservedthatgrowthratewasunaffectedat10kPa butdecreasedsemilogarithmicallywithloweringofpressureto 7.5and5kPa(Fig.1).Cellyieldat24hshoweda2-fold decreaseat10kPaandthenafurthersemilogarithmicdecreasewithloweringofpressureto7.5and5kPa(Fig.1). Growthwasessentiallyundetectableat2.5kPa,inagreement withpreviousresultsonsemisolidmedium(30). Growthinhibitionat5kPawasinvestigatedinfurtherdetail. Strain168wascultivatedinLBliquidmediumat101.3or5 kPa,andgrowthwasmonitoredasdescribedabove.At101.3 kPa,atypicalgrowthcurvewasobserved,featuringan 1-hlag phase,rapidexponentialgrowth,agradualentranceintostationaryphase,andovernightgrowthtoahighcelldensityof 335Klettunits(Fig.2A).Asexpected,thesamepatternwas repeatedupondilutionofthecultureintofreshLBmediumat 101.3kPaonthefollowingday(Fig.2B).Incontrast,when strain168wasgrownat5kPa,thecellsalsoexperiencedan 1-hlagperiod,butexponentialgrowthproceededatalower rateandthecellsabruptlyenteredstationaryphaseatalow celldensityof 40Klettunits;thislowdensitypersistedfor 24h(Fig.2A).Uponreturnoftheculturefrom5kPato101.3 kPaonthefollowingday,thecellsresumedexponentialgrowth immediately,withoutanydiscerniblelagphase,albeitata slowergrowthratethancellsprecultivatedat101.3kPa(Fig. 2B).Althoughthemolecularbasisforlow-pressuregrowth inhibitionispresentlyunknown,thedatasuggesttwoimportantfeatures.First,theimmediateresumptionofvegetative growthuponreturntoatmosphericpressureindicatestheexistenceofsometargetmolecule(s)withinthecellsthatcanbe FIG.1.Growthrate(doublings/hour;circles),andcultureyield (Klettunitsat24h;squares)asafunctionofpressure. B.subtilis 168 wasgrowninliquidLBatthepressuresindicated(101.3,10,7.5,and 5kPa[i.e.,1,013,100,75,and50mbar,respectively),andtheresults werenormalizedtothosefromstrain168grownatatmosphericpressure(101.3kPa),whichgrewtoanalOD660of334 2Klettunits withadoublingtimeof85 8min.Dataarepresentedasaverages standarddeviations(n 3). FIG.2.(A)Day1.Growthof B.subtilis strain168cultivatedat27CinLBmediumat101.3kPa(1,013mbar)(opencircles)or5kPa(50mbar) (lledcircles).(B)Day2.Strain168grownat101.3kPawasdiluted1:20intofreshLBmediumandcultivatedat101.3kPa,27C(opencircles). Theaskcontainingstrain168grownat5kPawassimplyrepressurizedto101.3kPaandcultivatedat27C(lledcircles). 7560NICHOLSONETAL. APPL.ENVIRON.MICROBIOL.
reversiblyinactivatedbypressurechangeswithoutbeingresynthesized denovo.Second,itappearedthatcellsgrownfor24h at5kPahadundergoneaphysiologicalalterationslowingtheir subsequentgrowthuponreturnto101.3kPa;furthermore,this alterationpersistedforatleast2doublingsafterreturnto 101.3kPa(Fig.2). Evolutionof B.subtilis toenhancedgrowthatlowpressure. Wereasonedthatgrowthinhibitionat5kPacouldbeusedas anenvironmentalstresstoselectforenhancedgrowthatlow pressureandfurthermorethatanalysisoflow-pressure-evolved stainscouldyieldinsightsintothemolecularmechanismsunderlyinggrowthatlowpressure.Therefore,thefollowingexperimentwasperformed.Congenicderivativesof B.subtilis strain168,strainsWN624(Spcr)andWN628(Cmr),were subjectedtolaboratoryevolutionat5kPaand101.3kPa, respectively,for1,000generations.Overthe1,000-generation courseoftheexperiment,aprogressiveincreasein24-hculture ODwasobservedinbothcultures(Fig.3AandB).Inboth cultures,theincreasewasobservedtooccurinastepwise manner:i.e.,cellswentthroughperiodsofnosignicant changeinOD,interspersedbyperiodscharacterizedbyincreasesinOD(Fig.3).Wehavepreviouslyobservedthispatternofâ€œpunctuatedequilibriumâ€(reviewedinreference10)in evolving B.subtilis cultures(17,18)andinterpretedthephenomenonassuccessivepopulationsweepsbyspontaneously arisingmutantscapableofincreasedgrowthundercontinued selectivepressure. Fitnessmeasurementsbycompetitionexperiments. Two single-colonyisolateswerestreakpuriedâ€”onefromeachculturegrownatnormalatmosphericpressure(101.3kPa)andat 5kPaâ€”andweredesignatedstrainsWN1105(Cmr)and WN1106(Spcr),respectively.Inordertoassesstherelative tnessoftheevolvedstrainsversustheirancestorsandeach other,pairwisecompetitionexperimentswereperformed. First,ancestralstrainsWN624andWN628wereinoculatedat equalcellnumbersandcompetedinLBmediumateither 101.3kPa(Fig.4A)or5kPa(Fig.4B)for 50generations. Neitherancestralstraindemonstratedaselectiveadvantage overtheotherateitherpressure,thusensuringthatthe amyE::spc or amyE::cat markersintheotherwiseidentical strainswereselectivelyneutral.Itwasimportanttoestablish thatancestralstrainsWN624andWN628showedthesame relativetness,becauseeachancestorwassubsequentlycompetedagainsttheevolvedstrainfromtheotherpopulation,to FIG.3.(A)EvolutionofstrainWN624toenhancedgrowthat5kPa.(B)EvolutionofstrainWN628toenhancedgrowthatatmospheric pressure(101.3kPa).Inbothpanels,theopticaldensity(Klettunits)of24-hcultureswasmonitored.Datapointsrepresenttheaveragesand standarddeviationsofdailycultureOD660valuesgroupedat1-week(50-generation)intervals.Datapointslinkedbyhorizontallinesbeneath lowercaselettersindicategroupsofdatathatwerenotsignicantlydifferentbyANOVA(P 0.05; n 7). FIG.4.CompetitionexperimentsbetweenancestralstrainWN624(opencircles)andWN628(lledcircles)propagatedtogetherinLBmedium withoutantibioticsat101.3kPa(A)or5kPa(B).Dataareaverages standarddeviationsfromduplicateexperiments. VOL.76,2010 B.SUBTILIS EVOLUTIONTOLOW-PRESSUREGROWTH7561
takeadvantageofthedifferenceintheirantibioticresistance markers. Inthenextsetofcompetitionexperiments,ancestralstrain WN628(Cmr)wascompetedagainstlow-pressure-evolved strainWN1106(Spcr)(Fig.5 ).Whenthetwostrainswere propagatedtogetherat101.3kPa,noselectiveadvantagewas observed(Fig.5A).However,whenthetwostrainswerecocultivatedat5kPa,strainWN1106showedacleargrowthadvantageatlowpressure,andancestralstrainWN628waslostfrom themixedpopulationatarateof1logper 10generations (Fig.5B).Thus,after1,000generationsofevolutionat5kPa, strainWN1106indeedappearedtohavegainedtnessfor growthatlowpressure. Inthenextsetofexperiments,ancestralstrainWN624 (Spcr)wascompetedagainststrainWN1105(Cmr),whichhad evolvedfor1,000generationsat101.3kPa.Improvedgrowthof WN1105inLBatatmosphericpressurelikelyresultedfrom improvedutilizationoftheresidualnutrientsinstationary phase,aswasobservedinpreviouslong-termevolutionexperiments(17).Asexpected,whenthecompetitionexperiment wasperformedat101.3kPa,strainWN1105exhibitedincreasedtnessovertheancestorstrainWN624,whichwaslost fromthemixedpopulationatarateof1logper 28generations(Fig.6A).However,whenthesamecompetitionexperimentwasperformedat5kPa,theevolvedstrainWN1105 showedadistinctdecreaseintnesscomparedtoancestral strainWN624,andstrainWN1105waslostfromthemixed populationatarateof1logper 16generations(Fig.6B). Theseresultsareinsharpcontrasttothoseobservedinthe competitionbetweenstrainsWN1106andWN628atnormal atmosphericpressure,wherenodistinctdifferenceintness wasobserved(Fig.5A).Theresultssuggestedthatprolonged evolutionunderconstantconditions(1,000generations,LB medium,27C,101.3kPa)mayhaveresultedinareductionin theabilityofstrainWN1105toadjusttoachangeinitsenvironmentalatmosphericpressure.Inpreviouslong-termevolutionexperiments,wealsoobservedalossofphenotypicplasticityduringlong-termpropagationunderconstantconditions; transcriptionmicroarrayexperimentsindicatedthatlossof phenotypicplasticitycouldbeattributedtoagloballossof transcriptomeexibilityinresponsetoenvironmentalchanges (16). Finally,bothevolvedstrainsWN1106(Spcr)andWN1105 (Cmr)werecompetedagainstoneanotherat101.3kPaorat5 FIG.5.CompetitionexperimentsbetweenancestralstrainWN628(lledcircles)andWN1106(opencircles)propagatedtogetherinLB mediumwithoutantibioticsat101.3kPa(A)or5kPa(B).Dataareaverages standarddeviationsfromduplicateexperiments. FIG.6.CompetitionexperimentsbetweenancestralstrainWN624(lledcircles)andWN1105(opencircles)propagatedtogetherinLB mediumwithoutantibioticsat101.3kPa(1,013mbar)(A)or5kPa(50mbar)(B).Dataareaverages standarddeviationsfromduplicate experiments. 7562NICHOLSONETAL. APPL.ENVIRON.MICROBIOL.
kPa(Fig.7).Theresultsobtainedshowedthateachstrainhad becomebetteradaptedforgrowthatthepressureforwhichit hadbeenevolved.At101.3kPa,strainWN1106waspreferentiallylostfromthemixedcultureatarateof1logper 11 generations(Fig.7A),andwhencocultivatedat5kPa,strain WN1105waslostfromthemixedcultureatarateof1logper 7generations(Fig.7B). Theresultspresentedinthispreliminarystudydemonstrate thatlowpressureinhibitsboththegrowthrateandnalgrowth yieldof B.subtilis cells.Whatcouldcauseinhibitionofbacterialcellgrowthatlowpressure,andhowmightstrainWN1106 haveevolvedtoovercomethisinhibition?Oneobviouspossibilityimmediatelypresentsitself:low-pressuregrowthinhibitionmayresultsimplyfromoxygenstarvation.Inourexperiments,loweringofpressureintheheadspaceabovetheculture from 101.3kPato5kPawouldleadtoacorresponding 20-folddecreaseinthepartialpressureofdissolvedgases (notablyoxygen)intheculture. B.subtilis iscapableofgrowth underoxygenlimitationviafermentationoranaerobicrespiration(reviewedinreference22),butLBmediumisapoor sourceofeitherfermentablecarbonsourcesoralternative electronacceptors.Therefore,boththegrowthrateandnal cellyieldof B.subtilis inLBmediumwouldbeexpectedtobe loweratthereduceddissolvedoxygenconcentrationsresulting fromlowpressure.Consideringthispossiblescenario,immediateresumptionofgrowthofwild-typestrain168bytransition from5kPato 101.3kPa(Fig.2)maymerelyresultfromthe returnofsufcientoxygentoanoxygen-starvedculture,and, furthermore,strainWN1106mayhaveevolvedanabilityfor enhancedlow-oxygengrowth,ratherthanlow-pressuregrowth perse.Inordertotestthisnotion,wecultivatedancestralstrain WN624andlow-pressure-evolvedstrainWN1106inliquidLB mediumunderoxygen-limitedconditionsandcomparedtheir growthproles(Fig.8).StrainWN1106wasclearlynotmore capableofanaerobicgrowththanstrainWN624inLBmedium (Fig.8A).NorwasstrainWN1106capableofenhancedgrowth underoxygenlimitationwhencultivatedinLBmediumcontainingeitherofthealternateelectronacceptorssulfate(Fig. 8B)ornitrate(Fig.8C)orbothsulfateandnitrate(Fig.8D). Thus,theevidencestronglysuggeststhatstrainWN1106has notevolvedsimplytoenhancedgrowthunderoxygen-limited conditions. Forinsightsintootherpossiblereasonsforlow-pressure growthinhibitionandtheevolutionofenhancedgrowthof strainWN1106atlowpressure,itmaybeinstructivetoturnto ourcurrentunderstandingoflifeattheoppositeextreme(i.e., highpressure).Fromthestudyofhigh-pressuremicrobiology, ithasbeenshownthatpressurechangescanexerteffectson thesynthesisandactivityofnumerouscellulartargets,includinglipids,enzymes,andnucleicacids(2).Forexample,the abilityofdeep-seapiezophilestogrowatextremehighpresFIG.7.Competitionexperimentsbetween5.0-kPa-evolvedstrainWN1106(lledcircles)and101.3-kPa-evolvedstrainWN1105(opencircles) propagatedtogetherinLBmediumwithoutantibioticsat101.3kPa(1,101mbar)(A)or5kPa(50mbar)(B).Dataareaverages standard deviationsfromduplicateexperiments. FIG.8.GrowthofstrainsWN624(opencircles)andWN1106 (lledcircles)underconditionsofoxygenlimitation.Cultureswere growninliquidLBmediumwithnoaddition(A),1mMsodiumsulfate (B),10mMsodiumnitrate(C),andboth1mMsodiumsulfateand10 mMsodiumnitrate(D).Dataareaverages standarddeviationsfrom duplicateexperiments. VOL.76,2010 B.SUBTILIS EVOLUTIONTOLOW-PRESSUREGROWTH7563
sureshasbeencorrelatedwiththepresenceintheirmembranesofalargepercentageofmono-andpolyunsaturated fattyacids(13).Piezophilescanvarytheratioofunsaturatedto-saturatedfattyacidsintheirmembranesinadirectrelationshipwithpressure(7),inordertomaintainahomeostatic membraneuidityandconsequentlytheactivityofvarious membrane-associatedproteinssuchastransporters,respiratoryenzymes,orenvironmentalsensors(13).Inaddition,pressure-relatedchangestothestabilityandactivityofvarious cytosolicenzymessuchasRNApolymerasehavealsobeen documented(13).Interestingly,low-pressuregrowthinhibition of B.subtilis 168appearedtobeimmediatelyreversibleupon returntonormalatmosphericpressure,butatalowerrate (Fig.2).Thisobservationsuggeststhatinhibitionmaybe causedbyinpartbyreversibleinactivationofsomecriticalcell componentsnotrequiring denovo synthesisandinpartby affectingsomecellcomponentsthatdorequirerecoverytime. Inthisscenario,strainWN1106mayharbormutationsrenderingsuchtargetslesssensitivetoinactivationbylowpressureor regulatorymutationsenhancingtheabilityofcellstocopewith low-pressurestress. Resultsfromourpreliminarystudiesdemonstrateforthe rsttimethatmicrobialevolutionispossibleatlowpressure, yieldingdescendantsthatareadaptedforenhancedgrowthat lowpressures.Howdoesthisndingimpacttheeldofastrobiology?AkeyunansweredquestionfortheroboticandhumanexplorationofMarsiswhetherterrestrialmicroorganisms canadapttoandacquirethecapabilityforactivemetabolism andreplicationat,the 0.7-kPaatmosphericpressureatthe martiansurface.Diversemicrobialcommunitiesincludingextremophilesandsporeformershavebeenrecoveredfrom spacecraftsurfacesandprocessingfacilitiespriortolaunchof spacecrafttoMars(9,15,31),andthesurvivalofsomeofthese speciesduringtheEarth-to-Marsinterplanetarytransitphase isquiteprobable(23,24,26).However,uponlandingonMars, atleast13separatebiocidalfactorsinadditiontolowpressure arelikelytobepresentonthemartiansurface(29,30).At presentitisunclearwhetherterrestrialmicroorganismscan overcometheentirecollectionofinteractingbiocidalfactorsin ordertoevolvethecapabilityforreplicationonMars.Results fromfutureresearchprobingthelowerlimitsofreplication andevolutionunderhypobaricconditionswilldirectlyconstrainplanetaryprotectionprotocolsforspacecraftandthe searchforanextantmartainmicrobiota(24)anddiscover whetherterrestrialmicroorganismswillbeabletoproliferate onthesurfaceornearsubsurfaceofMars(23). Probingthelow-pressurelimitforlifehasfundamentalimplicationsbeyondtheastrobiologyoflow-pressureenvironmentssuchasMars.Intheexperimentsdescribedinthiscommunication,weimposeda20-foldreductioninpressureon B. subtilis,abacteriumnormallyadaptedtoEarthsurfaceatmosphericpressureâ€”from101.3kPato5kPa.Thispressure changecouldbeconsideredanalogoustothehypotheticalremovalofadeep-seapiezophilefromtheMarianasTrench (10,911mbelowsealevelat 110MPa)toadepthofonly 540m( 5MPa).Ithaspreviouslybeendemonstratedthatthe growthrateofpiezophilesisdramaticallyslowedatprogressivelylowerpressures(5,20),inamanneranalogoustothat seenfor B.subtilis inFig.1.Therefore,itispossiblethatsimilar mechanismsareatworkinbothcasesandthatexploringthe low-pressurelimitofmicroorganismsnormallyadaptedto growthattheEarthâ€™ssurfacemightprovideinsightsintothe factorslimitinggrowthofpiezophilesatthelowerlimitsof theirownpressureranges.Inordertofurtherunderstandthe geneticresponseof B.subtilis tolow-pressurestress,current experimentaleffortsarebeingconcentratedonusingtranscriptionmicroarraystoidentifygeneswhosetranscriptionisalteredin(i)theancestralversuslow-pressure-evolvedstrains, (ii)incellspropagatedat101.3kPaversus5kPa,and(iii)in microaerophilicenvironmentsinducedbylowpressureversus thoseinducedbyoxygenlimitationatatmosphericpressure.ACKNOWLEDGMENTS Wethanktheanonymousreviewersfortheirinsightfulcomments. 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20. Miller,J.F.,N.N.Shah,C.M.Nelson,J.M.Ludlow,andD.S.Clark. 1988. Pressureandtemperatureeffectsongrowthandmethaneproductionofthe extremethermophile Methanococcusjannaschii.Appl.Environ.Microbiol. 54:3039. 21. Miller,J.M. 1972.Experimentsinmoleculargenetics.ColdSpringHarbor LaboratoryPress,ColdSpringHarbor,NY. 22. Nakano,M.M.,andP.Zuber. 2002.Anaerobiosis,p.393. In A.L. Sonenshein,J.A.Hoch,andR.Losick(ed.), Bacillussubtilis anditsclosest relatives:fromgenestocells.ASMPress,Washington,DC. 23. Nicholson,W.L. 2009.Ancientmicronauts:interplanetarytransportofendolithicmicrobesbycosmicimpacts.TrendsMicrobiol. 17:243. 24. Nicholson,W.L.,A.C.Schuerger,andM.S.Race. 2009.Migratingmicrobes andplanetaryprotection.TrendsMicrobiol. 17:389. 25. Nicholson,W.L.,andP.Setlow. 1990.Sporulation,germination,andoutgrowth,p.391. In C.R.HarwoodandS.M.Cutting(ed.),Molecular biologicalmethodsfor Bacillus.J.WileyandSons,Chichester,UnitedKingdom. 26. Schuerger,A.C. 2004.MicrobialecologyofthesurfaceexplorationofMars withhuman-operatedvehicles,p.363. In C.S.Cockell(ed.),Martian expeditionplanning.AmericanAstronauticalSocietypublicationAAS03â€“ 322.UniveltPublishers,SantaBarbara,CA. 27. Schuerger,A.C.,B.Berry,andW.L.Nicholson. 2006.Terrestrialbacteria typicallyrecoveredfromMarsspacecraftdonotappearabletogrowunder simulatedmartianconditions,abstr.1397.Abstr.37thLunarPlanetarySci. Conf.,Houston,TX,13to17March2006. 28. Schuerger,A.C.,R.L.Mancinelli,R.G.Kern,L.J.Rothschild,andC.P. McKay. 2003.Survivalofendosporesof Bacillussubtilis onspacecraftsurfacesundersimulatedmartianenvironments:implicationsfortheforward contaminationofMars.Icarus 165:253. 29. Schuerger,A.C.,D.W.Ming,andD.C.Golden. 2010.BiotoxcityofMars analogsoils:microbialdispersalintodesiccatedsoilsversusemplacementin saltoriceinclusions,abstr.5336.Abstr.2010Astrobiol.Sci.Conf.(AbSciCon2010),Houston,TX. 30. Schuerger,A.C.,andW.L.Nicholson. 2006.Interactiveeffectsofhypobaria, lowtemperature,andCO2atmosphereinhibitthegrowthofmesophilic Bacillus spp.undersimulatedmartianconditions.Icarus 185:143. 31. Venkateswaran,K.,M.Satomi,S.Chung,R.Kern,R.Koukol,C.Basic,and D.White. 2001.Molecularmicrobialdiversityofaspacecraftassemblyfacility.Syst.Appl.Microbiol. 24:311.VOL.76,2010 B.SUBTILIS EVOLUTIONTOLOW-PRESSUREGROWTH7565
1 HOW DOES BACILLUS SUBTILIS RESPOND AND ADAPT TO LOW PRESSURE GROWTH? By SAMANTHA MARIE WATERS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014
2 Â© 2014 Samantha Marie Waters
3 ACKNOWLEDGMENTS I thank my mother for providing a shining example of what inner strength and will power may accomplish in life. Sarah and S amuel, thank you for being supportive younger siblings. I would like to express my gratitude to Dr. Wayne L. Nicholson for granting me the opportunity to work in his lab. I would also like to thank my graduate committee, Drs. de Crecy, Kolaczkowski, Ferl a nd Romeo for their guidance during my research these past four years, as well as my graduate student cohort, Rafael Oliveir a. For their candid advice, I thank Drs. Mobberl ey, Foster, Mueller and Koenig. I thank the Computing Center for all the support with s oftware and Galaxy tool errors. A special thank you to Justin , for being super awesome. All of my friends who have been the solid ground under my feet: Christine, Samantha, Alia, Zinzi, Kimberly, Jon, Julia, and J ennifer; thank you for always believing in me. And to David, Monte, Josh and Beth, thank you for all the spectacular movie nights that were much appreciated and greatly needed to stay sane during these past few years.
4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 3 LIST OF TABLES ................................ ................................ ................................ ........................... 7 LIST OF ABBREVIATIONS ................................ ................................ ................................ .......... 9 ABSTRACT ................................ ................................ ................................ ................................ ... 10 1 LITERATURE REVIEW ................................ ................................ ................................ ....... 12 Pressure ................................ ................................ ................................ ................................ ... 12 High Hydrostatic Pressure: Protein Volume ................................ ................................ ........... 12 High Hydrostatic Pressure: Protein Denaturation ................................ ................................ ... 14 High Hydrostatic Pressure: Membrane Fluidity ................................ ................................ ..... 16 High Hydrostat ic Pressure: Mesophilic Organisms ................................ ................................ 18 Low Pressure ................................ ................................ ................................ .......................... 19 Bacillus subtilis Strain 168 ................................ ................................ ................................ ..... 20 Experimental Evolution ................................ ................................ ................................ .......... 21 2 EXPOSURE OF BACILLUS SUBTILIS TO LOW PRESSURE (5 KPA) INDUCES SEVERAL GLOBAL REGULONS INCLUDING THE SIGB MEDIATED GENERAL STRESS RESPONSE ................................ ................................ ................................ ............. 23 Introduction ................................ ................................ ................................ ............................. 23 Bacterial Strains, Media, and Growth Conditions ................................ ........................... 25 Isolat ion and Labeling of Total RNA ................................ ................................ .............. 26 Transcription Microarray Experiments ................................ ................................ ........... 26 Microarray Data Analysis and Normalization ................................ ................................ . 27 Galactosidase Assay ................................ ................................ ................................ ..... 27 Microarray Data Accession Number ................................ ................................ ............... 28 Results and Discu ssion ................................ ................................ ................................ ........... 28 Transcriptome Analysis of Strain WN624 at 5 kPa and ~ 101 kPa ................................ 28 Induction of ctc lacZ Expression by LP ................................ ................................ .......... 30 Inactivation of sigB Does Not Alter Fitness at ~ 101 kPa or at 5 kPa ............................. 31 Towards an Understanding of the LP Response. ................................ ............................. 33 3 MICROARRAY ANALYSIS OF DIFFERENTIAL GENE EXPRESSION IN A BACILLUS SUBTILIS STRAIN ADAPTED TO ENHANCED GROWTH AT 5 KPA ....... 60 Introduction ................................ ................................ ................................ ............................. 60 Material and Methods ................................ ................................ ................................ ............. 62 Bacillus subtilis , Media, and Growth Conditions ................................ ............................ 62 RNA Ext raction ................................ ................................ ................................ ............... 62 Microarray Experiments ................................ ................................ ................................ .. 63
5 Microarray Data Analysis and Normalization ................................ ................................ . 63 BLASTp Analysis of Unknown and Putative Function mRNA Signals ......................... 64 Sporulation Frequency ................................ ................................ ................................ ..... 64 galactosidase Assays ................................ ................................ ................................ .... 64 Competition Experiments ................................ ................................ ................................ 65 Microarray Data Accession Number ................................ ................................ ............... 65 Results ................................ ................................ ................................ ................................ ..... 66 Microarray Analysis of Global Gene Expression of Bacillus subtilis Strains WN624 and WN1106 at 5 kPa ................................ ................................ ................................ .. 66 Anaerobic Response at 5 kPa ................................ ................................ .......................... 67 Membrane Fluidity at Low Pressure ................................ ................................ ............... 69 Transporter Genes are Differentially Expressed at LP ................................ .................... 70 DNA Binding Proteins at LP ................................ ................................ ........................... 73 Genes of Unknown Function were Differentially Expressed at LP ................................ 74 The General Stress Response at LP ................................ ................................ ................. 75 Inactivation of sigB Does Not Alter Fitness at ~101 kPa or at 5 kPa .............................. 77 Sporulation at 5 kPa ................................ ................................ ................................ ......... 79 Discussion ................................ ................................ ................................ ............................... 79 4 WHOLE GENOME RE SEQUENCING REVEALS MUTATIONAL CHANGES IN BACILLUS SUBTILIS AFTER A 1,000 GENERATION 5 KPA EVOLUTION EXPERIMENT ................................ ................................ ................................ ..................... 126 Introduction ................................ ................................ ................................ ........................... 126 Materials and Methods ................................ ................................ ................................ ......... 127 Bacterial Strains, Media and Growth Conditions ................................ .......................... 127 DNA Extraction and Quality Control for Re Sequencing ................................ ............. 128 Whole Genome Re Sequencing and Mutation Identification ................................ ....... 128 In silico Analysis of Mutations ................................ ................................ ...................... 130 Rifampicin Resistance Assay ................................ ................................ ........................ 130 Competition Experiments ................................ ................................ .............................. 131 RNA Isolation ................................ ................................ ................................ ................ 131 Quantitative Reverse T ranscription Polymerase Chain Reaction (qRT PCR) ............. 132 Results and Discussion ................................ ................................ ................................ ......... 132 Whole Genome Re Sequencing and Alignment ................................ ............................ 132 Mutational Analysis During the 5 kPa Evolution Experiment ................................ ...... 133 bacD Analysis ................................ ................................ ................................ ............... 135 fliI Analysis ................................ ................................ ................................ ................... 136 parC Analysis ................................ ................................ ................................ ................ 137 ytoI Analysis ................................ ................................ ................................ .................. 138 resD and walK Analysis ................................ ................................ ................................ 139 rnjB Analysis ................................ ................................ ................................ ................. 142 Discussion ................................ ................................ ................................ ............................. 144 Bacillus sub tilis at 5 kPa ................................ ................................ ................................ 144 Genomic Changes in B. subtilis After 5 kPa Long Term Exposure .............................. 145 Transcription and Post Transcription Mutation al Strategies for Low Pressure Growth ................................ ................................ ................................ ....................... 145
6 5 SUMMARY OF RESEARCH ................................ ................................ .............................. 172 LIST OF REFERENCES ................................ ................................ ................................ ............. 176 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 193
7 LIST OF TABLES Table page 2 1 Details of microarrays used in Chapters 2 and 3 ................................ ................................ ..... 35 2 2 Strains and plasmids used in this study ................................ ................................ ................... 36 2 3 Summary of regulons with at least 10 target genes affected A ................................ ................. 37 2 4 B. subtilis genes significantly up regulated by exposure to 5 kPa ................................ .......... 38 2 5 B. subtilis genes significantly down regulated by exposure to 5 kPa ................................ ..... 46 2 6 Response of significantly and non significantly expressed General Stress Response ............ 50 3 1 Bacillus subtilis strains and plasmids used in this st udy ................................ ......................... 83 3 2 Expression data for microarray chips defined in Table 2 1 ................................ ..................... 84 3 3 SEED categories for genes with unknown function ................................ .............................. 120 3 4 Averages of CFUs, spore titers, and sporulation frequency ................................ .................. 121 4 1 Bacillus subtilis strains and plasmids used in this study ................................ ....................... 148 4 2 Oligonucleotides used to amplify, Sanger sequence and verifiy mutations .......................... 149 4 3 Mutations identified in WN1106 and the SNP occ uring in rnjB ................................ ........... 150 4 4 WN624 Mutational Calls Different from the Bacillus subtilis strain 168 ............................. 151 4 5 Rifampcin resistance assay to determine differences in mutation rate of strains WN1106, WN624 and WN1105 ................................ ................................ ................................ ...... 154 4 6 Flagstat data on mapped paired end reads ................................ ................................ ............. 155 4 7 Mutations called after mapping of WN1105 ................................ ................................ ......... 156 4 8 Differentially expressed signals of the ResDE and WalKR ................................ .................. 161
8 LIST OF FIGURES Figure page 2 1 Scatter plots of fluorescent intensity of Cy3 (X axes) vs. Cy5 (Y axes) ................................ 57 2 2 Venn diagram comparison of the set of genes belonging to the GSR regulon induced by: LP in WN624 ................................ ................................ ................................ ..................... 58 2 3 Determination of pressure induction of the SigB dependent GSR using a ctc::lacZ reporter fusion. ................................ ................................ ................................ ................... 59 3 1 Plots of Cy3 vs Cy5 log fluorescent intensity for microarrays. See text for details. ............. 122 3 2 Determination of pressure induction of the SigB dependent GSR using a ctc::lac Z reporter fusion ................................ ................................ ................................ .................. 123 3 3 Relative fitness values of congenic ancestral strains WN624 ................................ ............... 124 3 4 Average sporulation frequency at 5 k Pa and ~101 kPa of strains WN624 and WN1106 over three days. ................................ ................................ ................................ ................ 125 4 1 A) Proportion of fluorescent intensity of each mutant allele to total population in WN1106. ................................ ................................ ................................ .......................... 165 4 2 Representation of population percentages during the 5 kPa E.E. ................................ .......... 166 4 3 Alignment of rnjB fragments from WN1106 and the 5 kPa E.E. stocks ............................... 167 4 4 Moti lity agar showing growth of WN1106 and WN624 at either (i) 101 kPa and 37Â°C after 6 hour of incubation and (ii) 5 kPa and 27Â°C after 24 hours of incubation. ........... 168 4 5 q RT PCR of yweA, yocH, and fnr expression ratios in WN1106 to WN624 at 5 kP a. ........ 169 4 6 Visualization of the RnjA/RnjB heterodimer. ................................ ................................ ....... 170 4 7 Competitons of full gene deletions of rnjB mutants versus their wildtype strains. ............... 171
9 LIST OF ABBREVIATIONS 16S rRNA Small subunit of the bacterial and archaeal ribosome CFU Colony f orming units CO 2 Carbon dioxide Cm Chloramphenicol Erm Erythromycin GEO Gene Expression Omnibus GSR General stress response LB Miller LB media LIMMA Linear models for microarray analysis Microgram Micro liter mL Milliliter ng Nanogram Neo Neomycin nm Nanometer OD 660 Optical density at 660 nm wavelength RIN RNA integrity number rpm Revolutions per minute rRNA Ribosomal RNA Spc Spectinomycin
10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy HOW DOES BACILLUS SUBTILIS RESPOND AN D ADAPT TO LOW PRESSURE GROWTH? By Samantha Marie Waters August 2014 Chair: Wayne L. Nicholson Major: Microbiology and Cell Science Little is know n of how microorganisms respond and adapt to low pressure (LP) environments. It was previously reported that a strain of Bacillus subtilis , WN1106, which had evolved at 5 kPa for 1,000 generations, had an increased relative fitn ess and optical density compared to its ancestor strain, WN624. Transcriptional microarray experiments of strains WN1106 and WN624 revealed that B. subtilis exhibited a robust cellular response to LP growth involving a multitude of regulons including, but not limited, to: SigB dependent General Stress Response, Fnr, ResDE, Rex, Fur, and Ccp A. The microarray comparisons also revealed differences between the two strains involving regulons of: WalKR, ResDE, and SigD. Whole genome re sequencing of both strains showed a total of eight genomic changes , all occurring in coding regions, in LP evolved WN1106 compared to the ancestor, WN624. Seven of the mutations were single nucleotide polymorphisms (SNPs) and occurred in the following coding regions : bacD, fliI, par C, resD, walK, yvlD, and ytoI . The eight h mutation was a 9 nucleotide deletion resulting corresponding to a 3 amino acid in frame shift in the region of rnjB . This study investigated phenotypes that most likely result from each mutation. The transcriptiona l and mutational experiments, together, represent the first in depth analysis of a bacterium that has
11 undergone long term LP evolution and gives insight into how microorganisms may adapt to extremes in low pressure.
12 CHAPTER 1 LITERATURE REVIEW Pressure On Earth there are a number of harsh environments that challenge microbial life due to their extremes of physical factors: temperature, pH, osmolarity and pressure. Pressure is a fundamental thermodynamic parameter that affects biological processes at nearly every level: protein folding, biomolecule hydration state, enzyme reaction rates , membrane fluidity, and nucleic binding affinities of protein s (1 3) . Thus, it is of fundamental importance to understand the cellul ar and molecular mechanisms that enable microbes to grow and respond to extremes in ~101 kPa at sea level to ~ 10 kPa at a height of roughly 15 km (4) and is the most well characterized portion of the biosphere. However, regarding pressure, the known biosphere includes the stratosphere, tropospher e and oceanic zones. The oceanic zones possess a large portion of the biosphere ( i.e., the piezosphere) and experience an average pressure of ~ 38 MPa and at the deepest depths, the Mariana trench, a pressure of ~ 100 MPa (2) . Such pressure extremes (high hydrostatic pressure, HHP, or hyperbaria) have been compared to extremes of low temperature, however, there exist distinct thermodynamic differences between temperature and pressure: (i) temperature increases result in expanding volumes, in most cases, but an increase in pressure compresses volume; and (ii) temperature changes affect both internal energies and s ystem volumes, whereas pressure changes primarily affect volume (2) . High Hydrostatic Pressure : Protein Volume Current knowledge of microbial responses to pressure (or piezo , Greek for pr essure) is mainly confined to studies of high hydrostatic pressure, which is experienced by organisms (piezophiles) that dwell in the deep sea (piezosphere) environments. As stated, the average
13 pressure of the piezosphere is approximately ~38 MP a. Such an extreme of pressure, p , can be related to system volume changes in t ( d ln k / d ln p ) T = /RT (1 1) where k is the volumetric change during the formation of the activated reaction complex , also known as the reaction intermediate, (activation volume, mL/mol) (5, 6) . Equation 1 1 tells us that increasing pressures inhibit a reaction with a negative volume change, i.e. an increase in reaction volume, and favors a reaction with a positive volume change. In addition, when relating pressure to the Gibbs free energy, G, of a system, where S, E, and V respectiv ely represent the entropy, internal energy and volume; H is the enthalpy and equal to E + p V, where p is pressure: G = H TS = E + p V TS (1 2) and plotting changes of free energy with respect to changing temperature and pressure gives: d G = V dp S d T (1 3) (2) So at a constant temperature, d T = 0: ( d G/ dp ) T = V (1 4) (2) d G/ dp ) T (1 5) (2) Therefore, remember ing the le ChÃ¢telier Braun Principle, these equations relate how a change in pressure will shift ed either towards the larger or smaller volume of the reaction, i.e. increased pressure shifts the reaction to a smaller activation volume and decreases in pressure to a larger activation volume (7) .
14 High pressure favoring a decreased reaction volume is seen in a hyperbaric adaptation of Saccharomyces cerevisiae . The high affinity tryptophan permease, Tat2, has a positive activation volume (~ 50 mL/mol) and when present in the actively growing c ell confers resistance to elevated pressures that are typically inhibitory to growth (6) . The idea of hyperbaric adaptations favoring smaller protein volumes (i.e. positive activation volume changes) is evidenced by piezophilic p rotein analysis. When piezophiles are compared to their closest, non piezophilic relatives, analyses reveal changes in piezophilic protein structures favoring smaller, more compact proteins with decreases in beta strands and variable, unstructured regions, as well as differences in amino acid composition (8) ; the occurrence of proline and glycine residues are reduced in piezophilic proteins and there is an increase in acidic residues, the latter is most likely due to the high osmolarity of the deep sea (haloph ilic microorganisms also tend to favor acidic residues in proteins) (9) . High Hydrostatic Pressure : Prot ein Denaturation Extreme increases of pressure are also known to cause denaturation of proteins. Suzuki (10) observed at T < 30Â°C denaturation of proteins occurs as a first order kinetic reaction and proposed the following equation for pressure induced protein denaturation by hydration, whe re P is the native protein, n is the number of water molecules, P (H 2 O) n is the hydrated protein and P D the denature d protein: P + n H 2 O P (H 2 O) n P D (1 6) It was proposed that the formation of P (H 2 O) n is exothermic and has a volumetric decrease, which te nds to push the equilibrium towards the right as temperatures decrease and pressure increases, as would happen when bacteria sink in the ocean. This increased hydration of proteins causing the volume to decrease is understandable when considering the hydro phobicity and side group charges in the internal area of a protein, exposure of which to water molecules causes
15 volume decreases (2) . Adding to these, the internal cavities of proteins in vitro collapse with increases in pressure ( i.e. compressibility) res ulting in potentia l l y reduced volume. Despite all the in vitro considerations for extreme increases of pressure on protein volumes, only small negative values are actually evidenced in living systems; and extremes in pressure unfolding ar e dependent on oth er parameters, such as temperature and pH (11) . Therefore, un derstanding changes that occur to the whole organism when exposed to non optimal pressure conditions are of fundamental importance for understanding pressure responses and adaptations. Unlike temperature increases, a high pressure induced denatured protein may maintain secondary structural information (molten globule) and allows for temperature denatured aggregations to reform native structures in some cases at high pressure (~200 MPa) (12) . The pressures required for monomeric protein denaturation occurs in the range of 400 MPa to 800 MPa, which is four to eight times higher than the pressure at the Marina Trench (~100 MPa at the deepest depth of the oceans). While supermolecular structures (protein quaternary formations, or RNA protein complexes, e.g. ribosome) dissociate in the pressure range of 200 to 300 MPa, such high pressures, once again, do not occur in nat ure. But, these high pressure conditions not found in nature are used as food sterilization methods and studied to understand high pressure induced inactivation of bacterial endospores. When considering the whole organis m, biologically relevant increase s i n pressure (up to 100 MPa) are lethal to non piezophi lic microorganisms. This high pressure limit on b iological systems, which is lower than the upper pressure limit on protein structures, underlines the importance in vivo of other biologically relevant me chanisms and cellular structures that are affected by pressure.
16 High Hydrostatic Pressure : Membrane Fluidity Another reflection of the fundamentally different influences pressure and temperature have on cellular components is that of cellular membranes. In creases in pressure on lipids reflect decreases in temperature. Indeed, high pres sure affects the fluidity of lamellar phospholipid bilayers, which may undergo two phase transitions from gel to gel phase (L ) and a gel to liquid crystalline state (L ) (13) ; the lipid membrane is the most pressure sensitive structure of the cell (1 , 14) and the gelling effect (shift towards the L ) of high pressure decreases permeability and passage of water molecules, gases and ions important to cellular physiology (13) . There are many adaptations that reflect how crucial it is to maintain membrane fluidity across rather large fluctuations of pressure (14 16) . Membrane composition of saturated versus desaturated side chain acyl groups is one major route of adaptation (1, 15) . The unsaturated kinks in acyl groups may act to counter t he high packing and ordering lipids undergo during high pressure by creating void spaces and maintaining fluidity (13) ; increases in desaturation of fatty acid side chains and variance in membrane lipid compositions are also seen in cold temperature adapted microorganisms, i.e. psychrophiles, and are termed homeoviscous ad aptations (2) . Unsaturated fatty acids (UFAs) have been a major focus of HHP and psychrophilic research studies; one reason is they are relevant to biotechnology (creating polyunsaturated fatty acid s , PUFA s , as supplemental health products). PUFAs in the l ipid compositions of deep sea isolates was first described by DeLong and Yayanos (16) , a unique finding because up to this time PUFAs were thought to be a eukaryotic feature and not present in prokaryotes. The two predominantly used laboratory study genera for HHP research are Photobacterium sp p . and Shewanella sp p . Both deep sea isolated species of each genus produce the PUFA eicosapentaenoic acid (EPA), and the presence of this uncommon microbial membrane com pound is considered a piezoadaptation (1, 14) . Other deep sea isolates produce the PUFA
17 docosahexaenoic acid (DHA) , and Psychromonas kaikoae is the only known bacterium that produces both EPA and DHA (17) . It seems, however, that PUFAs are not required for HHP growth , rather it is monounsaturated fatty acids (MUFAs) that greatly influence maintenance of fluidity across a dynamic pressure range (15, 18) . It would seem from the literature that cold adapted microorganisms may have an advanta ge in a water column undergoing dynamic changes in pressure or on a sinking particle; many piezophiles are phylogenetically grouped within psychrophilic clades (19) . For example, Carnobacterium sp . AT7 , the first Gram (+) deep sea isolate (20) , is very closely related to Siberian permafrost and commercial freezer isolates (21) . And as mentioned, the two model piezophiles, P. profundum SS9 and Shewane lla violacea DSS12, are closely related to psychrophilic and psychrotolerant strains and species (20 ) . P iezosphere dwelling organisms most likely sunk over time to oceanic deepths (22) ; such a descent in a water colum n would be associated with pressure increases, but also, associate d with decreases in temperature. T hus, when considering the natural HHP environment at which microorganisms are exposed, pressure is rarely able to be isolated from temperature, as in the ab ove Eq. 1 4. This corresponds to the above examples linking pressure and cold temperature adaptations. But, piezophiles also have distinct genetic, expression, and physiological properties compared to their pressure mesophi lic cousins. An example of this i s seen from a 16S rRNA screening of deep sea samples and a subsequent phylogenetic alignment of helices 10 and 11, which revealed that deep sea species versus their closest psychrophilic relatives have insertions to the helical stems that is nearly exclusi ve to piezophiles (20) . Photobacterium profundum SS9 was the first organism in which differential e xpression of an outer membrane porin, ompH , was observed under high pressure, but not lowered pressure
18 ( in this case, standard atmospheric pressure) (9, 23) . It was determined that the differential pressure express ion of ompH and other pressure dependent expression changes of SS9 are regulated by the ToxR/ToxS two component regulatory system (24) . ToxR/ToxS system was the first described pressure regulation mechanism identified and works by turning off gene expression as pressures increase with a corresponding drop in abundance of these regulatory proteins as well (24) . The ToxR/ToxS system is sensitive t o membrane fluidity changes; when exposed to compounds increasing membrane fluidity at high pressure, the expression pattern is similar to lowered pressure expression (24) . Later, sequencing and transcriptional analysis revealed that SS9 has the most rRNA copies in a bacterial genome to date and that this, too, may lead to its uniquely robust and rapid response to wide pressure growth ranges (25, 26) . High Hydrostatic Pressure : Mesophilic Organisms Outsid e of the study of piezophiles, pressure mesophilic species (e.g. Escherichia coli and S. cerevisiae ) growth, cellular responses and adaptive mutations have been investigated at high pressure s. As mentioned above, the Tat2 tryptophan permease in S. cerevisi ae confers HHP growth resistance when present in growing cells (6, 27) . These studies are highly relevant to the food processing industry due to the practice of using HHP to decontaminate foodstuffs. Exposure of su ch organisms to moderate to high pressure has revealed numerous cellular processes that are affected by pressure (1, 28) . The model microeukaryote S. cerevisiae is killed in the high p ressure range of 100 to 200 MP a; however, brief exposure to such extreme elevated pressure is known to induce a number of stress related proteins, e.g. heat shock proteins (HSPs) and metallo oxidoreductases, as well as changes in carbohydrate utilization transcripts (29 31) . Pressure induced proteins (PIPs) in HHP studies of E. coli overlap with heat and cold shock proteins (28, 32) ; a total of 55 PIPs are
19 induced in E. coli and many of the proteins are only known to be expressed in response to high pressure (28) . It can be concluded from the literature that HHP stress on living organisms affects nearly all cellular processes and that piezo adapted organisms have evolved unique cellular adaptations to cope with the stress of deep sea living. However, just as low and high temperature adaptations together encompass how temperature extremes af fect organisms, there is a need to also understand how extremes in low pre ssure affect organisms to appreciate and understand the full range of biological adaptations to pressure . Low Pressure ude of ~15 km with a corresponding pressure of ~ 10 kP a. is found in the troposphere, therefore, the lowest naturally occurring pressure life is exposed to on Earth is roughly 10 kP a. There have been several studies on Bacillus spp., Serratia liquefaciens and E. coli growth at pressures down to 10 kPa that have shown little, if any, growth inhibition or physiological changes (33 36) . However, at pressures below 10 kPa, decreases in growth rates and eventual growth inhibition at pressures at or below 2.5 kPa (depending on the species) have been reported (34, 35, 37) . To date, only two examples of bacterial growth at pressures as low as 0.7 kPa have been reported: Carnobacterium spp. (38) and S. liquefaciens (37) . For the purpose of this report, low pressure (LP) refers to pressures below 10 kP a. While the examples of Carnobacterium spp. and S. liquefaciens show that bacteria may survive under a wide range of pr essure conditions ( Carnobacterium spp. also being isolated from the deep sea as mentioned above (20) ), the genome sequences of these organisms has only recently been reported (39, 40) . Therefore, the intrinsic factors allowing these organisms to grow at such lowered pressures is not currently known, nor are thes e organisms easily genetically
20 tractable or previously well characterized, retarding the study of cellular LP adaptations and responses. Other than natural terrestrial environments, LP conditions exist in (i) hypobaric storage facilities for plant and food products and (ii) on the surface of Mars. In the former environment, there have been reports of contaminants persisting at pressures as low as 0.7 kPa and are commonly occurring at pressures of 1.5 kPa (41) . A note, from literature on LP microbial growth and the LP storage of produce (e.g. apples, pears, legumes), LP seems to have a strongly inhibitory affect on microorganisms and on food decay. When organisms grown at LP are brought back up to higher pressures, growth with little to no die off is observed (33, 34) . This inhibitory e ffect of LP is in contrast to HHP, which has a more lethal affect on microbial growth. As mentioned above, the surface of Mars experiences extremes in LP conditions, with a surface pressure range of up to 1 kPa (average of 0.7 kPa) . The ability of any organism to g row under martian conditions is highly relevant to the fields of astrobiology and planetary protection. Currently, robotic missions sent to the surface of Mars are known to harbor terrestrial contaminants (42, 43) , and the se organisms have been shown to be capable of surviving the harsh conditions of interplanetary transit and deposition (44) ; together, these reports reveal the risk of terrestrial contamination of any extraterrestrial surface visited by robotic missi ons, i.e. a process termed forward contamination. Therefore, the study of LP responses and adapti ve changes in microorganisms is important for full y understanding the pressure range in which life may survive and grow. To this end, the study of a model labo ratory organism was conducted that had been experimentally evolved to grow better under a LP condition of 5 kP a. Bacillus subtilis Strain 168 Bacillus subtilis is the most well studied and characterized Gram (+) organism (45, 46) . B. subtilis is a ubiquitous soil bacterium, originally described in the late 1800s by Ferdinand
21 Cohn, and is a member of the genus Bacillus in the family Bacillaceae (47 ) . Bacilli are rod shaped bacteria capable of aerobic or facultative anaerobic growth and are most notable for their ability to form endospores (a dormant non vegetative cell capable of with standing numerous harsh environmental conditions) (47) . B. subtilis was the first species from this family, and first Gram (+) bacteria, to have its genome completely sequenced (48 ) ; since then, numerous Bacillus spp. have had genomes sequenced ( 47 ) . The 4.2 Mbp genome contains over 4,000 coding regions a nd a low G+C content (46, 48 , 49 ) . B. subtilis may also be mad e competent for genetic transformation, and a large number of molecular tools are available for its study. T his genetic tractability, its sequenced geno me, and the decades of molecular studies make it a suitable candidate for experimental evolutionary studies. Experimental Evolution Experimental evolution is a powerful tool that has allowed the real time study of various mechanisms of genetic change and a daptation in a wide variety of organisms that range from viruses to eukaryotes. Bacteria are especially suited for such experiments as they typically have short generation times and are haploid. This latter feature allows for minimizing the genetic noise o f allelic diversity when one wants to identify beneficial and novel mutations. In population genetics, experimental evolution has been used to study mutation rates, maintenance of haploids and diploids in a species, fitness, and the differences of negative , positive and neutral environmental selection on genes (reviewed in (50, 51 ) ). In addition to studying evolutionary mechanics, experimental microbial evolution may be utilized to study adaptive evolutionary change s, which occur during continuous exposure to a number of non optimal environmental conditions. These conditions have included: increased temperature (52 ) , dairy cult uring (53 ) , pa rasite host interactions (54 ) , low pH tolerance (55 ) , relaxed sporulation (56 ) , antibiotics (57 ) , increased salinity tolerance (58 ) , etc. Pairi ng
22 experimental evolution with next generation sequencing technologies has been truly revolutionary in the study of adaptive changes and their underlying genomic causes. In Bacillus subtilis , continuous culturing and ex perimental evolution studies have bee n used to investigate loss of sporulation (59, 60 ) , thermostability of enzymes (61 ) , and metabolic changes (62 ) . For the purpose of determining bacterial ada ptive strategies to LP , an evolution experiment was conducted for 1,000 generations at 27Â°C and 5 kPa using a wild type B. subtilis strain, WN624, as the starting inoculum (i.e. ancestor strain) (34) . During the 1,000 generations, weekly average optical densities increased multiple times (34) , possibly indicating punctuated evolutionary changes to LP growth during the experiment. A LP evolved strain was isolated from the terminus of this experiment, WN1106. The goal of this dissertation was to explo re the (1) transcriptional and (2) genomic changes between the ancestor WN624 and LP evolved WN1106 at 5 kPa, as well as to (3) describe how Bacillus subtilis transcriptionally responds to LP growth, which may indicate how LP affects microbial cellular pro cesses. The transcriptional investigations were conducted using microarray chips designed from the Bacillus subtilis strain 168 genome. For investigating the genomic changes that occurred and gave rise to the WN1106 strain, who le genome re sequencing was p erformed on both ancestor and evolved strain for the purpos e of comparing changes that had occurred during the 1,000 generations at 5 kP a. By exploring transcriptional responses to LP of the ancestor strain and the transcriptional and genomic changes that occur in LP evolved WN110 6, insights were gained into LP e ffects on bacterial cells and the possible genetic changes that may occur or may allow for growth at such low pressure extremes.
23 CHAPTER 2 EXPOSURE OF BACILLUS SUBTILIS TO LOW PRESSURE (5 KPA) INDU CES SEVERAL GLOBAL REGULONS INCLUDING THE SIGB MEDIATED GENERAL STRESS RESPONSE Introduction On Earth, there are a number of harsh environments that challenge microbial life due to extremes of fundamental physical factors such as temperature, pressure, pH, or osmolarity. It is thus of prime importance to understand the cellular and molecular mechanisms that enable microbes to grow, and indeed to flourish, at such physical extremes. Pressure is a fundamental thermodynamic parameter that affects biological pr ocesses at nearly every level, such as protein folding, hydration state of molecules, enzyme reaction kinetics, membrane fluidity, and protein nucleic acid binding affinity, to name but a few (63, 64 ) . High pressur e (HP) environments, ranging from sea level (~101 kPa) to the depths of the Mariana Trench (~100 MPa), are pressure, LP) environments on Earth are scarcely repr esented nearly all surface life is located at the bottom of the troposphere, where the lowest terrestrial barometric pressure, at the top of Mt. Everest, is ~34 kP a. However, recent reports of microbial metabolic processes occurring at high altitudes withi n clouds (65, 66 ) are beginning to challenge this paradigm. environments is gaining importance due to: (i) hypobaric chambers becoming increasingly used for long term storage of high value perishable agricultural commodities (41) ; (ii) microbial sampling of the limits of the upper atmosphere (66 70 ) ; and (iii) the astrobiological implications of terrestrial microorganisms capable of living in extreme LP environment s such as found on Mars (37, 38) .
24 Current knowledge of bacterial pressure responses is confined mostly to studies of (i) hyperbaric, or piezophilic, microbes and (ii) exposure to HP of pressure mesophiles such as Escherichia coli or spores of Bacillus spp. (3, 28, 71 ) . Ribosomal pyrosequencing analysis and culturing of deep sea samples revealed that numerous species of Bacteria, Archaea, and micro Eukarya exist at HP in the deep sea piezosphere (72 ) . Various mechanisms have been discovered or postulated that contribute to the adaptation of piezophilic microbes to growth at HP, such as: changes in composition of permeases i n the outer membrane (23) ; pressure sensing mechanisms (24) ; differential expression of termin al oxidases (73 ) ; differences in lipid composition of membranes (13, 74, 75 ) ; differ ences in the structure and amino acid composition of proteins (76 ) ; elongation of helical regions within 16S rRNA (20) ; and changes in enzymatic volumes (1, 32, 64, 77 ) . In contrast to the relative wealth of studies on how HP affects bacterial cells, there is a nearly complete lack of literature on mechanisms of microbial cellula r responses to LP. Just as both cold and heat tolerant organisms together encompass the entire range of extreme temperature ranges at which life persists, the study of HP on microbes alone does not fully describe the effects of pressure on cellular functi ons. The closest experimental analogue of microbial cellular response to LP was a comparison of the transcriptomes of the piezophilic organism Photobacterium profundum strain SS9 when grown at atmospheric pressure (~101 kPa) vs. HP (28 MPa ) (25) regulation of transcripts involved in amino acid and ion transport, amino acid metabolism, and a variety of other cellular processes (25) . To date, several bacterial species have been tested for growth and/or metabolism under various LP regimes (33, 36, 78, 79 ) . In most species tested, growth was observed to be
25 essentially normal from ~101 kPa down to 10 kPa (36) , but the growth of most microorganisms slowed dramatically at pr essures below 10 kPa and essentially ceased at 2.5 kPa (33, 37) . Two notable exceptions reported recently were six Carnobacterium spp. isolates from Siberian permafrost (38) and a strain of Serratia liquefaciens identified in a screen of multiple laboratory strains of bacteria (37) , bo in an anoxic, CO 2 dominated atmosphere (i.e., a simulation of the martian atmosphere). Growth of the model bacterium Bacillus subtilis is completely inhibited at 2.5 kPa and severely comp romised at 5 kPa (34, 37) . In order to probe the global response of B. subtilis to LP, we describe here transcription microarray experiments comparing transcript levels of cells cultivated at 5 kPa vs. ~101 kP a. Ma terials and Methods Bacterial Strains, Media, and Growth Conditions All strains and plasmids used in this study are listed in Table 2 1. Strain WN624 ( trpC2 amyE::spc ) has been described in detail previously (34, 80 ) . B. subtilis strains PB153 and PB344, harboring separate deletion insertion mutations inactivating sigB , were obtained from Chet Price (63 ) . B. subtilis strain BSM151 carrying a lacZ fusion to the sigB dependent , GSR induced ctc gene was obtained from Uwe VÃ¶lker (81 ) . Standard protocols were used for isolation of chromosomal DNA from donor strains (82) , preparation of competent B. subtilis cells, and DNA mediated transformation (83) . Miller LB liquid or agar medium (84) was used throughout and supplemented when necessary with the appropriate antibiotic (final concentration): chloramphenicol (Cm, 5 mg/mL); neomycin (Neo, 5Âµg/mL); spectinomycin (Spc, 100 Âµg/mL); or erythromycin (Erm, 1 Âµg/mL). Cells were grown under normal laboratory atmospheric pressure (~101 kPa) or low pressure (5 kPa) in an airtight dessicator attached to a programmable vacuum pump as described in detail previously (34, 86 ) . Cultures were shaken at moderate speed
26 Summerson photometer fitted with the No. 66 (660 nm; red) filter. Under these conditions, 100 Klett units = 1 OD660 = ~1 x 108 CFU pe r mL. Microscopic examination showed that cells grown at 5kPa were the same size and shape as those grown at ~101 kPa, thus an increase in optical density corresponded to an increase in total cells. Isolation and Labeling of Total RNA Equivalent masses of WN624 cells (obtained from 10 mL or 100 mL of overnight cultures grown at ~101 kPa or 5 kPa, respectively) were harvested by centrifugation, the supernatants removed by aspiration, and the cell pellets frozen at 70 C. Overnight cultures were used due to t he low optical density after 24 hr. of growth (~0.4 OD 660 ). Approximately 4 x 10 9 cells were obtained from each sample, estimated from culture optical densities determined before centrifugation. Total RNA was extracted from cells and treated with RNase fre e DNase using the sample concentrations and purity were determined by UV absorbance measurements at 260 and 280 nm (86) . RNA Integrity Numbers (RIN) were obtained using aliquots of total RNA using the RNA 6000 Nano Kit on an Agilent 2100 Bioanalyzer, (Agilent Technologies). The average RNA Integrity Number (RIN) (88 ) of total RNA samples was 9.71. Transcription Microarray Experiments Total RNA samples were sent to the University of Florida Interdisciplinary Center for Biotechnology Research (UF ICBR) for fluorescent labeling with Cy3 or Cy5 and microarray analyses. A custom glass slide microarray (GE 8x15K 60 mer; Agilent Technologies) was designed and built using the B. subtilis strain 168 genome sequence (90) . For each sample, approximately 12 ÂµL of RNA at a concentration of 500 ng/ ÂµL was loaded for a total RNA content of 6 Âµg.
27 Microarray Data Analysis and Normalization Gene names, locations and descriptions were cross ref erenced using the GenoList archive server ( http://genolist.pasteur.fr ). Each microarray chip yielded 15,209 data points equivalent to 4,103 genes with an average number of 3.7 measurements per gene. The raw data, green and red mean intensities, and dispersions were consistent across the 8 sample comparisons. Raw data scatter plots for each control comparison indicated the high quality of the data, i.e. a high level of correlation for control chips and a high level of dispersion for test chips (Figure 2 1). Loess normalization was applied to the microarray data to correct for bias caused by inconsistencies in the relative fluorescence intensity between the Cy3 and Cy5 dyes. Variations between the multiple microarray experiments were removed using quantile normalization. The analysis of (LIMMA, freely available at http://bioconductor.org ) package in the R programming language. The LIMMA package uses empirical Bayesian methods to provide stable results by moderating the standard errors of the estimated fold changes. Galactosidase Assay Constructed strains carrying the sigB dependent, GSR inducible ctc lacZ gene fusion (Table 2 galactosidase activity by ethanol, a known inducer of the GSR (63 ) e (~30 Klett units) then split. To one subculture was added ethanol to a final concentration of 5%, and incubation was continued for 45 min. To test for LP induction of ctc lacZ expression, cultures to early logarithmic phase (~20 Klett units). A zero time sample was taken, then the cultures were split into twelve 2 mL subcultures, of which 6 were incubated at ~101 kPa and 6 were incubated at various LP conditions (5, 10, 25, or 50 kPa) for a further 2.5 hr. Culture OD 660 values were determined, then a 1 mL sample from
28 each tube was centrifuged and the resulting cell pellet frozen at b galactosidase assay. Thawed cells were lysed and assayed for b galactosidase activity as describe d previously (88 ) . b galactosidase activity is expressed in Miller units (84) . Microarray Data Accession Number The complete set of microarray data has been deposited in the Gene Expression Omnibus (GEO) database at the National Center for Biotechnology Information ( http://www.ncbi.nlm.nih.gov/geo/ ) under accession n umber GSE50653. Results and Discussion Transcriptome Analysis of Strain WN624 at 5 kPa and ~ 101 kPa In order to gain a greater understanding of global gene expression changes occurring in response to LP, strain WN624 was studied using transcriptional micr oarrays. Total RNA was extracted from cells after 24 hours of growth at either 5 kPa or ~101 kPa, and RNA labeling and chip analyses were conducted as described in Materials and Methods. The data are summarized by 2 dimemsional plots (Figure 2 1) . To confi rm RNA integrity and consistency of probe labeling, a dye switch experiment was performed using the same total RNA labeled with Cy3 vs. Cy5, purified from strain WN624 cultured at ~101 kPa (Figure 2 1A) or 5 kPa (Figure 2 1B). In each case the data were ti ghtly clustered and displayed high correlation coefficients (R 2 ) of 0.9912 and 0.9959, respectively (Figure 2 1A, 2 1B). In contrast, microarrays probed with total RNA isolated from strain WN624 cultivated at 5 kPa vs. ~101 kPa exhibited much greater dispe rsion and a lower R 2 of 0.6114 (Figure 2 1C), indicating a profound alteration of the WN624 transcriptome in response to LP affecting the mRNA levels of hundreds of genes. In total, exposure of strain WN624 at 5 kPa resulted in identification of 223 signif icantly up regulated (Table 2 4) and 140 significantly down regulated (Table 2 5) genes, totaling approximately 9% of the Bacillus subtilis 168 genome.
29 Exposure to LP was found to lead to up and/or down regulation of target genes for a large number of glo bal regulons (Tables 2 4 and 2 5). The regulons found to be the most dramatically altered by exposure to 5 kPa are listed in Table 2 3. Exposure of B. subtilis to LP most notably induced the SigB mediated General Stress Response (GSR) (89 ) ; transition state regulons such as AbrB/Abh (90, 91 ) , CodY (92 ) , Rok (93 ) , SigH (94 ) , Spo0A (95 ) , and to a lesser extent, SigD (96 ) ; anaerobic regulons such as R esD (97 ) , and to a lesser extent Fnr (98 ) and Rex (99 ) ; the carbon catabolite repression regulator CcpA (100 ) ; and the IolR regulator of myo inositol catabolism (101 ) . Interestingly, a large number of tra nscripts (86 in total) belonging to no known regulons were both induced and repressed by LP exposure (Tables 2 3). Because LP exposure most strongly induced the SigB mediated GSR regulon, we chose to study LP induction of the GSR in further detail. The GSR regulon in B. subtilis consists of ~185 target genes, expression of which is induced in response to nutrient starvation and a variety of environmental stresses such as heat, cold, ethanol, or salt (89, 102, 103 ) . Transcriptional activation of GSR genes is under control of the alternate sigma factor sigma B ( B or SigB) encoded by the sigB gene (89 ) . The sigB gene is located within the rsbRSTUVWsigBrsbX operon, with a sigB dependent promoter embedded upstream of rsbV allowing for S igB dependent transcriptional induction of the last four genes in the operon including autoinduction of sigB . Activity of SigB is regulated by the RsbW anti sigma and RsbV anti anti sigma factor system in response to a complex system of regulators encoded by the upstream rsbRSTU operon and the rsbX gene, whose products are responsive to various environmental stresses (89 ) , and rsbPQ whose products are involved in energy stress activation of the SigB response (104 ) . Exposure of strain WN624 to 5 kPa resulted in a dramatic (~35 to 55 fold) up regulation of mRNA levels for genes of the rsbWVsigBrsbX operon (Table
30 2 4), but not of rsbRSTU or rsbPQ transcripts. Of the 185 target genes in the GSR regulon (listed in Table 2 6), 88 of these genes are known to be strictly depende nt upon SigB for their induction (108 110) (http://subtiwiki.uni goettingen.de/wiki) ( genes with underlined names in Table 2 6). Of these, 63 (70%) were up regulated in the microarray measuring mRNA from cells expo sed to 5 kPa vs. ~101 kPa (Figure 2 1C, Table 2 6). In order to compare GSR gene expression resulting from LP exposure to other physical stresses, we compared the LP mediated GSR of strain WN624 to the global GSR of B. subtilis exposed to ethanol, heat, o r salt stress in previous studies (105, 111, 109) (Figure 2 2). Very good concordance was observed between the two responses; LP exposure induced up regulation of 86 genes, 70 of which (81%) were also induced by ex posure to heat, ethanol, or salt (Figure 2 2). Notably, 30 ethanol/heat/salt inducible GSR genes were not induced by LP, and LP exposure induced expression of 16 genes not reported to be induced by ethanol/heat/salt stress (Figure 2 2), indicating that LP induced a GSR that partially overlapped with that induced by ethanol/heat/salt stress. Induction of ctc lacZ Expression by LP The above observations led us to the notion that exposure of B. subtilis cells to LP induces the SigB mediated GSR. Activation of the SigB dependent ctc lacZ reported gene fusion is a reliable and well established marker for GSR induction by all stresses studied to date (81 ) . We therefore tested expression of a ctc lacZ fusion in strains carrying either the wild type sigB gene (str ain WN1400) or the sigB D 3::spc knockout mutation (strain WN1407) (Figure 2 3). First, in order to assure that ctc lacZ expression was properly regulated by SigB in our strains we first induced the GSR by the classical treatment of exponentially growing cel ls with ethanol at 5% final concentration and assayed for b galactosidase activity after 45 min (Figure 2 3A). In strain WN1400 carrying the sigB + allele, expression of ctc lacZ was strongly induced (~7 fold)
31 by ethanol treatment (Figure 2 3A). In comparis on, basal expression the ctc lacZ in strain WN1407 carrying the sigB D 3::spc knockout mutation was lower, and induction of ctc lacZ by ethanol was much weaker (~2 fold) (Figure 2 3A). These results were expected, thus the ctc lacZ fusion appeared to be a re liable reporter of the SigB dependent GSR in our strains. Next the same two ctc lacZ reporter strains were tested for induction of the GSR by exposure to 5 kPa LP (Figure 2 3A). After exposure to 5 kPa for 2.5 hours, ctc lacZ expression was induced ~4 fold in strain WN1400 carrying the wild type sigB + allele (Figure 2 3A). In contrast, strain WN1407 carrying the sigB D 3::spc knockout mutation exhibited a much lower basal level of ctc lacZ expression and only very weak induction (<2 fold) by LP (Figure 2 3A). The results support the notion that LP induction of signals belonging to the GSR was indeed SigB dependent. We were interested in determining the level of LP required to trigger the SigB dependent GSR, so we measured expression of the ctc lacZ fusion in s train WN1400 ( sigB + ) at pressures of ~101, 50, 25, 10, and 5 kPa (Figure 2 3B). Strain WN1400 did not induce ctc lacZ expression until pressure was lowered to either 10 or 5 kPa (Figure 2 3B). Inactivation of sigB Does Not Alter Fitness at ~ 101 kPa or at 5 kPa The microarray data (Table 2 2, Figure 2 2) and the results of the ctc lacZ reporter experiments (Figure 2 3) indicated that the SigB dependent GSR was induced by exposure to LP in B. subtilis . We were interested in investigating what effect, if any, a sigB k nockout mutation would have on the relative fitness of B. subtilis at 5 kP a. Competition experiments, performed as described previously (34, 86 ) , between 2 congenic strains carrying either the wild type si gB + gene or a sigB D 2::cat insertion deletion mutation, showed that inactivation of sigB did not significantly change its relative fitness at either ~101 kPa or at 5 kPa (Figure 2 4). This result is consistent with previous experiments showing that a sigB n ull mutant had no noticeable disadvantage compared to wild type when grown under any of the stress conditions known to
32 induce the SigB mediated GSR (63, 89 ) . Therefore, as with other physical stresses, LP appeared to induce a subset of the SigB dependent GSR, however GSR induction did not improve growth under LP. Relatively little is known about how hypobaria affects cellular processes, or how microbial life is affected by and responds to LP. In this communication w e found by microarray experiments that Bacillus subtilis strain WN624 sensed and responded to LP stress by activation of at least a dozen known regulons including the SigB mediated GSR, and this notion was confirmed by the SigB dependent induction of ctc lacZ fusion expression by exposure of cells to LP. Non optimal pressure responses of piezophiles grown at standard atmospheric pressure (25) and pressure mesophiles grown at elevated pressures (28) are both known to induce stress response genes such as heat and cold shock proteins. In this communication we show that exposure of a pressure mesophile to LP up regulated several glob al regulons including the SigB dependent GSR. It was interesting to note that strain WN624 did not induce the GSR until pressure was lowered to 10 kPa, the equivalent of an altitude of ~18 km, over twice the height of Mt. Everest (8,848 meters). As stated previously, the known biosphere is contained within the troposphere, where the low pressure limit is ~10 kPa (4) . Interestingly, this is also the lower limit of pressure before significant decreases in growth rate and colony size were demonstrat ed in a number of Gram (+) and Gram ( ) bacterial species (36 38, 113) . Therefore it appears for most bacteria [with a few notable recent exceptions mentioned above (37, 38) ], there exists a low pressure limit at ~10 kPa below which cellular processes begin to be inhibited and the GSR is induced in B. subtilis . As observed previously with other environmental stresses (ethanol, heat, high salt), the SigB mediated GSR does not seem to be required for growth at 5 kP a. This does
33 not rule out that stress related, SigB independent gene(s) might be necessary for B. subtilis growth at LP. Future experiments to address this issue are in progress. Towards an Understanding of the LP Res ponse. A previous study compared growth kinetics of B. subtilis in LB medium at either ~101 kPa or 5 kPa (34) . At normal atmospheric pressure, cells grew to high density (>300 Klett units), but at 5 kPa growth ceased abruptly at ~40 Klett units and remained unchanged for 24 hours. Growth inhibition was apparently caused by s ome aspect of LP exposure and not by nutrient limitation, as cells immediately resumed exponential growth upon increase of pressure back to ~101 kPa (34) . In an attempt to integrate these prior observations with the results of the transcription microarrays, the following scenario might be envisioned as a preliminary working model, although others are certainly possible. One consequence of lowering the headspace pressure from ~101 to 5 kPa is to lower the partial pressure of oxygen in the medium by roughly a factor of 20; this might immediately lead to induction of the anaerob ic response involving the ResD, Fnr, and Rex regulons (Tables 2 4 and 2 5). These regulators in turn induce expression of a host of genes involved in fermentation and anaerobic respiration. However, because Miller LB medium lacks fermentable sugars or alte rnative electron acceptors such as nitrate, cessation of growth and entrance into stationary phase rapidly ensues. Entrance into stationary phase activates the SigB mediated GSR as well as a number of regulators of the transition state, such as AbrB/Adh, C odY, Rok, Spo0A, and SigH; in addition, CcpA has been shown to remodel carbon metabolism in the early stationary phase ( 110) . Induction of IolR by LP is difficult to fit into this scheme bec ause IolR is not a global regulator, but a specific regulator of genes involved in myo inositol catabolism (the operons iolABCDEFGHIJ and iolRS , and the iolT gene (101 ) . However, expression of this small regulon has also been documented to be strongly induced upon entrance into the stationary phase ( 111 ) .
34 In summary, exposure of B. subtilis to LP activates a large number of responses including the SigB dependent GSR. At present it is known that activation of sigB expression can occur by three pathways responsive to: energy stress ( 112 ) ; exposure to physical stresses such as ethanol, h eat, or high salt (63 ) ; or cold stress (81 ) . Determining which of these signaling pathway(s) are induced by LP will shed further insight into how the GSR is induced, directly or indirectly, by LP, and is a course for future work.
35 Table 2 1. Details of microarrays used in Chapters 2 and 3 Microarray Cy5 labelled probe Cy3 labelled probe Strain Pressure (kPa) Strain Pressure (kPa) 1 WN624 101 WN624 101 2 WN624 5 WN624 101 3 WN1106 101 WN624 101 4 WN624 5 WN6 24 5 5 WN1106 5 WN624 5 6 WN1106 101 WN1106 101 7 WN1106 5 WN1106 101 8 WN1106 5 WN1106 5
36 Table 2 2. Strains and plasmids used in this study Strain or Plasmid Genotype/Phenotype Source (reference) BSM151 trpC2, SPb::ctc lacZ ; Cm R , Erm R Uwe VÃ¶lker (82) PB344 Chet Price (81 ) WN624 trpC2, amyE::spc; Spc R , wild type, congenic with WN628 ( 113 ) WN628 trpC2, amyE::cat; Cm R , wild type, congenic with WN624 ( 113 ) WN1261 trpC2, amyE::neo, Neo R pECE73 628; NeoR/CmS WN1392 trpC2, amyE::neo sigBD3::spc; Neo R , Spc R PB344 WN1261; SpcR WN1400 lacZ; Neo R , Cm R , Erm R BSM151 WN1261; CmR, ErmR WN1407 lacZ; Neo R , Spc R , Cm R , Erm R BSM151 WN1392; CmR, ErmR pECE73 pCm::Neo antibiotic switching cassette BGSC ( 114 ) A Abbreviations: BGSC, Bacillus Genetic Stock Center; , t ransformation.
37 Table 2 3. Summary of regulons with at least 10 target genes affected A Regulator #Up regulated genes #Down regulated genes Total Response SigB 86 2 88 Transcription of General Stress Response genes Unknown 41 47 88 Unknown AbrB/Abh 42 16 58 Transition from growth to stationary phase CcpA 12 26 38 carbon catabolite repression Fur 1 25 26 regulation of iron homoeostasis ResD 22 0 22 Regulation of aerobic/anaerobic respiration CodY 19 1 20 Response to nutritional starvation Rok 13 1 14 Regulation of genetic competence Spo0A 13 0 13 Phosphorelay regulator of sporulation initiation SigH 11 1 12 Early stationary phase gene expression IolR 0 11 11 regulation of myo inositol catabolism A For detailed list of all regulons affected, refer t o Tables 2 4 and 2 5.
38 Table 2 4. B. subtilis genes significantly up regulated by exposure to 5 kPa BSU number Gene Name Fold Change P value Known Regulon Gene Description BSU00520 ctc 7.18 0.016 SigB ribosomal protein Ctc, binding 5S RNA BSU00830 ctsR 1 1.26 0.004 CtsR transcriptional regulator BSU00840 mcsA 8.82 0.009 CtsR, SigF activator of protein kinase McsB BSU00850 mcsB 5.29 0.036 CtsR, SigF protein tyrosine kinase BSU02100 cypC 7.90 0.013 SigB fatty acid beta hydroxylating cytochrome P450 BSU02 110 ybyB 76.68 <0.001 ? conserved hypothetical protein BSU02590 ycbP 14.75 0.002 SigB putative inner integral membrane protein BSU02830 ycdF 12.76 0.003 SigB putative dehydrogenase BSU02840 ycdG 6.14 0.025 ? putative glycosidase BSU03050 ldh 9.79 0.007 Rex L lactate dehydrogenase BSU03060 lctP 16.20 0.001 Rex L lactate permease BSU03200 ycgM 6.06 0.026 CodY, PutR, Spo0A proline oxidase BSU03300 nasD 5.87 0.028 NsrR, ResD, TnrA assimilatory nitrite reductase subunit BSU03760 yclK 4.83 0.044 ResD two component sensor histidine kinase [YclJ] BSU03940 ycnI 4.78 0.045 YcnK conserved hypothetical protein BSU03950 ycnJ 4.76 0.046 YcnK putative copper import protein BSU03960 ycnK 5.28 0.036 AbrB, YcnK putative transcriptional regulator (DeoR family) BSU0 4190 ydaD 6.73 0.019 SigB putative dehydrogenase BSU04200 ydaE 6.93 0.018 SigB conserved hypothetical protein BSU04220 ydaG 32.42 <0.001 SigB putative general stress protein BSU04240 ydzA 6.06 0.026 ? conserved hypothetical protein BSU04340 ydaP 19.14 0.001 SigB putative enzyme with pyruvate as substrate BSU04370 ydaS 9.33 0.008 SigB conserved hypothetical protein BSU04400 gsiB 90.26 <0.001 SigB, SigI general stress protein BSU04710 rsbV 36.63 <0.001 SigB anti anti sigma factor (antagonist of RsbW) BSU04720 rsbW 44.01 <0.001 SigB switch protein/serine kinase and anti sigma factor (inhibitory sigma B binding protein) BSU04730 sigB 56.94 <0.001 SigB RNA polymerase sigma 37 factor (sigma(B)) BSU04740 rsbX 49.01 <0.001 SigB serine phosphatase BSU05130 ydeB 6.71 0.02 ? putative transcriptional regulator BSU05790 ydhK 9.13 0.008 SigB hypothetical protein BSU05980 tatAY 5.66 0.03 ? component of the twin arginine pre protein translocation pathway BSU05990 tatCY 7.12 0.017 ? component of the twin arginin e pre protein translocation pathway BSU06590 yerD 5.50 0.033 SigB putative flavoenzyme BSU06640 yerI 8.57 0.01 AbrB putative kinase BSU06660 opuE 30.35 <0.001 SigB, CcpA proline transporter
39 Table 2 4. Continued BSU number Gene Name Fold Change P value Known Regulon Gene Description BSU06710 yerP 5.16 0.038 ? transporter involved in surfactin self resistance BSU06830 rapH 5.21 0.037 AbrB, ComK, RghR response regulator aspartate phosphatase BSU07380 yfmQ 6.94 0.018 ? conserved hypothetical protein BS U07550 yflT 31.64 <0.001 SigB heat stress induced protein BSU07750 yflA 45.79 <0.001 Abh, AbrB, SigB putative aminoacid transporter BSU07760 yfkT 24.75 <0.001 SigB putative spore germination integral inner membrane protein BSU07770 yfkS 13.26 0.003 SigB hypothetical protein BSU07780 yfkR 4.82 0.045 SigG putative spore germination protein BSU07850 yfkM 23.91 <0.001 Fur, SigB general stress protein 18 BSU07880 yfkJ 29.03 <0.001 SigB protein tyrosine phosphatase BSU07890 yfkI 21.62 <0.001 SigB conserved hypothetical protein BSU07900 yfkH 15.34 0.002 SigB putative integral inner membrane protein with ribonuclease fold BSU07920 yfkE 14.71 0.002 SigB, SigG putative H+/Ca2+ antiporter BSU07930 yfkD 7.91 0.013 SigB conserved hypothetical protein BSU08490 yfhD 9.35 0.008 SigB conserved hypothetical protein BSU08500 yfhE 5.70 0.03 SigB hypothetical protein BSU08510 yfhF 5.99 0.026 SigB putative nucleotide binding protein BSU08570 yfhK 89.64 <0.001 SigB, SigW conserved hypothetical protein BSU08580 yfhL 3 0.83 <0.001 SigB, SigW SdpC immunity factor BSU08590 yfhM 15.77 0.001 SigB, SigW putative hydrolase BSU08600 csbB 7.21 0.016 SigB, SigX putative glycosyl transferase BSU08990 yhbI 8.77 0.009 ? putative transcriptional regulator (MarR family) BSU09000 y hbJ 10.04 0.006 ? putative integral inner membrane protein; putative exporter subunit BSU09010 yhcA 6.50 0.021 ? putative exporter BSU09020 yhcB 5.10 0.039 ? putative oxidoreductase associated to oxygen stress BSU09140 yhcM 5.05 0.04 SigB, SigF, SigG hy pothetical protein BSU09190 yhcR 4.95 0.042 ? non specific extracellular endonuclease cleaving RNA and DNA BSU09230 yhcV 6.23 0.024 SigG putative oxidoreductase BSU09530 yhdN 28.76 <0.001 SigF, SigG aldo/keto reductase specific for NADPH BSU09690 nhaX 68.38 <0.001 SigB stress response protein, UspA family BSU09750 sspB 6.84 0.019 SigG, SpoVT small acid soluble spore protein (beta type SASP) BSU10130 hemH 5.09 0.039 ? ferrochelatase BSU10140 hemY 4.80 0.045 ? protoporphyrinogen IX and coproporphyrinog en III oxidase
40 Table 2 4. Continued BSU number Gene Name Fold Change P value Known Regulon Gene Description BSU10150 yhgD 6.40 0.022 ? putative transcriptional regulator BSU10230 yhfH 4.69 0.047 ? hypothetical protein BSU10430 yhxD 4.98 0.041 SigB put ative oxidoreductase BSU11490 yjbC 28.10 <0.001 PerR, SigB, SigM, SigW, SigX putative thiol oxidation management factor; putative acetyltransferase BSU11830 yjcE 19.45 0.001 SigB BG1315:unknown BSU11990 yjdB 92.88 <0.001 AbrB, PhoP putative exported pro tein BSU12080 ctaO 8.71 0.01 AbrB protoheme IX farnesyltransferase (heme O synthase) BSU12160 yjgC 7.65 0.014 SigB putative oxidoreductase BSU12270 yjlB 12.31 0.003 ? conserved hypothetical protein ; cupin family BSU12430 rapA 28.32 <0.001 CodY, ComA, Spo0A response regulator aspartate phosphatase BSU12440 phrA 31.67 <0.001 CodY, ComA, Spo0A secreted inhibitor of the activity of phosphatase RapA BSU13020 ykgA 38.11 <0.001 SigB putative aminohydrolase BSU13160 ykzA 24.31 <0.001 SigB organic hydroperox ide resistance reductase B BSU13170 guaD 8.66 0.01 PucR, SigB guanine deaminase BSU13850 ykvW 6.99 0.018 PerR Zn transporter BSU14660 ykzI 20.32 0.001 SigB conserved hypothetical protein BSU14890 ctaC 7.07 0.017 Abh, AbrB, CcpA, ResD cytochrome caa3 ox idase (subunit II) BSU14910 ctaE 4.81 0.045 Abh, AbrB, ResD cytochrome caa3 oxidase (subunit III) BSU14920 ctaF 5.07 0.04 Abh, AbrB, ResD cytochrome caa3 oxidase (subunit IV) BSU14930 ctaG 5.40 0.034 Abh, AbrB, ResD cytochrome aa(3) assembly factor BSU 15870 recG 7.74 0.013 ? branch migrating ATP dependent DNA helicase involved in DNA recombination and repair BSU15880 ylpC 5.70 0.03 ComA, FapR transcription factor BSU16180 flgB 5.02 0.04 CodY, SigD, Spo0A flagellar component of cell proximal portion of basal body rod BSU16190 flgC 5.80 0.029 CodY, SigD, Spo0A flagellar component of cell proximal portion of basal body rod BSU16200 fliE 5.15 0.038 CodY, SigD, Spo0A flagellar basal body protein BSU16210 fliF 5.95 0.027 CodY, SigD, Spo0A flagellar basal body M ring protein BSU16220 fliG 8.22 0.011 CodY, SigD, Spo0A flagellar motor switching and energizing component
41 Table 2 4. Continued BSU number Gene Name Fold Change P value Known Regulon Gene Description BSU16230 fliH 7.32 0.016 CodY, SigD, Spo0A fl agellar export apparatus component BSU16240 fliI 5.91 0.027 CodY, SigD, Spo0A flagellar specific ATPase BSU16250 fliJ 4.78 0.045 CodY, SigD, Spo0A flagellar synthesis chaperone BSU17100 pksC 6.25 0.024 AbrB, CodY malonyl CoA acyltransferase involved in polyketide synthesis BSU17110 pksD 7.65 0.014 AbrB, CodY enzyme involved in polyketide synthesis BSU17120 pksE 5.44 0.033 AbrB, CodY enzyme involved in polyketide synthesis BSU17130 acpK 4.63 0.049 AbrB, CodY acyl carrier protein BSU17180 pksJ 5.57 0.0 32 AbrB, CodY polyketide synthase of type I BSU17240 ymzB 4.97 0.041 AbrB, SigB conserved hypothetical protein BSU17690 yncM 24.09 <0.001 AbrB conserved hypothetical protein BSU17710 tatAC 17.94 0.001 ? component of the twin arginine pre protein translo cation pathway BSU18110 ynfC 11.12 0.005 ? conserved hypothetical protein BSU18310 ppsD 4.73 0.047 AbrB plipastatin synthetase BSU18320 ppsC 5.15 0.038 AbrB plipastatin synthetase BSU18350 dacC 5.85 0.028 SigH D alanyl D alanine carboxypeptidase BSU18 360 yoxA 5.45 0.033 SigH putative epimerase BSU18380 yoeB 7.97 0.012 WalR inhibitor of cell separation enzymes BSU18440 gltB 16.00 0.001 GltC, FsrA, TnrA glutamate synthase (small subunit) BSU18450 gltA 57.49 <0.001 GltC, FsrA, TnrA, Efp dependent Prote ins glutamate synthase (large subunit) BSU18510 yoxC 36.27 <0.001 SigB conserved hypothetical protein BSU18520 yoxB 27.18 <0.001 SigB conserved hypothetical protein BSU18530 yoaA 11.67 0.004 SigB putative N acetyltransferase BSU18920 phrK 6.80 0.019 Ab rB, SigH secreted regulator of the activity of phosphatase RapK BSU19030 yobO 7.09 0.017 AbrB, CcpA putative phage related pre neck appendage protein BSU19150 yocB 9.65 0.007 SigB conserved hypothetical protein BSU19310 dhaS 11.16 0.004 ? putative aldeh yde dehydrogenase BSU19410 yojL 6.53 0.021 Abh, AbrB, CcpA, SigD, SigH peptidoglycan hydrolase (cell wall binding d,l endopeptidase) BSU23120 resD 5.42 0.034 CcpA, PhoP, ResD two component response regulator
42 Table 2 4. Continued BSU number Gene Name F old Change P value Known Regulon Gene Description BSU23130 resC 4.72 0.047 CcpA, PhoP, ResD factor required for cytochrome c synthesis BSU23140 resB 5.96 0.027 CcpA, PhoP, ResD factor required for cytochrome c synthesis BSU23270 ribE 6.35 0.023 FMN box riboflavin synthase (alpha subunit) BSU23280 ribD 6.17 0.024 FMN box fused diaminohydroxyphosphoribosylaminopyrimidine deaminase; 5 amino 6 (5 phosphoribosylamino) uracil reductase BSU24000 bmrU 37.25 <0.001 SigB putative diacylglycerol kinase BSU24110 yqzF 4.99 0.041 ? conserved hypothetical protein BSU24550 gcvPB 11.10 0.005 Gly box glycine decarboxylase (subunit 2) (glycine cleavage system protein P) BSU24560 gcvPA 11.44 0.004 Gly box glycine decarboxylase (subunit 1) (glycine cleavage system protei n P) BSU24570 gcvT 9.45 0.007 Gly box aminomethyltransferase (glycine cleavage system protein T) BSU24740 yqxL 11.52 0.004 LexA, SigB putative CorA type Mg(2+) transporter BSU24750 yqhB 12.43 0.003 LexA, SigB putative membrane associated protein BSU247 60 yqhA 22.09 <0.001 SigB component of the piezosome (stressosome) BSU24770 yqgZ 94.89 <0.001 SigB, MgsR transcriptional regulator of stress BSU25080 yqfX 4.74 0.046 SigG conserved hypothetical protein BSU25220 antE 6.28 0.023 AbrB hypothetical protein BSU26620 yrdR 6.40 0.022 ? putative efflux transporter BSU26890 csn 82.13 <0.001 AbrB chitosanase BSU26900 yraL 4.83 0.044 ? conserved hypothetical protein BSU26920 yraJ 6.90 0.018 ? conserved hypothetical protein BSU26930 yraI 6.91 0.018 ? conserved hypothetical protein BSU28340 ysnF 6.25 0.024 SigB putative stress response protein BSU28550 ysiA 5.42 0.034 CcpA, FadR transcriptional regulator of fatty acids degradation (TetR/AcrR family) BSU28560 lcfA 6.41 0.022 CcpA, FadR long chain acyl CoA ligas e (degradative) BSU29260 ytpI 7.39 0.015 ? conserved hypothetical protein BSU29760 ytxJ 11.23 0.004 SigB, SigH conserved hypothetical protein BSU29770 ytxH 14.48 0.002 SigB, SigH conserved hypothetical protein BSU29780 ytxG 17.69 0.001 SigB, SigH conse rved hypothetical protein BSU30650 dps 11.35 0.004 SigB DNA protecting protein, ferritin BSU30660 ytkA 6.50 0.021 ? putative lipoprotein BSU30700 rpmE2 19.88 0.001 ? ribosomal protein L31 BSU30930 ytaB 20.41 0.001 SigB putative receptor BSU31280 yugU 6.17 0.025 SigB conserved hypothetical protein
43 Table 2 4. Continued BSU number Gene Name Fold Change P value Known Regulon Gene Description BSU31690 comP 6.50 0.021 ? two component sensor histidine kinase BSU31700 comX 7.87 0.013 ? competence pheromon e precursor (pheromone peptide aa 46 >55, modified) BSU31710 comQ 5.69 0.03 ? isoprenyl transferase (pre ComX modification) BSU31880 yukB 6.65 0.02 DegU BG1237:unknown; similar to unknown proteins BSU32890 yusQ 17.57 0.001 ? putative tautomerase BSU329 00 yusR 21.71 <0.001 SigE putative 3 oxoacyl acyl carrier protein reductase BSU32910 yusS 24.32 <0.001 ? putative 3 oxoacyl acyl carrier protein reductase BSU33200 yvrE 9.00 0.009 SigB conserved hypothetical protein BSU33240 oxdC 20.16 0.001 YvrI YvrH a oxalate decarboxylase BSU33410 yvgO 24.86 <0.001 AbrB, SigB conserved hypothetical protein BSU33530 yvaA 10.26 0.006 SigB putative oxidoreductase BSU33700 opuBD 5.20 0.037 GbsR, OpcR choline ABC transporter (permease) BSU33710 opuBC 22.06 <0.001 GbsR , OpcR choline ABC transporter (choline binding lipoprotein) BSU33720 opuBB 12.35 0.003 GbsR, OpcR choline ABC transporter (permease) BSU33730 opuBA 5.21 0.037 GbsR, OpcR choline ABC transporter (ATP binding protein) BSU33750 yvaW 5.92 0.027 AbrB, Rok, Spo0A export of killing factor BSU33760 yvaX 5.10 0.039 AbrB, Rok, Spo0A exporter of killing factor SpbC BSU33770 yvaY 12.75 0.003 AbrB, Rok, Spo0A killing factor SdpC BSU35050 yvnA 8.92 0.009 AbrB, CcpA putative transcriptional regulator BSU35060 cypX 7.61 0.014 AbrB putative monooxygenase (cytochrome P450) BSU35070 yvmC 5.46 0.033 AbrB conserved hypothetical protein BSU35180 csbA 10.33 0.006 SigB putative membrane protein BSU35310 yvyD 7.69 0.014 SigB, SigH ribosome associated sigma 54 modulation p rotein BSU35830 ywtG 9.85 0.007 SigB putative carbohydrate transporter BSU36460 ywoF 6.60 0.021 Abh, AbrB putative pectate lyase BSU36640 ureC 8.21 0.011 CodY, GlnR, PucR, SigH, TnrA urease (alpha subunit) BSU36650 ureB 18.29 0.001 CodY, GlnR, PucR, Si gH, TnrA urease (beta subunit) BSU36660 ureA 19.35 0.001 CodY, GlnR, PucR, SigH, TnrA urease (gamma subunit)
44 Table 2 4. Continued BSU number Gene Name Fold Change P value Known Regulon Gene Description BSU36670 csbD 16.92 0.001 SigB stress response pr otein BSU36720 ywmE 17.57 0.001 SigB hypothetical protein BSU37210 ywjC 44.45 <0.001 SigB conserved hypothetical protein BSU37240 ywiE 35.33 <0.001 SigB cardiolipin synthetase BSU37250 narI 57.08 <0.001 Fnr nitrate reductase (gamma subunit) BSU37260 n arJ 44.17 <0.001 Fnr nitrate reductase (protein J) BSU37270 narH 54.93 <0.001 Fnr nitrate reductase (beta subunit) BSU37280 narG 38.15 <0.001 Fnr nitrate reductase (alpha subunit) BSU37310 fnr 19.63 0.001 Fnr, NsrR, ResD transcriptional regulator (FNR/ CAP family) BSU37320 narK 10.14 0.006 Fnr, NsrR nitrite extrusion permease BSU37350 sboA 104.74 <0.001 AbrB, ResD, Rok subtilosin A BSU37360 sboX 118.20 <0.001 AbrB, ResD, Rok putative bacteriocin like product BSU37370 albA 84.14 <0.001 AbrB, ResD, Rok putative antilisterial bacteriocin (subtilosin) production enzyme BSU37380 albB 74.22 <0.001 AbrB, ResD, Rok putative membrane component involved in subtilosin production BSU37390 albC 56.75 <0.001 AbrB, ResD, Rok putative transporter involved in subtil osin production BSU37400 albD 61.89 <0.001 AbrB, ResD, Rok putative integral inner membrane protein involved in subtilosin production and immunity BSU37410 albE 116.47 <0.001 AbrB, ResD, Rok putative hydrolase involved in subtilosin production BSU37420 albF 102.31 <0.001 AbrB, ResD, Rok putative peptidase involved in subtilosin production BSU37430 albG 98.90 <0.001 AbrB, ResD, Rok putative integral inner membrane protein involved in subtilosin production and immunity BSU37440 ywhL 24.49 <0.001 ? conser ved hypothetical protein BSU37450 ywhK 5.03 0.04 ? factor interacting with DNA helicase PcrA BSU37670 ywfI 4.87 0.043 ? putative oxidoreductase/oxygenase/dismutase BSU38180 ywzA 26.75 <0.001 SigB conserved hypothetical protein BSU38430 gspA 69.26 <0.00 1 SigB putative glycosyl transferase (general stress protein) BSU38610 yxzF 5.85 0.028 SigB hypothetical protein BSU38630 katX 4.60 0.049 RsfA, SigB, SigF major catalase in spores BSU38730 cydD 13.06 0.003 CcpA, ResD, Rex ABC membrane transporter (ATP b inding protein) required for cytochrome bd function BSU38740 cydC 48.69 <0.001 CcpA, ResD, Rex ABC membrane transporter (ATP binding protein) required for cytochrome bd function
45 Table 2 4. Continued BSU number Gene Name Fold Change P value Known Regulon Gene Description BSU38750 cydB 40.17 <0.001 CcpA, ResD, Rex cytochrome bd ubiquinol oxidase (subunit II) BSU38760 cydA 27.64 <0.001 CcpA, ResD, Rex cytochrome bd ubiquinol oxidase (subunit I) BSU38920 pepT 4.96 0.042 ? peptidase T (tripeptidase) BSU38 930 yxjJ 5.37 0.035 DegU, SigB hypothetical protein BSU39040 yxiS 5.17 0.038 SigB hypothetical protein BSU39050 katE 29.22 <0.001 SigB catalase 2 BSU39330 yxiA 7.22 0.016 ? arabinan endo 1,5 alpha L arabinosidase BSU39810 csbC 20.29 0.001 SigB putative sugar transporter BSU39840 yxbG 7.12 0.017 SigB putative oxidoreductase BSU39940 yxaL 5.66 0.03 AbrB, Rok membrane associated protein kinase with beta propeller domain BSU40000 yxnA 10.68 0.005 SigB putative oxidoreductase BSU40660 yybF 37.30 <0.001 ? putative permease
46 Table 2 5. B. subtilis genes significantly down regulated by exposure to 5 kPa Accession BSU number Gene Name Fold Change P value Regulon Gene Description BSU02120 ybeC 7.73 0.013 ? putative H+/amino acid transporter BSU02230 purT 8.36 0.01 ? phosphoribosylglycinamide formyltransferase 2 BSU03370 yckA 4.89 0.042 ? putative ABC transporter (permease) BSU04000 ycsA 6.57 0.02 ? putative tartrate dehydrogenase BSU05680 ydgK 5.54 0.031 ? putative efflux transporter BSU06380 yebC 4.81 0.043 ? putative integral inner membrane protein BSU07160 yetH 5.37 0.033 ? putative lyase/dioxygenase BSU07340 yfnA 4.86 0.042 ? metabolite permease BSU09110 yhcJ 4.63 0.047 ? putative ABC transporter (binding lipoprotein) BSU09470 yhdH 6.55 0.02 ? putative sodium dependent transporter BSU10220 gltT 5.73 0.029 ? proton/sodium glutamate symport protein BSU10310 yhfO 4.82 0.043 ? putative N acetyltransferase BSU13430 ykoX 5.18 0.036 ? putative integral inner membrane protein BSU13960 ykw C 4.54 0.049 ? putative beta hydroxyacid dehydrogenase BSU14180 ykuQ 15.07 0.002 ? tetrahydrodipicolinate N acetyltransferase BSU14190 ykuR 6.15 0.024 ? N acetyl diaminopimelate deacetylase BSU14200 ykuS 5.28 0.035 ? conserved hypothetical protein BSU14550 ykrA 6.61 0.02 ? putative hydrolase BSU14820 ylaL 6.65 0.019 ? conserved hypothetical protein BSU17630 yncC 9.28 0.008 ? putative sugar transporter BSU19510 yojB 11.10 0.004 ? conserved hypothetical protein BSU19520 yojA 15.37 0.001 ? put ative H+/anion permease BSU27200 yrhG 12.18 0.003 ? putative formate/nitrite transporter BSU28900 ysbB 21.29 <0.001 ? antiholin factor BSU28910 ysbA 27.84 <0.001 ? antiholin factor BSU29480 ytxK 5.56 0.031 ? putative nucleic acid methyltransferase BSU29600 braB 5.20 0.036 ? branched chain amino acid Na+ symporter BSU31440 patB 5.23 0.036 ? C S lyase BSU31600 mrpA 5.82 0.027 ? Na+/H+ antiporter BSU31610 mrpB 6.80 0.018 ? Na+/H+ antiporter complex BSU31620 mrpC 6.26 0.023 ? component of Na+/ H+ antiporter BSU31630 mrpD 5.99 0.025 ? component of Na+/H+ antiporter BSU31640 mrpE 4.90 0.042 ? non essential component of Na+/H+ antiporter BSU32040 yuiF 4.96 0.04 ? amino acid transporter BSU33460 yvgT 6.20 0.023 ? putative integral inner memb rane protein BSU33560 yvaD 9.11 0.008 ? putative integral inner membrane protein BSU33570 yvaE 11.80 0.004 ? putative metabolite efflux transporter BSU33580 yvaF 7.71 0.013 ? putative transcriptional regulator BSU36360 mscL 4.95 0.04 ? large conduc tance mechanosensitive channel protein BSU36370 ywpB 4.99 0.04 ? (3R) hydroxymyristoyl [acyl carrier protein] dehydratase BSU36610 ywnC 6.99 0.017 ? putative integral inner membrane protein BSU37490 speB 5.98 0.026 ? agmatinase BSU37570 mmr 7.87 0. 012 ? toxic compound efflux transporter BSU38840 yxkD 4.86 0.042 ? efflux transporter
47 Table 2 5. Continued Accession BSU number Gene Name Fold Change P value Regulon Gene Description BSU39600 yxeC 7.22 0.016 ? putative integral inner membrane protein BSU39660 yxdJ 4.65 0.047 ? two component response regulator [YxdK] BSU39960 yxaI 4.79 0.044 ? putative integral inner membrane protein BSU37780 rocA 5.65 0.03 AbrB, AhrC, CodY, RocR, SigL delta 1 pyrroline 5 carboxylate dehydrogenase BSU35910 rbsR 5.80 0.028 AbrB, CcpA transcriptional regulator (LacI family) BSU09280 glpF 26.30 <0.001 AbrB, CcpA, GlpP glycerol permease BSU09290 glpK 4.82 0.043 AbrB, CcpA, GlpP glycerol kinase BSU34260 yvfB 6.20 0.023 AbrB, EAR, RemA, SinR BG1187:unknown BSU34 270 yvfA 6.47 0.021 AbrB, EAR, RemA, SinR BG1186:unknown BSU34320 yveP 5.91 0.026 AbrB, EAR, RemA, SinR putative glycosyltransferase involved in extracellular matrix formation BSU34330 yveO 4.65 0.047 AbrB, EAR, RemA, SinR putative glycosyltransferase BSU31960 dhbF 11.56 0.004 AbrB, Fur siderophore 2,3 dihydroxybenzoate glycine threonine trimeric ester bacillibactin synthetase BSU31970 dhbB 10.66 0.005 AbrB, Fur isochorismatase BSU31980 dhbE 14.72 0.002 AbrB, Fur 2,3 dihydroxybenzoate AMP ligase (enterobactin synthetase component E) BSU31990 dhbC 18.97 0.001 AbrB, Fur isochorismate synthase BSU32000 dhbA 16.64 0.001 AbrB, Fur 2,3 dihydro 2,3 dihydroxybenzoate dehydrogenase BSU32010 yuiI 11.58 0.004 AbrB, Fur, Efp dependent proteins bacilliba ctin trilactone hydrolase BSU34360 yveL 6.76 0.019 AbrB, RemA, SinR protein tyrosine kinase BSU34370 yveK 9.08 0.008 AbrB, RemA, SinR modulator of protein tyrosine kinase EpsB BSU23570 ansA 18.21 0.001 AnsR L aspartase (aspartate ammonia lyase) BSU2 3580 ansB 11.31 0.004 AnsR exported L asparaginase BSU23590 ansR 5.52 0.031 AnsR transcriptional regulator of ansAB (Xre family) BSU23550 mleA 21.44 <0.001 AnsR, CcpA NAD dependent malic enzyme (conversion of malate into pyruvate) BSU23560 mleN 30.5 4 <0.001 AnsR, CcpA malate H+/Na+ lactate antiporter BSU28710 cstA 4.69 0.046 CcpA carbon starvation induced membrane protein BSU30260 msmR 23.08 <0.001 CcpA transcriptional regulator (LacI family) BSU07610 citM 27.71 <0.001 CcpA, CitT transporter of divalent metal ions/citrate complexes BSU07620 yflN 11.63 0.004 CcpA, CitT putative metal dependent hydrolase BSU04470 dctP 38.15 <0.001 CcpA, FsrA C4 dicarboxylate transport protein BSU02140 glpT 19.12 0.001 CcpA, GlpP glycerol 3 phosphate permease BSU02130 glpQ 11.35 0.004 CcpA, GlpP, PhoP glycerophosphoryl diester phosphodiesterase BSU39670 fbaB 8.04 0.012 Ccpa, IolR 2 deoxy 5 keto D gluconic acid 6 phosphate aldolase BSU39680 iolI 9.17 0.008 Ccpa, IolR putative sugar phosphate epimerase/iso merase
48 Table 2 5. Continued Accession BSU number Gene Name Fold Change P value Regulon Gene Description BSU39690 iolH 8.10 0.011 Ccpa, IolR putative sugar phosphate epimerase/isomerase BSU39700 idh 7.02 0.017 Ccpa, IolR myo inositol 2 dehydrogenase BSU39710 iolF 9.80 0.006 Ccpa, IolR inositol transport protein BSU39720 iolE 10.20 0.006 Ccpa, IolR 2 keto myo inositol dehydratase BSU39730 iolD 7.84 0.012 Ccpa, IolR 3D (3,5/4) trihydroxycyclohexane 1,2 dione hydrolase BSU39740 iolC 8.75 0.009 Ccp a, IolR 2 deoxy 5 keto D gluconic acid kinase BSU39750 iolB 8.87 0.009 Ccpa, IolR 5 deoxy D glucuronic acid isomerase BSU39760 mmsA 7.95 0.012 Ccpa, IolR methylmalonate semialdehyde dehydrogenase BSU12000 manR 4.64 0.047 CcpA, ManR transcriptional an titerminator BSU07800 treP 43.61 <0.001 CcpA, PhoP, TreR phosphotransferase system (PTS) trehalose specific enzyme IIBC component BSU07810 treA 10.70 0.005 CcpA, PhoP, TreR trehalose 6 phosphate hydrolase BSU38040 sacA 5.07 0.038 CcpA, SacT sucrase 6 phosphate hydrolase BSU38050 sacP 6.20 0.023 CcpA, SacT phosphotransferase system (PTS) sucrose specific enzyme IIBC component BSU33940 gapA 7.18 0.016 CggR glyceraldehyde 3 phosphate dehydrogenase BSU34190 yvfH 6.97 0.017 ComA putative lactate perm ease BSU03270 ycgT 9.32 0.007 Fur putative thioredoxin reductase BSU03800 yclN 11.63 0.004 Fur putative iron siderophore ABC transporter (permease) BSU03810 yclO 11.41 0.004 Fur putative iron siderophore ABC transporter (permease) BSU03820 yclP 6.2 4 0.023 Fur putative iron siderophore ABC transporter (ATP binding protein) BSU07150 yetG 9.77 0.006 Fur putative monooxygenase BSU07520 yfmC 6.38 0.022 Fur iron dicitrate ABC transporter (binding lipoprotein) BSU08440 yfiY 15.59 0.001 Fur putative i ron(III) dicitrate transporter binding lipoprotein BSU08450 yfiZ 26.34 <0.001 Fur iron(III) siderophore transport permease BSU08460 yfhA 15.46 0.001 Fur iron(III) siderophore transport permease BSU08480 yfhC 5.16 0.037 Fur putative oxidoreductase (ni troreductase family) BSU10330 yhfQ 16.40 0.001 Fur putative iron(III) dicitrate binding lipoprotein BSU14150 ykuN 20.29 0.001 Fur short chain flavodoxin BSU14160 ykuO 20.35 0.001 Fur conserved hypothetical protein BSU14170 ykuP 22.00 <0.001 Fur sho rt chain flavodoxin BSU32940 yusV 4.57 0.049 Fur iron(III) siderophore transporter (ATP binding component) BSU33290 fhuC 9.60 0.007 Fur ferrichrome ABC transporter (ATP binding protein) BSU33300 fhuG 21.51 <0.001 Fur ferrichrome ABC transporter (perm ease) BSU33310 fhuB 25.37 <0.001 Fur ferrichrome ABC transporter (permease) BSU33320 fhuD 11.30 0.004 Fur ferrichrome ABC transporter (ferrichrome binding lipoprotein) BSU39610 yxeB 20.61 <0.001 Fur ABC transporter (ferrioxamine binding lipoprotein) BSU13890 ptsG 15.70 0.001 GlcT, Stringent Response phosphotransferase system (PTS) glucose specific enzyme IICBA component BSU06150 gutB 4.80 0.043 GutR glucitol (sorbitol) dehydrogenase BSU03450 hxlB 6.31 0.022 HxlR 6 phospho 3 hexuloisomerase (PHI) BSU03460 hxlA 6.33 0.022 HxlR 3 hexulose 6 phosphate synthase (HPS)
49 Table 2 5. Continued Accession BSU number Gene Name Fold Change P value Regulon Gene Description BSU06230 ydjK 33.70 <0.001 IolR myo inositol transporter BSU31580 maeN 9.66 0.007 M alR Na+/malate symporter BSU07700 nagP 5.29 0.035 NagR phosphotransferase system (PTS) N acetylglucosamine specific enzyme IICB component BSU29990 ytiP 5.10 0.038 PurR hypoxanthine/guanine permease BSU12280 yjlC 4.88 0.042 Rex, Stringent Response con served hypothetical protein BSU13490 ykrL 5.22 0.036 Rok, YkrK membrane protease BSU08760 spo0M 5.55 0.031 SigH, SigW sporulation control gene BSU14480 abh 5.89 0.027 SigM, SigX transcriptional regulator BSU12420 yjoB 4.68 0.046 SigW ATPase possib ly involved in protein degradation BSU29520 yteJ 7.58 0.014 SigW putative integral inner membrane protein BSU29530 sppA 8.06 0.011 SigW signal peptide peptidase BSU30000 ythQ 5.34 0.034 SigW putative ABC transporter (permease) BSU29490 tpx 5.07 0.0 38 Spx putative peroxiredoxin BSU02530 yczA 5.25 0.035 T box anti TRAP regulator BSU28080 folC 4.98 0.04 T box folyl polyglutamate synthase BSU28950 thrS 4.85 0.042 T box threonyl tRNA synthetase BSU28090 valS 5.13 0.037 T box, Efp dependent protei ns valyl tRNA synthetase BSU02540 ycbK 6.23 0.023 T box, TRAP putative efflux transporter BSU30990 yuaJ 8.24 0.011 Thi box thiamin permease BSU37940 ywdJ 4.56 0.049 TnrA putative purine/pyrimidine permease BSU22600 aroE 10.19 0.006 TRAP 3 phosphosh ikimate 1 carboxyvinyltransferase (5 enolpyruvoylshikimate 3 phosphate synthase) BSU22610 tyrA 7.11 0.016 TRAP prephenate dehydrogenase BSU22620 hisC 4.73 0.045 TRAP histidinol phosphate aminotransferase; tyrosine/phenylalanine aminotransferase BSU364 80 ywoD 5.70 0.029 YtrA putative efflux transporter
50 Table 2 6. Response of significantly and non significantly expressed General Stress Response genes to LP exposure (5 kPa). BSU number A Gene Name B,C Gene Description Ratio 5 kPa/ ~101 kPa P Value BSU0 0160 yaaH spore peptidoglycan hydrolase 1.8 0.2574 BSU00170 yaaI putative isochorismatase 4.1 0.0641 BSU00520 ctc ribosomal protein Ctc, binding 5S RNA 7.2 0.0164 BSU00530 spoVC peptidyl tRNA hydrolase 3.7 0.0799 BSU00660 yabT putative serine/threonine protein kinase 1.8 0.2608 BSU00890 yacL putative membrane protein 2.2 0.2019 BSU02100 cypC fatty acid beta hydroxylating cytochrome P450 8 0.0126 BSU02110 ybyB conserved hypothetical protein 76.7 <0.0001 BSU02540 ycbK putative efflux transporter 0.2 0 .023 BSU02580 ycbO putative Na+ driven exporter or maturation protein 1.3 0.3831 BSU02590 ycbP putative inner integral membrane protein 14.8 0.0018 BSU02790 ycdB putative hydrolase 0.7 0.3238 BSU02830 ycdF putative dehydrogenase 12.8 0.0029 BSU02840 y cdG putative glycosidase 6.1 0.0248 BSU02900 yceD putative stress adaptation protein 0.8 0.3981 BSU02910 yceE putative stress adaptation protein 0.8 0.3806 BSU02920 yceF putative stress adaptation transporter 0.6 0.2817 BSU02930 yceG conserved hypothet ical protein 1.4 0.3478 BSU03130 nadE ammonium dependent NAD+ synthetase 1 0.4807 BSU03910 gabD succinate semialdehyde dehydrogenase 1.5 0.3348 BSU04030 ycsD putative hydroxymyristoyl (acyl carrier protein) dehydratase 0.8 0.3977 BSU04190 ydaD putative dehydrogenase 6.7 0.0195 BSU04200 ydaE conserved hypothetical protein 6.9 0.0181 BSU04220 ydaG putative general stress protein 32.4 0.0001 BSU04340 ydaP putative enzyme with pyruvate as substrate 19.1 0.0007 BSU04370 ydaS conserved hypothetical protei n 9.3 0.0078 BSU04380 ydaT conserved hypothetical protein 3.9 0.072 BSU04400 gsiB general stress protein 90.3 <0.0001 BSU04420 ydbC conserved hypothetical protein 0.7 0.3244
51 Table 2 6. Continued BSU number A Gene Name B,C Gene Description Ratio 5 kPa/ ~ 101 kPa P Value BSU04430 ydbD putative manganese containing catalase 2.3 0.1806 BSU04710 rsbV anti anti sigma factor (antagonist of RsbW) 36.6 <0.0001 BSU04720 rsbW switch protein/serine kinase and anti sigma factor (inhibitory sigma B binding protein) 44 <0.0001 BSU04730 sigB RNA polymerase sigma 37 factor (sigma B) 56.9 <0.0001 BSU04740 rsbX serine phosphatase 49 <0.0001 BSU05150 ydeC putative transcriptional regulator (AraC/XylS family) 0.8 0.4186 BSU05360 ydfC putative permease 0.9 0.4652 BSU054 40 nap carboxylesterase NP 1.1 0.4512 BSU05490 mhqO putative dioxygenase 1.7 0.2844 BSU05790 ydhK hypothetical protein 9.1 0.0083 BSU06120 ydjB hypothetical protein 0.8 0.3873 BSU06220 ydjJ putative membrane associated potassium channel 1.7 0.2869 BS U06240 ydjL acetoin reductase/2,3 butanediol dehydrogenase 2.6 0.1513 BSU06310 gabP gamma aminobutyrate (GABA) permease 0.6 0.2567 BSU06350 yebA conserved hypothetical protein 1.3 0.3846 BSU06590 yerD putative flavoenzyme 5.5 0.0326 BSU06660 opuE proli ne transporter 30.4 0.0001 BSU06980 yesP rhamnogalacturonan permease 0.8 0.4234 BSU07260 yfnI exported glycerol phosphate lipoteichoic acid synthetase and anion binding protein 0.8 0.3903 BSU07540 yfmA unknown 0.8 0.4076 BSU07550 yflT heat stress induc ed protein 31.6 0.0001 BSU07680 yflH conserved hypothetical protein 1.4 0.3544 BSU07750 yflA putative aminoacid transporter 45.8 <0.0001 BSU07760 yfkT putative spore germination integral inner membrane protein 24.8 0.0003 BSU07770 yfkS hypothetical pro tein 13.3 0.0026 BSU07850 yfkM general stress protein 18 23.9 0.0003 BSU07880 yfkJ protein tyrosine phosphatase 29 0.0001 BSU07890 yfkI conserved hypothetical protein 21.6 0.0004 BSU07900 yfkH putative integral inner membrane protein with ribonuclease fold 15.3 0.0015 BSU07920 chaA putative H+/Ca2+ antiporter 14.7 0.0018
52 Table 2 6. Continued BSU number A Gene Name B,C Gene Description Ratio 5 kPa/ ~101 kPa P Value BSU07930 yfkD conserved hypothetical protein 7.9 0.0126 BSU08490 yfhD conserved hypothet ical protein 9.4 0.0077 BSU08500 yfhE hypothetical protein 5.7 0.0299 BSU08510 yfhF putative nucleotide binding protein 6 0.0264 BSU08570 yfhK conserved hypothetical protein 89.6 <0.0001 BSU08580 yfhL SdpC immunity factor 30.8 0.0001 BSU08590 yfhM put ative hydrolase 15.8 0.0014 BSU08600 csbB putative glycosyl transferase 7.2 0.0163 BSU09140 yhcM hypothetical protein 5.1 0.0399 BSU09390 ygxB putative integral inner membrane protein 3.8 0.0735 BSU09450 yhdF putative NAD(P) dependent dehydrogenase 3.5 0.0862 BSU09530 yhdN aldo/keto reductase specific for NADPH 28.8 0.0001 BSU09690 nhaX stress response protein, UspA family 68.4 <0.0001 BSU10430 yhxD putative oxidoreductase 5 0.0412 BSU10810 yisP putative squalene/phytoene synthase 3 0.1198 BSU11120 yitT putative integral inner membrane protein 2.1 0.2126 BSU11490 yjbC putative thiol oxidation management factor; putative acetyltransferase 28.1 0.0001 BSU11830 yjcE unknown 19.5 0.0006 BSU12010 manP phosphotransferase system (PTS) mannose specific e nzyme IIBCA component 0.3 0.1089 BSU12070 yjdJ conserved hypothetical protein 2.8 0.1363 BSU12150 yjgB hypothetical protein 1.7 0.283 BSU12160 yjgC putative oxidoreductase 7.7 0.0138 BSU12170 yjgD conserved hypothetical protein 4.4 0.0544 BSU12560 xpf putative RNA polymerase PBSX sigma factor like 1.1 0.4755 BSU12720 xkdS conserved hypothetical protein; putative PBSX prophage protein 0.8 0.3887 BSU13020 ykgA putative aminohydrolase 38.1 <0.0001 BSU13160 ohrB organic hydroperoxide resistance reductas e B 24.3 0.0003 BSU13170 guaD guanine deaminase 8.7 0.0097 BSU14210 ykuT putative small conductance mechanosensitive channel 2.9 0.1256 BSU14660 ykzI conserved hypothetical protein 20.3 0.0005
53 Table 2 6. Continued BSU number A Gene Name B,C Gene Descript ion Ratio 5 kPa/ ~101 kPa P Value BSU16640 ylxP conserved hypothetical protein 1.9 0.2534 BSU17240 ymzB conserved hypothetical protein 5 0.0414 BSU17850 lexA transcriptional repressor of the SOS regulon 0.8 0.3734 BSU18110 ynfC conserved hypothetical p rotein 11.1 0.0045 BSU18510 yoxC conserved hypothetical protein 36.3 <0.0001 BSU18520 yoxB conserved hypothetical protein 27.2 0.0002 BSU19150 yocB conserved hypothetical protein 9.7 0.007 BSU19240 yocK putative general stress protein 3.8 0.0737 BSU20 450 yorA putative capsid component; phage SPbeta 1.9 0.2505 BSU23060 ypzE hypothetical protein 1.1 0.4607 BSU23830 yqjL putative hydrolase 0.9 0.469 BSU23970 yqiY High affinity arginine ABC transporter (permease) 1.3 0.379 BSU24000 bmrU putative diacyl glycerol kinase 37.3 <0.001 BSU24010 bmr multidrug efflux transporter 1.5 0.3396 BSU24020 bmrR transcriptional regulator (MerR family) 2.6 0.1554 BSU24140 mmgD 2 methylcitrate synthase 1.3 0.3795 BSU24490 yqhQ conserved hypothetical protein 3.1 0.1127 BSU24740 yqxL putative CorA type Mg(2+) transporter 11.5 0.0041 BSU24750 yqhB putative membrane associated protein 12.4 0.0032 BSU24770 yqgZ putative transcriptional regulator of stress 94.9 <0.0001 BSU25020 sodA superoxide dismutase 0.5 0.1927 BSU261 80 yqbA putative phage capsid protein; skin element 1 0.4997 BSU26540 yrkE conserved hypothetical protein 1 0.4833 BSU27020 yraA general stress protein 1 0.4962 BSU27230 yrhD conserved hypothetical protein 1.5 0.3296 BSU27640 yrvC putative potassium tr ansport accessory component 0.5 0.1884 BSU27750 bofC Bypass of forespore C, intercompartmental signaling factor 3.1 0.1121 BSU27760 csbX putative permease 2.1 0.2072 BSU28180 ysxD putative integral inner membrane protein 0.4 0.1874 BSU28340 ysnF putati ve stress response protein 6.3 0.0237 BSU28450 sdhC succinate dehydrogenase (cytochrome b558 subunit) 0.2 0.0508
54 Table 2 6. Continued BSU number A Gene Name B,C Gene Description Ratio 5 kPa/ ~101 kPa P Value BSU28590 yshC DNA polymerase X 1.6 0.3109 BSU2 8810 abnA arabinan endo 1,5 alpha L arabinase 2.7 0.1382 BSU28830 ysdB conserved hypothetical protein 1.9 0.2434 BSU29410 ytkL putative metal dependent hydrolase 0.9 0.4641 BSU29760 ytxJ conserved hypothetical protein 11.2 0.0044 BSU29770 ytxH conserve d hypothetical protein 14.5 0.0019 BSU29780 ytxG conserved hypothetical protein 17.69 0.0009 BSU30020 ytzE putative transcriptional regulator (DeoR family) 1.9 0.2459 BSU30230 bioA lysine 8 amino 7 oxononanoate aminotransferase 1 0.4994 BSU30650 dps DN A protecting protein, ferritin 11.4 0.0042 BSU30700 rpmE2 ribosomal protein L31 19.9 0.0006 BSU30930 ytaB putative receptor 20.4 0.0005 BSU31280 yugU conserved hypothetical protein 6.2 0.0245 BSU31380 yuzA conserved hypothetical protein 2.1 0.2136 BSU 32320 yutC putative lipoprotein 1.1 0.465 BSU32520 yurG putative ureidoglycolate lyase (ureidoglycolase) 0.6 0.2864 BSU32880 yusP putative multidrug efflux transporter 2.3 0.1866 BSU33140 yvqJ putative efflux protein 0.7 0.3215 BSU33200 yvrE conserved hypothetical protein 9 0.0087 BSU33400 yvgN glyoxal/methylglyoxal reductase 0.6 0.3084 BSU33410 yvgO conserved hypothetical protein 24.9 0.0002 BSU33460 yvgT putative integral inner membrane protein 0.2 0.0233 BSU33530 yvaA putative oxidoreductase 10.3 0.0058 BSU33610 rnr ribonuclease R 1.5 0.3269 BSU33620 yvaK carboxylesterase 2.4 0.1769 BSU33660 yvaN transcriptional repressor 0.5 0.2084 BSU33710 opuBC choline ABC transporter (choline binding lipoprotein) 22.1 0.0004 BSU33720 opuBB choline ABC tr ansporter (permease) 12.4 0.0032 BSU34240 yvfD putative O acetyltransferase 0.3 0.0802 BSU34910 hisD histidinol dehydrogenase 1.2 0.423 BSU35180 csbA putative membrane protein 10.3 0.0057
55 Table 2 6. Continued BSU number A Gene Name B,C Gene Description R atio 5 kPa/ ~101 kPa P Value BSU35310 yvyD ribosome associated sigma 54 modulation protein 7.7 0.0136 BSU35670 gtaB UTP glucose 1 phosphate uridylyltransferase 1.6 0.3196 BSU35690 ggaA poly(glucosyl N acetylgalactosamine 1 phosphate) glucosyltransferase 3.1 0.1128 BSU35830 ywtG putative carbohydrate transporter 9.9 0.0066 BSU35970 ywsB conserved hypothetical protein 1.5 0.3426 BSU36670 csbD stress response protein 16.9 0.0011 BSU36720 ywmE hypothetical protein 17.6 0.0009 BSU36960 ywlB conserved hyp othetical protein 1.2 0.4134 BSU37210 ywjC conserved hypothetical protein 44.5 <0.0001 BSU37230 ywjA putative ABC lipid transporter (ATP binding protein) 0.8 0.4016 BSU37240 ywiE cardiolipin synthetase 35.3 0.0001 BSU37480 ywhH putative RNA binding pro tein 1 0.4908 BSU37620 rsfA prespore specific regulatory gene 0.9 0.4589 BSU37680 ywfH carrier protein reductase of bacilysin synthesis 1.5 0.3262 BSU38180 ywzA conserved hypothetical protein 26.8 0.0002 BSU38430 gspA putative glycosyl transferase (gen eral stress protein) 69.3 <0.0001 BSU38440 ywaF putative integral inner membrane protein 1 0.4946 BSU38600 licR transcriptional activator of the lichenan operon 1.4 0.3549 BSU38620 yxlJ 3 alkylated purines and hypoxanthine DNA glycosidase 2.3 0.1859 BS U38630 katX major catalase in spores 4.6 0.0494 BSU38720 yxkO putative carbohydrate kinase 1.5 0.3286 BSU38830 aldY putative aldehyde dehydrogenase 4.2 0.0593 BSU38930 yxjJ hypothetical protein 5.4 0.0346 BSU38960 yxjG putative methyltetrahydrofolate m ethyltransferase 0.4 0.1865 BSU39040 yxiS hypothetical protein 5.2 0.0378 BSU39050 katE catalase 2 29.2 0.0001 BSU39810 csbC putative sugar transporter 20.3 0.0006 BSU39840 yxbG putative oxidoreductase 7.1 0.0168 BSU40000 yxnA putative oxidoreductase 10.7 0.0052 BSU40020 yxaC unknown; similar to unknown proteins 1.1 0.4497 BSU40030 yxaB putative exopolysaccharide pyruvyl transferase 1.8 0.2664
56 Table 2 6. Continued BSU number A Gene Name B,C Gene Description Ratio 5 kPa/ ~101 kPa P Value BSU40450 yycD conserved hypothetical protein 1.7 0.2805 BSU40570 yybO putative permease 1.6 0.3024 BSU40810 yyaM putative efflux transporter 1.4 0.3694 A BSU numbers are gene identifiers from the Genolist database ( http://gen odb.pasteur.fr/ ). B Hatched cells denote genes with ratios of less than 0.22 and P <0.05 (i.e., significantly down regulated by at least 4.5 fold). C Underlined genes have been documented to be induced solely by a sigB dependent promoter ( http://subtiwiki.uni goettingen.de/wiki , (105, 111) .
57 Figure 2 1. Scatter plots of fluorescent intensity of Cy3 (X axes) vs. Cy5 (Y axes) labelled RNAs in microarray experime nts (log 2 scale). A) "Dye flip" control experiment of strain WN624 grown at ~101 kPa, labeled with Cy3 vs. Cy5. B) "Dye flip" control experiment of strain WN624 grown at 5 kPa, labeled with Cy3 vs. Cy5. C. Strain WN624 grown at ~101 kPa labeled with Cy3 vs . strain WN624 grown at 5 kPa labeled with Cy5.
58 Figure 2 2. Venn diagram comparison of the set of genes belonging to the GSR regulon induced by: LP in WN624; the classical GSR inducers ethanol (EtOH)/heat/salt, taken from ref. (111) ; both treatments; or neither treatment. Numbers of genes belonging to each category are in parentheses and gene names are listed. Underlined genes are strictly sigB dependent (see Table 2 6 and text for details).
59 Figure 2 3. Determination of pressure induction of the SigB dependent GSR using a ctc::lacZ reporter fusion. Relevant genotypes of strains WN1400 ( sigB + ) and WN1407 sigB::spc ) are denoted. A) Induction of ctc lacZ expression by 5% (v/v) ethanol or by exposure to 5 kPa (shaded bars) vs. the uninduced controls (open bars). B) Induction of ctc lacZ expression at ~101 kPa and various LPs (50, 25, 10, and 5 kPa) in strain WN1400 ( sigB + ) (shaded bars). Data are averages and standard deviations of triplicate samples taken from two independent experiments.
60 CHAPTER 3 M ICROARRAY ANALYSIS OF DIFFERENTIAL GENE EXPRESSION IN A BACILLUS SUBTILIS STRAIN ADAPTED TO EN HANCED GROWTH AT 5 KPA Introduction Current robotic missions to extraterrestrial environments, such as the Mars Science Laboratory (MSL), pose possible planetary protection concerns as outlined by international planetary protection regulations established by the Committee on Space Research (COSPAR) (42, 115 , 116 ) . The concern of a terrestrial microorganism surviving int erplanetary transit conditions on spacecrafts is reasonable, as several spore forming bacteria, in cluding species of the Bacillus genus, are common space craft assembly facility contaminants; these environments are themselves considered extreme in nature as they a re constantly exposed to low humidity, low nutrient conditions, including UV exposure (44, 117 ) . As the most visited body, Mars surface conditions are of primary focus in extreme extraterrestrial environmental survivability experiments; surface conditions on Mars range from temperature extremes ( 20 C t o 59 C), ~0.1 to 1 kPa surface pressure, atmospheric composition of ~97 % CO 2 , ~2.5 % N 2 , and other trace gases (33, 118 ) . In 2006, Schuerger and Nicholson exposed Bacillus spp., known to be spacecraft contaminants , to a range of pressures from ~ 101 kPa to 2 kPa to determine the effect of LP on grow th rates; below 10 kPa cells were significantly impaired in their ability to grow, and the lowest pressure where a growth rate was observed in the Bacillus spp. was ~ 2. 5 kPa (33) . Low pressure, hypobaria, is not an extreme environmental condition at which life is found on Earth; the pressure at the top of Mt. Everest is ~33 kP a. However, recently, two examples have been found of bacteria capable of growth at 0.7 kPa (the average martian surface pressure): Carnobacterium spp. (38) and Serratia liquefaciens (37) . However, these two organisms have
61 only recently had their genomes sequenced (39, 40) and are not well characterized (i.e. no transcriptional data under various growth conditions and lack of molecular biology tools ). Due to the lack of a natural hypobari c environment and no model organism with which to study LP responses, an evolution experiment was conducted to determine if a model microorganism, Bacillus subtilis , could adapt to a lowered pressure at which it initially grows poorly. Briefly, LB media wa s inoculated with Bacillus subtilis strain WN624 , carrying a spectinomycin resistance at the amyE locus (Table 3 1), and cultured at 5 kPa for ~1,000 generations (34) . Daily optical density measurements, in Klett units (1 OD 660 ~ 100 Klett units) showed an increase in growth ability during the course of 1,000 generations. At the termination of the experiment, a strain, WN1106, was isolated from the culture. Strain WN1106 was shown to be capable of o utcompeting the ancestor strain , WN624, at 5 kPa, but not 101 kPa (34) . Despite a complete lack of literature on hypobaric adapted microorganisms, parallels of adaption reported in the hyperbaric, de ep sea dwelling organisms (piezophiles of the piezosphere) provide an outline of how pressure exerts constraints on biological systems. Ribosomal pyrosequencing analysis and culturing of deep sea samples has identified numerous species of bacteria (predomi nantly gram negatives), archaea, and micro euk aryotes that are able to cope with the environmenta l condition of high hydrostatic pressure (HHP) (73) . Piezosphere m changes in permease composition of the outer membrane, differential expression of terminal oxidases, differences in lipid composition of membranes, structure and amino acid composition of proteins, elongation of 16S rRNA gene helices, and changes in enzy matic volumes (1, 8, 9, 20, 23, 25, 32, 65, 75, 119 ) . From the knowledge of adaption to hyperbaria, hypobaria is expected to have influences on protein supramolecular structure, membrane fluidity, enzymatic functio ns, and DNA/RNA
62 stability. It is reported here the results of several transcriptional microarray experiments comparing the ancestral strain WN624 and the LP evolved strain WN1106 at 101 kPa and 5 kPa (Table 2 1). Analysis of differential gene expression in both strains at 5 kPa compared to 101 kPa revealed genes involved in fatty acid synthesis, anaerobic growth, metal acquisition, respiratory chain components, SigB dependent General Stress Response, transporters, and a variety of unknown/hypothetical genes . Material and Methods Bacillus subtilis , Media, and Growth Conditions B. subtilis strains and plasmids used in this study are listed in Table 3 1. Ancestor strain WN624 ( trpC2, amyE::spc ), derived from the laboratory strain 168 ( 113 ) and WN1106 ( trpC2, amyE::spc ), which was derived after evolution of strain WN624 for 1,000 generations of growth at 5 kPa (34) have previously been described in detail. Strains PB153 and PB344 (Table 3 1) were generous gifts from Chet Price. Chromosomal DNA was isolated from these strains and used to trans form the ancestor strain. Strains were cultivated in Miller LB liquid medium (85) supplemented with spectinomycin (100 Âµ g/mL final concentration). Cells were grown under pressure conditions of 1 atmosphere (~101 kPa) or 5 kPa as described previously (34, 87) . Cultures were shaken at moderate speed (~170 rpm) on a rotary shaker at 27 was measured using a Klett Summerson photometer fitted with the No. 66 (660 nm; red) filter. Under these conditions, 100 Klett units = 1 OD 660 = ~ 1 x 10 8 cells per mL. RNA Extraction An equivalent mass of cells (10 mL or 100 mL from ov ernight cultures grown at 101 kP or 5 kPa, respectively) were harvested by centrifugation , the supernatants removed by aspiration, and the cell pellets frozen at 70 C. Approximately 4x10 9 cells were obtained from each sample, estimated from culture optica l densities determined before centrifugation. Total RNA was
63 extracted from cells and treated with RNase free DNase using the RiboPure Bacteria Kit purity were deter mined by UV absorbance measurements at 260 and 280 nm (88) . RNA Integrity Numbers (RIN) were obtained by running total RNA on the RNA 6000 nano assay on an Agilent 2100 Bioanalyzer, (Agilent Technologies). The ave rage RIN of our samples was 9.71. This number quantifies the (high) quality of our RNA samples more accurately than the 23S to 16S peak ratio (88 ) . Microarray Experiments Total RNA samples were sent to the University of Florida Interdisciplinary Cent er for Biotechnology Research (UF ICBR) for fluorescent labeling and microarray analysis. A custom glass slide microarray (GE 8X15K 60mer; Agilent Technologies) was designed and built using the B. subtilis strain 168 genome sequence (49) . The glass slide carried 8 separate microarrays that were probed as described in Table 2 1. For each sample, approximately 12 ÂµL at a concentration of 500 ng/ ÂµL was loaded for a total RNA content of 6 Âµg. Microarray Data Analysis and Normalization The genes names, locations and description s were cross referenced using the GenoList archive server (http://genolist.pasteur.fr). For each comparison, the microarray chips yielded ~ 15,209 data points equivalent to 4,103 genes with an average number of 3.7 measurements per gene. The raw data, gree n and red mean intensities, and dispersions were consistent across the 8 sample comparisons. Raw data scatter plots for each control comparison indicated the high quality of the data, i.e. high level of correlation for all control chips (1, 4, 6, & 8) and a high level of dispersion for all test chips (2, 3, 5, & 7) (Figure 3 1). Loess normalization was applied to the microarray data to correct for bias caused by inconsistencies in the relative fluorescence intensity between the Cy5 and Cy3 dyes. Variations between the multiple microarray
64 experiments were removed using quantile normalization. The analysis of gene differential available at http://bioconductor.org) package in the R programming language. The LIMMA package uses empirical Bayesian methods to provide stable results by moderating the standard errors of the estimated fold changes. Comparisons among the microarray chips were conducted graphically using the R progr am (Figure 3 2), and entered in to tables using Excel (Table 3 2). BLASTp Analysis of Unknown and Putative Function mRNA Signals All signals with unknown and putative functions were collected into respective fastA files, according to microarray conditions, from a GenBank file containing the full Bacillus subtilis strain 168 genome obtained through the NCBI website. The fastA files were run through BLASTp on the NCBI website using the non redundant protein sequences database. BLASTp results were imported into and analyzed by MEGAN, a metagenomic analyzer, and mapped in the program to a SEED functional role (Table 3 3). Sporulation Frequency Sporulation frequency determination was performed by standard methods in the lab (60) . Briefly, overnight cultures were used to inoculate 1 0 mL Spizizen minimal media containing 1x Sporulation salts at ~ 0.015 OD 660 , triplicate cultures were incubated at either 5 kPa or ~101 kPa at 27 Â° C. At 24, 48, and 72 hrs. 1 mL samples were taken, diluted, and plated before and after heat shock (80Â°C fo r 10 min.). Frequency of sporulation was determined by dividing spore titers (after heat shock samples) by total CF Us (before heat shock samples) . galactosidase Assays Constructed strains carrying the sigB dependent, GSR inducible ctc lacZ gene fusion (Table 3 galactosidase activity by ethanol, a known inducer of the GSR (81 )
65 units) then split. To one subculture was added ethanol to a final concentration of 5%, and incubation was continued for 45 min. To test for LP induction of ctc lacZ expression , cultures units). A zero time sample was taken, then the cultures were split into twelve 2 mL subcultures, of which 6 were incubated at ~101 kPa and 6 were incu bated at various LP conditions (5, 10, 25, or 50 kPa) for a further 2.5 hr. Culture OD 660 values were determined, then a 1 mL sample from each tube was centrifuged and the resulting cell pellet frozen at galactosidase assay. Thawed c e lls were lysed and assayed for galactosidase activity as described previously (87 ) . galactosidase activity is expressed in Miller units (85) . Competition Experiments Competition experiments were perfor med as described previously (34, 87) . Briefly, triplicate cultures of the two strains to be tested were co cultivated in 125 mL flasks containing either at ~101 kPa or at 5 kP a. On Day 0 (D 0 ), strains were each inoculated at an initial OD 660 of ~ 0.015. On each 10 mL of sterile LB. For each day of the c ompetition, including D 0 , viable counts were determined by plating serial tenfold dilutions made in PBS buffer (89 ) onto LB agar plates containing the appropriate antibiotic. Competition experiments were conducted for 7 days (~50 generations) and r elative fitness values were calculated as previously described ( 120 , 121 ) . Microarray Data Accession Number The complete set of microarray data has been deposited in the Gene Expression Omnibus (GEO) database at th e National Center for Biotechnology Information ( http://www.ncbi.nlm.nih.gov/geo/ ) under accession number GSE50653.
66 Results Microarray Analysis of Global Gene Expression of Bacillus subtilis Strains WN624 an d WN1106 at 5 kPa In order to gain a greater understanding of the global gene expression changes occurring in strains WN624 and WN1106 in response to LP, the two strains were studied using a series of transcriptional microarrays. Total RNA was extracted fr om cells after 24 hours of growth at either 5 kPa or ~101 kPa, and RNA labeling and chip analyses were conducted as described in Materials and Methods. The complete datasets of significantly up or down regulated transcripts are presented in Table 3 2, and the data are summarized in Fig. 3 1. First, to confirm RNA integrity and consistency of probe labeling, four control microarrays were run using the same RNA sample labeled with Cy3 vs. Cy5. In each case the data were tightly clustered and displayed high c orrelation coefficients (R 2 ) of 0.9912, 0.9959, 0.9964, and 0.9950, respectively (Fig. 3 1, panels A D). Two types of experimental microarrays were run (Table 2 1). The first type compared the transcriptomes of the same strain grown at normal pressure (~1 01 kPa) vs. LP (5 kPa) in ancestral strain WN624 (Fig. 3 1E) and LP evolved strain WN1106 (Fig. 3 1F). The data showed a profound alteration of the global transcriptomes of both strains affecting the mRNA levels of literally hundreds of genes. The large di spersion of the data was evident both visually and by the low R 2 values of 0.6114 and 0.5707, respectively (Figs. 3 1E, 3 1F). The second type of experiment compared the transcriptomes of ancestral strain WN624 vs. LP evolved strain WN1106 cultivated at th e same pressure, either ~101 kPa (Fig. 3 1G) or 5 kPa (Fig. 3 1H). Under both conditions considerable dispersion could also be visualized in the data, indicating that the global transcriptomes of strains WN624 and WN1106 differed substantially at ~101 kPa (R 2 = 0.8982) (Fig. 3 1G) and at 5 kPa (R 2 = 0.8969) (Fig. 3 1H).
67 All signals displaying a P value of < 0.05 were collected in Table 3 3. A total of 983 signals were significantly up regulated, down regulated, or both across the arrays, representing nearl y 25% of the B. subtilis genome. To compare the overlap between signals from each microarray, a V enn diagram of up and down regulated signals was made (Fig. 3 2). Of note, there was a far more robust up regulation in both strains at 5 kPa compared to down regulated signals with considerable overlap of down regulated signals that was not seen in up regulated signals. Anaerobic Response at 5 kPa In both strains, there is a robust anaerobic response due to the lowered partial pressure of dissolved oxygen in t he media under LP conditions. mRNA signals for genes involved in nitrate and nitrite respiration ( narIJHG , narK , nasD ) and genes involved in the hypoxic response ( fnr , resDC ) are up regulated in either one or both WN1106 and WN624 at 5 kPa (Table 3 2). Due to the induction of fnr and resD , both of which are regulator signals controlling the iron response in the case of the former and the low oxygen response by the latter (which also involves the regulation of fnr ), the corresponding signals they regulate ar e also differentially expressed. However, there is a significant difference in expression of some of these genes between the two strains; WN1106 up regulates the narIJHG operon seven fold greater than WN624 at 5 kP a. The presence of these signals in the mi croarray corresponds to the existence of a hypoxic condition at low pressure, as these genes are also differentially regulated in microarray experiments comparing low oxygen and aerobic gene expression in Bacillus subtilis (97 ) . Despite this, previous comparisons of WN1106 and WN624 under oxygen limiting conditions at 101 kPa indicated that WN1106 had a lowered relative fitness compared to WN624 under oxygen limiting conditions (86) or when nitrate was used as the term inal electron acceptor WN1106 did not grow better than WN624 (34) (Fig ure 3 3).
68 Considering the low oxygen condition when gr owing at LP , it is not surprising that several cytochrome gene transcrip ts, as well as their associated proteins, are differentially expressed during hypobaric growth; however, also considering pressure effects on protein stability, changes in respiratory chains of piezophilic microorganisms suggest that supramolecular structures may also be affected by pressure (32) . During aerobic growth , Bacillus subtilis has a branched electron transport chain, with up to four possible terminal oxidases: cytochromes caa 3 , aa 3 , bd , and YthAB ( 122 ) . Cytochromes caa 3 & bd are differentially expressed at LP, and expression of the former is also regulated by catabolite repression ( 123 ) . Cytochrome and associated genes that are up regulated at 5 kPa in both strains include cypC , ctaCDEFG (cytochrome caa 3 and its assembly factor), cccA , cypX , yvmC (a gene in the operon with cypX ), and cydDCBA (the bd type oxidase and its ABC transporter components; this operon is influenced by oxygen limiting conditions) ( 124 ) . Signals up regulated only in WN1106 at 5kPa include qcrCBA encoding the menaquinol:cytochrome c oxidoreductase ( 125 ) and this operon is under the control of the ResD/ResE response, which, as stated, is highly induced under low oxygen conditions, also regulates resBC ( 126 ) . ResBC , which is up regulated at LP only in the ancestral strain, WN624, are important cytochrome c assembly factors in B. subtilis ( 127 ) ; these signals also appear as genes down regulated in WN1106 vs. WN624 at 5 kPa due to the significant difference of the two strains in the expression of these signals [ resDCBA ]. Two cytochrome P 450 signals, cypC and cypX , are up regulated in both strains at 5 kPa; cytochromes P450 are sensitive to pressure changes and are a key point of study in piezophilic protein adaptations compared to pressure mesophiles ( 128 ) . In Bacillus subtilis , cypX catalyzes the oxidation of cyclic dipeptides, which are important for the formation of secondary metabolites, such as iron chelators ( 129 ) . In addition, cypC , formerly ybdT , is a fatty acid -
69 hydroxylating cytochrome P450 and under the control of the alternativ e sigma factor, sigB, which regulates the General Stress Response of Bacillus subtilis and other gram positive organisms (102 , 130 ) . It would appear that cytochrome P450s are involved in stress protection in bacter i a. Membrane Fluidity at Low Pressure Paralleling homeoviscous membrane adaptations documented in piezotolerant and piezophilic bacteria, hypobaria most likely also affects membrane lipid bilayers and their fluidity (2, 65, 75) . Regarding effects on membrane fluidity, increasing pressure correlates roughly with decreased temperature (13) . It stands to reason that a decrease in pressure may correspond to adaptations seen with increases in temperature, resulting in a more liquid crystalline (L ) state and higher disorder of acyl side chains, as is seen at high temperatures (13) . To note, however, wher eas temperature has effects on both volume and energy states of a system, pressure influences are purely volumetric (2) . In the piezosphere, hyperbaria exerts an extreme environmental constraint on lipid bilayers, which seems to be the most sensitive biolo gical system affected, effects of which range from water and ion transport, lowered phase transition enthalpy, as well as membrane thickness and lipid packing (Winter & Jeworrek, 2009). It may be expected that a lowered hydrostatic pressure on the membrane would allow for increased motion in the fatty acyl side chains due to an increase in system volumes, possibly having effects on membrane leakage of ions, transport of molecules and secreted proteins across the membrane, stability of supramolecular protein complexes, and integral membrane protein secondary and tertiary structure, affecting signaling proteins. Therefore, it is not unexpected that genes involved in fatty acid desaturation ( des , yocF [ desR ], yocG [ desK ]) are differentially expressed at LP in W N1106 (Table 3 2). However, recent investigation into the fatty acid saturation genes in LP adapted WN1106 did not reveal a change
70 at the genomic level to explain the increase of growth rate at 5 kPa (86) despite their lack of differential expression in the ancestral strain, WN624, at LP. And th ough a des mutant of WN1106 grew more poorly than wild type WN1106 at low pressure, the des mutant still had a higher fitness than WN624, indicating other changes in WN1106, outside of the fatty acid desaturase response, are responsible for its increased g rowth rate at 5 kPa (86) . It may be that genomic changes in other regions of WN1106 are affecting the differential expression of these signals. While polyketide pathways are important for some piezosphere dwelling microorganisms as a pathway to produce long chain PUFAs such as eicosapentaenoic a cid ( EPA ) and docosahexaenoic acid (DHA) (reviewed in (14, 131 ) , in Bacillus subtilis the polyketide pathway members coded for by the pksX operon are primarily involved in production of branch containing secondary metabolites ( 132 ) . The first polyketide pathway genes of the pksX operon are also differentially expressed under hypoxic conditions in Bacillus subtilis ; their presence in our pressure microarrays may be explained by the low oxygen conditions that correspond to hypobaric growth (97 ) . Transporter Genes are Differentially Expressed at LP Adaptations involving transporters are another mechanism that piezophilic microorganisms employ to cope with high hydrostatic pressure (65) . Until the isolation of Carnobacterium sp. strain AT7 from the deep sea ( 20) , most piezophilic isolates have been gram negative strains; a feature of gram negative outer membranes is the porin proteins, which aid in osmotic stress adaptation of halophiles. Therefore, it is not surprising that the first pressure regulated gene described was an acidic porin, OmpH (9, 23) . The microarrays revealed several transporters that are up regulated under LP conditions both in WN1106 and WN624:
71 opuE , ykvW , opuBD , opuBC , opuBB , opuB A . The gene opuE c odes for the proline osmoprotective uptake transporter, OpuE, which is under the regulation of the vegetative promoter, as well as a general stress promoter ( 133 ) , indicating possible osmotic stress at 5 kP a. Another indication of LP causing osmotic stress in B. subtilis , the up regulation of the high affinity choline ABC transp orter encoded by the opuB operon ( opuBD , opuBC , opuBB , and opuBA ). This transporter is important for osmoregulated uptake of choline, the precursor in the production of the osmoprotectant glycine betaine in Bacillus subtilis ( 134 ) and is also induced by heat stress (109) . The opuB gene appears to be induced at LP in both WN624 and WN1106. However, the opuC operon, also coding a high affinity choline ABC transporter with high homology to the opuB operon, but which additionally transports glycine betaine ( 134 ) , is not differentially expressed in either strain at LP. The gene formerly known as ykvW , now zosA , is P type metal transport ATPase and a member of the PerR regulon; this Zn( II) transporter is repressed by PerR and induced by hydrogen peroxide ( 135 ) . The up regulation of this gene indicates that cells may be experiencing oxidative stress under LP conditions; reactive oxygen and nitrogen species (RONS) have been reported in lung tissue of eukaryotes exposed to hypoxia as a result of hypobaric environmen ts corresponding to high elevations, ~ 34 kPa, the pressure at the top of Mt. Everest ( 136 , 137 ) . At 5 kPa, some transporter signals were seen to be down regulated in both strains WN1106 and WN624: ydjK , citM , yusV , fhuCGB , fhuD , yxeB . The gene formerly known as ydjK , iolT , is the major myo inositol uptake transporter in B. subtilis , and under the control of IolR repressor ( 138 ) . Its signal appearing as down relates to the down regulation of part of the inositol operon, iolCDEFHI , in both strains at 5 kPa; this operon also being under the control of IolR. However, WN1106 down regulates these genes even more intensely than WN624 at 5 kPa;
72 and iolRS expression is only down regulated in WN1106 at 5 kP a. When comparing strains WN1106 vs. WN624, at ~101 kPa, iolBCDEF and io lS are up regulated, indicating that at standard pressure WN1106 has a higher level of mRNA of the iol operon. The yusV gene codes for an ATPase that complexes with both the FeuABC and YfiYZ YfhA importers; these importers are responsible for transport of catecholate siderophores and growth on schizokinen/arthrobactin, respectively ( 139 ) . In accordance with YusV being down regulated, other Fur regulated genes are also down regulated; fhuD , a lipoprotein that binds ferrichrome and other siderophores, for transport by FhuBG and FhuC, transmembrane proteins and an ATPase, respectively, and whose mRNA signals are also down regulated; YxeB, a ferrioxamine substrate binding protein that also complexes with the ABC transporter, FhuBGC ( 139 ) . Transpo rter signals that were seen to be up regulated only in WN1106 were found in the genomic region of yxeNMLK . Though the yxeNMLK region o f the genome is annotated as coding for a putative amino acid ABC transporter, evidence strongly suggests that it transpor ts t he polar amino acid methionine and related molecules ( 140 ) . This correlates well with the observation that WN1106 also up regulates other sulfur related S box operons, such as the cys operon, at 5 kPa, whereas the ancestral strain WN624 does not. Three signals from the Opp system, an ATP dep endent oligopeptide transport system, oppCDF , were down regulated significantly only in WN1106 at 5 kPa; the Opp system is important for initiation of sporulation as well as genetic competence in Bacillus subtilis ( 141 ) . Despite both strains hav ing comparable relative fitness values to one another at ~101 kPa (34) , there are differences in expression levels of the two strains at standard pressure. At 101 kPa, WN1106 expressed signals yfmC , oppABCD , mntABC , and rbsD at least two fold over
73 WN624. YfmC is part of the yfmCDEF operon, described as part of the Fur regula tory response, which codes for a Fe citrate ABC transporter ( 139 ) ; this signal was also down regulated in WN624 and WN1106 at 5 kP a. The mntABC operon codes for a Mn 2+ ABC transporter and is under the control of MntR; this transporter has been show n to be up regulated strongly during cold shock in B. subtilis ( 142 ) . At 5 kPa, WN1106 showed an increase in the signal of mntH over WN624; MntH is a NRAMP (natural resistance associated macrophage protein) transporter of manganese and, like MntABC, it is under the control of MntR ( 143 ) . As mentioned previously, genes involved in the oxidative stress response were found to be differentially expressed at LP, and manganese is known to protect cells during oxidative stress ( 143 ) . Signals for nasA , y heIH , yhaSTU , ykkCD , amyDC , mmr , yxkD , and yxjA were at least two fold lower in WN1106 compared to WN624, at ~101 kPa, supporting further that WN1106 has an altered expression pattern at ~101 kPa when compared to the ancestor. And at 5 kPa, WN1106 increase s the signal of mntH over WN624 and signals decreased compared to WN624 were lmrB , appABC , oppCDF , yqiX , amyDC , tagH , nrgA , and nupC . Together, these transcriptional differences highlight that changes occurred in the 5 kPa evolution experiment that gave ri se to WN1106 from the ancestor strain WN624. DNA Binding Proteins at LP piezophile P. profundum SS9 at lowered pressure (i.e. standard pressure) compared to its optimal dee p sea pressure range (32) ; the hypobaric response of the deep sea dwelling SS9 resulted in an increased expression of DNA repair proteins when exposed to 0.1 MP a. HP stabilizes hydrogen bond ing interactions ( 144 , 145 ) , and protein nucleic acid associations are expected to be one of the processes most heavily influenced by pressure fluctuations (8, 32, 65) . Mic roarray analysis indicated several DNA associated genes that were differentially expressed at LP. In both strains,
74 dps , which codes for a DNA protective protein during starvation events, was up regulated at 5 kPa; dps is also a B dependent gene ( 146 ) . Another dps homolog, mrgA , was up regulated only in WN1106 at LP; mrgA codes for a stress response DNA binding protein which forms stable complexes with DNA and protects against oxidative cell death ( 147) . At 5 kPa, WN624 up regulated recG (now known as recU ); recU codes for a highly conserved gram positive protein that is involved in recombinase activity, binds three and four stranded DNA structures, has endonuclease activity at Holliday junctions, forms asymmetrical nicks, and exhibits annealing capabilities ( 148 ) . When comparing the expre ssion pattern of the two strains at ~101 kPa, WN1106 up regulated several genes encoding DNA associated proteins: dnaX , perA , smf , dnaK , ruvB , and dnaB . The only down regulated signal corresponding to a DNA associated protein was that of dnaC , which was do wn when comparing WN1106 to WN624 at 5 kP a. Genes of Unknown Function were Differentially Expressed at LP Despite the extensive use of Bacillus subtilis in the laboratory as a common gram positive model organism, a large portion of its genome remains uncha racterized. Numerous (> 500) mRNA signals belonging to genes of unknown and putative functions were differentially expressed across the microarrays. To investigate their relevance to understanding the response of the cells to changes in pressure, the prote in sequences were analyzed by BLASTp (using non redundant protein sequence database); the BLASTp reports were further analyzed by MEGAN, a genomic analyzer, where the results were assigned to a SEED functional role (Table 3 3). The SEED classifications rev ealed ~ 170 sequences differentially expressed corresponding to carbohydrate utilization, RNA/DNA associated and metabolism pathways, virulence, sulfur and nitrogen metabolism, cell division, amino acid and secondary metabolism,
75 respiration and more. These gene category hits are in line with signals of known function; however, a large portion (~350) of the sequences analyzed were still unable to be classified by when type condition also saw differential expression of a number of unknown function signals. And again, when E. coli is exposed to HP conditions, there is a robust differential expression of signals with unknown f unctions (28) as well as stress response signals. The General Stress Response at LP As mentioned above, in HP studies of pressure mesophil es, it has been reported that mutations in RpoS of Escherichia coli conferred increased high hydrostatic pressure (HHP) resistance ( 149) . Indeed, when E. coli experienced a press ure upshift from atmospheric pressure to ~55 MPa, several cold and heat shock stress response proteins were increased; these proteins were termed pressure induced proteins (PIPs) (28) . Stress proteins are also induced when piezophilic microorganisms are grown at lowered and atmospheric pressures (respectively, 0.3 and 0.1 MPa), such as in Thermococcus barophilus , which induced a stress respo nse protein under these conditions that was not expressed at its optimal growth pressure (~35 MPa) ( 150 ) . As mentioned above, SS9, a well studied piezophile, also activated several stress response genes when grown at 0.1 MPa, specifically, chaperone proteins involved in proper protein folding, suggesting that lowered pressure conditions did not convey proper protein folding in this bacterium (25) . In parallel with high pressure microbes grown at lowered pressure (i.e. 101 kPa), B. subtilis strains WN624 and WN1106 both induced stress related proteins belonging to th e General Stress Response (GSR) when exposed to LP. As previously reported, nearly one third of the up regulated genes (87 out of 298) of either strain WN624 or WN1106 when compared at 5 kPa vs. ~101 kPa showed that exposure to
76 LP resulted in significant u p regulation of the signal for sigB ( 151 ) ; this is nearly one half of the known sigB dependent genes, no down regulated genes involved in the GSR were detected. Thus the microarrays reve aled that exposure of B. subtilis cells to LP induced the GSR in both the ancestral and LP evolved strains. The GSR regulon in B. subtilis consists of over 150 target genes, expression of which is induced in response to starvation and a variety of environm ental stresses (89, 102, 103 ) . Transcriptional activation of GSR genes is under control of the alternate sigma factor sigma B ( B ) encoded by the sigB gene (89 ) . Of the genes described to be in the GSR, ~ 83 are known to have thei r induction solely dependent upon SigB regulation; of these 83, there are 63 that are up regulated in at least one of the pressure microarrays. Microarrays in which gene expression of strains WN624 vs. WN1106 were compared either at ~101 kPa or at 5 kPa re vealed that very few genes of the GSR regulon were differentially expressed in the two strains at either atmospheric pressure or LP indicating that LP induced a similar subset of SigB dependent GSR genes in both WN624 and WN1106. This lab has previously re ported on induction of the GSR by LP in WN624 ( 151 ) . Expanding upon this work, we investigated the expression of the SigB dependent ctc lacZ reporter gene fusion (82) in ancestral and LP evolved strains carrying either the wild type sigB gene or the sigB 3::spc knockout mutation (Fig. 3 3). In order to assure that ctc lacZ expression was properly regulated by SigB in our strains (Table 3 1), we first induced the GSR by the classical treatment of exponentially growing cells with ethanol at 5% final concent ration and assayed for b galactosidase activity after 45 min (Fig. 3 3A). In both ancestral strain WN1400 and LP evolved strain WN1447, each carrying the sigB + allele, expression of ctc lacZ was significantly induced by ethanol treatment (Fig. 3 3A), where as ethanol induction of ctc lacZ expression was much weaker or absent (~1 to 2 fold) in
77 the analogous strains WN1407 and WN1377, each carrying the sigBD3::spc knockout mutation (Fig. 3 3A). Thus, the ctc lacZ fusion appeared to be a reliable reporter of t he SigB dependent GSR in our strains. Next the ctc::lacZ reporter strains were tested for induction of the GSR by exposure to 5 kPa LP (Fig. 3 3B). After exposure to 5 kPa for 2.5 hours, ctc lacZ expression was induced in both ancestral strain WN1400 and L P evolved strain WN1447 each carrying the wild type sigB + allele (Fig. 3 3B). In contrast, no LP induction of ctc lacZ fusion expression was observed in the analogous ancestral and LP evolved strains WN1407 and WN1377 carrying the sigBD3::spc knockout muta tion (Fig. 3 3B). We noted that at normal atmospheric pressure (~101 kPa), the background level of ctc lacZ fusion expression was ~2 fold higher in LP evolved strain WN1447 than in ancestral strain WN1400 (Fig. 3 3B). We were thus interested in determining the level of LP required to trigger the SigB dependent GSR in the ancestral vs. the LP evolved strain. Expression of the ctc::lacZ fusion was monitored in ancestral WN1400 and LP evolved strain WN1447, both carrying the sigB + allele, at pressures of ~101, 50, 25, 10, and 5 kPa (Fig. 3 3C). As previously reported, ancestral strain WN1400 did not induce ctc lacZ expression until pressure was lowered to 10 or 5 kPa ( 151 ) (Fig. 3 3C). In con trast, it was striking to note that ctc lacZ fusion expression in LP evolved strain WN1447 was strongly induced at higher pressures of 50 and 25 kPa (Fig. 3 3C). Thus it appeared that the LP evolved strain had become more sensitive to GSR induction by LP e xposure. Inacti v ation of sigB Does Not Alter Fitness at ~101 kPa or at 5 kPa The microarray data (Table 3 2) and the results of the ctc lacZ reporter experiments (Fig. 3 3) indicated that the SigB dependent GSR was induced by exposure to LP both in ancestr al and LP evolved strain backgrounds. We were interested in investigating what the significance, if
78 any, a sigB knockout mutant would have on the relative fitness of B. subtilis ancestral or LP evolved strains at 5 kP a. It has been previously shown that a sigB null mutant has no noticeable disadvantage compared to wild type when grown at any of the stress conditions (starvation or environmental) known to induce the SigB mediated GSR (81 ) . Therefore, as with the othe r inducing conditions of the GSR, LP could be causing a redundant protective state that has no fitness advantage. We therefore constructed congenic derivatives of ancestral strain WN624 and LP evolved strain WN1106, each carrying a sigBD2::cat insertion de letion mutation, resulting in strains WN1232 and WN1233, respectively (Table 3 1), which were used to assess their relative fitnesses at ~101 kPa and at 5 kPa (Fig. 3 4). To compare relative fitnesses, all constructed strains were used in pair wise competi tion experiments against a common strain, WN1261, which is congenic to ancestral strain WN624 but which carries an amyE::neo marker (Table 3 1). Extensive control experiments verified that the antibiotic resistance markers inserted at the amyE locus were s electively neutral [ (34) and data not shown]. Competition of ancestral strain WN624 vs. the congenic reference strain WN1261 confirmed that these two strains showed no difference in their relative fitness at either ~101 and 5 kPa (Fig. 3 4). Furthermore, introduction of the sigB 2::cat mutation into strain WN624 (i.e., strain WN1232) did not alter its fitness relative to strain WN1261 (Fig. 3 4). When the same experiment was performed using LP evolved strain WN1106 vs. reference strain WN1261, it was observed that strain WN1106 w as significantly more fit at 5 kPa, but not at ~101 kPa (Fig. 3 4), in good agreement with previous observations (34) . Introduction of the sigBD2::cat mutation into strain WN1106 (i.e., strain WN1233) also did not significantly affect its fitness relative to reference strain WN1261 at either ~101 or 5 kPa (Fig. 3 4). The dat a thus indicated that the SigB mediated GSR exerted no
79 significant effect on the competitive fitness of either ancestral or LP evolved B. subtilis strains at either normal atmospheric pressure or at an LP of 5 kP a. This is in line with previous observation s that a sigB null mutant has little to no growth disadvantage under other environmental stresses; indeed, in our studies with ethanol induction test of our ctc::lacZ constructs, there was no adversity on growth in the cell lines also containing a sigB del etion (data not shown). Sporulation at 5 kPa It has been previously suggested that the induction of the GSR is one avenue for the cell to cope with non optimal environmental conditions as opposed to or when the more extreme process of undergoing sporulatio n is limited (81 ) . Because LP seems to inhibit cellular processes important for normal growth and division, we decided to briefly investigate if the process of sporulation is inhibited at 5 kP a. To this end, a simp le sporulation frequency experiment was conducted in both WN624 and WN1106 to compare their ability to form spores at ~101 kPa and 5 kPa (Fig. 4, Table 3 4). Both strains were capable of forming spores after 3 days at 5 kPa, however, this ability was great ly reduced from ~101 kPa total spore titers and sporulation frequency (Fig. 4, Table 3 4). While both strains showed a comparable ability to form spores at ~101 kPa, there was a difference in their growth at 5 kPa in Spizizen media (Table 3 4). This may be an underlining cause for the reduced number of spores in the WN1106 cultures and the order of magnitude decrease in sporulation frequency compared to WN624 cultures (Fig. 4, Table 3 4). Discussion Relatively little is known of how hypobaria affects cellul ar processes, or how microbial life responds to LP. In this communication, transcriptional microarray analyses were conducted on strains WN1106 and WN624 to determine (i) genes differentially expressed during hypobaric
80 growth, (ii) different LP response be tween the ancestor and evolved strains, and (iii) genes that may be important for the LP phenotype of WN1106. The strains differentially express genes involved in nitrogen respiration, lipid synthesis, respiration chain components, sulfur metabolism, trans porters, SigB General Stress Response, and carbohydrate utilization pathways when grown at 5 kP a. The differences between the hypobaric evolved WN1106 and ancestral strain WN624 responses at low pressure include pathways involved in sulfur metabolism, tran sporters, motility, polyketide biosynthesis, respiratory chain components, and iron acquisition. Our investigation of the phenotypic difference between GSR activation at various lowered pressures by reporter gene fusions in both strains revealed that WN110 6 induces the GSR at higher pressures than WN624. Strain WN1106 strongly induced the GSR at 50 kPa, the equivalent of ~5,000 meters above sea level, the approximate altitude of the city of La Rinconada, Peru (pop. 30,000). In sharp contrast, ancestral stra in WN624 induced the GSR at 10 kPa, the equivalent of an altitude of ~16,000 meters, nearly twice the height of Mt. Everest (8,848 meters). It is possible that WN1106 during the 5 kPa experimental evolution culturing underwent a genomic change in one or mo re of the genes involved in regulating the GSR ( rsbV, rsbW, rsbX, sigB, rsbS, rsbT, rsbU, rsbP, rsbQ ). Currently, whole genome re sequencing analysis is being conducted on WN1106, this should reveal underlying genomic changes that account for differences i n transcriptional regulation at 5 kPa compared to WN624 (See Chapter 4) . However, the SigB dependent GSR does not confer an increased fitness to LP when compared to a knock out sigB strain in either WN624 or WN1106 [this work and ( 151 ) ]. Nor does the sigB deletion in WN1106 decrease its relative fitness over the ancestor, WN624.
81 Investigation into sporulation at LP revealed that WN624 has a greater ability to grow in Spizizen minimal medi um compared to WN1106. This in turn may be the cause of difference between the two strains when comparing their sporulation frequency. It is also interesting to note because the 5 kPa evolution experiment that gave rise to WN1106 was conducted in LB (34) and other medias have not been tested at LP for either strain. The over all transcriptional differences in the LP response of the two strains when grown at 5 kPa indicates that there exists genomic alterations in the hypobaric evolved strain that may be affecting transcriptional and/or post transcriptional processes; these alt erations, however, are difficult to pin point in the microarray analysis due to the large number of differentially expressed genes. However, the anaerobic response of both, which was highly induced by LP exposure, was previously investigated by competition experiments under standard pressure hypoxic conditions and revealed that WN1106 is less fit at these conditions than WN624 (86) , highlighting a possible change that may have occurred in the anaerobic response of WN1106. Also, any changes to the genome which do not correspond to a differential e xpression pattern at 5 kPa compared to 1 atm will not be identifiable by microarray analysis. As stated, future whole genome resequencing of the two strains for genomic comparisons is underway. Resequencing data is capable of detecting single nucleotide po lymorphisms (SNPs) and areas where insertion or deletion (INDELs) events have occurred. This may uncover such mutations in transcriptional and/or post transcriptional machinery and explain why certain signals are highly induced in the evolved WN1106, e.g. des , without changes having occurred to promoter sequences of such signals (86) . This is one of the first studies of conditions, which has relevance to understanding the limits of Earth microorganisms
82 survivability to extreme extraterrestrial environments. The data reveals the plasticity of long term adapted terrestrial, mesophilic organisms to non optimal extreme environments and the rather rapid changes (here ~ 3 months) that occur during such exposure. These results further advance the knowl edge of how a microorganism senses and responds to changes in pressure, and compliments previous experimental data on piezophilic organism adaptations.
83 Table 3 1. Bacillus subtilis strains and plasmids used in this study Strain or Plasmid Genotype/Phenoty pe Source (reference) BSM151 trpC2, SP ctc::lacZ ; Cm R , Erm R Uwe VÃ¶lker (Brigulla et al., 2003) PB153 2 ::cat Chet Price (Boylan et al., 1993) PB344 Chet Price (Boylan et al., 1993) WN624 trpC2, amyE::spc ; S pc R , Ancestral strain (Maughan et al., 2006) WN628 trpC2, amyE::cat ; Cm R , Ancestral strain (Maughan et al., 2006) WN1106 trpC2, amyE::spc ; Spc R . Evolved for 1,000 generations to enhanced growth at 5 kPa (Nicholson et al., 2010) WN1232 trpC2, amyE::spc, sigB 2:: cat ; Spc R , Cm R in WN624 background PB153 WN624; Cm R (this study) WN1233 trpC2, amyE::spc 2:: cat ; Spc R , Cm R in WN1106 background PB153 WN1106; Cm R (this study) WN1261 trpC2 , amyE::neo ; Neo R in WN628 background pECE73 WN628; Neo R (this study) WN1278 trpC2 , amyE::neo ; Neo R in WN1106 background pECE141 WN1106; Neo R (this study) WN1377 trpC2 , amyE::neo , sigB 3::spc , ctc::lacZ; Neo R , Spc R , Cm R , Erm R in WN1106 backgroun d BSM151 WN1393; Cm R , Erm R (this study) WN1392 trpC2, amyE::neo sigB 3::spc; Neo R , Spc R in WN628 background PB344 WN1261; Spc R (this study) WN1393 trpC2, amyE:: neo sigB 3::spc; Neo R , Spc R in WN1106 background PB344 WN1278; Spc R (this study) WN1400 trpC2 , amyE::neo; ctc::lacZ ; Neo R , Cm R , Erm R in WN628 background BSM151 WN1261; Cm R , Erm R (this study) WN1407 trpC2, amyE::neo; sigB 3::spc; ctc::lacZ ; Neo R , Spc R , Cm R , Erm R in WN628 background BSM151 WN1392; Cm R , Erm R (this study) WN1447 trpC2, amyE: ctc::lacZ; Neo R , Cm R , Erm R in WN1106 background BSM151 WN1278; Cm R , Erm R (this study) pECE73 pCm::Neo antibiotic switching cassette BGSC (Steinmetz and Richter, 1994) pECE141 pSpc::Neo antibiotic switching cassette BGSC (Steinmetz and Richter, 1994) A Abbreviations: BGSC, Bacillus Genetic Stock Center; , transformation.
84 Table 3 2. Expression data for microarray chips defined in Table 2 1 Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU00090 guaB 1.21 2 2.25 1.04 inosine monophosphate dehydrogenase BSU00190 dnaX 1.22 3.54 1.42 3.04 DNA polymerase III (gamma and tau subunits) BSU00400 yabE 2.37 1.22 1.2 2.27 putative cell wall shaping enzyme BSU00490 spoVG 2.89 6.91 1 2.44 regulator required for spore cortex synthesis (stage V sporulation) BSU00520 ctc 7.18 10.05 1.49 1.19 ribosomal protein Ctc, binding 5S RNA BSU00530 spoVC 3.67 9.62 3 1.1 peptidyl tRNA hydrolase BSU00540 yabK 2.36 5.46 2.71 1.16 conserved hypo thetical protein BSU00580 yabN 1.42 2.86 2.1 1.01 putative fusion methylase and nucleotide pyrophosphohydrolase BSU00670 yacA 2.29 1.12 1.04 2.2 tRNAile lysidine synthetase BSU00680 hprT 1.35 1.86 1.13 2.38 hypoxanthine guanine phosphoribosyltra nsferase BSU00720 yacD 1.19 1.74 1.02 2.27 putative protein secretion PrsA homolog BSU00730 cysK 1.36 1.83 2.9 1.08 cysteine synthase BSU00830 ctsR 11.26 6.11 1.02 1.89 transcriptional regulator BSU00840 mcsA 8.82 4.82 1.04 1.81 activator of protein kinase McsB BSU00850 mcsB 5.29 3.3 1.06 1.57 protein tyrosine kinase BSU00930 cysE 1.47 2.13 1.47 2.21 serine acetyltransferase BSU00940 cysS 1.59 1.72 1.26 2.2 cysteinyl tRNA synthetase BSU00950 yazC 1.87 1.59 1.23 2.42 ribonuclease for 23S RN A maturation BSU00960 yacO 1.91 1.66 1.2 2.44 23S rRNA methyltransferase BSU01060 ybxB 2.16 1.74 2.28 1.72 ribosomal RNA methyltransferase BSU01160 rplC 1.38 1.83 1.55 2.18 ribosomal protein L3 (BL3) BSU01170 rplD 1.35 1.92 1.63 2.12 ribosomal protein L4 BSU01190 rplB 1.6 1.56 1.54 2.15 ribosomal protein L2 (BL2) BSU01200 rpsS 1.96 1.56 1.7 2.24 ribosomal protein S19 (BS19) BSU01210 rplV 2.18 1.33 1.55 2.44 ribosomal protein L22 (BL17) BSU01220 rpsC 2.3 1.34 1.4 3.02 ribosomal protei n S3 (BS3) BSU01230 rplP 1.81 1.61 1.64 2.49 ribosomal protein L16 BSU01240 rpmC 1.85 1.56 1.52 2.36 ribosomal protein L29 BSU01250 rpsQ 1.64 1.59 1.55 2.25 ribosomal protein S17 (BS16) BSU01260 rplN 1.65 1.49 1.44 2.4 ribosomal protein L14 BS U01520 ybaK 1.1 2.41 2.63 1.21 putative alkylated deoxynucleotide triphosphohydrolase
85 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU0 1530 cwlD 1.16 1.94 2.37 1.04 N acetylmuramoyl L alanine amidase BSU01550 gerD 2.47 1.38 1.34 2.33 rapid response to nutrient germinants BSU01560 kbaA 2.17 1.54 2.17 3.02 inner membrane protein involved in activa tion of the KinB signaling pathway to sporulation BSU01600 ybbA 3.29 7.76 1.38 1.65 putative iron chelator esterase BSU01610 feuC 3.83 8.78 1.46 1.64 iron uptake protein BSU01620 feuB 3.57 12.06 2.26 1.9 iron uptake protein BSU01630 feuA 2.04 14.28 3.26 4.08 iron hydroxamate binding lipoprotein BSU01640 ybbB 2.67 4.91 1.12 2.06 putative transcriptional regulator (AraC/XylS family) BSU01700 murQ 1 2.64 1.13 2.95 D lactyl ether N acetylmuramic 6 phosphate acid etherase BSU01760 ybbR 2.2 8 1.69 1.06 3.79 conserved hypothetical protein BSU01780 glmS 1.2 2.25 1.05 2.13 L glutamine D fructose 6 phosphate amidotransferase BSU01890 ybcL 1.86 1.32 1.04 2.69 putative efflux transporter BSU01910 ybcO 1.46 7.96 1.99 2.82 sporulation killi ng factor A BSU02040 ybdN 2.99 6.13 1.55 1.55 putative phage protein BSU02050 ybdO 2.21 2.99 1.56 2.56 putative phage protein BSU02100 cypC 7.9 11.87 1.3 1.12 fatty acid beta hydroxylating cytochrome P450 BSU02110 ybyB 76.68 47.24 1.17 2.41 conserv ed hypothetical protein BSU02120 ybeC 7.73 8.08 2.12 1.75 putative H+/amino acid transporter BSU02130 glpQ 11.35 19.24 1.12 2.34 glycerophosphoryl diester phosphodiesterase BSU02140 glpT 19.12 28.13 1.06 2.29 glycerol 3 phosphate permease BSU 02230 purT 8.36 5.42 1.06 1.45 phosphoribosylglycinamide formyltransferase 2 BSU02340 gltP 2.19 6.55 1.08 3.17 proton/glutamate symport protein BSU02350 gamP 3.66 6.87 1.66 1.04 phosphotransferase system (PTS) glucosamine specific enzyme IICBA c omponent BSU02530 yczA 5.25 2.78 1.26 1.98 anti TRAP regulator BSU02540 ycbK 6.23 2.89 1.12 2.37 putative efflux transporter
86 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa W N1106 to WN624 at 101kPa Gene Description BSU02580 ycbO 1.32 4.99 3.43 1.13 putative Na+ driven exporter or maturation protein BSU02590 ycbP 14.75 18.45 1.3 1.25 putative inner integral membrane protein BSU02670 lmrB 1.52 2.35 2.58 1.56 efflux tran sporter; drug export protein BSU02680 lmrA 1.13 1.8 2.2 1.42 transcriptional repressor of lmrAB and yxaGH operons BSU02830 ycdF 12.76 11.54 1.2 1.47 putative dehydrogenase BSU02840 ycdG 6.14 5 1.16 1.29 putative glycosidase BSU02950 yceI 2.68 1.02 1.17 2.46 putative transporter BSU03020 ycgA 1.86 3.01 1.62 2.58 putative integral inner membrane protein BSU03050 ldh 9.79 37.61 9.96 2.09 L lactate dehydrogenase BSU03060 lctP 16.2 192.18 19.73 1.07 L lactate permease BSU03080 ycgE 3.42 2.61 4.7 4 2.05 putative transcriptional regulator BSU03090 ycgF 3.82 1.66 3.05 1.95 putative aminoacid export permease BSU03180 cah 3.07 1.27 2.85 1.16 S deacylase BSU03200 ycgM 6.06 2.39 2.66 1 proline oxidase BSU03210 ycgN 4.3 1.97 3.16 1.37 putativ e 1 pyrroline 5 carboxylate dehydrogenase BSU03220 ycgO 1.19 1.79 3.52 2.33 putative proline/ornithine permease BSU03270 ycgT 9.32 6.7 1.18 1.63 putative thioredoxin reductase BSU03300 nasD 5.87 5 1.06 1.33 assimilatory nitrite reductase subunit BSU03330 nasA 3.95 1.8 1.03 2.49 nitrate transporter BSU03370 yckA 4.89 5.29 1.49 1.25 putative ABC transporter (permease) BSU03380 yckB 3.72 5.16 1.64 1.01 putative ABC transporter (binding lipoprotein) BSU03450 hxlB 6.31 12.73 1.12 2.4 1 6 phospho 3 hexuloisomerase (PHI) BSU03460 hxlA 6.33 11.28 1.17 2.82 3 hexulose 6 phosphate synthase (HPS) BSU03490 srfAB 1.6 1.04 1.66 3.22 surfactin synthetase BSU03670 yclF 2.33 2.53 3.08 2.68 di tripeptide proton ABC symporter BSU03760 yc lK 4.83 7.18 1.51 1.08 two component sensor histidine kinase [YclJ] BSU03800 yclN 11.63 10.22 1.45 1.42 putative iron siderophore ABC transporter (permease) BSU03810 yclO 11.41 13.05 1.36 1.11 putative iron siderophore ABC transporter (permease)
87 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU03820 yclP 6.24 10.62 1.26 1.55 putative iron siderophore ABC transporter (ATP binding protein) BSU03830 yclQ 3.54 9.12 1.53 2.3 putative iron siderophore ABC transporter (binding lipoprotein) BSU03940 ycnI 4.78 1.3 2.49 1.25 conserved hypothetical protein BSU03950 ycnJ 4.76 1.03 2.87 1.42 putative copper import protein BSU03960 yc nK 5.28 1.07 3.42 1.45 putative transcriptional regulator (DeoR family) BSU04000 ycsA 6.57 5.09 1.15 1.44 putative tartrate dehydrogenase BSU04050 ycsF 1.29 1.71 2.29 1.02 putative nitrogen containing heterocycle degradation enzyme BSU04060 ycsG 1.35 2.37 3.46 1.06 putative branched chain amino acids transporter BSU04070 ycsI 1.5 2.3 3.3 1.09 conserved hypothetical protein BSU04080 kipI 1.64 2.39 3.33 1.27 putative inhibitor of the autophosphorylation reaction of KinA BSU04090 kipA 1.09 2.92 2.73 1.1 putative hydrolase subunit antagonist of KipI BSU04100 kipR 1.02 3.87 3.45 1.14 transcriptional regulator (IclR family) BSU04140 pbpC 2.69 1.42 1.67 2.3 penicillin binding lipoprotein 3 BSU04190 ydaD 6.73 8.63 1.48 1.28 putative deh ydrogenase BSU04200 ydaE 6.93 6.78 1.44 1.38 conserved hypothetical protein BSU04210 ydaF 2.41 6.71 2.24 1.19 putative ribosomal protein N acetyltransferase BSU04220 ydaG 32.42 26.27 1.04 1.17 putative general stress protein BSU04240 ydzA 6.06 8.85 1 .46 2.24 conserved hypothetical protein BSU04320 ydaO 1.27 4.03 7.01 1.34 putative metabolite transporter BSU04340 ydaP 19.14 9.92 1 1.82 putative enzyme with pyruvate as substrate BSU04350 ydaQ 2.36 4.95 3.84 2.07 BG1206:unknown BSU04360 mntH 2.8 6 1.12 2.66 1.02 manganese transporter BSU04370 ydaS 9.33 9.08 1.2 1.12 conserved hypothetical protein BSU04400 gsiB 90.26 25.11 1.8 7.47 general stress protein BSU04470 dctP 38.15 18.49 2.41 2.69 C4 dicarboxylate transport protein BSU04500 ydbK 3 .22 3.66 2.13 1.73 putative efflux ABC transporter (permease component) BSU04510 ydbL 1.6 3.92 1.2 2.26 conserved hypothetical protein
88 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5 kPa WN1106 to WN624 at 101kPa Gene Description BSU04520 ydbM 3.46 26.4 4.84 1.51 putative acyl CoA dehydrogenase BSU04530 ydbN 1.62 2.6 3.72 1.09 conserved hypothetical protein BSU04540 ydbO 1.13 3.31 2.34 1.59 putative cation efflux system BSU0462 0 acpS 1.49 1.16 1.19 2.13 holo acyl carrier protein synthase BSU04650 ydcD 2.13 1.32 1.36 2.19 antitoxin EndoAI BSU04710 rsbV 36.63 26.7 1.16 1.89 anti anti sigma factor (antagonist of RsbW) BSU04720 rsbW 44.01 30.44 1.1 1.85 switch protein/serine kinase and anti sigma factor (inhibitory sigma B binding protein) BSU04730 sigB 56.94 35 1.1 2 RNA polymerase sigma 37 factor (sigma(B)) BSU04740 rsbX 49.01 26.91 1.04 1.72 serine phosphatase BSU04780 ydcI 1.01 1.72 1.41 2.37 putative RNA helicase BSU05130 ydeB 6.71 4.73 1.25 1.4 putative transcriptional regulator BSU05160 ydeD 2.89 1.24 1.32 2.32 putative permease BSU05200 ydeH 1.21 2.35 1.12 2.32 putative integral inner membrane protein BSU05230 ydeK 1.87 2.7 3.53 1.64 putative permease BSU05580 ydgC 2.1 1.14 1.13 2.14 putative transcriptional regulator BSU05590 ydgD 2.05 1.06 1.11 2.23 conserved hypothetical protein BSU05620 ydgF 4.14 1.34 1.49 2.25 putative amino acid permease BSU05640 ydgG 4.03 1.61 6.39 1.12 putative transcriptional regulator (MarR family) BSU05650 ydgH 2.29 1.74 7.44 1.77 putative membrane component BSU05680 ydgK 5.54 3.43 1.24 2.04 putative efflux transporter BSU05690 ydhB 2.16 1.8 2.69 1.4 putative integral inner membrane protein BSU05 790 ydhK 9.13 12.23 1.23 1.14 hypothetical protein BSU05810 ydhM 1 3.11 1.12 3.68 oligo alpha mannoside phosphotransferase system enzyme IIB BSU05820 ydhN 1.28 3.11 1.1 4.24 oligo alpha mannoside phosphotransferase system enzyme IIA BSU05830 ydhO 1 .66 3.2 1.67 3.44 oligo alpha mannoside phosphotransferase system enzyme IIC BSU05840 ydhP 1.73 2.62 1.24 3.87 mannoside phospho beta d glucosidase BSU05850 ydhQ 1.36 2.58 1.21 3.13 transcriptional regulator (GntR family)
89 Table 3 2. Continued Acce ssion BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU05860 ydhR 1.19 3.63 1.26 2.72 putative carbohydrate kinase BSU05870 ydhS 1.24 4.11 1.34 2.82 phosphohexomut ase; cupin family BSU05980 tatAY 5.66 16.69 1.87 1.62 component of the twin arginine pre protein translocation pathway BSU05990 tatCY 7.12 16.12 2.13 1.03 component of the twin arginine pre protein translocation pathway BSU06080 ydiQ 1.86 2.53 2.12 2. 16 BG1278:unknown BSU06150 gutB 4.8 3.03 1.05 1.81 glucitol (sorbitol) dehydrogenase BSU06230 ydjK 33.7 28.24 1.57 1.66 myo inositol transporter BSU06250 ydjM 1.83 2.94 5.45 1.03 conserved hypothetical protein BSU06260 ydjN 1.28 5.59 7.23 1.0 2 putative membrane protein BSU06270 ydjO 1.25 1.38 1.22 2.47 conserved hypothetical protein BSU06350 yebA 1.32 1.8 1.07 2.23 conserved hypothetical protein BSU06380 yebC 4.81 2.87 1.62 2.61 putative integral inner membrane protein BSU06590 ye rD 5.5 10.5 2.03 1.09 putative flavoenzyme BSU06610 pcrA 1.03 2.3 1.04 2.24 ATP dependent DNA helicase BSU06640 yerI 8.57 12.3 1.27 1 putative kinase BSU06650 sapB 3.02 5.06 1.79 1.02 membrane component BSU06660 opuE 30.35 28.65 1.06 1.16 proline tr ansporter BSU06680 gatA 2.26 4.91 1.77 1.32 glutamyl tRNA(Gln) amidotransferase (subunit A) BSU06710 yerP 5.16 1.41 1.9 1.88 transporter involved in surfactin self resistance BSU06790 yeeD 2.17 1.76 2.22 2.55 conserved hypothetical protein BSU06800 yezA 1.34 2.05 2.21 1.63 hypothetical protein BSU06810 yeeF 1.72 2.55 2.19 1.54 BG1282:unknown; similar to unknown proteins from B. subtilis BSU06830 rapH 5.21 9.79 1.09 1.94 response regulator aspartate phosphatase BSU06850 yeeK 1.66 2.35 2.98 1.25 spore associated protein BSU06940 yesL 2.54 1.59 1.41 2.63 putative permease BSU07150 yetG 9.77 8.06 2.15 2.41 putative monooxygenase BSU07160 yetH 5.37 2.57 1.08 2.23 putative lyase/dioxygenase BSU07190 yezD 1.06 2.99 2.76 1.07 conserved hypothetical protein BSU07200 yetJ 3.87 1.33 1.36 4.11 putative integral inner membrane protein BSU07210 yetK 4.18 1.2 1.36 5.38 putative efflux transporter BSU07320 yfnC 1.54 1.1 1.94 3 putative efflux transporter BSU07340 yfnA 4.86 3.5 1.05 1.58 metabolite permease
90 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU07350 yfmT 1.2 1.25 3.58 2.52 putative aldehyde dehyd rogenase BSU07360 yfmS 1.13 1.4 4.08 3.75 putative chemotaxis sensory transducer BSU07380 yfmQ 6.94 9.7 1.16 1.69 conserved hypothetical protein BSU07470 yfmH 1.02 5.64 1.54 3.78 BG1295:unknown BSU07520 yfmC 6.38 10.73 1.07 2.24 iron dicitrate ABC transporter (binding lipoprotein) BSU07550 yflT 31.64 22.42 1.34 1.87 heat stress induced protein BSU07560 pel 1.36 1.97 1.43 2.09 pectate lyase BSU07610 citM 27.71 18.89 1.09 1.4 transporter of divalent metal ions/citrate complexes BSU07620 yflN 11.63 9.74 1.05 1.03 putative metal dependent hydrolase BSU07700 nagP 5.29 3.86 1.38 2.19 phosphotransferase system (PTS) N acetylglucosamine specific enzyme IICB component BSU07710 yflE 1.41 2.21 3.06 1.06 putative exported enzyme and ani on transporter BSU07750 yflA 45.79 34.3 1.1 1.2 putative aminoacid transporter BSU07760 yfkT 24.75 16.4 1.1 1.26 putative spore germination integral inner membrane protein BSU07770 yfkS 13.26 10.24 1.34 1.23 hypothetical protein BSU07780 yfkR 4.82 3 .48 1.2 1 putative spore germination protein BSU07800 treP 43.61 14.42 1.09 5.11 phosphotransferase system (PTS) trehalose specific enzyme IIBC component BSU07810 treA 10.7 6.01 1.08 1.93 trehalose 6 phosphate hydrolase BSU07850 yfkM 23.91 20.96 1.38 1.26 general stress protein 18 BSU07860 yfkL 2 2.54 2.61 2.09 efflux transporter BSU07880 yfkJ 29.03 14.6 1.05 1.87 protein tyrosine phosphatase BSU07890 yfkI 21.62 11.42 1.07 1.66 conserved hypothetical protein BSU07900 yfkH 15.34 9.13 1.18 1.38 putative integral inner membrane protein with ribonuclease fold BSU07920 yfkE 14.71 14.06 1.1 1.25 putative H+/Ca2+ antiporter BSU07930 yfkD 7.91 6.96 1.2 1.04 conserved hypothetical protein BSU07990 yfjR 2.49 1.67 1.5 2.97 putative beta hydro xyacid dehydrogenase BSU08030 yfjN 2.7 3.69 2.21 1.74 tRNA dihydrouridine synthase 2 BSU08120 yfjF 1.33 1.64 2.6 1.1 putative membrane protein
91 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU08130 yfjE 1.01 2.03 2.88 1.43 putative integral inner membrane protein BSU08140 yfjD 1.21 1.83 2.5 1.44 putative integral inner membrane protein BSU08150 yfjC 1.31 1.73 2.51 1.76 hypothetical protein BSU08160 yfjB 1.22 1.48 2.3 1.71 hypothetical protein BSU08390 yfiT 1.9 1.55 1.44 2.19 metal dependent hydrolase BSU08440 yfiY 15.59 25.31 2.63 1.12 putative iron(III) dicitrate transporter binding lipoprotein BSU08450 yfiZ 26.34 18.3 1.72 2.55 iron(III) siderophore transport permease BSU08460 yfhA 15.46 13.83 1.72 1.8 iron(III) siderophore transport permease BSU08480 yfhC 5.16 4.38 1.24 1.29 putative oxidoreductase (nitroreductase family) BSU08490 yfhD 9.35 5.1 1.32 1.43 c onserved hypothetical protein BSU08500 yfhE 5.7 4.02 1.16 1.69 hypothetical protein BSU08510 yfhF 5.99 4.58 1.37 2.01 putative nucleotide binding protein BSU08520 recX 2.3 1.31 1.38 2.26 regulatory protein RecX BSU08570 yfhK 89.64 96.78 1.07 1.24 con served hypothetical protein BSU08580 yfhL 30.83 44.46 1.21 1.11 SdpC immunity factor BSU08590 yfhM 15.77 22.42 1.25 1.3 putative hydrolase BSU08600 csbB 7.21 9.18 1.1 1.24 putative glycosyl transferase BSU08630 yfhQ 1.06 3.82 1.34 2.74 A/G specif ic adenine glycosylase or DNA (apurinic or apyrimidinic site) lyase BSU08640 yfhS 1.57 3.7 1.06 5.78 conserved hypothetical protein BSU08650 fabL 1.76 4.32 1.4 2.14 enoyl acyl carrier protein reductase III BSU08660 sspE 2.83 2.68 3.11 2.36 small acid soluble spore protein (gamma type SASP) BSU08740 ygzB 1.12 1.96 1.18 2.12 putative membrane protein BSU08760 spo0M 5.55 3.34 1.1 2.02 sporulation control gene BSU08820 katA 2.1 3.71 2.35 1.24 vegetative catalase 1 BSU08990 yhbI 8.77 1.51 7.28 1.59 putative transcriptional regulator (MarR family) BSU09000 yhbJ 10.04 1.23 5.92 1.77 putative integral inner membrane protein; putative exporter subunit BSU09010 yhcA 6.5 1.51 7.52 1.24 putative exporter BSU09020 yhcB 5.1 1.61 6.74 1.13 p utative oxidoreductase associated to oxygen stress BSU09030 yhcC 2.33 1.44 3.57 1.02 hypothetical protein BSU09110 yhcJ 4.63 4.83 1.02 1.16 putative ABC transporter (binding lipoprotein) BSU09130 yhcL 2.21 2.76 3.55 1.75 sodium cystine symporter
92 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU09140 yhcM 5.05 6.96 1.43 1.07 hypothetical protein BSU09190 yhcR 4.95 2.88 1 1.48 non specific extracellular endonuclease cleaving RNA and DNA BSU09230 yhcV 6.23 1.62 8.11 1.05 putative oxidoreductase BSU09280 glpF 26.3 20.82 1.65 2.47 glycerol permease BSU09290 glpK 4.82 2.19 1.22 1.98 glycerol kinase BSU09350 yhdB 1.28 3.56 3.33 1.1 conserved hypothetical protein BSU09360 yhdC 1.07 3.15 2.85 1.04 putative exported protein BSU09370 lytF 1.42 1.02 2.67 1.97 gamma D glutamate meso diaminopimelate muropeptidase (major autolysin) BSU09420 lytE 1.43 2.24 1.59 2.48 cell wall hydrolase; phosphatase associated protein (major autolysin) BSU09430 citR 2.26 1.29 2.65 1.14 transcriptional regulator CitR (LysR family) BSU09440 citA 1.37 1.15 2.15 1.71 citrate synthase I BSU09470 yhdH 6.55 4.67 1.77 2.48 putative sodium dep endent transporter BSU09490 yhdJ 1.97 1.9 2.12 1.7 putative acetyltransferase BSU09530 yhdN 28.76 26.54 1.33 1.74 aldo/keto reductase specific for NADPH BSU09580 yhdS 1.61 2.29 2.23 1.33 BG1302:unknown BSU09590 yhdT 1.47 4 2.44 1.07 putative membran e protein BSU09630 yhdX 4.44 7.12 1.67 1.21 conserved hypothetical protein BSU09690 nhaX 68.38 82.89 1.05 1.18 stress response protein, UspA family BSU09710 yheI 1.91 1.73 1.06 3.75 ABC transporter (ATP binding protein) involved in the signalling pa thway that activates KinA during sporulation initiation BSU09720 yheH 1.98 2.14 1.27 3.81 ABC transporter (ATP binding protein) involved in the signalling pathway that activates KinA during sporulation initiation BSU09750 sspB 6.84 1.03 6.29 1.15 sma ll acid soluble spore protein (beta type SASP) BSU09850 yhaU 3.37 1.97 1.37 2.74 transporter involved in K+ efflux BSU09860 yhaT 2.8 1.67 1.62 2.6 K+/H+ antiporter for K+ efflux
93 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/1 01kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU09870 yhaS 1.39 1.15 2.02 4.08 K+/H+ antiporter for K+ efflux BSU09880 yhaR 1.82 3.35 1.29 2.53 putative dehydratase BSU09950 prsA 1.54 1.48 1.05 2.6 8 molecular chaperone lipoprotein BSU10020 serC 3.18 1.98 2.53 1.45 phosphoserine aminotransferase BSU10100 yhgC 1.52 1 1.33 2.2 conserved hypothetical protein BSU10130 hemH 5.09 8.68 1.86 1.16 ferrochelatase BSU10140 hemY 4.8 8.56 1.85 1.07 pro toporphyrinogen IX and coproporphyrinogen III oxidase BSU10150 yhgD 6.4 2.07 1.64 1.81 putative transcriptional regulator BSU10220 gltT 5.73 4.12 1.65 2.79 proton/sodium glutamate symport protein BSU10230 yhfH 4.69 4.1 1.31 1.52 hypothetical protei n BSU10310 yhfO 4.82 3.39 1 1.77 putative N acetyltransferase BSU10330 yhfQ 16.4 15.06 1.11 1.02 putative iron(III) dicitrate binding lipoprotein BSU10380 hemAT 2.19 1.14 2.97 1.52 haem based dioxygen sensor BSU10430 yhxD 4.98 2.89 1.47 1.08 putative oxidoreductase BSU10440 yhjA 2.44 1.37 1.64 2.18 conserved hypothetical protein BSU10480 yhjE 2.03 1.06 1.21 2.47 putative integral inner membrane protein BSU10560 yhjM 2.34 6.77 1.57 1.87 transcriptional regulator of the ntd operon; (Lac I family) BSU10570 yhjN 2.61 8.18 2.29 1.48 putative integral inner membrane protein BSU10730 yisI 1.47 1.55 1.52 3.9 Spo0A P phosphatase BSU10770 wprA 1.12 3.17 2.38 1.62 cell wall associated protease BSU10820 yisQ 4.23 2.81 1.51 2.4 putative Na +driven efflux transporter BSU10850 yisS 2.88 2.53 2.1 1.96 putative dehydrogenase BSU10860 yisT 4.53 7.29 1.2 1.93 conserved hypothetical protein BSU11260 yjzC 2.24 4.61 3.12 1.26 conserved hypothetical protein BSU11330 fabHA 2.39 1.93 2.53 1.74 beta ketoacyl acyl carrier protein synthase III BSU11340 fabF 1.27 2.34 2.23 1.25 beta ketoacyl acyl carrier protein synthase II BSU11380 appA 3.06 1.75 2.2 1.19 BG1108:oligopeptide ABC transporter (oligopeptide binding protein) BSU11390 appB 1.6 5 1.01 2.33 1.39 oligopeptide ABC transporter (permease)
94 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU11400 appC 1.66 1.02 2.39 1 .4 oligopeptide ABC transporter (permease) BSU11430 oppA 1.35 2.71 1.56 2.52 oligopeptide ABC transporter (binding lipoprotein) BSU11440 oppB 1.43 4.61 1.62 4.14 oligopeptide ABC transporter (permease) BSU11450 oppC 1.25 6.86 2.31 3.01 oligopepti de ABC transporter (permease) BSU11460 oppD 1.33 6.85 2.45 2.86 oligopeptide ABC transporter (ATP binding protein) BSU11470 oppF 1.83 6.29 2.31 1.75 oligopeptide ABC transporter (ATP binding protein) BSU11490 yjbC 28.1 12.4 1.16 2.64 putative thio l oxidation management factor; putative acetyltransferase BSU11500 spxA 3.23 5.39 1.51 1.19 redox sensitive regulator enzyme BSU11510 yjbE 1.98 6.04 3.3 1.02 putative transporter component BSU11560 yjbI 2.22 1.82 2.07 2.25 putative thiol management oxi doreductase component BSU11570 yjbJ 1.16 1.48 1.56 2.27 putative murein lytic transglycosylase BSU11720 fabI 1.11 1.44 2.48 1.65 enoyl acyl carrier protein reductase BSU11820 yjcD 4.4 5.12 1.3 1.11 putative ATP dependent DNA helicase BSU11830 yjcE 19.45 19.24 1.08 1.2 BG1315:unknown BSU11860 yjcH 3.89 1.17 1.14 3.65 putative hydrolase BSU11990 yjdB 92.88 136.8 1.04 1.37 putative exported protein BSU12000 manR 4.64 4.45 1.02 1.02 transcriptional antiterminator BSU12010 manP 3.09 7.51 1.19 4.07 phosphotransferase system (PTS) mannose specific enzyme IIBCA component BSU12020 manA 3.14 7.72 1.3 4.07 mannose 6 phosphate isomerase ; cupin family BSU12040 yjdG 1 2.19 1.03 2.15 putative acetyltransferase BSU12080 ctaO 8.71 6.2 1.53 1.19 protoheme IX farnesyltransferase (heme O synthase) BSU12100 yjeA 2.87 2.8 4.88 1.26 secreted deoxyriboendonuclease BSU12110 yjfA 1.36 1.13 2.61 2.36 conserved hypothetical protein BSU12120 yjfB 1.18 1.84 4.72 8.67 conserved hypothetical prote in BSU12160 yjgC 7.65 3.78 1.13 1.77 putative oxidoreductase BSU12270 yjlB 12.31 9.32 1.17 1.11 conserved hypothetical protein ; cupin family
95 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN62 4 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU12280 yjlC 4.88 6.21 1.27 1.02 conserved hypothetical protein BSU12400 yjnA 1.12 2.48 2.35 1.2 putative integral inner membrane protein BSU12410 yjoA 1.17 1.04 2.74 2.28 conserved hypotheti cal protein BSU12420 yjoB 4.68 4.04 1.41 1.75 ATPase possibly involved in protein degradation BSU12430 rapA 28.32 21.6 2.11 1.6 response regulator aspartate phosphatase BSU12440 phrA 31.67 21.18 2.16 1.23 secreted inhibitor of the activity of ph osphatase RapA BSU12470 yjqA 3.54 1.86 1.16 2.15 conserved hypothetical protein; PBSX phage BSU13020 ykgA 38.11 28.54 1.04 1.38 putative aminohydrolase BSU13090 ykkC 2.03 1.57 1.52 2.22 efflux transporter BSU13100 ykkD 2 1.39 1.53 2.34 effl ux transporter BSU13120 proB 1.61 2.47 1.73 2.64 glutamate 5 kinase BSU13160 ykzA 24.31 26.34 1.46 1.28 organic hydroperoxide resistance reductase B BSU13170 guaD 8.66 22.39 2.25 1.13 guanine deaminase BSU13300 ykoK 2.54 4.4 2.94 1.54 magnesium transporter BSU13340 ykoM 1.34 1.21 1.39 2.39 putative transcriptional regulator (MarR family) BSU13430 ykoX 5.18 4.27 1.26 1.46 putative integral inner membrane protein BSU13490 ykrL 5.22 2.77 1 2.25 membrane protease BSU13520 ykrP 1.49 4.2 2 2.66 1.31 putative integral inner membrane protein; putative acyltransferase BSU13640 spo0E 2.6 10.67 1.36 2.66 negative regulatory phosphatase acting on Spo0A P (sporulation) BSU13680 motB 2 1.55 2.65 2.1 motility protein B; MotB component of the stator flagellum complex BSU13690 motA 1.67 1.23 3.13 2.44 motility protein A; MotA component of the stator flagellum complex BSU13850 ykvW 6.99 13.25 1.58 1.21 Zn transporter BSU13890 ptsG 15.7 14.85 1.42 1.34 phosphotransferase system (PTS) glu cose specific enzyme IICBA component BSU13920 splA 1.16 3.24 5.07 1.23 TRAP like transcriptional regulator BSU13930 splB 1.12 2.38 3.38 1.14 spore photoproduct (thymine dimer) lyase BSU13950 mcpC 1.73 1.15 3.99 2.02 methyl accepting chemotaxis prot ein
96 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU13960 ykwC 4.54 1.29 4.29 1.09 putative beta hydroxyacid dehydrogenase BSU14010 ch eV 1.42 1.24 2.96 2.65 coupling protein and response regulator for CheA activity in response to attractants (chemotaxis) BSU14150 ykuN 20.29 10.82 1.21 2.13 short chain flavodoxin BSU14160 ykuO 20.35 12.28 1.03 2.24 conserved hypothetical protei n BSU14170 ykuP 22 12.46 1.07 2.06 short chain flavodoxin BSU14180 ykuQ 15.07 10.98 1.4 1.23 tetrahydrodipicolinate N acetyltransferase BSU14190 ykuR 6.15 3.6 1.5 1.02 N acetyl diaminopimelate deacetylase BSU14200 ykuS 5.28 3.4 1.35 1.05 c onserved hypothetical protein BSU14220 ykuU 2.24 5.53 1.08 2.32 putative 2 cys peroxiredoxin BSU14230 ykuV 2.11 5.02 1.01 2.35 thiol disulfide isomerase BSU14350 yknX 2.22 1.97 1.66 2.77 putative efflux permease BSU14360 yknY 2.01 1.99 1.62 2.4 9 putative ABC transporter (ATP binding protein) BSU14440 ykpB 1.71 3.07 3.21 1.6 ketopantoate reductase BSU14480 abh 5.89 2.51 1.04 2.3 transcriptional regulator BSU14490 kinC 1.74 1.02 1.16 2.08 two component sensor histidine kinase BSU14520 ad eC 2.05 1.97 2.83 1.47 adenine deaminase BSU14550 ykrA 6.61 2.58 1.35 2.02 putative hydrolase BSU14570 ykyA 1.99 1.02 1.13 2.62 putative chromosome partitioning protein BSU14580 pdhA 2.19 2.22 2.93 4.87 pyruvate dehydrogenase (E1 alpha subunit) BSU14590 pdhB 2.43 2.3 3.15 3.73 pyruvate dehydrogenase (E1 beta subunit) BSU14600 pdhC 1.94 1.6 3.67 3.68 pyruvate dehydrogenase (dihydrolipoamide acetyltransferase E2 subunit) BSU14610 pdhD 2.57 1.08 6.38 3.09 dihydrolipoamide dehydrogenase E3 su bunit of both pyruvate dehydrogenase and 2 oxoglutarate dehydrogenase complexes BSU14630 speA 1.97 2.57 3.98 1.24 arginine decarboxylase BSU14640 yktA 1.52 2.55 2.79 1.24 conserved hypothetical protein BSU14660 ykzI 20.32 32.45 1.42 1.08 conserved hypothetical protein BSU14670 yktC 3.67 8.33 1.27 1.72 inositol monophosphatase BSU14680 ykzC 2.42 5.79 1.04 2.2 conserved hypothetical protein BSU14820 ylaL 6.65 2.93 1.01 2.59 conserved hypothetical protein BSU14870 ctaA 2.93 4.03 1.58 2.33 he me A synthase
97 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU14890 ctaC 7.07 15.52 1.16 1.84 cytochrome caa3 oxidase (subunit II) BSU1 4900 ctaD 4.48 15.67 1.01 3.58 cytochrome caa3 oxidase (subunit I) BSU14910 ctaE 4.81 16.19 1.04 3.5 cytochrome caa3 oxidase (subunit III) BSU14920 ctaF 5.07 18.74 1.06 3.48 cytochrome caa3 oxidase (subunit IV) BSU14930 ctaG 5.4 12.55 1.4 3.48 cy tochrome aa(3) assembly factor BSU14940 ylbA 2.1 5.48 1.11 2.82 conserved hypothetical protein BSU15100 ylbP 1.19 1.18 2.61 2.49 putative acetyltransferase BSU15140 mraW 3.16 1.19 1.04 2.95 S adenosyl dependent methyltransferase active on membrane located substrates BSU15160 pbpB 1.37 2.29 1.34 2.24 penicillin binding protein 2B BSU15190 mraY 1.93 4.6 2.13 1.27 phospho N acetylmuramoyl pentapeptide transferase BSU15250 ylxW 1.6 1.38 1.08 2.08 conserved hypothetical protein BSU15300 bpr 2 .09 5.27 3.24 1.2 bacillopeptidase F BSU15480 pyrP 1.27 4.36 3.82 1.53 uracil permease BSU15490 pyrB 1.13 3.16 3.3 1.17 aspartate carbamoyltransferase BSU15500 pyrC 1.09 2.74 2.93 1.11 dihydroorotase BSU15570 cysH 1.7 12.58 5.88 1.19 (phospho)adenos ine phosphosulfate reductase BSU15580 cysP 1.4 17.09 8.08 1.26 sulfate permease BSU15590 sat 1.53 23.66 12.79 1.05 sulfate adenylyltransferase BSU15600 cysC 1.51 22.23 9.19 1.33 adenylylsulfate kinase BSU15610 ylnD 1.36 24.18 10.44 1.35 uroporphyri nogen III and precorrin 1 C methyltransferase BSU15620 ylnE 1.1 5.81 4.29 1.53 sirohydrochlorin ferrochelatase BSU15630 ylnF 1.21 4.81 3.43 1.6 Precorrin 2 dehydrogenase BSU15720 def 1.58 1.63 1.09 2.46 polypeptide deformylase BSU15730 fmt 1.6 1 .55 1.02 2.45 methionyl tRNA formyltransferase BSU15740 yloM 1.44 1.36 1.17 2.51 RNA binding Sun protein; 16S rRNA m5C967 methyltransferase, S adenosyl L methionine dependent BSU15760 prpC 1.27 1.4 1.16 2.08 phosphorylated protein phosphatase BSU15770 prkC 1.16 1.91 1.16 2.49 protein kinase BSU15800 yloS 1.99 1.14 2.18 1.06 thiamine pyrophosphokinase BSU15870 recG 7.74 4.56 1 1.75 branch migrating ATP dependent DNA helicase involved in DNA recombination and repair
98 Table 3 2. Continued Accession B SU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU15880 ylpC 5.7 2.87 1.31 1.78 transcription factor BSU15940 smc 1.33 1.33 1.35 2.28 chromosome condensation and segrega tion SMC ATPase BSU15960 ylqB 1.89 8.56 3.61 15.29 conserved hypothetical protein BSU15980 ffh 2.95 1.27 1.16 2.17 signal recognition particle like (SRP) GTPase BSU16060 rnhB 3.6 1.17 2.44 1.26 ribonuclease HII BSU16070 ylqG 3.16 1.01 2.85 1.08 p utative glycosyltransferase BSU16080 ylqH 1.77 1.79 2.36 1.33 putative flagellar biosynthesis protein BSU16110 smf 3.78 1.06 1.54 2.39 DNA processing Smf single strand binding protein BSU16180 flgB 5.02 1.44 1.77 2.18 flagellar component of cell pr oximal portion of basal body rod BSU16190 flgC 5.8 1.81 1.55 2.4 flagellar component of cell proximal portion of basal body rod BSU16200 fliE 5.15 1.62 1.74 2.11 flagellar basal body protein BSU16210 fliF 5.95 1.59 1.45 2.88 flagellar basal body M ri ng protein BSU16220 fliG 8.22 1.76 1.25 4.06 flagellar motor switching and energizing component BSU16230 fliH 7.32 1.61 1.3 3.83 flagellar export apparatus component BSU16240 fliI 5.91 1.66 1.19 3.47 flagellar specific ATPase BSU16250 fliJ 4.78 1.59 1.11 3.11 flagellar synthesis chaperone BSU16260 ylxF 3.47 1.33 1.22 2.4 putative kinesin like protein BSU16270 fliK 2.7 1.28 1.03 2.13 flagellar hook length control protein BSU16400 flhF 2.06 1.21 1.2 2.09 GTPase involved in the export of flagell a BSU16410 ylxH 2.3 1.32 1.16 2.57 essential component of the flagellar assembly machinery BSU16420 cheB 2.19 1.71 1.25 2.88 methyl accepting chemotaxis proteins (MCP) glutamate methylesterase BSU16510 pyrH 1.22 2.22 1.33 2.17 uridylate kinase BS U16590 ylxS 2.18 1.88 1.79 2.3 putative RNA binding protein BSU16600 nusA 2.31 1.77 1.63 3.02 transcription translation coupling factor involved in Rho dependent transcription termination BSU16610 ylxR 2.49 1.77 1.71 2.68 putative RNA binding prote in; putative new fold BSU16620 ylxQ 2.18 2 1.89 2.74 ribosomal protein L7Ae BSU16700 ylxY 1.18 2.2 3.27 1.76 putative sugar deacetylase
99 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5 kPa WN1106 to WN624 at 101kPa Gene Description BSU16710 mlpA 1.51 1.74 3.2 1.3 specific processing protease BSU16790 tepA 1.12 5.25 6.11 1.21 protein export enhancing factor BSU16820 ymfD 1.3 3.54 1.92 2.47 BG1342:unknown; similar to multidrug resist ance protein BSU16830 ymfE 1.74 2.09 1.81 2.38 BG1342:unknown; similar to multidrug resistance protein BSU16870 fabG 1.18 1.53 2.26 1.17 putative oxidoreductase BSU16910 ymfM 1.83 1.07 1.14 2.54 conserved hypothetical protein BSU16920 pgsA 1.77 1. 29 1.24 3.11 CDP diacylglycerol glycerol 3 phosphate 3 phosphatidyltransferase BSU16930 cinA 1.89 1.06 1.49 3.24 competence damage inducible regulator BSU16950 pbpX 1.37 2.55 2.29 1.63 penicillin binding endopeptidase X BSU16980 spoVS 1.23 1.34 1.5 8 3 regulator required for dehydratation of the spore core and assembly of the coat (stage V sporulation) BSU17100 pksC 6.25 6.88 1.04 1.01 malonyl CoA acyltransferase involved in polyketide synthesis BSU17110 pksD 7.65 13.73 1.44 1.14 enzyme involved in polyketide synthesis BSU17120 pksE 5.44 14.29 2.17 1.11 enzyme involved in polyketide synthesis BSU17130 acpK 4.63 15.33 2.39 1.18 acyl carrier protein BSU17140 pksF 3.41 13.42 2.59 1.41 decarboxylase converting malonyl S AcpK to acetyl S AcpK fo r polyketide synthesis BSU17150 pksG 1.9 5.9 2.3 1.59 acetyl S AcpK beta ketothioester polyketide intermediate transferase BSU17160 pksH 1.84 5.75 2.14 1.66 dehydratase for polyketide biosynthesis BSU17180 pksJ 5.57 6.38 1.06 1.11 polyketide synthase of type I BSU17240 ymzB 4.97 5.71 1.19 1.12 conserved hypothetical protein BSU17280 ymaD 1.92 1.2 1.22 2.23 putative peroxiredoxin related protein BSU17410 cwlC 1.04 2.34 2.13 1.28 N acetylmuramoyl L alanine amidase BSU17420 spoVK 1.13 2.32 2.1 8 1.04 mother cell sporulation ATPase BSU17430 ynbA 2.41 1.03 1.19 2.71 putative GTP binding protein protease modulator BSU17440 ynbB 1.64 1.37 1.07 2.15 putative C S lyase
100 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN11 06 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU17450 glnR 2.37 1.97 1.73 2.71 transcriptional regulator (nitrogen metabolism) BSU17630 yncC 9.28 10.91 2.19 1.53 putative sugar transporter BSU17690 yncM 24.0 9 30.51 1.15 1.79 conserved hypothetical protein BSU17710 tatAC 17.94 19.91 1.02 1.18 component of the twin arginine pre protein translocation pathway BSU17890 tkt 1.15 1.79 1.04 2.2 transketolase BSU17900 yneE 1.19 3.33 2.77 1.05 conserved hypothet ical protein BSU18000 citB 1.41 11.77 8.13 1.64 aconitate hydratase (aconitase) BSU18010 yneN 1.92 16.92 8.67 1.75 putative membrane bound proteins with a thioredoxin like domain BSU18110 ynfC 11.12 9.17 1.13 1.05 conserved hypothetical protein BSU18120 alsT 4.24 5.88 4.16 2.57 amino acid carrier protein BSU18150 ynfF 2.94 5.49 1.43 1.27 endo xylanase BSU18160 xynD 3.48 5.24 1.48 1.02 endo 1,4 beta xylanase (xylanase D) BSU18190 yngC 1.24 1.91 1.16 2.58 putative integral inner membra ne protein BSU18310 ppsD 4.73 2.41 1.77 1.14 plipastatin synthetase BSU18320 ppsC 5.15 2.74 1.41 1.25 plipastatin synthetase BSU18350 dacC 5.85 5.9 1.11 1.14 D alanyl D alanine carboxypeptidase BSU18360 yoxA 5.45 5.28 1.14 1.25 putative epimerase BS U18370 yoeA 1.1 3.81 1.01 3.39 putative efflux transporter BSU18380 yoeB 7.97 3.09 2.44 1.08 inhibitor of cell separation enzymes BSU18440 gltD 16 2.3 3.98 1.53 glutamate synthase (small subunit) BSU18450 gltA 57.49 7.51 4.8 1.57 glutamate synthase (large subunit) BSU18500 fabG 3.43 1.92 1.59 3.01 putative oxido reductase BSU18510 yoxC 36.27 42.02 1.04 1.05 conserved hypothetical protein BSU18520 yoxB 27.18 24.99 1 1.08 conserved hypothetical protein BSU18530 yoaA 11.67 15.33 1.07 1.09 pu tative N acetyltransferase BSU18610 yoaH 1.61 1.14 3.51 2.04 putative methyl accepting chemotaxis protein BSU18620 yoaI 1.64 2.75 2.14 1.01 putative 4 hydroxyphenylacetate 3 hydroxylase BSU18830 pps 2.03 1.11 2.4 1.13 putative phosphoenolpyruvate s ynthase BSU18840 xynA 1.91 2.01 1.33 2.97 endo 1,4 beta xylanase BSU18860 yozH 1.6 1.05 1.23 2.08 hypothetical protein BSU18910 rapK 4 6.09 1.1 1.54 response regulator aspartate phosphatase
101 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU18920 phrK 6.8 11.9 1.38 1.98 secreted regulator of the activity of phosphatase RapK BSU19030 yobO 7.09 8.04 1.28 1.47 putative phage related p re neck appendage protein BSU19150 yocB 9.65 17.89 1.54 1.16 conserved hypothetical protein BSU19170 yocD 1.1 4.23 2.67 1.41 putative carboxypeptidase BSU19180 des 4.19 17.18 5.98 1.25 fatty acid desaturase BSU19190 yocF 1.82 15.29 8.15 1.04 two comp onent sensor histidine kinase [DesR] BSU19200 yocG 1.42 10.17 5.13 1.22 two component response regulator [DesK] BSU19210 yocH 1.76 4.32 4.8 1.9 putative exported cell wall binding protein BSU19250 yocL 1.08 2.01 2.8 1.28 hypothetical protein BSU19260 yocM 1.02 2.05 2.4 1.16 putative spore coat protein BSU19310 dhaS 11.16 9.78 2.02 1.99 putative aldehyde dehydrogenase BSU19340 yocR 2.2 1.19 1 2.88 putative sodium dependent transporter BSU19350 yocS 1.33 5.13 5.06 1.42 putative sodium depende nt transporter BSU19410 yojL 6.53 5.98 1.42 1.25 peptidoglycan hydrolase (cell wall binding d,l endopeptidase) BSU19420 yojK 2.96 1.18 4.27 1.76 putative glycosyltransferase BSU19510 yojB 11.1 9.09 1.32 1.7 conserved hypothetical protein BSU195 20 yojA 15.37 10.5 1.39 1.97 putative H+/anion permease BSU19530 yodA 3.66 7 2.33 1.14 putative tautomerase BSU19550 yodC 3.95 3.29 1.75 2.42 putative oxidoreductase BSU19580 yodF 3.46 1.64 1.07 2.51 putative Na+/metabolite permease BSU1 9640 yodL 1.17 1.07 1.49 2.24 conserved hypothetical protein BSU19650 yodM 2.65 1.81 1.84 2.5 putative phospholipid phosphatase BSU19750 cgeE 1.38 1.19 2.36 2.14 protein involved in maturation of the outermost layer of the spore BSU20420 yorD 1 .29 2.88 1.61 3.3 hypothetical protein; phage SPbeta BSU20560 yoqO 1.17 1.57 1.15 2.21 putative membrane protein; phage SPbeta BSU20580 yoqM 48.21 134.28 3.36 1.11 putative membrane bound protein; phage SPbeta BSU20770 yopT 1.16 1.25 2.03 2.17 hyp othetical protein; phage SPbeta BSU20780 yopS 1.44 1.96 2.16 1.68 putative transcriptional regulator; phage SPbeta
102 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 1 01kPa Gene Description BSU21010 yonS 1.19 1.22 2.19 2.28 putative glycosyl hydrolase lipoprotein; phage SPbeta BSU21480 sunA 7.87 28.34 1.16 3.86 sublancin 168 lantibiotic antimicrobial precursor peptide in SPBeta prophage BSU21520 yolC 2.67 5.11 1 .22 1.56 SP beta phage protein BSU21530 yolB 1.04 2.7 1.54 3.89 conserved hypothetical protein; phage SPbeta BSU21540 yolA 1.01 2.1 1.9 4.17 putative exported protein; SPbeta phage BSU21780 yplP 1.92 3.44 2.29 1.37 transcriptional enhancer BSU218 50 ypiP 1.2 2.49 2.34 1.27 putative methyltransferase BSU21930 cspD 2.52 2.82 1.53 4.54 cold shock protein, molecular chaperone, RNA helicase co factor BSU21940 degR 2.01 4.03 1.21 2.3 activation of degradative enzymes (aprE, nprE, sacB) production or activity BSU22030 ypbR 1.64 3.73 1.08 2.09 putative GTP binding protein BSU22260 yppF 1.02 3.43 2.75 1.06 putative site specific integrase BSU22380 ypmB 1.26 2.48 1.19 2.62 conserved hypothetical protein BSU22390 ypmA 1.13 1.86 1.27 2.56 co nserved hypothetical protein BSU22510 ypjC 1.94 1.43 1.41 2.26 putative integral inner membrane protein BSU22530 ypjA 1.94 1.6 1.98 2.31 putative integral inner membrane protein BSU22540 qcrC 3.09 8.76 1.01 3.31 menaquinol:cytochrome c oxidore ductase (cytochrome cc subunit) BSU22550 qcrB 2.63 6.81 1.33 3.37 menaquinol:cytochrome c oxidoreductase (cytochrome b subunit) BSU22560 qcrA 3.21 6.68 1.29 2.75 menaquinol:cytochrome c oxidoreductase (iron sulfur subunit) BSU22600 aroE 10.19 6.2 2.11 1.07 3 phosphoshikimate 1 carboxyvinyltransferase (5 enolpyruvoylshikimate 3 phosphate synthase) BSU22610 tyrA 7.11 6.71 1.97 2.39 prephenate dehydrogenase BSU22620 hisC 4.73 4.37 2.58 2.27 histidinol phosphate aminotransferase; tyrosine/phenyla lanine aminotransferase BSU22630 trpA 2.94 4.31 2.23 3.28 tryptophan synthase (alpha subunit)
103 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Descript ion BSU22720 cheR 2.11 1.26 1.62 2.62 methyl accepting chemotaxis proteins (MCPs) methyltransferase BSU22730 ndk 2.28 1.84 1.58 2.2 nucleoside diphosphate kinase BSU22760 hepS 1.85 2.31 1.46 2.84 heptaprenyl diphosphate synthase component I BSU22 800 spoIVA 1.26 2.1 2.18 1.13 morphogenetic stage IV sporulation protein BSU22900 ypfB 1.1 2.57 1.92 2.12 conserved hypothetical protein BSU23080 aroD 1.2 1.63 2.4 1.19 3 dehydroquinate dehydratase BSU23120 resD 5.42 1.97 2.37 1.23 two compone nt response regulator BSU23130 resC 4.72 1.62 2.48 1.06 factor required for cytochrome c synthesis BSU23140 resB 5.96 1.05 2.83 2.15 factor required for cytochrome c synthesis BSU23150 resA 4.51 1.29 3.43 1.64 extracytoplasmic thioredoxin involved i n cytochrome c maturation (lipoprotein) BSU23250 ribH 2.76 2.44 1.98 2.34 6,7 dimethyl 8 ribityllumazine synthase, beta subunit BSU23270 ribE 6.35 2.82 1.77 3.85 riboflavin synthase (alpha subunit) BSU23280 ribD 6.17 2.57 1.68 3.38 fused diaminohydroxyp hosphoribosylamin opyrimidine deaminase; 5 amino 6 (5 phosphoribosylamino) uracil reductase BSU23360 ppiB 3.62 1.64 1.16 2.16 peptidyl prolyl isomerase BSU23370 ypuA 2.14 1.26 1.55 2.57 putative exported protein BSU23550 mleA 21.44 19.16 1.53 1.37 NAD dependent malic enzyme (conversion of malate into pyruvate) BSU23560 mleN 30.54 25.29 1.72 1.38 malate H+/Na+ lactate antiporter BSU23570 aspA 18.21 20.6 1.68 1.4 L aspartase (aspartate ammonia lyase) BSU23580 ansA 11.31 19.31 1.84 2. 47 exported L asparaginase BSU23590 ansR 5.52 4.62 1.07 1.25 transcriptional regulator of ansAB (Xre family) BSU23670 yqkA 1.47 1.6 1.19 2.71 conserved hypothetical protein BSU23680 yqjZ 1.4 1.47 1.22 2.41 putative degradation enzyme (oxygenase) B SU23690 yqjY 1.29 1.5 1.04 2.14 putative acetyltransferase
104 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU23850 zwf 2.62 1.11 2.31 1. 35 glucose 6 phosphate 1 dehydrogenase BSU23860 yqjI 2.04 1.15 2.24 1.23 NADP+ dependent 6 P gluconate dehydrogenase BSU23900 yqjF 4.14 5.15 1.55 1.2 conserved hypothetical protein BSU23980 yqiX 1.58 1.35 2.12 1.05 High affinity arginine ABC transp orter binding lipoprotein BSU23990 yqiW 1.98 1.12 1.12 2.28 conserved hypothetical protein BSU24000 bmrU 37.25 37.25 1.1 1 putative diacylglycerol kinase BSU24020 bmrR 2.56 5.63 1.86 1.11 transcriptional regulator (MerR family) BSU24030 bkdB 1.52 1.84 1.41 2.13 branched chain alpha keto acid dehydrogenase E2 subunit (lipoamide acyltransferase) BSU24050 bkdAA 1.12 2.68 1.51 2.36 branched chain alpha keto acid dehydrogenase E1 subunit BSU24060 lpdV 1.15 3 1.23 3.27 branched chain alpha keto acid dehydrogenase E3 subunit (dihydrolipoamide dehydrogenase) BSU24070 buk 1.06 4.94 1.33 5.12 branched chain fatty acid kinase BSU24080 bcd 2.26 2.04 1.29 4.11 branched chain amino acid dehydrogenase BSU24090 ptb 1.55 2.91 1.37 3.62 phosphate bu tyryl coenzyme A transferase BSU24100 bkdR 2.61 5.39 1.2 2.68 transcriptional regulator BSU24110 yqzF 4.99 4.71 1.27 1.09 conserved hypothetical protein BSU24300 xseA 2.16 1.29 1.21 2.47 exodeoxyribonuclease VII (large subunit) BSU24350 accB 1.62 3.35 2.22 2.54 acetyl CoA carboxylase subunit (biotin carboxyl carrier subunit) BSU24550 gcvPB 11.1 7.1 1.12 1.32 glycine decarboxylase (subunit 2) (glycine cleavage system protein P) BSU24560 gcvPA 11.44 6.63 1.11 1.59 glycine decarboxylase (subuni t 1) (glycine cleavage system protein P) BSU24570 gcvT 9.45 7.11 1.11 1.43 aminomethyltransferase (glycine cleavage system protein T) BSU24740 yqxL 11.52 17.46 1.2 1.03 putative CorA type Mg(2+) transporter
105 Table 3 2. Continued Accession BSU number Gen e Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU24750 yqhB 12.43 14.5 1.01 1.07 putative membrane associated protein BSU24760 yqhA 22.09 36.43 1.85 1.24 component of the piezosome (s tressosome) BSU24770 yqgZ 94.89 68.8 1.23 2.23 transcriptional regulator of stress BSU24780 yqgY 1.61 3.34 1.52 3.07 conserved hypothetical protein BSU24790 yqgX 2.97 1.26 1.29 2.25 putative metal binding hydrolase BSU24800 yqgW 2.28 4.17 1.78 3 .45 conserved hypothetical protein BSU24850 glcK 2.86 1.12 1 2.75 glucose kinase BSU24860 yqgQ 2.28 1.03 1.11 2.83 conserved hypothetical protein BSU24930 yqzD 1.05 3.28 2.31 1.69 conserved hypothetical protein BSU25020 sodA 2.21 1.17 1.01 2.88 s uperoxide dismutase BSU25050 yqgA 1.82 1.21 1.32 2.15 hypothetical protein BSU25080 yqfX 4.74 1.79 3.15 1.09 conserved hypothetical protein BSU25100 zur 1.9 1.16 1.77 3.02 transcriptional regulator (Fur family) BSU25110 yqfU 1.41 1.3 2.05 2.49 putative integral inner membrane protein BSU25190 cccA 3.91 6.22 1.02 1.6 cytochrome c550 BSU25220 antE 6.28 1.69 1.34 4.99 hypothetical protein BSU25250 yqzB 2.74 1.28 1.06 3.05 negative regulator of gluconeogenesis BSU25270 glyQ 1.22 3.22 1.19 2.42 glycyl tRNA synthetase (alpha subunit) BSU25370 yqfB 3.16 1.38 1 4.26 conserved hypothetical protein BSU25380 yqfA 3.44 1.29 1.16 4.88 conserved hypothetical protein BSU25390 yqeZ 1.76 1.5 1.05 2.62 putative membrane bound hydrolase BS U25470 dnaK 1.37 4.42 1.43 2.46 molecular chaperone BSU25480 grpE 1.15 4.6 1.23 3.75 nucleotide exchange factor for DnaK activity BSU25650 yqeI 1.09 2.09 1.08 2.24 putative RNA binding protein BSU25660 aroE 1.14 2.08 1.03 2.2 shikimate 5 dehyd rogenase BSU25670 yqeH 1.22 1.52 1.21 2.15 GTPase involved in ribosome 30S assembly BSU25680 yqeG 1.13 1.9 1.07 2.27 putative hydrolase BSU25740 yqeB 1.19 1.73 1.12 2.12 conserved hypothetical protein BSU25840 phrE 3.46 6.98 1.22 2.43 regulator o f the activity of phosphatase RapE BSU25880 yqxJ 3.17 12.45 2.83 1.13 hypothetical protein; skin element BSU25890 yqxI 4.37 18.08 2.73 1.24 hypothetical protein; skin element BSU26240 yqaO 5.33 5.24 1.01 1.16 conserved hypothetical protein; skin elem ent
106 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU26620 yrdR 6.4 2.04 4.9 1.08 putative efflux transporter BSU26640 trkA 1.02 1.84 1.57 2.09 putative oxidoreductase BSU26650 czcD 1.18 1.94 1.77 2.65 potassium/proton divalent cation antiporter BSU26820 yrpD 4.46 11.51 1.85 1.27 putative lipoprotein BSU26890 csn 82.13 128.8 2.24 1.19 chitosanase BSU26900 yraL 4.83 3.85 1.03 1.29 conserved hypothetical protein BSU26920 yraJ 6.9 7.92 1.86 1.93 conserved hypothetical protein BSU26930 yraI 6.91 8.41 1.62 1.81 conserved hypothetical protein BSU27020 yraA 1 2.67 1.2 3.19 general stress protein BSU27100 yrhP 1.1 1.73 2.01 3.75 putative efflux transporter BSU27190 yrzI 1.63 1.89 1.23 4.27 conserved hypothetical protein BSU27200 yrhG 12.18 10.07 1.26 1.43 putative formate/nitrite transporter BSU27220 yrhE 1.6 2.78 1.02 4.14 putative oxido reductase BSU27230 yrhD 1 .51 2.36 1.1 3.67 conserved hypothetical protein BSU27240 yrhC 1.17 2.42 2.58 1.02 conserved hypothetical protein BSU27250 yrhB 1.19 4.07 4.25 1.3 cystathionine gamma lyase and homocysteine gamma lyase for reverse transsulfuration pathway BSU27260 yrhA 1.08 3.77 4.05 1.04 cystathionine beta synthase for the reverse transsulfuration pathway BSU27280 yrrT 1.36 4.21 4.38 1.43 putative AdoMet dependent methyltransferase BSU27410 alaS 1.75 6.69 1.92 2.31 alanyl tRNA synthetase BSU27470 yrrD 3.32 1.7 2.35 1.12 conserved hypothetical protein BSU27490 yrrB 1.06 3 1.49 2.22 putative tetratricopeptide repeat family protein BSU27550 aspS 3.86 1.17 3.68 1.08 aspartyl tRNA synthetase BSU27560 hisS 1.9 1.13 3.39 1.96 histidyl tRNA synthetase B SU27590 yrvI 1.83 1.06 2.54 1.37 D Tyr tRNATyr deacylase BSU27600 relA 1.79 1.1 2.22 1.26 GTP pyrophosphokinase (RelA/SpoT) BSU27730 ruvB 1.58 2.62 1.23 2.08 Holliday junction DNA helicase, ATP dependent component BSU27780 yrzF 4.11 6.26 1.04 1.8 BG1381:unknown BSU27790 yrzG 4.31 7.41 1.06 1.64 BG1381:unknown BSU27930 spo0B 1.98 1.08 1.2 2.49 sporulation initiation phosphotransferase BSU28040 radC 1.12 1.92 1.01 2.09 putative DNA repair protein BSU28080 folC 4.98 3.41 1.15 1.18 folyl po lyglutamate synthase BSU28090 valS 5.13 4.85 1.02 1.09 valyl tRNA synthetase BSU28110 spoVID 1.18 3.75 3.43 1.03 morphogenetic spore protein (stage VI sporulation)
107 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kP a/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU28240 ysoA 1.98 1.38 1.35 3.4 putative hydrolase BSU28300 ilvH 1.28 2.04 2.82 1.47 acetolactate synthase (acetohydroxy acid synthase) (small subunit) BSU28310 ilvB 1.38 3.06 4.24 1.8 acetolactate synthase (acetohydroxy acid synthase) (large subunit) BSU28340 ysnF 6.25 8.65 1.35 1.21 putative stress response protein BSU28400 ysmB 1.17 1.48 1.21 2.14 putative transcriptional regulator (MarR family) BSU28410 gerE 1.54 1.48 2.11 2.22 transcriptional regulator BSU28500 trxA 1.03 2.11 1.02 2.14 thioredoxin BSU28550 ysiA 5.42 1.78 1.5 1.96 transcriptional regulator of fatty acids degradation (TetR/AcrR family) BSU28560 lcfA 6.41 2.25 1.52 1.79 long chain acyl CoA ligase (degradative) BSU28580 mutSB 2.04 1.49 1.05 2.76 putative DNA mismatch repair enzyme BSU28640 pheS 1.25 2.77 1.09 2.08 phenylalanyl tRNA synthetase (alpha subunit) BSU28680 ysfC 2.44 16.41 1 11.36 glycolate oxidase subunit BSU28690 ysfD 3.31 12.55 1.12 6.79 glycolate oxidase iron sulfur subunit BSU28710 cstA 4.69 1.46 1.27 2.85 carbon starvation induced membrane protein BSU28890 yscB 3.69 1.38 4.08 1.9 putative lipoprotein BSU28900 ysbB 21.29 15.68 4.75 7.77 antiholin factor BSU28 910 ysbA 27.84 24.34 7.24 10.19 antiholin factor BSU28950 thrS 4.85 6.2 1.19 1.79 threonyl tRNA synthetase BSU28990 dnaB 1.25 1.06 1.6 2.16 helicase loading protein; replication initiation membrane attachment protein BSU29110 phoP 2.38 1.5 1.44 2.48 two component response regulator BSU29140 citZ 1.5 4.43 4.42 1.62 citrate synthase II BSU29160 ytvI 1.12 1.06 1.98 2.43 putative permease BSU29190 pfkA 1.42 1.94 1.35 2.15 6 phosphofructokinase BSU29260 ytpI 7.39 6.63 1.39 1.02 conserved hy pothetical protein BSU29400 ytlI 1.13 5.07 5.03 1.04 transcriptional regulator (LysR family) BSU29480 ytxK 5.56 2.84 1.94 1.2 putative nucleic acid methyltransferase BSU29490 tpx 5.07 2.44 1.18 2.25 putative peroxiredoxin BSU29520 yteJ 7.58 8 1.46 1.15 putative integral inner membrane protein
108 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU29530 sppA 8.06 9.89 1.58 1.08 si gnal peptide peptidase BSU29570 sspA 3.93 1.43 4.03 1.09 small acid soluble spore protein (alpha type SASP) BSU29600 braB 5.2 4.18 1.53 1.87 branched chain amino acid Na+ symporter BSU29670 tyrS 2.31 3.25 1.64 2.29 tyrosyl tRNA synthetase BSU29 680 acsA 1.54 5.37 1.82 2.15 acetyl CoA synthetase BSU29750 aroA 1.82 1.48 2.61 1.02 3 deoxy D arabino heptulosonate 7 phosphate synthase; chorismate mutase isozyme 3 BSU29760 ytxJ 11.23 8.88 1.1 1.49 conserved hypothetical protein BSU29770 ytxH 14. 48 10.71 1.24 1.91 conserved hypothetical protein BSU29780 ytxG 17.69 11.17 1.01 1.74 conserved hypothetical protein BSU29810 ytpS 1.34 2.54 1.42 2.28 BG1390:unknown; similar to DNA translocase BSU29990 ytiP 5.1 3.5 1.33 1.95 hypoxanthine/guanine p ermease BSU30000 ythQ 5.34 3.41 1.89 2.85 putative ABC transporter (permease) BSU30010 ythP 4.11 3.07 1.77 2.51 putative ABC transporter (ATP binding protein) BSU30020 ytzE 1.9 5.23 1.24 3.08 putative transcriptional regulator (DeoR family) B SU30050 ytgP 2.13 2.97 2.82 1.97 putative enzyme involved in polysaccharide biosynthesis BSU30260 msmR 23.08 6.42 1.18 5.66 transcriptional regulator (LacI family) BSU30270 msmE 1.04 1.59 4.45 3.05 multiple sugar binding lipoprotein BSU30280 amyD 1.19 1.32 3.87 4.42 carbohydrate ABC transporter (permease) BSU30290 amyC 1.32 1.26 3.58 4.2 maltose and multiple sugars ABC transporter (permease) BSU30300 melA 1.7 1.11 1.95 3.63 alpha D galactoside galactohydrolase BSU30350 yttB 2.5 8 3.09 2.46 2.05 putative efflux transporter BSU30570 ytmB 3.92 24.45 6.34 1.03 conserved hypothetical protein BSU30580 ytmA 1.61 15.8 8.38 1.02 putative hydrolase BSU30590 ytlA 1.19 4.04 3.66 1.08 BG1387:unknown BSU30600 ytlB 1.1 2.92 3.28 1.02 BG1 387:unknown BSU30610 ytlC 1.04 2.03 2.19 1.06 putative ABC transporter component, ATP binding BSU30620 ytlD 1.14 1.52 2.1 1.21 putative permease of ABC transporter BSU30640 ytkC 1.81 6 3.56 1.11 putative autolytic amidase
109 Table 3 2. Continued Access ion BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU30650 dps 11.35 18.96 1.62 1.07 DNA protecting protein, ferritin BSU30660 ytkA 6.5 8.8 1.33 1.02 putative lipoprote in BSU30670 luxS 1.32 2.59 2.64 1.37 S ribosylhomocysteine lyase BSU30700 rpmE2 19.88 22.27 1 1.2 ribosomal protein L31 BSU30750 mntC 1.67 1.79 1.88 2.56 manganese ABC transporter (permease) BSU30760 mntB 1.67 2.15 1.93 2.95 manganese ABC transpo rter (ATP binding protein) BSU30770 mntA 1.53 2.29 1.92 3.29 manganese ABC transporter (manganese binding lipoprotein) BSU30930 ytaB 20.41 15.79 1.27 1.05 putative receptor BSU30990 yuaJ 8.24 9.47 1.38 1.18 thiamin permease BSU31080 yuaB 2.52 5.53 5.79 2.73 conserved hypothetical protein BSU31160 yubA 1.79 1.06 1.23 2.15 putative integral inner membrane protein BSU31210 yulB 1.02 2.97 1.13 2.1 putative transcriptional regulator (DeoR family) BSU31220 yuxG 1.38 1.89 1.15 2.18 putativ e sugar phosphate dehydrogenase BSU31240 mcpA 2.14 1.8 7.49 1.9 methyl accepting chemotaxis protein BSU31260 mcpB 1.6 1.52 3.77 1.72 methyl accepting chemotaxis protein BSU31280 yugU 6.17 7.81 1.1 1.33 conserved hypothetical protein BSU31310 yu gP 1.08 2.8 1.18 2.45 putative metal dependent protease/peptidase BSU31340 yugM 1.42 1.58 1.13 2.56 putative transporter BSU31360 yugK 2.32 1.2 2.11 1.37 putative NADH dependent butanol dehydrogenase BSU31440 patB 5.23 5.13 1.47 1.44 C S lyase BSU31460 kapB 4.45 2.83 1.18 2.26 factor required for KinB signal transduction and activation of the phosphorelay to sporulation BSU31480 yuxJ 1.83 2.22 2.26 1.58 putative exporter BSU31510 yufK 3.54 3.18 1.93 2.12 putative integral inner mem brane protein BSU31580 maeN 9.66 5.64 1.18 2.27 Na+/malate symporter BSU31600 mrpA 5.82 4.75 1.33 1.54 Na+/H+ antiporter BSU31610 mrpB 6.8 5.11 1.52 1.72 Na+/H+ antiporter complex BSU31620 mrpC 6.26 5.85 1.53 1.68 component of Na+/H+ an tiporter BSU31630 mrpD 5.99 3.95 1.08 1.66 component of Na+/H+ antiporter
110 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU31640 mrpE 4.9 2.85 1.11 1.61 non essential component of Na+/H+ antiporter BSU31690 comP 6.5 2 1.21 3.85 two component sensor histidine kinase BSU31700 comX 7.87 1.97 1.13 5.3 competence pheromone precursor (pheromone peptide aa 46 >55, modified) BSU31710 comQ 5.69 1.54 1.04 3.7 isoprenyl transferase (pre ComX modification) BSU31730 yuzC 1.03 2.49 1.05 2.66 inner spore coat protein BSU31790 yueG 2.48 1.44 1.39 2.21 putative spore germination protein BSU31800 yueF 2.53 1.56 1.5 2.52 putative integr al inner membrane protein BSU31880 yukB 6.65 3.13 1.2 2.19 BG1237:unknown; similar to unknown proteins BSU31890 yukC 1.65 1.06 1.66 2.57 putative membrane associated enzyme involved in bacteriocin production BSU31900 yukD 1.29 1.5 2.44 2.05 putative bac teriocin BSU31910 yukE 2.49 2.48 2.5 2.82 conserved hypothetical protein BSU31930 ald 1.48 2.05 1.54 2.19 L alanine dehydrogenase BSU31940 yuxI 2.11 5.48 1.18 2.26 BG1046:unknown BSU31950 yukJ 2.15 4.63 1.16 2.33 BG1238:unknown BSU31960 dhbF 11 .56 7.66 1.44 1.12 siderophore 2,3 dihydroxybenzoate glycine threonine trimeric ester bacillibactin synthetase BSU31970 dhbB 10.66 17 1.41 1.3 isochorismatase BSU31980 dhbE 14.72 18.1 1.34 1.12 2,3 dihydroxybenzoate AMP ligase (enterobactin synth etase component E) BSU31990 dhbC 18.97 22.96 1.68 1.12 isochorismate synthase BSU32000 dhbA 16.64 15.86 1.2 1.05 2,3 dihydro 2,3 dihydroxybenzoate dehydrogenase BSU32010 yuiI 11.58 13.35 2.78 2.02 bacillibactin trilactone hydrolase BSU32040 yuiF 4.96 2.11 2.11 1.12 amino acid transporter BSU32710 yurY 1.2 1.68 1.03 2.11 sulfur mobilizing ABC protein, ATPase BSU32870 yusO 2.97 1.58 3.69 1.25 putative transcriptional regulator (MarR family) BSU32880 yusP 2.29 1.4 3.84 1.37 putativ e multidrug efflux transporter BSU32890 yusQ 17.57 8.36 1.73 1.04 putative tautomerase
111 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU 32900 yusR 21.71 8.36 1.88 1.18 putative 3 oxoacyl acyl carrier protein reductase BSU32910 yusS 24.32 9.1 1.93 1.24 putative 3 oxoacyl acyl carrier protein reductase BSU32940 yusV 4.57 5.01 1.88 1.65 iron(III) siderophore transporter (ATP binding component) BSU32990 mrgA 2.57 6.2 3.55 1.34 metalloregulation DNA binding stress protein BSU33200 yvrE 9 3.41 1.23 2.92 conserved hypothetical protein BSU33210 yvrG 1.68 1.27 1.32 2.58 two component sensor histidine kinase YvrG innvolved in cell wall processes [YvrH] BSU33230 yvrI 2.69 1.14 2.89 1.01 alternative sigma factor BSU33240 oxdC 20.16 2.87 5.75 1.08 oxalate decarboxylase BSU33290 fhuC 9.6 9.87 1.42 1.32 ferrichrome ABC transporter (ATP binding protein) BSU33300 fhuG 21.51 15.75 1.42 1.81 ferrichrome ABC transporter (permease) BSU33310 fhuB 25.37 15.43 1.34 1.71 ferrichrome ABC transporter (permease) BSU33320 fhuD 11.3 15.13 1.53 1.31 ferrichrome ABC transporter (ferrichrome binding lipoprotein) BSU33410 yvgO 24.86 42.8 1.53 1.12 conserved hypothetical protein BSU33430 yvgQ 1.17 1.17 2.46 1.74 sulfite reductase (hemoprotein beta subunit) BSU33440 yvgR 1.23 1.36 2.47 3.59 sulfite reductase (flavoprotein alpha subunit) BSU33460 yvgT 6.2 2.67 1.42 1.76 putative int egral inner membrane protein BSU33490 yvgW 1.77 1.13 2.8 2.09 copper(I) transporting ATPase BSU33510 yvgY 3.19 5.81 1.57 1.03 copper insertion chaperone and transporter component BSU33530 yvaA 10.26 5.01 1.24 1.62 putative oxidoreductase BSU33560 y vaD 9.11 1.32 2.55 3.14 putative integral inner membrane protein BSU33570 yvaE 11.8 1.24 3 3.09 putative metabolite efflux transporter BSU33580 yvaF 7.71 1.06 3.49 2.66 putative transcriptional regulator BSU33700 opuBD 5.2 6.71 1.17 1.54 chol ine ABC transporter (permease) BSU33710 opuBC 22.06 25.18 1.08 1.17 choline ABC transporter (choline binding lipoprotein)
112 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN 624 at 101kPa Gene Description BSU33720 opuBB 12.35 11.04 1.2 1.25 choline ABC transporter (permease) BSU33730 opuBA 5.21 3.56 1.52 1.19 choline ABC transporter (ATP binding protein) BSU33750 yvaW 5.92 10.41 1.51 1.15 export of killing factor BSU3 3760 yvaX 5.1 12.26 1.61 1.24 exporter of killing factor SpbC BSU33770 yvaY 12.75 25.53 1.58 1.26 killing factor SdpC BSU33870 yvbI 2.74 4.9 2.96 1.67 putative permease BSU33910 pgm 1.04 2.51 1.15 2.71 phosphoglycerate mutase BSU33920 tpiA 1.0 3 2.84 1.15 2.9 triose phosphate isomerase BSU33930 pgk 1.07 2.5 1.18 2.41 phosphoglycerate kinase BSU33940 gapA 7.18 12.94 1.4 1.82 glyceraldehyde 3 phosphate dehydrogenase BSU34080 yvfS 1.25 2.54 2.86 1.29 putative ABC transporter (permease) BSU34090 yvfR 1.22 4.1 4.58 1.13 putative ABC efflux transporter (ATP binding protein) BSU34100 rsbQ 2.03 1.57 1.75 2.11 regulator of RsbP phosphatase BSU34110 rsbP 2.15 1.88 2.01 2.2 serine phosphatase BSU34190 yvfH 6.97 3.39 1.18 2.86 putative lactate permease BSU34210 yvfG 2.34 1.58 1.71 2.35 conserved hypothetical protein BSU34260 yvfB 6.2 4.96 1.05 1.14 BG1187:unknown BSU34270 yvfA 6.47 3.95 1.13 1.15 BG1186:unknown BSU34320 yveP 5.91 4.25 1.18 1.16 putative glycosyltransfer ase involved in extracellular matrix formation BSU34330 yveO 4.65 4.12 1.04 1.16 putative glycosyltransferase BSU34360 yveL 6.76 4.14 1.2 1.34 protein tyrosine kinase BSU34370 yveK 9.08 5.8 1.15 1.32 modulator of protein tyrosine kinase EpsB B SU34390 pnbA 1.03 1.59 1.31 2.42 para nitrobenzyl esterase (intracellular esterase B) BSU34440 pbpE 4.32 2.82 1.36 2.42 penicillin binding protein 4* BSU34540 clpP 1.36 1.12 1.38 2.42 ATP dependent Clp protease proteolytic subunit BSU34640 yvd D 1.62 1.96 1.12 2.75 conserved hypothetical protein BSU34650 yvdC 1.02 4.47 1.58 2.7 putative pyrophosphohydrolase BSU34760 yvcK 2.67 1.95 1.47 2.13 gluconeogenesis factor BSU34790 trxB 1.05 4.75 4.04 1.26 thioredoxin reductase BSU34800 yvcE 1. 38 1.02 2.22 1.66 secreted cell wall DL endopeptidase BSU34950 yvpA 1.34 1.43 2.09 1.03 secreted pectate lyase BSU35050 yvnA 8.92 7.74 1.12 1.02 putative transcriptional regulator
113 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101k Pa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU35060 cypX 7.61 11.67 1.18 1.34 putative monooxygenase (cytochrome P450) BSU35070 yvmC 5.46 11.01 1.23 1.74 conserved hypothetical protein BSU35180 csbA 10. 33 11.16 1.09 1.11 putative membrane protein BSU35220 minJ 2.01 1.69 2.05 2.54 topological determinant of cell division BSU35230 yvzD 1.71 1.23 2.63 1.73 swarming motility protein fragment; C terminal part of swrAA BSU35240 yvjB 1 2.37 2.97 1.0 5 PDZ containing carboxyl terminal protease processing protease BSU35280 yvjA 1.52 1.32 1.21 2.38 putative integral inner membrane protein BSU35300 secA 2.45 1.22 1.18 2.54 translocase binding subunit (ATPase) BSU35310 yvyD 7.69 17.41 1.15 2.55 rib osome associated sigma 54 modulation protein BSU35320 fliT 1.42 1.17 2.43 2.13 flagellar assembly protein FliT involved in control of flagella expression BSU35330 fliS 1.34 1.22 2.64 2.09 flagellar assembly protein FliS BSU35340 fliD 1.74 1.32 2.27 1.74 flagellar hook associated capping protein 2 (HAP2) BSU35350 yvyC 1.51 1.19 2.65 1.91 putative flagellar protein BSU35360 hag 1.34 1.68 6.64 15.49 flagellin protein BSU35410 flgK 1.59 1.26 2.16 1.72 flagellar hook filament junction BSU3545 0 comFC 2.87 1.64 1.36 2.34 putative component of the DNA transport apparatus BSU35620 lytC 1.21 1.97 2.16 1.37 N acetylmuramoyl L alanine amidase (major autolysin) BSU35690 ggaA 3.07 8.78 2.46 1.24 poly(glucosyl N acetylgalactosamine 1 phosphate) g lucosyltransferase BSU35700 tagH 1.76 2.92 2.24 1.27 ATP binding teichoic acid precursor transporter component BSU35710 tagG 2.17 2.72 2.12 1.6 teichoic acid precursors permease BSU35750 tagA 3.8 1.8 1.23 2.76 N acetylmannosamine (ManNAc) C4 hyd roxyl of a membrane anchored N acetylglucosaminyl diphospholipid (GlcNAc pp undecaprenyl, lipid I) glycosyltransferase BSU35780 lytD 2.46 1.16 2.41 1.13 exported N acetylglucosaminidase (major autolysin) (CWBP90)
114 Table 3 2. Continued Accession BSU numb er Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU35820 gerBC 1.01 3.73 3.57 1.09 lipoprotein component of the germination receptor B BSU35830 ywtG 9.85 20.08 1.92 1.05 putative carbohydrate transporter BSU35850 ywtE 3.38 1.15 2.15 1.57 putative hydrolase BSU35860 ywtD 1.87 1.87 3.98 4.04 gamma DL glutamyl hydrolase (PGA depolymerase) BSU35910 rbsR 5.8 6.53 1.36 1.49 transcriptional regulator (LacI family) BSU35930 rbs D 2.06 3.22 1.21 2.32 ribose ABC transporter (membrane bound ribose binding) BSU35980 ywsA 1.58 14.28 9.99 1.25 hypothetical protein BSU35990 ywrO 2.13 4.88 3.34 3 nitroreductase BSU36080 ywrF 1.42 1.03 1.4 2.17 conserved hypothetical protein B SU36120 ywrB 2.99 1.84 1.36 2.22 putative anion transporter BSU36130 ywrA 2.8 1.89 1.27 2.33 putative anion transporter BSU36190 ywqJ 1.67 2.27 2.38 1.9 putative transposase or phage integrase BSU36200 ywqI 1.49 2.33 2.85 1.9 conserved hypotheti cal protein BSU36210 ywqH 1.55 1.99 2.47 1.72 conserved hypothetical protein BSU36230 ywqF 1.67 2.14 2.55 1.51 UDP glucose dehydrogenase BSU36240 ywqE 1.4 4.28 3.06 1.18 protein tyrosine phosphatase BSU36360 mscL 4.95 2.38 1.07 2.51 large conduc tance mechanosensitive channel protein BSU36370 ywpB 4.99 3.02 1.16 1.85 (3R) hydroxymyristoyl [acyl carrier protein] dehydratase BSU36460 ywoF 6.6 8.03 1.59 1.34 putative pectate lyase BSU36480 ywoD 5.7 2.54 1.48 3.07 putative efflux transporte r BSU36510 nrgA 1.01 1.73 2.1 1.43 ammonium transporter BSU36610 ywnC 6.99 5.64 1.45 1.83 putative integral inner membrane protein BSU36640 ureC 8.21 4.05 1.68 1.08 urease (alpha subunit) BSU36650 ureB 18.29 10.07 1.66 1.14 urease (beta subun it) BSU36660 ureA 19.35 10.33 1.62 1.22 urease (gamma subunit) BSU36670 csbD 16.92 19.87 1.23 1.08 stress response protein BSU36720 ywmE 17.57 20.17 1.36 1.17 hypothetical protein BSU36750 spoIID 1.19 4.84 3.11 1.21 autolysin required for complet e dissolution of the asymmetric septum (stage II sporulation) BSU36770 ywmB 3.13 1.37 2.15 1.1 conserved hypothetical protein BSU36780 ywzB 2.23 1.78 2.23 1.58 conserved hypothetical protein BSU36800 atpC 1.44 1.07 1.29 2.19 ATP synthase (subunit epsilon, F1 subunit)
115 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU36880 atpI 1.2 1.17 2.01 2.21 ATP synthase (subunit i) BSU37150 pyrG 4.3 10.03 2.49 1.01 CTP synthetase BSU37210 ywjC 44.45 42.27 1.25 1.38 conserved hypothetical protein BSU37240 ywiE 35.33 27.59 1.12 1.48 cardiolipin synthetase BSU37250 narI 57.08 246.83 7.68 1.01 nitrate reductase (gamma subunit) BSU37260 n arJ 44.17 208.21 7.17 1.01 nitrate reductase (protein J) BSU37270 narH 54.93 228.4 6.67 1.04 nitrate reductase (beta subunit) BSU37280 narG 38.15 199.34 8.19 1.07 nitrate reductase (alpha subunit) BSU37310 fnr 19.63 20.15 1.55 1.56 transcriptional re gulator (FNR/CAP family) BSU37320 narK 10.14 22.45 1.66 1.24 nitrite extrusion permease BSU37340 ywiB 1.25 1.54 1.19 2.49 conserved hypothetical protein BSU37350 sboA 104.74 214.19 1.36 1.15 subtilosin A BSU37360 sboX 118.2 95.17 1.84 1.12 putati ve bacteriocin like product BSU37370 albA 84.14 51.33 2.22 1.35 putative antilisterial bacteriocin (subtilosin) production enzyme BSU37380 albB 74.22 61.26 1.82 1.22 putative membrane component involved in subtilosin production BSU37390 albC 56.75 4 0.13 2.05 1.46 putative transporter involved in subtilosin production BSU37400 albD 61.89 40.8 2.26 1.43 putative integral inner membrane protein involved in subtilosin production and immunity BSU37410 albE 116.47 68.63 2.18 1.1 putative hydrolase i nvolved in subtilosin production BSU37420 albF 102.31 58.17 2.37 1.04 putative peptidase involved in subtilosin production BSU37430 albG 98.9 47.23 2.55 1.11 putative integral inner membrane protein involved in subtilosin production and immunity BSU3 7440 ywhL 24.49 10.13 2.19 1.01 conserved hypothetical protein BSU37450 ywhK 5.03 2.01 2.73 1.04 factor interacting with DNA helicase PcrA BSU37490 speB 5.98 2.34 2 1.49 agmatinase BSU37570 mmr 7.87 2.61 1.05 2.78 toxic compound efflux transport er BSU37670 ywfI 4.87 11.29 1.45 1.66 putative oxidoreductase/oxygenase/dismutase
116 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU37680 ywfH 1.52 4.94 2.47 1.3 carrier protein reductase of bacilysin synthesis BSU37760 rocC 4.16 3.43 6.71 2.18 arginine/ornithine permease BSU37770 rocB 2.04 6.5 11.02 1.14 putative N deacylase involved in arginine and ornithine utilization BSU37780 r ocA 5.65 1.78 7.81 1.45 delta 1 pyrroline 5 carboxylate dehydrogenase BSU37790 rocG 2.85 1.03 6.43 2.64 glutamate dehydrogenase BSU37800 yweA 1.6 6.64 3.98 2.73 member of the processed secretome BSU37900 spsB 1.04 2.64 2.86 1.06 putative dTDP gly cosyl/glycerophosphate transferase BSU37910 spsA 1.09 2.53 2.51 1.06 spore coat dTDP glycosyltransferase BSU37920 ywdL 1.01 2.6 2.51 1.01 spore coat protein BSU37940 ywdJ 4.56 3.02 1.39 2.04 putative purine/pyrimidine permease BSU38040 sacA 5.07 4.28 1.08 1.04 sucrase 6 phosphate hydrolase BSU38050 sacP 6.2 6.09 1 1.18 phosphotransferase system (PTS) sucrose specific enzyme IIBC component BSU38060 ywcJ 3.83 5.44 1.32 1.01 formate/nitrite transporter BSU38100 ywcH 2.17 1.22 1.1 2.32 puta tive monooxygenase BSU38110 nfrA 2.49 1.21 1.13 2.89 FMN containing NADPH linked nitro/flavin reductase BSU38180 ywzA 26.75 19.77 1.01 1.47 conserved hypothetical protein BSU38230 ywcB 3.75 1.76 1.39 2.24 putative phage protein (superinfection i mmunity) BSU38260 ywbN 1.93 1.43 1.77 2.21 iron dependent peroxidase convert ferric iron into ferrous iron BSU38270 ywbM 2.16 9.04 1.08 5.4 lipoprotein binding ferrous or ferric iron for transport BSU38280 ywbL 4.11 11.76 1.14 3.21 ferrous ion per mease BSU38340 ywbF 2.74 1.44 1.16 2.36 putative sugar permease BSU38410 sacX 1.15 2.87 2.82 1.31 negative regulator of SacY BSU38420 sacY 3.17 17.84 4.74 1.25 transcriptional antiterminator BSU38430 gspA 69.26 55.32 1.56 1.98 putative glycosyl transferase (general stress protein) BSU38550 ywaA 1.54 1.79 3.13 1.08 branched chain amino acid aminotransferase BSU38590 licB 4.29 5.52 1.68 1.1 phosphotransferase system (PTS) lichenan specific enzyme IIB component
117 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU38610 yxzF 5.85 8.17 1.55 1.14 hypothetical protein BSU38630 katX 4.6 2.47 1.2 1.45 major catalase in spores BSU38720 yxkO 1.52 5.09 3.09 1.28 putative carbohydrate kinase BSU38730 cydD 13.06 31.86 1.66 1.21 ABC membrane transporter (ATP binding protein) required for cytochrome bd function BSU38740 cydC 48.69 83.98 1.33 1.23 ABC membrane transporter (ATP binding pro tein) required for cytochrome bd function BSU38750 cydB 40.17 42.31 1.19 1.26 cytochrome bd ubiquinol oxidase (subunit II) BSU38760 cydA 27.64 27.67 1.01 1.01 cytochrome bd ubiquinol oxidase (subunit I) BSU38770 yxkJ 2.5 1.04 4.91 2.23 citrate/mal ate/H+ symporter BSU38830 aldY 4.23 5.26 1.19 1.07 putative aldehyde dehydrogenase BSU38840 yxkD 4.86 2.22 1.05 2.11 efflux transporter BSU38850 yxkC 2.68 5.8 7.95 4.18 conserved hypothetical protein BSU38920 pepT 4.96 1.46 1.05 2.84 peptidas e T (tripeptidase) BSU38930 yxjJ 5.37 23.83 1.06 4.78 hypothetical protein BSU39020 yxjA 1.51 2.7 1.65 3.01 purine nucleoside transporter BSU39030 yxiT 3.29 1.42 1.01 2.26 hypothetical protein BSU39040 yxiS 5.17 2.7 1.04 1.8 hypothetical protein BSU39050 katE 29.22 11.42 1.24 1.92 catalase 2 BSU39070 bglS 2.16 1.22 2.11 1.27 endo beta 1,3 1,4 glucanase BSU39100 yxiO 1.77 2.68 1.34 2.24 putative efflux transporter BSU39220 yxxG 1.44 4.13 2.95 1.18 hypothetical protein BSU39230 wapA 1.1 3 4.48 2.72 1.45 cell wall associated protein precursor BSU39250 yxiE 4.57 7.38 1.06 1.63 phosphate starvation protein (universal stress protein A family) BSU39280 yxxE 1.7 1.2 2.22 1.06 conserved hypothetical protein BSU39290 yxxD 1.7 1.14 2.1 1.2 conserved hypothetical protein BSU39310 yxiC 1.08 1.75 2.76 1.37 conserved hypothetical protein BSU39320 yxiB 1.01 1.54 2.11 1.43 conserved hypothetical protein BSU39330 yxiA 7.22 3.38 1.56 1.14 arabinan endo 1,5 alpha L arabinosidase BSU39350 hutH 2.28 1.28 2.43 1.12 histidine ammonia lyase (histidase) BSU39360 hutU 2.95 1.15 4.29 1.08 urocanase BSU39370 hutI 3.36 1.01 4.16 1.02 imidazolone 5 propionate hydrolase BSU39380 hutG 3.8 1.37 3.04 1.12 formiminoglutamate hydrolase BSU39390 h utM 3.17 1.49 3.24 1.19 histidine permease
118 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU39400 pdp 2.14 2.4 2.28 2.39 pyrimidine nucleoside phosphorylase BSU39410 nupC 3.52 4.85 2.79 1.94 pyrimidine nucleoside Na+(H+) cotransporter BSU39460 yxeQ 1.18 2.3 2.11 1.24 putative catabolic enzyme BSU39470 yxeP 1.06 3.48 3.36 1.12 putative amidohydrolase BSU39480 yxeO 1.34 4.58 4 .22 1.13 putative ABC transporter (ATP binding protein) BSU39490 yxeN 1.3 5.17 5.59 1.18 putative ABC transporter (permease) BSU39500 yxeM 1.36 6.05 6.06 1.31 putative ABC transporter (binding lipoprotein) BSU39510 yxeL 1.36 5.27 4.82 1.03 putative acet yltransferase BSU39520 yxeK 1.23 5.19 5.51 1.11 putative monooxygenase BSU39580 yxeE 1.53 1.46 1.09 2.36 spore coat protein BSU39600 yxeC 7.22 4.17 2.09 3.15 putative integral inner membrane protein BSU39610 yxeB 20.61 20.85 1.33 1.13 ABC tran sporter (ferrioxamine binding lipoprotein) BSU39660 yxdJ 4.65 5.62 1.18 1.07 two component response regulator [YxdK] BSU39670 fbaB 8.04 18.43 4.39 1.52 2 deoxy 5 keto D gluconic acid 6 phosphate aldolase BSU39680 iolI 9.17 23.97 5.32 1.97 putat ive sugar phosphate epimerase/isomerase BSU39690 iolH 8.1 22.43 6.09 2.05 putative sugar phosphate epimerase/isomerase BSU39700 idh 7.02 20.78 5.97 2.85 myo inositol 2 dehydrogenase BSU39710 iolF 9.8 23.72 6.13 2.45 inositol transport protein BSU39720 iolE 10.2 26.11 5.88 2.52 2 keto myo inositol dehydratase BSU39730 iolD 7.84 19.55 4.22 3.52 3D (3,5/4) trihydroxycyclohexane 1,2 dione hydrolase BSU39740 iolC 8.75 18.93 4.32 3.5 2 deoxy 5 keto D gluconic acid kinase BSU39750 iolB 8. 87 19.61 3.48 2.92 5 deoxy D glucuronic acid isomerase BSU39760 mmsA 7.95 17.96 2.71 2.8 methylmalonate semialdehyde dehydrogenase BSU39770 iolR 2.22 6.6 1.66 1.87 transcriptional regulator (DeoR family) BSU39780 iolS 2 4.99 1.28 2.08 aldo ke to reductase BSU39810 csbC 20.29 20.31 1.12 1.02 putative sugar transporter BSU39830 yxcA 2.3 1.18 1.11 2.38 hypothetical protein BSU39840 yxbG 7.12 8.94 1.46 1.28 putative oxidoreductase
119 Table 3 2. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106 to WN624 at 5kPa WN1106 to WN624 at 101kPa Gene Description BSU39880 yxbC 2.99 6.39 1.44 1.76 conserved hypothetical protein BSU39920 asnH 1.28 3.09 2.73 1.03 asparagine synthetase (glutamine hydrolyzing) BSU399 30 yxaM 2.1 12.71 5.26 1.16 putative efflux transporter BSU39940 yxaL 5.66 23.18 1.43 3.14 membrane associated protein kinase with beta propeller domain BSU39950 yxaJ 4.3 1.53 1.49 4.33 putative integral inner membrane protein BSU39960 yxaI 4.79 2.3 1.47 1.54 putative integral inner membrane protein BSU40000 yxnA 10.68 9.63 1.17 1.13 putative oxidoreductase BSU40040 yxaA 1.15 1.9 2.1 1.3 glycerate kinase BSU40180 yydF 2.92 12.1 1.55 2.54 peptide controlling LiaRS BSU40310 phrG 1.81 4.22 1.0 8 2.19 secreted regulator of the activity of phosphatase RapG BSU40420 purA 1.38 3.51 2.56 1 adenylosuccinate synthetase BSU40440 dnaC 2.71 4.03 2.18 1.38 replicative DNA helicase BSU40470 yycC 1.08 2.16 1.54 3.34 conserved hypothetical prote in BSU40480 yycB 1.26 2.22 1.16 2.29 putative anion ABC transporter (permease) BSU40510 yybT 1.37 2.59 2.9 1.38 putative phosphodiesterase BSU40520 yybS 1.36 1.61 2.34 1.78 putative integral inner membrane protein BSU40550 ppaC 1.71 1.17 2. 18 1.55 inorganic pyrophosphatase BSU40660 yybF 37.3 18.86 3.05 1.3 putative permease BSU40830 yyaK 1.71 1.79 1.05 2.9 putative integral inner membrane protein BSU40980 yyaB 1.73 1.73 1.97 2.1 putative integral inner membrane protein BSU41000 gid B 1.18 3.42 1.31 2.33 7 methylguanosine methyltransferase specific for the 16S rRNA BSU41010 gidA 1.01 3.53 1.41 2.45 tRNA uridine 5 carboxymethylaminomethyl modification enzyme BSU41050 rnpA 1.15 3.38 2.55 1.14 protein component of ribonuclease P (RNase P) (substrate specificity) Underlined values are not significant ( p value > 0.05). Genes that are italiced are significant only in WN624 5 kPa/101 kP a. Genes that are bolded are significant only in WN1106 5 kPa/101 kP a.
120 Table 3 3. SEED catego ries for genes with unknown function significantly expressed in microarray studies SEED Category: WN1106/WN624 WN1106 & WN624 WN1106/WN624 at 101 kPa at 5 kPa/101 kPa at 5 kPa Down Up Down Up Down Up Carbohydrates 4 3 6 13 5 4 Co factors, Vitamins , Prosthetic groups, Pigments 1 1 2 2 Virulence 3 5 7 8 7 4 Sulfur Metabolism 2 1 1 Stress Response 4 3 3 1 Protein Metabolism 2 1 2 1 1 RNA Metabolism 1 2 3 2 1 1 Motility and Chemotaxis 1 1 1 3 Secondary Metabolism 1 Amino Acids an d Derivatives 1 2 4 2 4 Nitrogen Metabolism 2 Fatty acids, Lipids, and Isoprenoids 2 2 1 Nucleosides & Nucleotides 1 2 Cell Wall & Capsule 2 1 2 DNA Binding 1 Metabolism of Aromatic Compounds 1 1 1 DNA Metabolism 2 1 Pha ges, Prophages, Transposable elements 1 1 1 Membrane Transport 1 1 Respiration 2 2 1 Transcriptional Accessory Proteins 2 Cell Division and Cell Cycle 2 Regulation and Cell Signaling 1 Dormancy and Sporulation 1 1 Phosph orous Metabolism 2 1 1 1 Not Assigned 86 36 32 101 43 60 Total Genes: 112 59 60 145 71 82
121 Table 3 4. Averages of CFUs, spore titers, and sporulation frequency from triplicate cultures of WN624 and WN1106 at 5 kPa and ~101 kPa in Spizizen minimal m edia Day Strain Pressure Ave. CFUs / mL St. Dev. Ave. Spores / mL St. Dev. Ave. Spore Frequency St. Dev. 1 WN1106 101 kPa 720000000 69282032.3 0 0 0 0 5 kPa 26666666 7 61101009 0 0 0 0 WN624 101 kPa 400000000 20000000 0 0 0 0 5 kPa 10866666 7 38279 672 0 0 0 0 2 WN1106 101 kPa 60666666 7 64291005 36666666 7 75718777 0.61 0.13 5 kPa 37333333 9865765 9800 8834.03 0 0 WN624 101 kPa 47333333 3 80829037 320000000 40000000 0.68 0.03 5 kPa 41333333 13613718 450000 363455 0.01 0.01 3 WN1106 101 kPa 56 0000000 0 48666666 7 100664459 0.87 0.18 5 kPa 36000000 8717797 2066666 7 808290 0.06 0.01 WN624 101 kPa 653333333 41633320 480000000 40000000 0.74 0.1 5 kPa 72000000 5291502 1226666 7 3002221 0.17 0.03
122 Figure 3 1. Plots of Cy3 vs Cy5 log fluore scent intensity for microarrays. See text for details.
123 Figure 3 2. Determination of pressure induction of the SigB dependent GSR using a ctc::lacZ reporter fusion. Strains used and their relevant genotypes are denoted in the figure. A ) Induction of c tc lacZ expression by 5% (v/v) ethanol (filled bars) vs. the uninduced control (open bars). B ) Induction of ctc lacZ expression by LP (5 kPa) (filled bars) vs. the uninduced control (open bars). C ) Induction of ctc lacZ at ~101 kPa and various LPs (50, 25, 10, and 5 kPa) in strains WN1400 (ancestor; sigB + ) (open bars) and WN1447 (LP evolved; sigB + ) (filled bars). Data are averages and standard deviations of triplicate samples taken from duplicate experiments.
124 Figure 3 3. Relative fitness values of conge nic ancestral strains WN624 ( amyE::spc ) and WN1232 ( amyE::spc , sigBD2::cat ) and congenic LP evolved strains WN1106 ( amyE::spc ) and WN1233 ( amyE::spc , sigBD2::cat ) at ~101 kPa (open circles) or 5 kPa (filled circles). Each strain was competed against wild type strain WN1261 ( amyE::neo ) for 50 generations and relative fitness values computed as described in Materials and Methods and previously (Nicholson et al., 2010) .
125 Figure 3 4. Average sporulation frequency at 5 kPa and ~101 kPa of strains WN624 and WN1106 over three days.
126 CHAPTER 4 WHOLE GENOME RE SEQUENCING REVEALS MUTATIONAL CHANGES IN BACILLUS SUBTILIS AFTER A 1,000 GENERATION 5 KPA EVOLUTION EXPERIMENT Introduction The adaptive ability of microorganisms to survive and even thrive in a wide range of harsh environments has been an area of profoun d interest for decades. Despite the extensive study of extreme environments on Earth, one extreme environment, that of low pressure (LP), remains relatively unknown in terms of cellular effects and microbial adaptations. The lowest terrestrial barometric p ressure is ~34 kPa atop the highest peak of the Himalayas. However, low pressure environments, and the ability of microorganisms to live and adapt in them, is of relevance to (i) the hypobaric plant and food storage industry, (ii) aeromicrobiology and (iii ) the field of astrobiology. Extending microbial pressure research to include low pressure microbial response and adaptation is also important because it complements high pressure microbial studies; just as together psychro and thermo adaptations comprise the range of extreme temperature adaptations. Due to the lack of natural terrestrial LP environments in which to sample organisms to study, experimental evolut ion (E.E.) is a powerful tool to investigate how microorganisms may adapt to LP growth. In a pre vious report we communicated one such evolution experiment of Bacillus subtilis for 1,000 generations at 5 kPa in LB media (34) . During this experiment, average weekly increases in optical density measurements indicated periods of possible punctuated evolutionary changes. At the terminus of the experiment, a strain was isola ted, WN1106 (Table 4 1), which was reported to have an increased relative fitness compared to the ancestor strain, WN624, of the 5 kPa E.E. at LP but not standard pressure (~101 kPa). Previous transcriptional studies comparing transcripts of (i) WN1106 at 5 kPa and 101 kPa, (ii) WN624 at
127 5 kPa and 101 kPa, (iii) WN1106 to WN624 at 5 kPa and 101 kPa revealed significant (87, 152 ) . It is described here experi ments used to determine the underlying genomic changes that may have occurred during the 5 kPa E.E. and give rise to the phenotypic and transcriptional differences of WN1106 and WN624 at 5 kP a. Whole genome re sequencing was used to study differences betwe , of which there were 8 confirmed mutations in WN1106 . Back stocks collected at ~ 50 generation intervals during the 5 kPa E.E. were analyzed for the presence of these mutations occurring in WN1106 and used for competition studies a gainst congenic strains of WN624 and WN1106 at 5 kPa to determine correlations between mutations and phenotypic changes . Materials and Methods Bacterial Strains, Media and Growth Conditions All strains are listed in Table 4 1. Ancestor strains WN624 ( trpC2 amyE::spc ) and WN628 ( trpC2 amyE::cat ) , and the two evolved strains, WN1105 ( trpC2 amyE::cat ; 1,000 gen. at 101 kPa) and WN1106 ( trpC2 amyE::spc ; 1,000 gen. at 5 kPa) have been described previously in detail (34, 1 13 ) . Strain GP45 carrying the rnjB::spc knockout mutation was a generous gift from Jorg Stulke. Transformation of B. subtilis was performed by standard methods of our lab (84) . Miller LB liquid or agar medium (85) were used throughout and supplemented when necessary with the appropriate antibiotic (final concentr ation): chloramphenicol (Cm, 5 Âµ g/mL); neomycin (Neo, 5 Âµ g/mL); s pectinomycin (Spc, 100 Âµg/mL). As previously described for pressure growth, cells were grown under normal lab atmospheric pressure (~101.3 kPa ), low pressure (5 kPa), or low O 2 (34, 87, 153 ) . Cultures were shaken at moderate speed (~150 rpm) on a rotary Summerson photometer fitted with the No. 66 (660 nm; red) filter and under these conditions: 100 Klett units = 1 OD 660 = approx 1
128 x 10 8 cells per mL. Motility experiments were conducted as previously described ( 120 ) ; briefly, 0.3% agar LB plates with Spc were spotted with 1 ÂµL mid log phase (~0.6 OD 660 ) culture of either WN624 or WN1106 and allowed to incubate at 37 temperature and 5 kPa for 24 hours. DNA Extraction and Quality Control for Re Sequencing Strains WN624, WN1105, and WN1106 were each grown up overnight, in LB with by centrifugation (10 min. at 10 krpm) and DNA was extracted using a standard protocol (84, 85) . DNA concentration was measured and 280 nm absorption ratio for DNA purity was determined by UV spectrophotometry to be above a ratio of 1.8 as required by the Vanderbilt Technologies for Advanced Genomics center. Samples for each strain were submitted to Vanderbilt Gen omics center in TE buffer at 5 Âµ g in a volume of 30 Âµ L. Whole Genome Re Sequencing and Mutation Identification Whole genome re sequencing of the ancestral strain WN624 and evolved strains WN1105 and WN1106 was performed by the Illumina HiSeq 2000 system (Illumin a Inc, San D iego, CA) . Raw left and right end reads were analyzed using the Galaxy platform ( 153 155 ) and run through the following tools before mapping: FASTQ Groomer ( 156 ) , FASTQC, and Ren ame Sequences (based on the FASTX toolkit by Assaf Gordon) ( 156 ) . For mapping the right and left hand reads, BWA ( 157 ) was used and the reference Bacillus subtilis strain 168 genome, downloaded from the NCBI genome website. The resulting SAM files were converted to BAM format using SAMtoo ls ( 158 ) ( 159 ) and plotting the data using the R program; this revealed a high coverage >100x with a
129 mean of ~5000x. SAMstat ( 160 ) and flagstat ( 158 ) were run to determine mapping quality, total number of mapped reads and number of properly paired reads that were mapped. SNPs and InDels were called using the Unified Genotyper Tool ( 161 ) . For quality control, two separate mappings were conducted with the reads from each strain: a mapping without filtering reads and one with the reads filtered based on quality before mapping. Filter FASTQ ( 161 ) (minimum quality 28, maximum quality 50, maximum bases allo wed outside of quality range 5) was used in the latter case a nd resulted in 41.09% and 43.28% of the left and right hand reads being kept, respectively for WN1106, 49.07% and 44.51% of the respective left and right hand reads of WN624, and 47.8% and 43.2% of the respective left and right hand reads of WN1105. Both f iltered and unfiltered mappings resulted in the same high quality SNP and InDel calls; however, the percentage of reads that were properly paired for the filtered data was very low (<1%), but still resulted in ~ 10x coverage of the genome [data not shown]. SNPs and InDels with high mapping quality (>1,000) were checked by BAMview, a visualization tool for viewing BAM files, and confirmed the presence of eight mutations (seven SNPs and one deletion event) to be further confirmed by Sanger re sequenc ing. The SNPs and the deletion found in WN1106 were further verified by Sanger sequencing (University of Florida Core Sequencing Facility) of a ~500 nt fragment containing the SNP/InDel region from both strains WN624 and WN1106; primers used for these purp oses are listed in Table 4 2. This confirmed that the mutations identified in WN1106 were present only in WN1106, these are listed in Table 4 3. To further determine when each mutation occurred during the 5 kPa evolution experiment that resulted in strain WN1106 (34) , each mutation was amplified from stock cultures that wer e taken weekly during the experiment: a total of 20 stocks collected approximately every 50 generations. Chromatographs from the amplified regions of the 5 kPa evolution experiment re -
130 sequencing were used to estimate when mutations arose and, if at all, th ey dominated, by taking the proportion of the mutant nucleotide fluorescence over the total fluorescence at the position (Figure 4 1). SNPs and InDels were identified to be present in all strains, which differed from the B. subtilis 168 reference genome , a nd these are listed in Table 4 4. These were not verified by th is study, however, those listed in boldface type have been verified by other independent studies. In silico Analysis of Mutations Secondary structure predictions were performed using the online CFSSP tool at www.biogem.org ( 162 , 163 ) . Tertiary structure predictions were conducted using Swiss Model Work space for RnjB, BacD, ResD, ParC and FliI mutant protein sequences with closest PDB structure available used as reference, 3ZQ4, 3VMM, 1B00, 1ZVU and 2DPY, respectively. Structural alignments were conducted using Pymol. ResD was aligned and structure was d etermined based on two transcriptional response regulators whose structures have been determined: Escherichia coli PhoB and Mycobacterium tuberculosis Mtr A. Alignmen ts were performed using Clustal Omega (www.ebi.ac.uk/Tools/msa/clustalo/) . Rifampicin Resis tance Assay To determine mutation frequencies of WN1105, WN624 and WN1106, a rifampicin resistance assay was conducted using a standard protocol described previously ( 113 ) . Briefly, cultures of strains WN1105, WN624, and WN1106 were inoculated in duplicate cultures and 1 mL samples were taken to determine total CFUs and Rif resistant CFUs. After 24 hours of gr owth, another 1 mL of cells was taken to determine total CFUs and Rif resistance CFUs (Table 4 5).
131 Competition Experiments To determine the relative fitness gains or losses which occurred during the 5 kPa evolution experiment (34) , each of the 5 kPa 50 generation back stocks were individually competed against congenic strains of WN624 and WN1106 in duplicate at 5 kPa (Table 4 1, Figure 4 1) by standard methods previously described by our lab (34, 153 ) and relative fitness values were calculated ( 121 ) . Briefly, each individual strain or whole population was cultured overnight in Miller LB media with appropriate antibiotics , at 27 Âº C and 170 rpm. The overnight cultures were used to inoculate 10 mL Miller LB media at ~ 0.015 OD 660 (as determined by UV spectrophotometry) per strain. Day zero (D 0 ) population ratios were determined by plating serial 10 fold dilutions on the appropriat e antibiotic LB agar plates for respective strains. Ratios of the cell populations were calculated each day for up to a week or until one of the strains was no longer detected in the mixed culture . Fitness coefficients, S , were calculated as followed: S = log([E/A] N /[E/A] N 1 )/6.6. Where E/A is the population ratio of the evolved strain to the ancestor strain, N is day, N 1 the previous day, and 6.6 is the approximate number of generations that occurred from one day to the next . The relative fitness value wa s determined by adding 1 to the S coffecient; values greater than 1 indicate an increase in fitness compared to the ancestor strain, less than 1 indicates a decrease in fitness compared to the ancestor, and a value of 1 indicates no noticeable fitness diff erence between the evolved or ancestor strains. RNA Isolation Cells from cultures grown for 24 hours at either 5 kPa or 101 kPa were rapidly centrifugated and immediately processed for total RNA extraction using the RiboPur e Bacteria kit (Ambion) following each sample was measured by UV absorbance at 260 and 280 nm (88) .
132 Quantitative Reverse Transcription Polymerase Chain Reaction (qRT PCR) qRT P CR was conducte d by standard methods as described previously ( 153 ) . Briefly, primers (Sigma) were used to amplify targe t genes from cultures grown under conditions of: 101 kPa or 5 kP a . cDNAs were generated and amplified using the SuperScript III Platinum SYBR Green One Step qRT PCR kit (Invitrogen) according t Reactions were performed in a MiniOpticon real time PCR detection system (Bio Rad). The expression changes were calculated from the delta delta threshold cycle (C T ) values ( 164 ) ; each product was confirmed by melting curve analysis. R esults and Discussion Whole Genome Re Sequencing and Alignment Ancestral strain WN624 was subjected to experimental evolution for 1,000 generations at either 5 kPa or ~ 101 kPa (34). To determine genom ic changes that occurred in evolved strain s WN1106 and WN1105 whole genome re sequencing using the Illumina platform was performed. The sequencing reads were mapped to the Bacillus subtilis strain 168 reference genome, obtained from the NCBI website. Mappi ng statistics for each strain are listed in Table 4 6. SNP/InDel calls were analyzed based on (i) presence in evolved strain but not the ancestor, (ii) high mapping quality as determined by the Unified Genotyper tool, and (iii) visual confirmation from BamView, a mapping visualization tool. During the mutation al analysis of WN1105, more than 100 mutations were called, nearly a magnitude higher than the amount of mutations for WN1106. During analysis of this list, it was noticed tha t a putative deletion was identified in the coding region of the mutL gene (Table 4 7) , whose product is involved in the DAM dependent mismatch repair pathway ( 165 ) . Mutations in mutL have been reported to cause a mutator phenotype in B. subtilis resulting in an increase in spontaneous mutations in the genome ( 165 ) . Mutator phenotypes have been previously reported
133 during long term evolution experiments of B. subtilis and E. coli ( 113 , 166 ) . Be cause WN1105 was evolved at standard pressure alo ng side the 5 kPa evolution experiment that gave rise to WN1106, its purpose was to act as a control for LB and flask growth conditions. However, a mutator phenotype would negate its purpose. Therefore, to simply answer if WN1105 had indeed gained a mutato r phenotype, a rifampicin resistance (R i f R ) assay was preformed according to the Methods section. The results are shown in Table 4 5 and reveal that WN1 105 has an increased occurance of spontaneous R i f R mutantions compared to both the ancestor strain WN624 , and LP evolved strain WN1106 . These data s uggest that strain WN1105 during the course of t he 101 kPa evolution experiment had gained a mutator phenotype, and this mutation is supposedly in the coding region of mutL . However, further confirmation through Sanger re sequencing of WN1105 mutations listed in Table 4 7 was not performed in this investigation, which primarily is focused on LP related mutations. For WN1106, 7 single nucleotide polymorphisms and 1 deletion event , all occurring within coding region s, were selected for further confirmation by PCR amplification and Sanger resequencing. The primers from the amplification of mutation regions are listed in Table 4 2. All 8 mutations in WN1106 were confirmed, further analyzed and are discussed below (Tabl e 4 3). Mutational Analysis During the 5 kPa Evolution Experiment To determine when each of the mutations occurring in WN1106 arose during the 5 kPa evolution experiment, each mutation region was amplified from the twenty back stocks each representing ~ 50 generations per stock. The proportion of muta nt allele to the population was estimated as described in Methods from the sequencing chromatographs for each mutation at 50 generation intervals (Figure 4 1A). Based upon the proportions of mutant allele fluor escence for the 8 mutations in WN1106, it becomes apparent that the mu tations could be grouped into (i) mutations occurring early during the evolution experiment ( rnjB 9 nt deletion, ytoI, parC, and
134 fliI ) and (ii) mutations detected towards the end of the evolution experiment ( walK, yvlD, resD, and bacD ) (Fig. 4 1A). In the former group, parC and ytoI mutations are a sub population (referred to as population B) of the first dominant rnjB 9 nt deletion, here on referred to as population A (Figure 4 2). It is unknown if other mutations co occur with the four early mutations as sequencing was only conducted on the mutation regions of interest from WN1106. From the sequencing of the back stocks, there is evidence that the culture had two sweeps of the 9 nt delet ion in rnjB ; the first sweep (population A and subpopulation B) dominated by ~450 generations but was overtaken by another population, designated here as population X. The X population, which dominated by ~800 generations was serendipitously determined to possess a SNP resulting in an early termination codon in the coding region of rnjB (Table 4 3). This rnjB SNP was detected due to its being in the same 500 nt region amplified to detect the 9 nt deletion of rnjB (Fig. 4 3) and its fluorescent proportion wa s determined and plotted (Fig. 4 1C). Population X was subsequently over taken by a population referred to as B1; this population is most probably the re emergence of the sub population B from the first rnjB deletion sweep but with the presence of addition al mutations: bacD, resD, walK, and yvlD (Fig. 4 1A). Again, it is unknown the complete mu tational diversity that occurred during the 5 kPa E.E.; future studies could be conducted utilizing deep sequencing of the back stock populations to determine semi qu antitatively mutational proportions in the culture and population dynamics, as well as mutations which co occur with one another . Future work will also further analyze population X by isolating a strain containing the truncated rnjB coding region and condu cting further mutational analysis as well as fitness competitions. To determine the fitness gains that occurred during the 5 kPa E.E., each 50 generation back stock was competed in duplicate at 5 kPa against a congenic ancestor (WN628) or LP -
135 evolved (WN127 9) strain ( Table 4 1, Figure 4 1C). The 0 generation mark refers to the ancestor WN624, which was the starting population at the beginning of the 5 kPa E.E. Interestingly the competition of the 50 generation population against the congenic ancestor strain indicated a significant fitness increase after only a week of exposure to 5 kPa (Fig. 4 1C); the fitness increased at a near ly linear rate until ~ 350 generations where the values reached a plateau in both competition studies. To note, there was some minor fluctuation in relative fitness values towards the last ~300 generations that roughly corresponds to the rise and sharp decline of population X. bacD Analysis The SNP in the coding region of bacD causes a residue change of E97Q located in the N terminal r egion of the BacD enzyme. BacD is an L amino acid ligase, which in vivo is responsible for the ATP dependent ligation of L alanine and L anticapsin and the production of the antibiotic bacilysin ( 167 ) . In vitro th is enzyme is promiscuous in its substrate recognition ( 167 ) and its s tructure has been determined ( 168 ) . A structural alignment between wild type and mutant protein indicates that there are no obvious structural disruptions in this stretch of residues ; however, as would be expected the surface charge in this region is ch anged , becoming less acidic . Due to the mutation lying in the stretch of residues between two loops (loops 2 & 3), which are involved in orientation of substrates and catalysis within the binding pocket, it is possible this charge difference could cause sl ight structural alterations and affect the catalysis , but this supposition requires further biophysical studies. I t is unknown how a mutation in BacD would affect growth of B. subtilis at LP. This mutation swept through the 5 kPa evolution experiment popul ations at ~ 900 generations and could be a neutral hitchhiker mutation as it co occurs with the mutations in resD, walK and yvlD . All four of these mutations occur red during the second sweep of the rnjB deletion (Fig. 4 1A).
136 Further analysis of the role of wild type and mutant BacD are needed at LP to determine if this mutation is neutral, beneficial or detrimental . fliI Analysis The gene fliI is located within the large (>26 kbp) fla/che operon of B. subtilis and codes for the cytoplasmic flagellar specifi c ATPase of the Type III secretion system responsible for flagella component export ( 169 171 ) . It is thought that secretion of the flagella subunits are exported in an ATP dependent unfolding process facilitated by FliI in a complex with the chaperone, FliJ, and the regulator of both, FliH ( 169 , 171 ) . fliI mutants are known to be deficient in flagella ( 169 , 172 174 ) , resulting in a non motile phenotype. Therefore, a motility phenotype was investigated for WN1106 compared to the ancestor, WN624. First, the two strain s were compared under phase contrast microscopy ; WN624 cells exhibite d aerotaxis, i.e. cells swam to wards trapped air bubbles, whereas the WN1106 cells appeared non motile when viewed. A subsequent standard 0.3% mo tility agar spot test was performed, confirming the motility of the ancestor and a non motile phenotype of the LP evolved WN1106 when grown for 6 hr. at 37Â°C (Fig. 4 4A). As seen from the figure, WN624 has a much more pronounced sized colony compared to th e colony of WN1106, this indicates that the mutation in fliI could result in a decrea sed motility phenotype. It should be noted here , the fliI mutation is in a background with the other 7 mutation s of WN1106 and it is unclear how and if these mutations int eract with one another and may affect motility . We were interested in testing to see if either of the strain s was motile at 5 kPa and let a motility agar plate spotted with both strains incubate at 5 kPa for 24 hours. N either of the two strain s show ed moti lity (Fig. 4 4A), indicating that LP may have an inhibitor y affect on one or more motility processes in B. subtilis . The fliI SNP does not occur in the highly conserved Walker A and B motifs, involved in nucleotide binding of ATPases ( 175 , 176 ) , but in the N terminal domain region at the inter -
137 subunit interface ( 177 ) . F liI is known to oligomerize to a homo hexameric ring ( 178 ) that is subunit of F 1 ATPases and the ATPase EscN from E. coli ( 171 , 179 ) . The first 100 N terminal amino acids of FliI forms a six barrel that interacts with FliH ( 180 ) and may be responsible for oligomerization ( 177 ) . Okabe et al. investigated with in frame deletion analysis the first 100 a. a. of Salmonella FliI; deletions of the region 20 40 a. a. caused non motility and evidence suggested that amino acids in this region were involved in ATPase suppression and docking interaction between the export gat e and the FliH FLiI complex ( 177 ) . Based on this previous study, the mutation of B. subtilis P35T in this r egion could be affecting oligomerization and/or docking effi ciency with the export pore . It has been found previously that long duration continuous culturing of B. subtilis may lead to mutations resulting in loss of motility due to lack of chemical gradien ts in a shaken flask (57) ; loss of motility has been reasoned to be beneficial to bacterial life in a flask due to energy costs (63) , flagellar motion and assembly are energetically expensive ( 181 ) . A s the SNP in fliI arose and became dominate early in all detectable cells lines from re se quencing the 5 kPa E.E. stocks (Fig. 4 1A), it is thought that the loss of motility, due to the P35T mutation, gave a higher fitness advantage during the 5 kPa evolution experimen t not due to overcoming the inhibitive nature of LP but by decreasing unnecce ssary cellular energy expenditure. parC Analysis The SNP in the parC coding region changes an aspartate to histidine at position 205 (D205H). The parC gene codes for ParC, the topoisomerase IV subunit A, an essential gene ( 182 ) involved in decatenation and also, evidence suggests, replication fork movement ( 183 , 184) . Mutational targeting of DNA supercoiling has been shown to occur in evolution experiments with E. coli (185 ) ; the mutation in parC therefor may indicate that this mutation
138 does not necessarily have a role in LP adaptation but is in agreement with what is seen in other long term E.E. T he mutation occurs in the tower domain of the protein, whose primary function is believed to be structural, but which also contributes to DNA binding. Sequencing analysis of when this mutation occurred during the 5 kPa evolution experiment revealed that a minority of the population possessed this mutation during the first sweep of t he rnjB deletion, which is referred to as popu lation B (Fig. 4 1A and 4 2) and also possessed the ytoI and rnjB deletion mutations. It is reasoned that population B most likely included the ancestral cell line for WN1106. It is unknown how this mutation mi ght influence growth at LP of B. subtilis . Further studies are needed to elucidate the role of this mutation in B. subtilis at 5 kP a. ytoI Analysis The mutation in ytoI results in a valine to phenylalanine change at residue 77 (V77F). This mutation occurre d ear ly during the 5 kPa E.E. in population B, co occurring in the population with fliI , parC and rnjB deletion mutations (Fig. 4 1A and 4 2), and as stated above is a sub population of population A (defined by the first sweep of the rnjB 9 nt deletion eve nt). As stated previously , it is bel ieved that this subpopulation most likely contains the ancestor population that resulted in WN1106. YtoI is an uncharacterized protein with similarities to other uncharacterized proteins. It is unknown how this mutation affects ability to grow at LP and as it co occurred with fliI , parC , and the rnjB deletion may be a neutral hitchhiking mutation; further investigations into the ytoI mutation in a clean background (i.e. with no other mutations) at 5 kPa are c urrently underway to investigate its putative role in LP growth of B. subtilis .
139 resD and walK Analysis Two of the mutations occurred in different two component systems (TCS): WalKR and ResDE (Table 4). ResDE is the anaerobic TCS in B. subtilis ( 186 ) , with ResD functioning as the response regulator; and WalKR is an essential TCS in control of cell wall metabolism ( 187 ) , WalK being the sensor kinase of this system. The mutant forms of walK and resD co occur during the 5 kPa E.E., first appearing towards the end of the experiment at ~ 900 950 generations (Fig. 4 1A). This is when the second rnjB deletion sweep appears, termed population B1, as i t also corresponds to a re emergence of the parC and ytoI mutations (Fig. 4 1A and 4 2). Population B1 sharply overtook population X as compared to the first rnjB deletion sweep of population A, indicating that one, or more, of the four mutations first det ected in this population ( yvlD, resD, bacD, and walK ) may have increased the relative fitness of a cell line in population B singularly or by additive effects with another mutation (s). As stated previously, bacD and yvlD mutations may be neutral, thus poi nting towards a more pertinent role of either the walK or resD (or both) mutations on LP growth. Transcriptional arrays of both WN1106 and WN624 at 5 kPa and 101 kPa (Chapters 2 and 3) reveal ed a pronounced response of both strains to LP and differences be tween the two strains when exposed to LP, resp ectively ( Table 4 8 , 152 ). These differences included signals that are regulated by either ResDE or WalKR (Table 4 8). To further confirm that mRNAs whose expression is dependent on either ResDE or WalKR are di fferentially expressed between WN1106 and WN624, q RT PCR analysis was utilized to determine relative signal abundance at 5 kPa and 101 kPa in both strains (Fig. 4 5). For the WalKR system yocH was chosen due to its relatively well characterized regulation by WalKR ( 186 , 188 ) and for ResDE system fnr was used. The use of fnr to determine regulation differences between the two strain s at 5 kPa is
140 discussed below. Primers for each of the q RT PCR products are listed i n Table 4 2 and the results are discussed in relation to the respective mutation. Both yocH and fnr express ion levels were significantly higher in WN1106 compared to WN624 at 5 kPa, but not at 101 kPa (Fig. 4 5). These results, along with the differences s een in the transc riptional microarrays (Table 4 8), indicate that the mutations in WalK (T195M) and ResD (P110Q) may be influencing functionality of the respective TCAs at 5 kP a. As stated above, ResD is the response regulator in the anaerobic TCS of B. su btilis and belongs to the OmpR like response regulator family ( 189 ) . We previously reported that WN1106 has a decreased relative fitness compared to the ancestor under hypoxic and normal atmospheric conditions, indicating that any LP advantage WN1106 gained during the 5 kPa evolution experiment was most likely not a result of anaerobic adaptive changes ( 153 ) despite a pronounced low oxygen response by both WN624 and WN11 06 at LP ( Table 4 8 , 152 ). Two OmpR like response regulators which have had their structures determined were used to analyze the mutation in ResD: PhoB from E. coli ( 190 ) and MtrA from Mycobacterium tuberculosis ( 191 ) . Structural alignments revealed that the mutation of a proline to glutamine at position 110 in ResD corresponds to a stretch of amino acids involved in protein protein interactions when homodimerization occurs in OmpR like response regulators ( 190 ) . H owever, ResD, unlike other OmpR like response regulator s , does not form homodimers either in its phosphorylated or unpho sphorylated form ( 192 ) . Though ResD does not form homodimers, there are multiple binding sites for ResD at some ResD dependent promoters, such as the promoter of ctaA (three binding sites) ( 192 ) , which are required for full expression. Transcription of fnr was chosen for q RT PCR analysis because fnr only possesses one ResD binding site at its promoter region. The reason for this choice was that if there were multiple effects of the mutation, i.e. molecule -
141 molecule interactions as well as DNA binding and signaling interaction s , a gene whose regulation was only dependent on one copy of ResD binding the promoter region would indicate if the P110Q change affected signaling . The expr ession ratio of fnr between WN1106 and WN624, though not highly pronounced is still significant as determined by ANOVA pairwise comparison , does indicate that there is a low oxygen response differentiation between the two strains when grown at 5 kPa and th at the underlying reason could be the mutational change in ResD. Further work is underway to clearly characterize the mutant ResD in a clean background , without the presence of the other mutations, at LP to determine its role. WalK is the transmembrane se n sor kinase of the essential TCS WalKR that regulates cell wall metabolism in B. subtilis ( 187 ) . Genes whose transcription is dependent upon WalR are most highly expressed during exponential phase growth ( 187 ) , including yocH . WalK fu nction is believed to be modulated by two integral membrane proteins YycI and YycH, each of which possesses a transmembrane helix that has been reported to have interactions with the two transmembrane helices of WalK and when these contacting residues are mutated have been shown to affect yocH transcript levels ( 188 ) . The T195M mutation occurs in the second of these helices (transmembrane helix 2, TM2) and may affect the intermolecular contacts that regulate WalK function. For these reasons, yocH expression levels were compared in WN110 6 and WN624 grown at 5 kPa (Fig. 4 5). The expression ratio indicates that yocH levels are indeed significantly increased in strain WN1106 at 5 kPa, and this is further supported by the microarray data ( Table 4 8 , 152 ). These data imply that the mutatio nal change in WalK has a phenotype at LP in WN1106 similar to previous work that affected the modulation of WalK by YycI ( 188 ) . H owever, more work is needed to investigate this mutation in a clean background and determine its role on fitness and growth of B. subtilis at LP.
142 rnjB Analysis the coding region of rnjB ; this resulted in residues, an alanine, lysine and isoleucine, being deleted in a 3 amino acid in frame shift. rnjB codes for RNase J2, which to gether with RNase J1 (also known as RnjA) forms a complex (RnjA/RnjB complex) that is an important component of the major mRNA global processing systems in Bacillus subtilis (reviewed in ( 193 ) ). The known primary function of RnjB is that of an endoribonuclease ( 194 , 195 ) with RnjA, the essential riboenzyme in the complex, having the main exoribonuclease function as well as an endoribonuclease function ( 193 , 195 , 196 ) . Both RnjA and RnjB are zinc metallo hydrolases of the metallo CASP proteins, which act on RNA and DNA substrates (reviewed in ( 197 ) ). The crystal structure of RnjA has been resolved ( 198 ) . RnjB shares ~ 48% sequence homology to RnjA, however, the overall surface charge difference between the two enzymes is significant: RnjA has a pI of 6.5 point is 9 ( 198 ) . This charge difference is thought to be a contributing factor to the difference in enzymatic activity betwee n the two (i.e. endo versus exo nuclease); it has also been shown that despite the fact that RnjB is non essential , the protein does exert an endonuclease specificity role on RnjA, as when RnjA is complexed with itself and not RnjB the cut site of certai n RNA substrates differs, the reverse is also true of RnjB endoribonuclease recognition sites in vitro ( 195 ) . CASP subfamily of proteins and their protein structures . The 9 nt deletion event in rnjB CASP domain ( 198 ) ; therefore the deletion was studied in silico for structural perturbations to this region. Secondary and tertiary structure predictions were performed (see Methods and
143 Materials sectio no longer forms and in its place an unstructured region exists (Fig. 4 6). This region is predicted to act as a structural hinge between the metallo CASP domains ( 198 ) . Transcriptional analysis of RnjB knock out, RnjA knock down, and double RnjB/RnjA mutants have been reported ( 196 , 199 ) . Our own transcriptional studies (Chapters 2 and 3) ( 152 ) were analyzed for differences in signals known to be affected by the previous transcriptional study of a RnjB mutant (Table 4 8) ( 196 ) . Noteably , the Mader et al. 2008 study showed a decrease decay rate of two transcripts, yweA and spoVG , whose steady state levels were higher in a double RnjA/R njB mutant ( 196 ) . Both yweA and spoVG signals were also found to be upregulated in WN1106, but not WN624, at 5 kPa (Table 4 8). To further determine if the deletion in RnjB affe cts mRNA signals of known target s of the RNase J1/J2 complex , yweA transcrip t l evels in strains WN1106 and WN624 were compared using q RT PCR of total RNA isolated from cultures grown at 5 kPa and 101 kPa (Fig. 4 5). It was observed that yweA mRNA levels were significantly higher in WN1106 com pared to WN624 at 5 kPa, thus the deletio n in rnjB may be altering the function of the RnjA/RnjB complex at 5 kP a. Based on the evidence from the microarry and q RT PCR transcriptional studies, the deletion in rnjB may have a similar p henotype to a full gene disruption . To investigate the relativ e fitness of wild type strains t rnjB strains, congenic strains of WN624 and WN1106 were transformed with gDNA from a B. subtilis strain harboring an rnjB::spc knockout mutation as described in the Methods section (Table 4 1). The competition experiments revealed that wild type WN1106 harboring the 9 nt deletion in rnjB showed no difference in relative fitness with the full deletion strain at ei ther 5 kPa or ~101 kPa (Fig. 4 7 ). However, the full deletion of rnjB in the ancestor strain did increase its fitness at both p ressures
144 studied compa red to wild type WN624 (Fig. 4 7 ). These results indicate that in the LP evolved WN1106, the 9 nt deletion has a similar fitness phenotype to a full rnjB deletion; however, as evidenced by the full gene deletion of rnjB in the ancestr al WN624 being more fit than wild type WN624 at 5 kPa and 101 kPa, the 9 nt deletion may not be comparable to a full deletion. Further study into the role the 9 nt deletion in WN1106 has on the functionality of the RnjA/RnjB complex at 5 kPa and 101 kPa is currently underway. Discussion Bacillus subtilis at 5 kPa In 2010 we reported on a strain of B. subtilis , WN1106, that had evolved for 1,000 generations at 5 kPa and resu lted in a LP phenotype, which was define d as an increase in (i) optical de nsity and ( ii) relative fitness compared to the ancestor strain at 5 kPa (34) . He re we identified genomic changes that occurred in the LP evolved strain WN1106, determined the population kinetics of mutation appearance , and determined the fitness increase gains during the 5 kPa E.E . From the fitness competitions of 50 generation back st ocks from the 5 kPa E.E. versus the ancestor strain WN624 , it was observed that by the first 50 generations (i.e. first week) the culture had exhibited enhanced fitness at 5 kPa (Fig. 4 1B). It is however unknown if this phenotype corresponds to specific g enomic changes as (i) whole population sequencing of the back stocks was not conducted and (ii) none of the mut ations in WN1106 were detected by our methods at 50 generations. The fitness increases in the 5 kPa E.E. stocks compared to the ancestor revealed a plateau in values at about 350 generations, and this coincided with a plateau in the competitions of the 5 kPa E.E. stocks against WN1106 (Fig. 4 1B). A similar flattening out of fitness gains has been reported during long term evolution of Escherichia coli ( 166 ) and
145 indicates the rate of fitness increases decelerated over time of the experiment despite the dynamic population fluctuations that a re evidenced by mutation analysis (Fig. 4 1A). Genomic Changes in B. subtilis After 5 kPa Long Term Exposure Whole genome re sequencing was conducted to investigate genomic changes in WN1106 that could be linked to the LP phenotype described above as well as to establish causation with differences in the transcriptional studies previously r eported ( Chapters 2 and 3, 152 ). T he genomic analysis of WN1106 revealed 8 mutations that all occurred in coding regions with no mutations determined to be in non coding regions (Table 4 3). Of these, it is believed that half may be neutral hitchhiker alleles that arose with beneficial mutations: bacD, yvlD, ytoI and parC . A fifth mutation, fliI , possibly affects motility though further analysis in a clean background with no other mutations is still needed to confirm no further involvement of the other mutations in motility. It is thought that this fliI mutation may reduce unnecessary energy expenditures in the cell as flagellar export and motilit y are both energetically ex pensive ( 181 ) . Mutations that resu lt in decreased or lack of motility have been reported previously in long term evolution experiments of B. subtilis (57, 63) . It may be that for long duration growth in flasks, motility is a first step to advantage ous growth no matter the environmental condition being stud ied . Transcription and Post Transcription Mutational Strategies for Low Pressure Growth Three of the mutations occurring in WN1106, two SNPs ( resD and walK ) and one 9 nt deletion event ( rnjB ), targ eted transcriptional and post transcriptional processes, respectively. The former two mutation s, both members of separate TCS , co occurred late in the 5 kPa E.E. in population B 1, which was the second rise to dominance of the rnjB 9 nt deletion subpopulati on B (harboring the fliI, parC and ytoI mutations) (Fig. 4 1A). It may be that one of these two mutations, or both, are the cause of the sweep of populat ion B1 in the growth culture, and the
146 other two mutations that also appear late in the experiment, yvlD and bacD could be hitchhiker mutations. Investigation into the resD mutation found that the region harboring the mutation is involved in protein protein interactions in homologous proteins that dimerize ( 190 , 200 ) . ResD, does not apparently form homodimers in solution. However, it can bind to multiple contiguous binding si tes at ResD dependent promoter regions. I t is thought that ResD monomers may bind cooperatively via intermolecular interactions at promoter regio ns with more than one binding site. While WN1106 has decreased fitness at low oxygen and standard pressure growth conditions compared to WN624 (86) , this does not rule out that the mutation in resD could be beneficial in low oxygen conditions coupled with low pressure. Further studies are needed to investigate this. WalK has been reported to respond to fluctuations in membrane fluidity and depletion of Wa lK in the cell activates the TCS DesKR, resulting in increased des transcription ( 187 ) . It was rep orted previously that WN1106 showe d increased transcription of des at 5 kPa but it was unclear what the underlying reason for this increase was, as localized sequencing of des and desKR did not reveal any mutations (86) . It may be that the underlying genomic explanation for up regulation of des at 5 kPa in WN1106 is due to the m utation in walK . The mutation in walK occurs in one of two transmembr ane helix regions (TM2) that makes intermolecule contacts with regulators of WalK kinase activity, YycI and YycH. Deletion of YycH results in increased expression of a target ( yocH lacZ ) of the WalKR system as evidenced by reporter gene fusion studies ( 188 ) . Under normal cellular conditi ons, WalKR targets are activated or repressed, depending on the target, during exponential growth. We have previously reported that at 5 kPa B. subtilis reaches a stationary phase like stage much earlier in
147 growth , but when cells are returned to standard pressure, there is an immediate return to exponential growth with no lag phase (34) . This could indicate that one or more exponential growth processes are being inhibited at LP; the mutation in walK could be a cellular target to overcome this inhibitory ef fect. Further work is still needed to fully clarify th e role each mutation in the TCSs play at LP. It is of int erest to note, that both TCS , ResDE and WalKR, have interactions and cross talk with, and in the case of ResDE transcription even requires, the p hosphate TCS PhoPR in B. subtilis ( 201 , 202 ) . The 9 nt deletion in rnjB , which results in the 3 amino acid in frame deletion in a highly helix region of the protein, could have a phenotype similar to a full gene deletion (Fig. 4 6). However, a full deletion of rnjB may not be as fit when grown at LP, suggested by the SNP in rnjB that results in a truncated gene (Fig . 4 1C). P opulation X, which overtook population A (Fi g. 4 1A and 4 1C), dominated the evolving LP culture by 800 generations. However, there was a sharp decline in the population X with the re emergence of a population carrying the rnjB deletion, populati on B1 (Fig. 4 1A and 4 1C). It would be fruitful for future work to determine the mutation (s) that were co occurred with the rnjB SNP, as any conjecture in relation to LP growth for this mutation alone may not currently be made.
148 Table 4 1. Bacillus subti lis strains and plasmids used in this study Strain or Plasmid Genotype/Phenotype Source (reference) WN624 trpC2, amyE::spc ; Spc R , Ancestral Strain (Maughan et al. 2006) WN628 trpC2, amyE::cat ; Cm R , Ancestral Strain (Maughan et al. 2006) WN1105 trpC2, amyE::cat ; Cm R , Evolved to enhanced growth at 101 kPa (Nicholson et al. 2010) WN1106 trpC2, amyE::spc ; Spc R . Evolved to enhanced growth at 5 kPa (Nicholson et al. 2010) WN1261 trpC2, amyE::neo ; Neo R , Ancestral Strain pECE141 WN624 (tf); this study WN1278 trpC2 , amyE::neo ; Neo R in WN1106 background pECE141 WN1106 (tf); this study WN1279 trpC2 , amyE::cat ; Cm R in WN1106 background pDAG32 WN1106 (tf); this study WN1518 trpC2 , amyE::neo, rnjB::spc ; Neo R , Spc R in WN624 ba ckground GP45 WN1261 (tf); this study WN1519 trpC2 , amyE::neo, rnjB::spc ; Neo R , Spc R in WN1106 background GP45 WN1278 (tf); this study GP45 trpC2, rnjB::spc ; Spc R Laboratory of J. Stulke 50gen 1000gen amyE::spc ; Spc R , Generations 50 1000 of the 5 kP a Evolution Experiment stocks [each 50 generations is equivalent to 1 week]; evolved from ancestor WN624 (Nicholson et al. 2010) pECE73 pCm::Neo antibiotic switching cassette BGSC (Steinmetz and Richter 1994) pECE141 pSpc::Neo antibiotic switching casset te BGSC (Steinmetz and Richter 1994) pDAG32 pSpc::Cm antibiotic switching cassette BGSC (Steinmetz and Richter 1994)
149 Table 4 2. Oligonucleotides used to amplify, Sanger sequence and verifiy mutations that were called during mapping; olignonucleotide u sed for q RT PCR Oligonucleotide bacD F3871584 AGAGCAGCACGGAAATCTTCA bacD R3872064 TCATCGCTGATCTTGGAGGC fliI F1695745 TGCTGATGAAAAAGCTCAAAAAGG fliI R1696207 GCGGTTCTCCAAAAGCATCG parC F1935866 ACACGGTTGAATTTGTGCCG parC R1936322 TCACGA ACCTCTGAGATGCC resD F2417342 GAAGCTTTTCCCGATCATACACC resD R2417791 ATGGTGATGAAGCCATTGCC rnjB+261F AACAAGCTGTCCGTTCCAGT rnjB+771R TTCCGGCTACGGCAATCTTT walK F4152887 CAGAGGCTAGCTTTCTGCGT walK R4153352 GTGGCTGGGAAACAAACGAC yto F2998178 TCATCCGTGTTAAAGC CCCC yto R2998587 ATCGATTCACTGCCTGTCGG yvlD F3606521 GCACAACAGAGGGAGTGCAA yvlD R3607016 GCCAGCCTCATTTTATCGATCTT yweA F GGATGCGCCTAACAAATCA yweA R GCTTTCTCTTGGAGCGGTAG fnr F GTGCGTGCTCATCCATTT fnr R CGGAAAAGACCTGACGCT yocH F GCCCAGTCGTGGTTGTT yocH R CGGAGCTCACGCTTCTG
150 Table 4 3. Mutations identified in WN1106 and the SNP occuring in rnjB Gene Name Position on Genome Mutation Amino Acid Change fliI 1695979 C A P 35 T rnjB 1749958 AGATCGCCA AKI parC 1936060 G C D 205 H resD 2417575 G A P 110 Q ytoI 2998392 C A V 77 F yvlD 3606764 A T Stop to K bacD 3871798 C T E 97 N walK 4153105 G A T 195 M rnjB 1749949 C A C to Stop
151 Table 4 4. WN624 Mutational Calls Different from the Bacillus subtilis strain 168 reference genome, b olded text corresponds to confirmed mutations identified in Dr. Ziegler's re sequencing of Bacillus subtilis ancestor strains Position on Chromosome Reference nucleotide Alternate Base Call Mapping Quality Score 52646 C T 9244.82 165747 ATC A 10025.58 1 65751 C T 8194.06 166037 A +T 10253.67 166343 AG A 4473.72 376017 AG A 7400.34 490580 GT G 1625.65 557865 G GT 8282.74 608214 G GA 5500.48 774697 CA C 6688.83 1073117 C A 8183.26 1073872 AT A 7564.19 1224524 T G 9080.62 1264284 T A 9563.02 1317 152 G GGTC 11709.7 1317153 C T 8466.93 1412484 T G 6992.04 1424639 T G 8686.59 1610896 T C 8782.54 1618068 C T 8242.85 1675849 C T 8492.49 1741603 AG A 7581.04 1756603 G A 8069.56 1764558 C T 8750.74 1841168 C CA 7328.45 2011091 G A 7529.73
152 Tab le 4 4. Continued Position on Chromosome Reference nucleotide Alternate Base Call Mapping Quality Score 2041099 A G 7510.75 2064175 T C 1646.16 2064176 C T 1583.7 2064263 C T 7250.25 2064314 T C 7206.08 2064395 C T 4123.26 2064413 A C 3109.67 20645 53 A G 6920.07 2064560 T C 8293.95 2096245 TA T 7775.71 2097080 C CA 7297.35 2174751 ACT A 10736.66 2201408 A G 8871.3 2271424 T C 7823.84 2271505 C T 7489.52 2271523 A C 7067.95 2366016 T C 9095.72 2421606 T C 8282.96 2480646 T A 6007.65 24806 53 CT C 10341.93 2480666 GT G 9997.16 2546153 TC T 5557.73 2560902 C T 9227.67 2581726 G GT 7136.33 2619105 C T 8527.75 2814468 C T 8298.04 2893906 G A 7607.76 2982417 T A 8788.13
153 Table 4 4. Continued Position on Chromosome Reference nucleotide Al ternate Base Call Mapping Quality Score 2982437 T C 8627.47 3051461 G A 7547.29 3178443 T C 4806.05 3253956 T C 7704.27 3319154 C T 8335.98 3391676 A G 7317.36 3527377 G A 6411.94 3696869 T C 6769.24 3770058 G GA 6711.24 3902306 C A 8525.92 3935 822 AT A 9154.91 3993539 G T 7496.79 4095811 C T 8998.6 4155390 C CA 6986.58
154 Table 4 5. Rifampcin resistance assay to determine differences in mutation rate of strains WN1106, WN624 and WN1105 Strain Repetition #RIF50 colonies/mL cells Frequency of Mutation (r/N) per 10^8 cells 624 A 1 0.16 B 5 0.83 C 0 0 1105 A 344 68.8 B 364 62.76 C 224 53.3 3 1106 A 5 1.56 B 20 3.70 C 32 4.10
155 Table 4 6. Flagstat data on mapped paired end reads Strain WN624 WN1105 WN1106 Total Reads 104979183 + 0 in total (QC passed reads + QC failed reads) 88573325 + 0 in total (QC passed reads + QC failed reads) 106043431 + 0 in total (QC passed reads + QC failed reads) Duplicate Reads 0 + 0 duplicates 0 + 0 duplicates 0 + 0 duplicates Mapped Reads 102249189 + 0 mapped (97.40%: nan%) 85924396 + 0 mapped (97.01%: nan%) 101084682 + 0 mapped (95.32%: nan%) Paired Reads 104979183 + 0 paired in sequencing 88573325 + 0 paired in sequencing 106043431 + 0 paired in sequencing Left Reads 52473457 + 0 read1 44268929 + 0 read1 53013438 + 0 read1 Right Reads 52505726 + 0 read2 44304396 + 0 read2 53029993 + 0 read2 Properly Paired Reads 99079843 + 0 properly paired (94.38%: nan%) 83801182 + 0 properly paired (94.61%: nan%) 99688928 + 0 properly paired (94.01%: nan%) Pair ed Reads Mapped 101628499 + 0 with itself and mate mapped 85308186 + 0 with itself and mate mapped 100387188 + 0 with itself and mate mapped Single Mapped Reads 620690 + 0 singletons (0.59%: nan%) 616210 + 0 singletons (0.70%: nan%) 697494 + 0 singletons (0.66%: nan%)
156 Table 4 7. Mutations called after mapping of WN1105; mutations were not confirmed. MutL is bolded Locus Position on Chromosome Mutation Amino Acid Change dnaA 799 T C H 130 H spoIIE 72999 A G Q 821 R yabT 74042 C T S 78 S ybbB [Btr Fe BB uptake system mediator] 184622 A G W 128 R ybbD 187877 G A G 168 G skfE 217129 A frameshift ybeC 231553 +G frameshift ybfF upstream 238499 A G ybfH 242745 C T L 31 L yceD 313054 T C V 92 A nin (comJ) 371902 T frameshift srfAB 388262 A G A 173 A ubiD/yclC 414360 A G T 402 A ydaN 485635 A G A 597 A rsbT 520965 G A A 120 A upstream ydzM 556161 T C upstream ydzN ( 8) inserted T 557865 +T ydeD 562933 T frameshift purB 700301 T C L 24 L ligA 723203 G A E 531 K yefA 738200 T C W 200 R yeeA 740651 G A D 122 N yetK 788815 A G S 60 S yfmJ 818307 C T G 175 S yflA 845583 G A G 272 R
157 Table 4 7. Continued Locus Position on Ch romosome Mutation Amino Acid Change yfjF 886074 C T G 34 S yfiN 908689 C T A 241 V trnD His/tRNA His 952491 T C yhcA 978794 C T L 340 L glpF 1003041 G A G 181 S glpD 1005343 T C I 123 I spoVR 1016668 A G E 341 G lytE 1019186 G A V 72 V yhaR 1062168 C T R 226 R pbpF 1084478 T C Y 210 H yhfS 1109920 A G L 188 S gerPE 1148864 C T G 94 G asnO 1158817 CA frameshift yitJ 1180303 A G V 50 A yjgC 1286640 T C F 350 F xkdE 1328273 TC frameshift downstream ykbA 1351299 A G mgtE/ykoK 1396947 A G Q 312 P ykrV 1426390 C T G 250 G kinD 1432492 A G V 170 A zosA/ykvW 1451758 A C T 181 P ykuL 1485555 C T H 35 Y ylxX 1595261 G A G 18 S cysP 1631887 A G T 265 A smc 1667527 A G E 323 G nusA 1732291 A G E 4 G mutL 1778783 T frameshift
158 Table 4 7. Continued Locus Position on Chromosome Mutation Amino Acid Change pksH 1791461 A G Y 90 C pksJ 1805756 T frameshift pksL 1817725 G A A 3269 T pksM 1828956 G A P 2468 P pksM 1832264 G A W 3571 Sto p thyA 1902940 A G E 241 G yndE 1909586 C T G 167 G citB 1927798 T C I 373 I ppsC 1977830 C T L 1573 L ppsA 1994262 C T W 1232 Stop yoeC 2003494 T C I to M proH 2016969 G A A 257 V yoaG 2028214 A G D 122 D downstream yozV 2055750 G A yobD 2056350 G A G 25 R upstream yobJ 2069810 T C yobT 2082249 T C H 69 R yocB 2086899 G A A 209 A yozD 2137716 A G L 21 L yomI 2250007 T C L 285 S ypgR 2303133 G A A to V yppC 2339807 T C K 319 E upstream asnC 2347596 G A ypbB 2408581 C T E 295 K yqkF 2459877 G A P 184 P bkdR 2505241 G A R 543 W accC 2530873 A G Y 288 Y
159 Table 4 7. Continued Locus Position on Chromosome Mutation Amino Acid Change downstream pbpA ( 8) 2581763 T yqeZ 2619105 C T A225A yr kN 2707590 C T P 155 L aapA 2767814 T C F 419 F yrhG 2779794 T C K 157 E yrhB 2785922 T C S 73 S spoOB 2854276 T C E 95 G yslB 2909201 T C W 58 R ysfB 2932814 C T T 280 M gapB 2967582 G A A 158 V ytvI 2983850 A G A 229 A ytpS (sf tA) 3051461 G A P 375 S ytjP 3068708 G A P 35 L ytpB 3121673 G A G 323 G upstream trnB Glu 3171701 A yubB (bacA) 3195223 T yuxJ 3232805 T C W 56 R yufQ 3244474 T C L 273 P comQ 3256067 T upstream guaC 3302852 G A yutJ 330956 0 G A G 232 S pucL 3334295 T C Y 378 Y yurH 3342469 G A R 401 R fhuD 3418621 G A G 50 R yvsG 3421814 A G T 15 A yvaQ 3458644 A G A 193 A yvfH 3511041 +T frameshift
160 Table 4 7. Continued Locus Position on Chromosome Mutation Amino Acid Cha nge epsN 3515638 +C frameshift yvdJ 3552010 G A S 118 S ggaB 3668454 T frameshift upstream alsR 3711441 G A ywqF 3730056 G A A 252 V ureA 3769012 G A P 33 S spoIID 3777268 G A A 162 A ywkA (maeA) 3802096 T frameshift fadF/ywjF 381617 5 C T A 109 T clsB/ywjE 3817672 T C F 340 S ywjA 3820368 T C K 372 E narI 3823608 G A P 208 S rocB 3878252 G A P 214 L sacA 3903337 T C S 105 G ywcC 3922961 C T Start M 1 I downstream ywaF 3945466 A menA 3951195 G A P 156 L yxlH 3967270 T C W 176 R yxjH 3998078 A frameshift yxiD 4037586 +T frameshift upstream yxiB 4039197 T C hutM 4048336 T C W 267 R yxeL 4060761 T C Q 15 R yxbG 4092622 G A G 260 R yxaH 4105950 G A A 63 V upstream rocR 4145579 C T
161 Ta ble 4 8. Differentially expressed signals of the ResDE and WalKR two component system and of the RnjA/RnjB complex (underlined values indicate a P value > 0.05) Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106/WN624 at 5kPa WN1106/WN624 at 101kPa Gene Description ResDE dependent Signals BSU03300 nasD 5.87 5 1.06 1.33 assimilatory nitrite reductase subunit BSU07380 yfmQ 6.94 9.7 1.16 1.69 conserved hypothetical protein BSU10130 hemH 5.09 8.68 1.86 1.16 ferrochelatase BS U10140 hemY 4.8 8.56 1.85 1.07 protoporphyrinogen IX and coproporphyrinogen III oxidase BSU14870 ctaA 2.93 4.03 1.58 2.33 heme A synthase BSU14890 ctaC 7.07 15.52 1.16 1.84 cytochrome caa3 oxidase (subunit II) BSU14900 ctaD 4.48 15.67 1.01 3.58 cy tochrome caa3 oxidase (subunit I) BSU14910 ctaE 4.81 16.19 1.04 3.5 cytochrome caa3 oxidase (subunit III) BSU14920 ctaF 5.07 18.74 1.06 3.48 cytochrome caa3 oxidase (subunit IV) BSU14930 ctaG 5.4 12.55 1.4 3.48 cytochrome aa(3) assembly factor BSU 17690 yncM 24.09 30.51 1.15 1.79 conserved hypothetical protein BSU21480 sunA 7.87 28.34 1.16 3.86 sublancin 168 lantibiotic antimicrobial precursor peptide in SPBeta prophage BSU23120 resD 5.42 1.97 2.37 1.23 two component response regulator BSU231 30 resC 4.72 1.62 2.48 1.06 factor required for cytochrome c synthesis BSU23140 resB 5.96 1.05 2.83 2.15 f actor required for cytochrome c
162 Table 4 8. Continued Accession BSU number Gene Name WN624 at 5kPa/101kP a WN1106 at 5kPa/101kPa WN1106/WN 624 at 5kPa WN1106/WN624 at 101kPa Gene Description BSU23150 resA 4.51 1.29 3.43 1.64 extracytoplasmic thioredoxin involved in cytoc hrome c maturation BSU30660 ytkA 6.5 8.8 1.33 1.02 putative lipoprotein BSU35310 yvyD 7.69 17.41 1.15 2.55 ribosome associated sigma 54 modulation protein BSU37250 narI 57.08 246.83 7.68 1.01 nitrate reductase (gamma subunit) BSU37260 narJ 44.17 208.21 7.17 1.01 nitrate reductase (protein J) BSU37270 narH 54.93 228.4 6.67 1.04 nitrate reductase (beta s ubunit) BSU37280 narG 38.15 199.34 8.19 1.07 nitrate reductase (alpha subunit) BSU37310 fnr 19.63 20.15 1.55 1.56 transcript ional regulator (FNR ) BSU37320 na rK 10.14 22.45 1.66 1.24 nitrite extrusion permease BSU38060 ywcJ 3.83 5.44 1.32 1.01 formate/nitrite transporter BSU38730 cydD 13.06 31.86 1.66 1.21 ABC membrane t ransp orter required for cytochrome bd function BSU38740 cydC 48. 69 83.98 1.33 1.23 ABC membrane t r ansporter req uired fo r cytochrome b d BSU38750 cydB 40.17 42.31 1.19 1.26 cytochrome bd ubiquinol oxidase (subunit II) BSU38760 cydA 27.64 27.67 1.01 1.01 cytochrome bd ubiquinol oxidase (subunit I) BSU39940 yxaL 5.66 23.18 1.43 3.14 membrane ass ociated protein kinase
163 Table 4 8. Continued Accession BSU number Gene Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106/WN624 at 5kPa WN1106/WN62 4 at 101kPa Gene Description WalKR Regulat ed Signals BSU06250 ydjM 1.83 2.94 5.45 1.03 conserved hypothetical protein BSU09420 lytE 1.43 2.24 1.59 2.48 cell wall hydrolase; phosphatase associated protein (major autolysin) BSU12100 yjeA 2.87 2.8 4.88 1.26 secreted deoxyriboendonuclease BSU 14170 ykuP 22 12.46 1.07 2.06 short chain flavodoxin BSU18380 yoeB 7.97 3.09 2.44 1.08 inhibitor of cell separation enzymes BSU19180 des 4.19 17.18 5.98 1.25 fatty acid desaturase BSU19210 yocH 1.76 4.32 4.8 1.9 putative exported cell wall binding protein BSU31960 dhbF 11.56 7.66 1.44 1.12 siderophore 2,3 dihydroxybenzoate glycine threonine trimeric ester bacillibactin synthetase BSU34800 yvcE 1.38 1.02 2.22 1.66 secreted cell wall DL endopeptidase BSU39230 wapA 1.13 4.48 2.72 1.45 cell w all associated protein precursor RnjB Related Signals BSU00490 spoVG 2.89 6.91 1 2.44 regulator required for spore cortex synthesis (stage V sporulation) BSU09280 glpF 26.3 20.82 1.65 2.47 glycerol permease BSU09370 lytF 1.42 1.02 2.67 1.97 gamma D glutamate meso diaminopimelate muropeptidase BSU10380 hemAT 2.19 1.14 2.97 1.52 haem based dioxygen sensor BSU12120 yjfB 1.18 1.84 4.72 8.67 conserved hypothetical protein
164 Table 4 8. Continued Accession BSU number Ge ne Name WN624 at 5kPa/101kPa WN1106 at 5kPa/101kPa WN1106/WN624 at 5kPa WN1106/WN624 at 101kPa Gene Description BSU13690 motA 1.67 1.23 3.13 2.44 motility protein A; MotA component of the stator flagellum complex BSU14010 cheV 1.42 1.24 2.96 2.65 cou pling protein and response regulator for CheA activity in response to attractants (chemotaxis) BSU16080 ylqH 1.77 1.79 2.36 1.33 putative flagellar biosynthesis protein BSU19750 cgeE 1.38 1.19 2.36 2.14 protein involved in maturation of the outermost layer of the spore BSU31260 mcpB 1.6 1.52 3.77 1.72 methyl accepting chemotaxis protein BSU33770 yvaY 12.75 25.53 1.58 1.26 killing factor SdpC BSU35360 hag 1.34 1.68 6.64 15.49 flagellin protein BSU37800 yweA 1.6 6.64 3.98 2.73 member of th e processed secretome
165 Figure 4 1. A) Proportion of fluorescent intensity of each mutant allele to total population in WN1106; determined from re sequencing chromatograms as described in materials and me thods section of this chapter. B) Relative fitne ss of ancestor strain (open circles) and LP evolved strain (filled circle) to each of the 50 generation back stocks from the 5 kPa E.E. Error bars are the standard deviation of averages of duplicate cultures. C) The SN P in rnjB fluorescent proportion chang es in the 5 kPa E.E.
166 Figure 4 2. Representation of population percentages during the 5 kPa E.E. Population A refers to the cell line (s) representing the first rnjB 9 nt deletion sweep. Population B refers to the subpopulation of Population A, which con tains, in addition to the 9 nt rnjB deletion, the parC and ytoI SNPs. Population X refers to the cell line (s) that overtook Populations A and B, containing the rnjB SNP resulting in a truncated coding region. Population B1 represents the cell line (s) tha t represent the second sweeping dominance of the 9 nt rnjB deletion and also possessing the mutations: parC, fliI, resD, bacD, yvlD, ytoI, and walK .
167 Figure 4 3. Alignment of rnjB fragments from WN1106 and the 5 kPa E.E. stocks: g refers to generations. Image shows the 9 nucleotide deletion and reveals a SNP at position 1749949 during 800 generations (yellow box).
168 Figure 4 4 . Moti lity agar showing growth of WN1106 and WN624 at either (i) 101 kPa and 37Â°C after 6 hour of incubation and (ii) 5 kPa and 27Â°C after 24 hours of incubation.
169 Figure 4 5. q RT PCR of yweA, yocH, and fnr expression ratios in WN1106 to WN624 at 5 kPa (gray bars) and 101 kPa (striped bars). Error bars represent standard error of averages of technicial triplicates of experimenta l duplicates. All differences in expression of the same gene at the two different pressures were significant (ANOVA; P < 0.05).
170 Figure 4 6. Visualization of the RnjA/RnjB heterodimer: RnjA (cerulean) and RnjB (orange). Helix 5 of each protein is highli ghted (RnjA yellow, RnjB green); the RnjB protein contains the mutation and reveals a disruption to the formation of helix 5.
171 Figure 4 7. Competitons of rnjB ::spc knockout mutants versus their wildtype strains of both A) WN624 and B) WN1106. Error bars represent standard deviations of triplicate average values.
172 CHAPTER 5 SUMMARY OF RESEARCH Studies of how microorganisms respond to pressure have been limited mostly to the extreme high pressures of the deep sea (i.e., the piezosphere). In contrast -despi te the fact that the growth of most bacteria is inhibited at pressures below ~2.5 kPa -little is known of microbial responses to low pressure (LP). To study the global LP response, we performed transcription microarrays on Bacillus subtilis cells grown und er normal atmospheric pressure (~101 kPa) vs. a near inhibitory LP (5 kPa), equivalent to an altitude of ~20 km. Microarray analysis revealed altered levels of 363 transcripts belonging to several global regulons (AbrB, CcpA, CodY, Fur, IolR, ResD, Rok, Si gH, Spo0A). Notably, the highest number of up regulated genes, 86, belonged to the SigB mediated General Stress Response (GSR) regulon. This is the first report of transcriptome changes resulting from exposure of ba cterial cells to LP, and gives insights i nto cellular processes that may respond to LP exposure. As previously described in Nicholson et al., 2010, a strain of Bacillus subtilis 168, isolated from a experimental evolution growth culture exposed to 5 kPa for ~1,000 generations, WN1106. The LP evol ved strain was shown to have an increased growth rate and fitness coefficient c ompared to the ancestral st rai n of the experiment, WN624. Evidence reported here reveals , by transcriptional microarray experiments, how the LP evolve d WN1106 differentially res ponded to growth at 5 kPa compared to the ance stor WN624. In total, over 1,000 signals coding for known and unknown functions were dif ferentially expressed across the microarrays. Functions of these signals correspond to what has previously been shown in t ranscriptional studies of non optimal pressure growth: (i) pressure mesophiles grown at high pressure and (ii) piezophiles (microorgansism of the deep sea, i.e. piezosphere) grown at standard atmospheric pressure. These differential expression signals incl ude anaerobic respiration, oxidative stress,
173 changes in respiration chain expression, permease expression, deoxy and ribonucleotide associated proteins, stress proteins (including those belonging to the SigB dependent General Stress Response), fatty acid pathway prot eins, metal uptake, and a large number of proteins with unknown functions. Because we were interested in the role of the SigB dependent GSR induction at LP, further investigation was conducted by monitoring the expression of the sigB dependent ctc lacZ reporter fusion in wild type strains of ancestor WN624 and LP evolved WN1106, as well as strains harboring a sigB::spc knockout mutation . It was also determined that LP evolved strain WN1106 exhibited a more sensitive induction of the response at higher pressures than the ancestor strain WN624 did . The underlying reasons behind this difference in induction pattern are currently unknown, but are the concern of future research. One conjecture is that ch anges in membrane fluidity could be the initial signaling pathway for induction of the GSR at LP ; if this is the case, then any mutation affecting membrane signaling and composition (i.e. WalK) might be an underlying reason behind differences in LP induction of the GSR between the two strains as pressur e is lowered . In addition to the LP response, k nowledge of how microo rganisms adapt to LP environm ents is limited. During a 1,000 generation evolution experiment at 5 kPa, Bacillus subtilis evolved to low pressure growth as evidenced by increases in weekly average optical density readings . G enomic differences in WN1106 were investigated using whole genome re sequencing . This resulted in the identification of 8 geno mic changes in strain WN1106 , all in coding regions, compared to the ancestor WN624: 7 single nucleotide polymorphisms (SNPs) and one 9 nucleotide deletion event. Four of the mutations ( fliI, parC, ytoI, and rnjB 9nt)
174 occurred early in the 5 kPa E.E.; the other four mutations ( walK, resD, yvlD, and bacD ) occurred late, only being detected during th e last 150 generations of the experiment. Based on the proportion of mutant fluorescent intenties throughout the experiment, it is known that there was a cell line (s) which harbored all four of the early mutations by approximately 550 generations. This p opulation was not dominant early in the E.E. and only rose to dominance during the last 150 generations of the experiment with the corresponding sweeps of the late occuring four mutations. It stands to reason that one or more of these latter mutations was the underlying reason for the sweep. Previous experimental evolution studies have shown that the two most influential factors on mutational sweeps in haploid populations are population size and mutational fitness coefficien ts (203, reviewed in 204 ); geneti c drift, alone, does not explain the rise of the mutations in resD, walK, yvlD and bacD . Neither does the assumption that all of the four mutations may be neutral or deleterious hitchhikers; at least one of these mutations must have conferred some sort of fitness advantage to allow for a rapid rise to dominance. Competition analysis would determine the imp ortance of each individual late occu rring mutation (or combinations of mutations) in a cell line harboring the early four mutations in regards to LP growt h. Two of the se late occuring SNPs arose in sepa rate two component systems (ResDE and WalKR); and the 9 nt deletion resulted in a 3 amino acid in frame deletion within RnjB, a protein involved in global mRNA processing in B. subtilis . These three mutation s , along with the identification of a sepa rate cell line (s) harboring the additional rnjB SNP, seem to indicate that transcriptional and post transcriptional processes as possible targets of LP adaptation. One avenue of thought for this involves the possi ble decrease in translation efficiency caused by LP effects on the quaternary structure of ribosomes . A decreased translation rate could possibly be
175 offset by an increase in mRNA pools resulting from mutations in the RNase J1/J2 complex . It is known that h igh pressure destabilizes quate r a ny structures, such as the ribosome (3). If LP also causes molecule molecule interactions to be weakened in the ribosome, this could reduce the translation into protein products of a number of important mRNA substrates. Mea suring the translation efficiency at LP would test the validity of this argument, as well as doing proteomic studies to compare increases in mRNA pools with corresponding protein product levels. In the case of the WalK mutation, it has been reported previo usly that at LP B. subtilis prematurely enters stationary phase, as indicated by growth curves (34). The WalKR TCS is active during exponential growth phase and essential to the cell. It is thought that LP has a number of inhibitory effects on microbial pr ocesses, and one or more of these inhibitory effects may affect exponential growth. The mutation in WalK may interfer e with binding of YycI and subsequent modulating interactions . If so, testing a deletion mutation of YycI in the ancestor strain WN624 at L P for relative fitness gains and the mRNA levels of yocH lacZ would support this reasoning to the mutation being beneficial in B. subtilis at LP. Overall, m utations were analyzed in silico and in some instances, efforts were made to link observed phenotyp es of WN1106 with the newly identified mutations. For example, one SNP occurred in the N terminal coding region of the flagellar ATPase, fliI , which is in mutational analysis of Salmonella indicate this region to be important for proper flagella export. Mo tility experiments reveal ed that WN1106 exhibited decreased motility compared to WN624, consistent with an impaired FliI. The results suggest that continued exposure of B. subtilis for only 3 months (i.e. 1,000 generations) to a LP at which it initially gr ows poorly was sufficient to induce genomic changes resulting in better LP growth.
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193 BIOGRAPHICAL SKETCH Samantha Waters has always held an interest in the field of as trobiology, and more specifically on the focus of extreme organisms, their environment, and evolutionary changes that ove rcome the extreme conditions; t his le d to her contacting Dr. Wayne Nicholson at the University of Florida with interest in his lab, and after accepting a PhD position to work on how a model microorganism transcriptionally responds to and genomically adapts to low pressure growth. Samantha successfully obtained a NASA Earth and Space Science Fellowship from 2013 2014 to fund her research. As a young career researcher, she has represented her field at the Astrobiology Graduate Student Conference (2011 2013), internationally at Astrobiology Science Conference (2012), and regionally at the Southeastern and Florida Branches of the American Soci ety of Microbiology (2011 and 2012, respectively). Samantha received her PhD from the University of Florida in the summer of 2014.