CATECHOL 2,3 DIOXYGENASE ASSISTED CLEAVAGE OF AROMATICS BY ANAEROBIC TERMITE GUT SPIROCHETES AND GENOMIC EVIDENCE OF A COMPLETE META PATHWAY

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1 CATECHOL2,3 DIOXYGENASE ASSISTEDCLEAVAGEOFAROMATICSBY ANAEROBIC TERMITEGUTSPIROCHETESANDGENOMICEVIDENCEOFA COMPLETEMETA PATHWAY Thesisby KaitlynShaeLucey InPartialFulfillmentoftheRequirements FortheDegreeof DoctorofPhilosophy CaliforniaInstituteofTechnology Pasadena,California 2014 (DefendedJune13,2013)

2 i

3 ii 2014 KaitlynShaeLucey AllRightsReserved

4 iii ACKNOWLEDGMENTS AsayoungchildIgrewupinSouthBoston,orSouthie.MyfamilyandIlivedina brickhousethatstoodonthetopof PillHill (namedforthedoctorsanddentists whobuilthomesandpracticedtherearoundtheturnofthelastcentury)atthe centerofthemainstreetoftheneighborhood,broadway.dependingonawesterly oreasterlydirection,icouldfollowbroadwaytothepublichousingprojectsorto Bostonproper anepicenterofcultural,artistic,andscholarlydiversity.myhouse wasapivotalpointfromwhichmylifebegan,andmyearlychildhoodinsouthieleft anindeliblemarkmotivatingmetocontinuallychallengemyselfandstrivefor betterwhilesimultaneouslygivingback. Accordingly,firstandforemostIwouldliketothankmymother,father,andyounger brother,gerald,whohavebelievedinmypotential,givenmetherightbalanceof guidanceandfreedomtochoosemyownpath,andbeengenuinelyinvestedinmy successfromthestart.membersofmyextendedfamilyhavealsobeenunparalleled rolemodelsandsupporters,andiwouldliketothankmygrandparents,william SpencerandJanetVanDyke,mylategrandmother,BettyLucey,myGodfather, MichaelOwens,andmyAuntCarole,UncleJim,AuntLynn,UncleGreg,UncleDave, anddaveroberts. BeginningatWellesleyCollege,Iembarkeduponapathinthesciencesinhopesof bothcontributingsolutionstothestem relatedchallengesoursocietyand

5 iv environmentisfacing,andinspiringotherstoengageinstemalso.myprofessors andmentorsatwellesleycollege,specificallyprofessordaveellerbyandprofessor MaryAllen,wereintegralinhelpingmeinitiatemypathinthesciences.Iwould alsoliketothankwellesleycollege,ingeneral,forfocusingonundergraduate learning,includingalaboratorycomponentwithnearlyeveryscienceclass,and providingundergraduateswithoriginalresearchopportunitiesandprominent responsibilityinthelab.myundergraduateexperiencehasplayedasignificantrole inwhereiamtodayandwhereiamgoing. AtCaltech,formerlabmembersLizOttesenandEricMatsonalsohelpedkick star myresearchbysharingmanymolecularandphysiologicalskillsets.iwouldalso liketothankbothxinningzhangandadamrosenthalwho,inadditiontosharing withmemuchknowledge,havebeenstellarmentors.andofcourse,iwouldliketo thankjaredleadbetter.ihavelearnedsomuchaboutbeingagoodscientistanda goodteacherworkinginyourlabandwithyou.ta ingmicrobialphysiology,the Asilomarconference,andpreparingmypublicationhavebeenparticularly significantlearningexperiences.iamgrateful,too,tovictoriaoprhan,sarkis Mazmanian,andDianneNewmanforservingonmythesiscommittee.Ithasbeen anabsolutepleasureworkingwithyouandwithstudentsandstaffinyourlabs,and Iamthankfultohavehadtheseopportunities. IalsohavedevelopedsomeamazingfriendshipsatCaltechthatIhopewillextend farbeyondourtimehere.first,iwouldliketothankelitzatocheva,nathan

6 v Dalleska,andFelicaHuntforbeingsuchgreatmentorsandrolemodels,inaddition tofriends.iwouldalsoliketothankchinnyidigo,megschwamb,mandygrantz, andkateschillingfortheirfriendshipsincepracticallydayonehereatcaltech,as wellasmynewestfriendsmollyandlunafortheirencouragingandsupportive purrs.lastly,iwouldliketothanklosangeles.fromthemajesticsangabriel Mountains,totheunpredictablePacificCoast,Iamveryfortunatetohavespentthe pastfiveyearsinsuchaninspirationalsetting. Iamgratefulforthetop notcheducationandlaboratoryresearchopportunitiesi havehadtodate,andwillcontinuetostrivetomovethefieldofenvironmental microbialforwardaswellasteachandmentorthenextgenerationofmicrobial ecologists.attheveryleast,ihopetoinstillinothersatechnologicalliteracyand appreciationforscienceeveniftheydonotalsopursuestemcareers.thosewho havebeenwithmealongthisjourneyhavebeenexcellentteachers,mentors, scientists,androlemodels,andiaspiretoserveotherslikeyouallhaveinspiredme.

7 vi theterrestrialenvironment,andrepresentsanarrayofmicrobialmetabolic diversity.decipheringtheintricaciesofthismicrobialcommunitytoobtainas completeapictureaspossibleofhowitfunctionsasawhole,requiresa combinationofvarioustraditionalandcutting edgebioinformatic,molecular, physiological,andculturingapproaches.isolatesfromthisecosystem,including Treponemaprimitiastr.ZAS 1andZAS 2aswellasT.azotonutriciumstr.ZAS 9, havebeensignificantresourcesforbetterunderstandingthetermitesystem.while notallfunctionspredictedbythegenomesofthesethreeisolatesaredemonstrated invitro,theseisolatesdohavethecapacityforseveralmetabolismsuniqueto spirochetesandcriticaltothetermitesystem srelianceuponlignocellulose.inthis thesis,workculturing,enrichingfor,andisolatingdiversemicroorganismsfromthe termitehindgutisdiscussed.additionally,strategiesofmembersofthetermite hindgutmicrobialcommunitytodefendagainsto2 stressandtogenerateacetate, dioxygenaseandothermeta cleavagecatabolicpathwaygenesaredescribedinthe anaerobic termitehindgutspirochetest.primitiastr.zas 1andZAS 2,andthe firstevidenceforaromaticringcleavageinthephylum(division)spirochetesisalso ABSTRACT Thetermitehindgutmicrobialecosystemfunctionslikeaminiaturelignocellulosemetabolizingnaturalbioreactor,hassignificantimplicationstonutrientcyclingin the biofuel ofthetermitesystem,areproposed.inparticular,catechol2,3 presented.theseresultssuggestthatthepotentialforo2 dependent,yetnonrespiratory,metabolismsofplant derivedaromaticsshouldbere evaluatedin termitehindgutcommunities.potentialfutureworkisalsoillustrated.

8 vii TABLEOFCONTENTS Acknowledgments... iii Abstract...vi TableofContents....vii ListofTables.....viii ListofFigures.....ix Chapter1:Researchwithtermitehindgutmicroorganisms&background Abstract..1 1 Introduction MaterialsandMethods Results Discussion References Appendix 1 44 Chapter2:Exploringgenomicevidenceofacetogenicdemethylationby Treponemaazotonutriciumstr.ZAS Abstract..2 1 Introduction MaterialsandMethods Results Discussion References Appendix 2 26 Chapter3:Catechol2,3 dioxygenaseandothermeta cleavagecatabolic pathwaygenesinthe anaerobic termitegutspirochetetreponemaprimitia Abstract..3 1 Introduction MaterialsandMethods.3 4 Results Discussion References Appendix 3 57 Chapter4:Conclusions.4 1 References

9 viii LISTOFTABLES Chapter1 Table1 1:Appearanceofbaker syeastandappearanceandodorofcorresponding yeastautolysateandautolysatesupernatant. Table1 2:GrowthratesofTreponemaprimitiastr.ZAS 2on4YAComedium preparedfromyeastautolysatesofdifferentbrandsandpackagingtypes. Table1 3:Treponemaprimitiastr.ZAS 2enzymaticdefensesagainstoxidative stress. Chapter2 Table2 1:MethylatedcompoundstestedforgrowthonbyTreponema azotonutriciumstr.zas 9. Chapter3 Table3 1:Treponemaprimitiastr.ZAS 1andZAS 2meta cleavagepathwaygenes andpfamfeatures. Table3 2:SelectionpressureanalysisofTreponemaprimitiastr.ZAS 1andZAS 2 meta cleavagepathwaygenes. Table3 3:MonoaromaticcompoundcleavagebyTreponemaprimitiastr.ZAS 1. Table3 4:AveragedepthofO2penetrationinTreponemaprimitiastr.ZAS 1and ZAS 2O2/aromaticgradientcultures.

10 ix LISTOFFIGURES Chapter1 Fig.1 1:Aphylogenetically lower termite,adampwoodtermitezootermopsis nevadensisworker,andadissectedintestinaltractofanotherz.nevadensisworker specimen. Fig.1 2:Phylogramof higher and lower termitefamiliesaswellashousehold andwoodroaches. Fig.1 3:Aphylogenetically higher termite,agnathamitermessp.worker,anda dissectedintestinaltractofanotherworkergnathamitermessp.specimen. Fig.1 4:Anexampleofthestructureoflignin. Fig.1 5:Generalschemeunderlyingthesymbiosisbetweenwood feeding lower termitesandtheirmutualistichindgutmicrobiota. Fig.1 6:RadialprofileofO2(andH2)inthehindgutofthewood feedingtermite Reticulitermesflavipes(figurefromBrune&Friedrich2000). Fig.1 7:Treponemaazotonutriciumstr.ZAS 9(a)andT.primitiastr.ZAS 2(b). Fig.1 8:Spirochete likemorphologiesthatdominatedliquidenrichmentcultures foracetogenicspirochetes.(a) Floppy morphology.(b)whatwouldbeisolated astreponemaprimitiastr. ZNS 1. Fig.1 9:Proposedmechanismsforcombatingoxidativestressinthetermite hindgut. Fig.1 10:GeneneighborhoodsofenzymesinvolvedinO2transformation. Chapter2 Fig.2 1:Acetyl CoA(Wood Ljungdahl)pathwayforCO2 reductiveacetogenesis. Fig.2 2:Treponemaazotonutriciumstr.ZAS 9geneneighborhoodswithboth putativemethyltransferasesandhomologsoftheacetyl CoA(Wood Ljungdahl) pathwayofacetogenesis. Fig.2 3:GrowthofTreponemaazotonutriciumstr.ZAS 9withvarious meth(ox)ylatedcompounds.

11 x Chapter3 Fig.3 1:Catechol2,3 dioxygenase basedmeta cleavagepathwayofaromatic metabolismandshunts. Fig.3 2:Geneneighborhoodsrepresentingcompletecatechol2,3 dioxygenasebasedmeta cleavagepathways. Fig.3 3:(a)PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 catechol2,3 dioxygenase(pf00903,step1).(b)exampleofmusclealignment usedtocreatetrees. Fig.3 4:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 2 hydroxymuconicsemialdehydehydrolase(pf00561,step2). Fig.3 5:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 22 oxopent 4 enoatehydratase(pf01557,step3). Fig.3 6:MicrooxicgrowthofTreponemaprimitiastr.ZAS 1. Fig.3 7:Treponemaprimitiastr.ZAS 1O2/catecholgradientculturesandcontrols.

12 1 1 CHAPTER1 Researchwithtermitehindgutmicroorganisms&background ABSTRACT: Microbialecologyresearchrequiresacombinationoftraditionalandcutting edge bioinformatic,culturing,physiological,andmolecular,aswellasinvitro,invivo,and insituapproaches.severalofthesetechniqueswereutilizedtoobtaintheresults presentedhere,whichprovideafoundationforresearchdiscussedinsubsequent chapters.first,toimprovegrowthratesofthetreponematermitehindgutisolates thathavebeencontinuouslypassagedforoveradecadein4yacomedium,growth wasevaluatedonmediapreparedwithyeastautolysatefromdifferentbrandsand packagingofbaker syeast.whiledoublingtimeonmediumwithredstaryeast autolysatehadincreasedfrombetween22and35hrstobetween54and77hrssince thetreponemaisolateswereinitiallyobtained,growthratesonmediumwith Fleischmann syeastautolysatemarkedlyimprovedto47hrsfort.primitiastr.zas 2.Next,inanefforttoobtainadditionaltermitehindgutmicrobialcommunity membersinpureculture,successfulenrichmentculturesofmethanogenswere preparedinitiallyasaprimerintheenrichmentandisolationprocedure.thena spirochete,t.primitiastr. ZNS 1, wasisolatedfromthezootermopsisnevadensis termitehindgut.thisstrainismorphologicallysimilartot.primitiastr.zas 1and ZAS 2,shares100%16SrRNAsequenceidentitywithT.primitiastr.ZAS 2and99% 16SrRNAsequenceidentitywithT.primitiastr.ZAS 1,andexhibitspreliminary physiologicalevidenceofh2+co2acetogenesis.effortstoisolatethetermite hindgutmicrobialcommunitymemberencodingthe ZnD2Sec phylotyope,the

13 1 2 phylotypethatisresponsibleforthemajorityoftheformatedehydrogenase expressioninthetermitehindgut,however,wereunsuccessful.nevertheless, enrichmentculturestargetingthisorganismweresuccessfullyusedastemplatesfor microfluidicdigitalpcrandhelpedcorrelatethe ZnD2Sec phylotypewitha deltaproteobacteriaandnotaspirocheteaspreviouslythought.finally,t.primitia str.zas 1andZAS 2lackhallmarkmechanismsofdealingwithoxidativestress, specificallycatalaseandsuperoxidedismutase.itislikelythattheyhaveother strategiesandrelationshipswitho2intheirenvironmentthatareexploredhereand alsorelevanttoworkdiscussedinchapter3.muchoftheresearchpresentedinthis chapterhasthepotentialforcontinuedworkinthefuture. INTRODUCTION: Termitesandtheirmutualistichindgutmicrobiota Betrayingtheirsmallsize,termitesareorganismswithabigimpact.Theirinfluence onecosystemsarisesfromboththeiruniquebehaviorpatternsandtheirnumerical abundance(wood&sands1978).photosyntheticfixationofco2yieldstheearth s mostabundantformofbiomass,lignocellulosicplantmaterial(breznak&brune 1994).Termites,aswellasonlyafewotherarthropods,havetheuniqueabilityto metabolizelignocellulosefromlivingplantsandplantmaterialsatvariousstagesof decomposition(wood1976;brune1998a).thisincludeswood,grasses,roots,and soilorganicmateramongotherdiversesubstrates(wood1976;brune1998a). Moreover,whiletermitesareentirelyterrestrialandarerestrictedtowithin45 N andslatitudes,aridregions,andaltitudesunder3000m,theycovermorethanhalf oftheworld slandsurface(wood&johnson1986).therefore,termiteshavebeen

14 1 3 recognizedasecosystemengineers,facilitatingthedecompositionoftheplanet s mostabundantyetenzymatically recalcitrantbiopolymersandotheraspectsofsoil function(breznak&brune1994;sugimotoetal.2000). Earlystudiesinvestigatinghowtermitesmetabolizelignocelluloseintonutrients andenergyobservedthataconsiderableportionofthetermite sbiomassisdevoted toitsintestinaltractasistypicalofanimalsthatconsumematerialsthataredifficult todigest(fig.1 1)(Wood&Johnson1986). Fig.1 1:Aphylogenetically lower termite,adampwoodtermitezootermopsis nevadensisworker,andadissectedintestinaltractofanotherz.nevadensisworker specimen.thetermiteintestinaltractrepresentsalargeportionoftheinsect s biomass(wood&johnson1986).specimencollectedfromsangabrielmountains, Pasadena,CA.Scalebarrepresents25mm.Bothimagesaretothesamescale. (FigurecourtesyoftheLeadbetterLab). Withinthisintestinaltract,andespeciallyinthelargestandmostdilatedregion,the hindgut,acomplex,obligate,andnutritionally symbiotichindgutmicrobial

15 1 4 communitywasdiscovered(fig.1 1)(Leidy1877;Cleveland1926;Hungate1955; Yamin&Trager1979;Yamin1980).Althoughtermitesareinfamousfortheir abilitytodevourwoodanddocontributeenzymestolignocellulosedigestion,itis theircomplexmicrobialcommunitycomprisingupwardsof10 6 microorganisms andrepresentingover200speciesthatisintegraltometabolizinglignocelluloseinto nutrientsandenergytosupportthetermitesystem(leidy1877;cleveland1926; Hungate1955;Yamin&Trager1979;Yamin1980).Whilethesymbioticassociation oftermiteswithmicroorganismscomprisesdifferentlevelsofinteraction,ranging fromtheextracorporalcultivationoffungusgardenstothemostintimate associationswherebacteriaresideintracellularly,themajorityoftheprokaryotic symbiontsoftermitesarelocatedintheintestinaltracteitherfree living,attached tothegutepithelium,orassociatedwithintestinalprotozoa(brune2006). Astermitesarerecognizedfortheirunusualabilitytometabolizelignocellulose,the metaboliccapabilitiesofthesemicroorganismsarealsounique.together,termites andtheirhindgutmicrobiotaareprimeexamplesofminiaturenaturalbioreactorsin theterrestrialenvironment(brune1998a). Higher and lower termitelineages TermitesbelongtotheorderIsopteraconsistingofover2000livingspecies representedbysevenfamilieswhosebiology,behavior,andnutritionarediverse (Breznak&Brune1994).Membersofthefirstsixfamiliesarereferredto collectivelyasthe lower termitesandmembersoftheseventhfamilyarereferred toasthe higher termites(fig.1 2).

16 1 5 Fig.1 2:Phylogramof higher and lower termitefamiliesaswellashousehold andwoodroaches.phylogenetically higher termites,themostspecies richand abundantlineageoftermites,arerepresentedbyonefamily,and lower termites arerepresentedbysixfamilies.muchmoreisknownabout lower termitesthan higher termites(wood&johnson1986;breznak&brune1994).treeadapted from,andbasedupon,phylogeneticanalysesreportedbyinwardetal Although higher termitescompriseonlyonefamily,theyarethemostspecies rich andabundantofthetermitelineages. Higher termitesaremoreevolvedthantheir lower termitecounterparts(fig.1 2),anddistinctionsarealsoseenwithrespect tointestinaltractarchitecture(figs.1 2and1 3),gutmicrobialcommunity composition,andfoodpreference(wood&johnson1986;breznak&brune1994).

17 1 6 Fig.1 3:Aphylogenetically higher termite,agnathamitermessp.worker,anda dissectedintestinaltractofanotherworkergnathamitermessp.specimen.the intestinaltractsof higher termitesaremorecompartmentalizedthanthoseof lower termites(wood&johnson1986;breznak&brune1994).specimen collectedfromsubterraneannestsatjoshuatreenationalpark,ca(ottesen& Leadbetter2011).Scalebarrepresents25mm.Bothimagesaretothesamescale. (FigurecourtesyoftheLeadbetterLab). First,thegutarchitectureof higher termitesismorecomplexthanthatof lower termites,withtheintestinaltractsof higher termitesbeingmore compartmentalizedthanthoseof lower termites(figs.1 2and1 3).Also,thegut microbialcommunityof lower termitesconsistsofmembersrepresentingallthree domainsoflife,thebacteria,archaea,andeukaryasuchascellulolyticoxymonad, trichomonad,andhypermastigoteprotozoa(wood&johnson1986).incontrast, thegutmicrobialcommunityofmost higher termitesismadeupofbacteriaand archaeaonly.thesedifferencesarealsoreflectedingeneraldiettrendswith lower termitesgenerallyconsuminglignocellulosicplantmaterialsatearlystages

18 1 7 ofdecomposition,and higher termitesgenerallyconsuminglignocellulosicplant materialsthathavebeenmodified(wood&johnson1986).somespeciesof phylogenetically higher termitesalsometabolizelignocellulose,butwithout polysaccharide fermentingprotozoalsymbionts(wood&johnson1986;warnecke etal.2007).muchlessisknownabout higher termitesthan lower termites, particularlyconcerninghowtheymetabolizediverselignocellulosicsubstrates. Lignocellulose Lignocelluloseisthestructuralpolymerinthecellwallsandmiddlelamellaeofmost higherplants(breznak&brune1994).thiscompoundisalsotheearth smost abundantformofbiomass,andrepresentsaconsiderablereservoirofcarbon.soil invertebrates,suchastermites,helpcyclelignocellulosethroughphysical dispersionandinitialdissimilationofthispolymer.microorganisms,however,play thelargestroleinthecyclingoflignocelluloseviafurtherdegradationand, ultimately,respiration(breznak&brune1994). Themetabolismoflignocelluloseisnontrivial,asthecompound scomplex biochemicalstructurerendersitdifficulttodegrade.themajorconstituentsof lignocellulosearethreepolymers:cellulose,hemicelluloses,andlignin(breznak& Brune1994).Inparticular,celluloseisahighly ordered,crystalline,homopolymer ofβ linkedglucosemolecules.asaresultofintra andinter molecularhydrogen bondingwithinthemolecule,celluloseconferstolignocelluloseinelasticity,tensile strength,andresistancetohydrolysis.hemicelluloseisalinearorbranched heteropolysaccharidecomprisedmainlyofd xylose,d mannose,l arabinose,

19 1 8 and/ord galactose.hemicellulosecovalentlylinkswithligninandformsan intricatematrixthatsurroundstheorderlycellulosemicrofibrilsandimpedesthe enzymaticdegradationoftheentirelignocellulosemolecule.lignin,thethirdand mostresistantofthethreecomponentstoenzymaticdegradation,isanaromatic polymerconsistingofnon repetitivephenylpropanesubunitsrandomlylinedby variousc Candetherbonds.Ligninisintimatelyinterspersed,andcovalently linkedatvariouspoints,withhemicellulosetoformamatrixsurroundingthe orderlycellulosemicrofibrilsandtoprotectthemfromenzymatichydrolysis(fig.1 4)(Breznak&Brune1994). Fig.1 4:Anexampleofthestructureoflignin.Ligninisanaromaticpolymer consistingofnon repetitivephenylpropanesubunitsrandomlylinkedbyvariousc Cbonds.Ligninisalsothecomponentoflignocellulosemostresistanttoenzymatic degradation.thisisduetothestabilityimpartedbyitsaromaticrings(breznak& Brune1994)(figurefromwww.freepatentsonline.com).

20 1 9 Theproportionsofthesethreeconstituentsvarydependingonthetypeofplant material,andcellulosecanrepresentanywherefrom30 50%ofthelignocellulose molecule,withhemicelluloseandlignineachconstituting20 30%(Breznak&Brune 1994).Woodyplantscontainahigherportionofligninrelativetootherplanttypes, andthereisaninversecorrelationbetweenthelignincontentofplantmaterialand itsdigestibility(breznak&brune1994).theabilitytomodifyand/ormetabolize thelignincomponentoflignocellulose,therefore,iscentraltocarbonturnoverinthe naturalenvironment. Lignocellulosemetabolismby lower termites Researchwithwood feeding,phylogenetically lower termiteshasgenerateda nearcompletepictureofhow lower termitesandtheirmicrobialsymbionts collectivelymetabolizelignocelluloseintonutrientsandenergy(fig.1 5). Fig.1 5:Generalschemeunderlyingthesymbiosisbetweenwood feeding lower termitesandtheirmutualistichindgutmicrobiota.acetateisthe biofuel ofthe termitesystem(odelson&breznak1983;breznak&switzer1986;breznak1994;

21 1 10 Leadbetteretal.1999;Tholen&Brune2000;Graber&Breznak2004;Graberetal. 2004).First,symbiotichindgutprotozoahydrolyzewoodpolysaccharidesand fermenttheresultingsugarmonomersintoacetateaswellasco2andh2. MethanogenicarchaeaconvertsomethisCO2andH2intomethane,butthemajority ofthisco2andh2isconvertedintoadditionalacetatebyco2 reducing homoacetogens(cleveland1926;hungate1955;yamin&trager1979;yamin 1980;Odelson&Breznak1983;Breznak&Switzer1986;Breznak1994;Leadbetter etal.1999).thefateoflignininthesystem,however,remainscontentious(butler &Buckerfield1979;Cookson1987;Pastietal.1990;Kuhnigketal.1994).(Figure courtesyoftheleadbetterlab). Initially,symbiotichindgutprotozoahydrolyzewoodpolysaccharides,suchas celluloseandhemicellulose,andfermenttheresultingsugarmonomersintoacetate, CO2,andH2accordingtothereaction: C6H12O6+2H2O 2CH3COOH+2CO2+4H2 (Fig.1 5)(Odelson&Breznak1983;Breznak&Brune1994;Brune1998a).Next, prokaryoticmicroorganismsinthehindguteitherusesomeoftheresultingh2and CO2toproducemethane(asisthecasewithmethanogenicarchaea),orconvert mostofthath2andco2intoanadditionalacetatemolecule(asisthecasewith homoacetogenicbacteriasuchasspirochetes)accordingtothefollowing: 4H2+2CO2 CH3OOH+2H2O (Fig.1 5)(Braumanetal.1992;Leadbetteretal.1999).Theseproductsarethen absorbedandoxidizedbythetermitehostforbothnutritionandenergy(odelson& Breznak1983;Braumanetal.1992;Brune1998a).Acetateisanoxidizableenergy sourceaswellasaprecursorofaminoacids,hydrocarbons,andterpenes(brauman etal.1992).

22 1 11 Althoughthetermitesystemhasbeenfoundtodissimilate75 99%ofcelluloseand 65% 90%ofthehemicellulose,muchlessisknownaboutthefateoflignininthe termitesystem,andresearchtothatendiscontentious(butler&buckerfield1979; Cookson1987;Pastietal.1990;Kuhnigketal.1994).ReliablymeasuringC 14 labelledligninsbefore,during,andafterpassagethroughthetermiteguttractis challenging(butler&buckerfield1979;cookson1987;pastietal.1990;kuhnigket al.1994).manystudieshavehypothesizedthato2isanecessaryco substratefor thecompletedegradationofligninaromaticmonomers(brune1998b). Physicalparametersoftermitehindgutenvironment Environmentalconditionsinsidethetermiteintestinaltractaffectmicrobial metabolismandlignocellulosedigestion(zimmer&brune2005).likewise, microbialactivityalsopartlydeterminesphysiologicalgutconditionssuchasph level(appel1993;zimmer&topp1997),redoxpotential(bignell1984;kappler& Brune2002),andoxygenconcentration(Bignell1984;Bruneetal.1995aandb). Combined,physicalparametersinnatetothetermiteintestinaltractandimparted bytheinhabitingcomplexmicrobialcommunitymayhaveadaptedover evolutionarytimeforefficientmetabolismoflignocellulose(zimmer&brune2005). Initialinvestigationsintothephysiochemicalconditionsofthegutdeemedthe environmentananoxichabitatinwhichanaerobicmicroorganismsfermentwood polysaccharidesintonutrientsandenergy(cleveland1926;hungate1955;brune 1998a).Nevertheless,atermitehindgutissurroundedbytissuesaeratedbythe insect strachealsystem,andsubsequentstudiesdeterminedthatasaresultofits

23 1 12 smallsizeandrelativelylargesurface area to volumeratio,notonlyisthehindgut environmentpartiallyoxic,butthemajorityofitsvolume,approximately60%, containssomelevelofoxygenbetween50to100µmatthehindgutepithelium beforereachinganoxiawithinabout150to200µmofthewall(fig.1 6)(Bruneetal. 1995aandb;Tholenetal.1997;Brune1998aandb;Graber&Breznak2004; Zimmer&Brune2005). Fig.1 6:RadialprofileofO2(andH2)inthehindgutofthewood feedingtermite Reticulitermesflavipes(figurefromBrune&Friedrich2000).Approximately60%of thehindgut svolumecontainssomelevelofoxygenbetween50to100µmatthe hindgutepitheliumbeforereachinganoxiawithinabout150to200µmofthewall. Thischaracteristichasimplicationstomicrobialmetabolismsinthehindgut(Brune etal.1995aandb;tholenetal.1997;brune1998aandb;graber&breznak2004; Zimmer&Brune2005).

24 1 13 Inparticular,aradialoxygengradientspansfromthehindgut speripherytoits center,withoxygenconcentrationsnearthehindgutepithelium50to100µmand decreasingsteeplytoanoxiawithinabout150to200µmofthewall(fig.1 6) (Bruneetal.1995a).Thediscoverythatthehindgutcontainsvariouslevelsof oxygenandonlycentralportionsofthehindgutareanoxicopenedthepossibility thatthetermitegutcommunityconsistsofmicroorganismswithvaryingdegreesof oxygentoleranceandmetabolisms(bruneetal.1995b;tholenetal.1997;wenzel etal.2002;zimmer&brune2005;wertz&breznak2007a;wertz&breznak 2007b). More recentstudiesofthegutmicroflorahaveconfirmedthepresenceoflarge numbersofmicroorganismswithvaryingdegreesofo2 tolerance(tholenetal. 1997;Wenzeletal.2002;Wertz&Breznak2007a;Wertz&Breznak2007b).Given thatproductsofincompletereductionofo2,suchassuperoxideradical(o2 ), hydrogenperoxide(h2o2),hydroxylradical(ho ),andsingletoxygen(*o2),can seriouslydamageintracellularmacromoleculesaswellaswholecells,someofthe moreaerotolerantmembersmaypossessenzymaticadaptationstocombat oxidativestressandmaintainanoxicconditionsnecessaryforthesurvivaland functioningofthemicrobialhindgutcommunity(brune1998aandb;brioukhanov &Netrusov2007).

25 1 14 Lower termitehindgutisolatestreponemaprimitiastr.zas 1andZAS 2and T.azotonutriciumstr.ZAS 9 Spirochetesarehighlymotile,spiralorundulatebacteriaandareoneofthemost abundant,consistentlypresent,andmorphologicallydistinctgroupsofprokaryotes intermitehindguts(breznak&brune1994;graber&breznak2004).thehindguts oftermites,infact,hostadiversityofspirochetesunparalleledbyanyotherhabitat onearth(leadbetteretal.1999).threespirochetesfromphylogenetically lower termiteshavebeenisolatedinpureculture:treponemaprimitiastr.zas 1andZAS 2andT.azotonutriciumstr.ZAS 9,fromwhichmetabolicactivitieshitherto unknowntobeperformedbyspirocheteshavebeenfound(fig.1 7aandb) (Leadbetteretal.1999;Graberetal.2004). a b Fig.1 7:Treponemaazotonutriciumstr.ZAS 9(a)andT.primitiastr.ZAS 2(b).T. primitiastr.zas 1ismorphologicallyindistinguishablefromT.primitiastr.ZAS 2. Scalebarsrepresent5µm(figurefromGraberetal.2004).

26 1 15 T.primitiastr.ZAS 1andZAS 2areabletoderiveacetatefromH2plusCO2 accordingtothewood Ljungdahlpathwayofcarbonfixation,andT.azotonutricium str.zas 9iscapableofN2fixation(Graber&Breznak2004).Bothofthese processesareimportantinthedeliveryofcarbon,nitrogen,andenergytotermites (Odelson&Breznak1983).Equallysignificantaretheimplicationsfor lignocellulosedegradationaswellascarbonturnoverintheenvironmentasaresult oftheseorganisms metabolisms(warneckeetal.2007). Leveragingdiverselaboratorytoolstostudycomplexmicrobialcommunities Itiswidelyknownthatthevastmajorityofmicroorganismsinthenatural environmenthaveyettobeobtainedinpureculture.consequently,inthelast decadesthemicrobiologyfieldhaswitnessedanupsurgeinthedevelopmentof bioinformaticandmoleculartechniquesandtoolstobetterunderstandwhothese microorganismsareandhowtheyfunctionintheirnaturalenvironments(ottesen etal.2006). Multiplexmicrofluidicdigital PCRisanexcellenttooldevelopedintheLeadbetter Labforlinkingfunctionalgenesto16SrRNAgenesforbacterialidentificationto betterunderstandwhoinamixedsampleiscapableofdoingwhat(ottesenetal. 2006).Inaddition,the62MbP3hindgutregionmetagenomefromthe higher termite,nasutitermessp.hasbeenasignificantresourceininvestigatingthe functionspotentiallyimpartedbythemutualistichindgutmicrobialcommunityin the higher termitesystem(warneckeetal.2007).moreover,thismetagenomic datasetcanbeusedtoprovidehintsatobtainingmicroorganismsfromthe higher

27 1 16 termiteinpureculture,aswellascomparedandcontrastedagainstwhatisknown aboutthe lower termitesystem(warneckeetal.2007). Astechnologiescontinuetodevelop,manyresearchersarecombiningvarious bioinformatic,molecular,physiological,andculturingtoolstoaskandanswerwho, what,where,when,how,howmuch,withwhom,andwhy(rosenthaletal.inreview). MATERIALS&METHODS: Termitecollectionandstorage Workerspecimensofthephylogenetically lower dampwoodtermite,zootermopsis nevadensis,werecollectedinthesangabrielmountains,pasadena,ca.inlabthese specimensweremaintainedinplasticboxeswithwoodfromwhichtheywere collected,andboxeswerestoredatroomtemperature,at95%humidity,andinfoilcoveredglassaquaria. Phylogenetically higher termitespecimenswerecollectedfromjoshuatree NaturalPark,CA.Inlab,thesespecimenswerealsomaintainedinplasticboxeswith woodand/orsoilfromwhichtheywerecollected,andboxeswerestoredatroom temperatureandhumidity,andinthedark.ifanisolatefromthegutmicrobial communityofoneofthese higher termiteshadbeenobtained,thetermiteswould havebeenclassifiedmorphologicallyandtheiridentitywouldhavebeen determinedviacytochromeoxidasegenesequencing(warneckeetal.2007).

28 1 17 Mediaandcultivation RoutineinvitrogrowthandmaintenanceofTreponemaprimitiastr.ZAS 1andZAS 2aswellasT.azotonutriciumstr.ZAS 9,wasin5mL4YAColiquidmediumin25mL butylrubber stopperedbalchtubesaspreviouslydescribed(leadbetteretal.1999; Graber&Breznak2004;Graberetal.2004).Afterisolation,T.primitiastr. ZNS 1, (seebelow)wasgrowninthesamemediaandunderthesameconditionsast. primitiastr.zas 1andZAS 2.Allcultureswereincubatedinthedark,atroom temperature,inahorizontalposition,andwithoutagitation. Yeastautolysate WhilemaximumgrowthyieldsofTreponemaprimitiastr.ZAS 1andZAS 2aswell ast.azotonutriciumstr.zas 9,reachingbetweenOD600nm=0.8and1.0in4YACo mediumundernormalcultureconditions,hadnotchangedmarkedlysincetheir isolationgrowthrateshadincreased,andthusimprovingthegrowthratesofthese isolateswasthefocusoftheworkpresentedhere(leadbetteretal.1999;graber& Breznak2004;Graberetal.2004).Inanefforttoimprovethegrowthratesofthese isolatesin4yacomedium,growthrateswerecomparedonmediacomprisedof yeastautolysatepreparedfromdifferentbrandsanddifferentpackagingtypes withinthesamebrandofbaker syeast.severalyeastautolysatepreparationswere consideredbecauseyeastfromdifferentbrandsanddifferentpackagingtypes withinthesamebrandlookedandsmelleddifferent(table1 1).GrowthratesofT. primitiastr.zas 2,specifically,wereevaluatedontheassumptionthatyeast autolysatepreparationsthatledtogrowthrateimprovementsofthisstrainwould alsoleadtogrowthrateimprovementsoftheothertreponemaisolates.

29 1 18 Autolysatewaspreparedaspreviouslydescribedandthedifferentbatcheswere preparedsimultaneouslyandidentically(leadbetteretal.1999).yeastautolysate waspreparedfromfleischmann sbrandactivedryyeast(fromlargemedium,and smallpackages),safbrandactivedryyeast(fromlargeandsmallpackages),and RedStarbrandactivedryyeast(fromlargeandsmallpackages).Largepackages rangedfrom454gto908g,smallpackagesrangedfrom7gto21g,andmedium packagingdescribesajarof113g.allbrandsandpackagetypescontainedsorbitan monostearate. Enrichmentsandisolation Methanogenswereenrichedforfromphylogenetically lower (Zootermopsis nevadensisworkers)termites,andacetogenicspirocheteswereenrichedforfrom both lower and higher termites.4yacoliquidmediumwasusedforboth spirocheteandmethanogenenrichments,andwaspreparedwithyeastautolysate madefromjarsoffleischmann sbrandactivedryyeastasthisbrandandpackaging typeofbaker syeastwasfoundtoimprovegrowthratesoftreponemaprimitiastr. ZAS 2(Table1 2)(Leadbetteretal.1999;Graber&Breznak2004;Graberetal. 2004).ThisliquidmediumwassupplementedwithallTreponemamediavitamins andcofactors,amixtureofb12vitaminformulations(100mg/300mleachof vitaminb12(icnbiomedicalsinc.),hydroxocobalaminacetate(sigma),and hydroxocobalamin HCl(Sigma)),1.875g/50mLxylan,andasugarcocktailof 3.25g/Lcellobioseand1geachofD maltose,d sucrose,d trehalose,d glucose,dfructose,d xylose,d mannose,l rhamnose,d galactose,l arabinose,d arabinose, L sorbose,d mannitol,d ribose,l fucose,sodiumd glucuronate,andsodiumd

30 1 19 galactoronateperliter(leadbetter&breznak1996;leadbetteretal.1998;graber &Breznak2004;Graberetal.2004). Severaldilution to extinctionenrichmentsfromwholegutcontentsof lower (Zootermopsisnevadensisworkers)and higher termitesunder80%h2/20%co2 (vol/vol)wereperformed.enrichmentculturesfromwholegutcontentsof higher termitescouldonlybesuccessfullytransferredtwoorthreetimes,however,and higher termitespecimensranoutorexpired(typicallyafterthreeweeksinlab) beforesuccessfulenrichments,andsubsequentlyisolates,couldbeobtained.even theadditionof200ulofthesupernatantofspundowndysgonomonasstr.jt5 1 cultures(arecentisolatefromthe higher termitehindgut)toenrichmentcultures didnotleadtosuccessfulenrichmentsnorisolations. Successfuldilution to extinctionliquidenrichmentsformethanogensand spirochetesfromthegutcontentsofz.nevadensiswerefollowedbythreesuccessive single colonypicksfromagardilutionseriesforisolation(leadbetter&breznak 1996,Leadbetteretal.1999;Graber&Breznak2004;Graberetal.2004).Agar dilutionseriesforisolationwereperformedin10mlofthe4yacomedium describedabove(alsoin25mlbutylrubber stopperedbalchtubes)solidifiedby incorporating3%ultrapureagarose.sterile,de oxygenatedneedleswereusedto transfersubsurface,well isolatedcoloniesfromonesuccessiveisolationtoanother. Allenrichmentandisolationcultureswereincubatedinthedark,atroom temperature,inahorizontalposition,andwithoutagitation.

31 1 20 Methanogens ToenrichformethanogensspecificallyfromZootermopsisnevadensisgutcontents, the4yacomediumusedforenrichmentsdescribedabovewasamendedwiththe antibacterialdrugsrifamycinsvandcephalothin(leadbetter&breznak1996). Manymethanogensarenaturallyresistanttotheseantibiotics(Leadbetter& Breznak1996). InliquidculturesmethanogenswereobservedasaresultoftheiruniqueF420 fluorescence.negativeheadspacepressuregeneratedinliquidculturesunderan 80%H2/20%CO2headspace,aswellasincreasedgrowthclosetothe80%H2/20% CO2headspaceinagardilutionseries,alsosuggestedgrowthofmethanogens. Acetogenicspirochetes ToenrichforacetogenicspirochetesspecificallyfromZootermopsisnevadensisgut contents,the4yacomediumusedforenrichmentsdescribedabovewasamended withtheantibacterialdrugsrifamycin,phosphomycin,andbromoethanesulfonate, aswasusedtoisolatetreponemaprimitiastr.zas 1andZAS 2aswellasT. azotonutriciumstr.zas 9(Leadbetteretal.1999;Graberetal.2004).Many spirochetesarenaturallyresistanttotheseantibiotics,andthelatterisknownto inhibitgrowthofh2 consumingmethanogens(leadbetteretal.1999). Inliquidculturesspirocheteswereobservedasaresultoftheirdistinctmorphology. Negativeheadspacepressuregeneratedinliquidculturesunderan80%H2/20% CO2headspace,aswellasincreasedgrowthclosetothe80%H2/20%CO2 headspaceinagardilutionseries,alsosuggestedgrowthofacetogens.

32 1 21 AspirocheteT.primitiastr. ZNS 1 wasisolatedfromadilution to extinction enrichmenttubethatreceivedtheequivalentof0.1gutcontents.afterisolationt. primitiastr. ZNS 1 wascultivatedunderthesameconditionsasdescribedfor routinemaintenanceoft.primitiastr.zas 1andZAS 2(seeabove)(Leadbetteret al.1999;graber&breznak2004;graberetal.2004). Identifying ZnD2Sec Toidentifythetermitehindgutmicrobialcommunitymemberencodingthe ZnD2Sec phylotyope,thephylotypethatisresponsibleforthemajorityofthe formatedehydrogenaseexpressioninthetermitehindgut,enrichmentsforthis organismwerepreparedinthesamemannerasenrichmentsforacetogenic spirochetesasdescribedabove,ontheassumptionthatbecausethisorganismis responsibleforthemajorityofformatedehydrogenaseexpressionthatitisan acetogen(rosenthaletal.inreview).itwashopedthatthisorganismcouldbe isolatedandthatitsgenomecouldbeobtainedforfurtherresearch.althoughan isolatewasnotobtained,theseenrichmentculturesweresuccessfullyusedas templatesformicrofluidicmultiplexdigitalpcrexperiments.beforeusingthese enrichmentsastemplatesformicrofluidicmultiplexdigitalpcr,however,qrt PCR wasemployedtoexaminerelativeabundanceoforganismswiththe ZnD2Sec phylotyperelativetotheotherorganismsintheenrichmentculture.

33 1 22 qrt PCR qrt PCRreactions(25µLtotal)containediTaqSYBRGreenSupermixwithROX (Bio RadLaboratories,Irvine,CA)(12.5µL/rxn),ZNO1636F(1µL/rxn),ZNO1729R (1µuL/rxn),PCRwater(9.5µL/rxn),andenrichmentculturetemplate(1µL/rxn). TheSYBRGreenprotocolwasusedontheBio RadDNAEnginethermocycler (Chromo4realtimedetector)andwashotstart95 Cfor3minfollowedby44cycles of95 Cfor15secand60 Cfor30sec. MicrofluidicmultiplexdigitalPCR Microfluidicchipexperimentswereperformedasdescribedpreviously,andwith degeneratepcrprimerandprobesetstargetingfdh(fdhf)andbacterialssurrna (AppendixTable1A 1)(Ottesenetal.2006;Tadmoretal.2011;Rosenthaletal.in review).samplesweremanuallyretrievedfromchipchambersforsequencing,also asdescribedpreviously(ottesenetal.2006;tadmoretal.2011;rosenthaletal.in review). 16SrRNAandfdhFPCR For16SrRNAidentificationinenrichmentandisolatecultures,oncecultures reachedod600nm=0.5,200µlofculturefluidwasremoved,centrifugedat 13,000rpmfor1min,andresuspendedin50µL1xTAE.Alternatively,cultureswere harvestedatod600nm=0.5fordnaextractionusingadneasyextractionkit (QIAGEN,Valencia,CA).ThissamplewasthenusedasatemplateforsimplexPCR reactionsonamastercyclermodel5331thermocyclerandwithagarosegel electrophoresis.pcrreactions(20µltotal)containedfailsafepcrpremixd

34 1 23 (EpicentreBioechnologies,Madison,WI)(10µL/rxn),Prok27Fprimer(1µL/rxn), Prok1492Rprimer(1µL/rxn),HiFidelityTaqPolymerase(0.2µL/rxn),PCRwater (5.8µL/rxn),andtemplate(2µL/rxn).Benchtopthermocyclingconditionswerelid 105 C,hotstart95 Cfor2min,(denaturation95 Cfor30sec,annealing50 Cfor 30sec,andextension72 Cfor1.5minrepeated30X),andfinalextension72 Cfor 5min. Samplesretrievedfrommicrofluidicchipexperimentswerescreenedforboth16S rrnaandfdhfgeneproductsalsoviasimplexpcronamastercyclermodel5331 thermocyclerandwithagarosegelelectrophoresis.pcrreactions(50µlrxntotal, broughttofinalvolumewithpcrwater)containediqmultiplepowermix(bio Rad Laboratories), nMofeachprimer(Prok533FandGen1100Rfor16SrRNA, ZNO1204FandZNO1792RforfdhF;AppendixTable1A 1),and2.5µLoftemplate. Benchtopthermocyclingconditionswerelid105 C,hotstart95 Cfor2min, (denaturation95 Cfor15sec,annealing60 Cfor1min,andextension72 Cfor1min repeated30or35x),andfinalextension72 Cfor10min(Rosenthaletal.inreview). Productsfromsamplesthatyielded16SrRNAamplicons(andfdhFampliconsas wasthecaseformicrofluidicchip derivedsamples)werepcrpurified(qiaquick PCRpurification,QIAGEN).16SrRNAPCRproductswereclonedinTOPO TA vectors(topo TAcloningkit,Invitrogen).Plasmidsfromrandomlychosenclones werepurified(qiaprepspinminiprep,qiagen).16srrnapcrproductsand plasmidsweresequencedwithgenerict3andt7primers,andfdhfproductswere sequencedwithzno1204fandzno1729r(appendixtable1a 1).Allsequencing

35 1 24 reactionswereperformedatlaragen,inc.(losangeles,ca)andblasthitsto sequenceswereobtainedtoidentifymicrofluidicchip derivedsamples(geeretal. 2010). TreponemaprimitiaenzymaticdefensesagainstO2 stress ForidentificationofenzymesrepresentingdefensesagainstO2 stressintreponema primitiastr.zas 1andZAS 2,genomicdataforthestrainswereobtainedfromRAST (Azizetal.2008),JGIIMG/M(Markowitzetal.2006),andNCBIBLAST(Geeretal. 2010).Genomeswerethensearchedforenzymesrecordedintheliteratureas beingactiveagainsto2 stress(dollaetal.2006;brioukhanov&netrusov2007; Rochaetal.2007;Imlay2008b;LeFournetal.2008;Sundetal.2008;Riebeetal. 2009;Lakhaletal.2011;Xiaoetal.2011;Figueiredoetal.2012). RESULTS: Yeastautolysateformediaandcultivation TheisolationofTreponemaprimitiastr.ZAS 1andZAS 2aswellasT. azotonutriciumstr.zas 9,wasnon trivial,andonemediacomponentthataidedin theiracquisitioninpureculturewasayeastautolysatepreparedfreshfrombaker s yeast(leadbetteretal.1999).interestingly,commercialyeastextractscouldnotbe usedinlieuofthisfreshlypreparedyeastautolysate(leadbetteretal.1999).from thetreponemaorganisms initialisolationin YACo medium,however,afterbeing passagedforoveradecade,itsgrowthrateshadincreasedfromadoublingtime initiallyof22,29,and35hrsfort.primitiastr.zas 1,T.primitiastr.ZAS 2,andT. azotonutriciumstr.zas 9,respectively,toadoublingtimebetween54to77hrs

36 1 25 currentlyforeachtreponemaisolate(leadbetteretal.1999;graber&breznak 2004). InanefforttoimprovethegrowthratesoftheTreponemaisolatesin4YACo medium,onefactorthatwarrantedre evaluationwasthevitalyeastautolysate mediacomponent(leadbetteretal.1999).forexample,store boughtbaker syeast containssorbitanmonostearate asurfactantwithemulsifyingproperties in additiontotheyeastitself.thisadditivecaninhibitmicrobialgrowth,and consequently,4yacomediumcomprisedofbaker syeastautolysatepreparedfrom differentbrands,anddifferentpackagingwithinthesamebrand,werepreparedand growthratesoft.primitiastr.zas 2werecomparedoneachasaproxyforgrowth bytheotherisolates.asafirstobservation,yeastfromdifferentbrandsand differentpackagingtypeswithinthesamebrandlookedandsmelleddifferentso autolysatepreparedfromeachwastested(table1 1).

37 1 26 Table1 1:Appearanceofbaker syeastandappearanceandodorofcorresponding yeastautolysateandautolysatesupernatant. Brand Packaging Dryyeast Autolysate Autolysate Autolysate appearance RedStar a large long cylindrical pellet;tan SAF small small cylindrical pellet;dark tan SAF large long cylindrical pellet; cream Fleischmann s small small cylindrical pellet;light tan Fleischmann s large small spheres; lightbrown appearance lighttan odor strong sour supernatant orange/brown tan sweet darkyellow cream cream very sweet bread (sweet) orange yellow darktan sweet darkyellow a RedStarbrandactivedryyeastwasusedfororiginalyeastautolysatepreparations(Leadbetteret al.1999) AfterexamininggrowthratesofT.primitiastr.ZAS 2obtainedon4YAComedium preparedfromfleischmann sbrandactivedryyeast(fromlargemedium,andsmall packages),safbrandactivedryyeast(fromlargeandsmallpackages),andredstar brandactivedryyeast(fromlargeandsmallpackages),growthratesimprovedthe mostonmediawithyeastautolysatepreparedfromjarsoffleischmann sbrand activedryyeast(table1 2).Specifically,thiswas47hrscomparedtobetween54 and77hrspreviously,and29hrsuponisolation(table1 2)(Leadbetteretal.1999; Graber&Breznak2004).Fromthispointon,therefore,routinegrowthaswellas enrichmentandisolationattemptsoccurredin4yacoliquidmediumpreparedwith yeastautolysatemadefromjarsoffleischmann sbrandbaker syeast.

38 1 27 Table1 2:GrowthratesofTreponemaprimitiastr.ZAS 2on4YAComedium preparedfromyeastautolysatesofdifferentbrandsandpackagingtypes. Brand Packaging Doublingtime b +/ std.error (hrs) RedStar a large 74+/ 1 SAF small 85+/ 1 SAF large 111+/ 12 Fleischmann s small 47+/ 0 Fleischmann s large 50+/ 4 a RedStarbrandactivedryyeastwasusedfororiginalyeastautolysatepreparations(Leadbetteret al.1999) bdoublingtimeisanaverageoftriplicatecultures Enrichment Methanogens Asanexerciseintheenrichmentandisolationprocedure,methanogenswere targetedfromphylogenetically lower termites(workersofzootermopsis nevadensis).thisisbecause,similartoacetogens,theyarealsoh2+co2consumers andwoulddemonstratesimilarrelationshipswiththeir80%h2/20%co2(vol/vol) headspaceatmosphereasacetogens.ingeneral,however,fromwhatisknown aboutspirocheteandmethanogenisolatesobtainedfromthetermitesystemthus far,methanogensappeartogrowfasterthanspirochetes(35to40hrs)inthe mediumusedhereandsuccessorproblemsintheprocedureareabletobedetected faster(leadbetter&breznak1996;leadbetteretal.1998). MethanogenswereenrichedforinliquidcultureandnotedbytheirF420 fluorescence.inliquidculture,methanogensappearedbothfilamentousaswellas cocciandrod shaped.negativeheadspacepressurewasalsogeneratedbythese liquidculturesunderan80%h2/20%co2headspace.adilution to extinction

39 1 28 enrichmenttubethatreceivedtheequivalentof0.1gutcontentswasusedto inoculateanagardilutionseriesforsubsequentisolation.withinthefirstofthree agardilutionspreferentialgrowthofcolonieswasobservedclosetothemeniscusof theculturesattheagar/headspaceinterface.presumablythiswasbecause methanogenswereintheculturesandweregrowingclosetotheirsubstrate,the 80%H2/20%CO2(vol/vol)headspace.Distinctcoloniescouldbereadily transferredfromtheseagarculturesthatdisplayedf420fluorescenceandwere consistentinmorphologywithwhathadalreadybeenobservedinenrichmentsas methanogens.becausemethanogenenrichmentsandisolationsweresimplytrials, however,effortsweredirectedtowardsacetogenicspirocheteenrichmentsand isolations. Acetogenicspirochetes Acetogenicspirocheteswereenrichedforinliquidculturefromboth lower and higher termites,andnotedbytheiruniquemorphologies.inliquidculture, spirochetesappeareddiverseinmorphologywithtwomorphologiesdominatingthe cultures(fig.1 8aandb).Negativeheadspacepressurewasalsogeneratedbythese liquidculturesunderan80%h2/20%co2headspace.adilution to extinction enrichmenttubethatreceivedtheequivalentof0.1gutcontentswasusedto inoculateanagardilutionseriesforsubsequentisolation.withinthefirstofthree agardilutions,preferentialgrowthofcolonieswasobservedclosetothemeniscusof theculturesattheagar/headspaceinterface.presumablythiswasbecause acetogenicspirocheteswereintheculturesandweregrowingclosetotheir substrate,the80%h2/20%co2(vol/vol)headspace.distinctcoloniescouldbe

40 1 29 readilytransferredfromtheseagardilutionseriesculturesandwereconsistentin morphologywithwhathadalreadybeenthoughttobespirochetesinliquid cultures.coloniesweredisc likeinappearance.noacetogenicspirochetes,norany othermicroorganismsforthatmatter,wereobtainedfromdilution to extinction enrichmentsfromjoshuatreenationalpark higher termitesamples. a b Fig.1 8:Spirochete likemorphologiesthatdominatedliquidenrichmentcultures foracetogenicspirochetes.(a) Floppy morphology.(b)whatwouldbeisolated astreponemaprimitiastr. ZNS 1. 4YAComediumforliquidenrichmentcultures waspreparedwithyeastautolysatemadefromjarsoffleischmann sbrandactive dryyeastsupplementedwithalltreponemamediavitaminsandcofactors,amixture ofb12vitaminformulations(100mg/300mleachofvitaminb12(icnbiomedicals Inc.),hydroxocobalaminacetate(Sigma),andhydroxocobalamin HCl(Sigma)), 1.875g/50mLxylan,andasugarcocktailof3.25g/Lcellobioseand1geachofDmaltose,D sucrose,d trehalose,d glucose,d fructose,d xylose,d mannose,lrhamnose,d galactose,l arabinose,d arabinose,l sorbose,d mannitol,d ribose, L fucose,sodiumd glucuronate,andsodiumd galactoronateperliter(graber& Breznak2004;Graberetal.2004).Toenrichforacetogenicspirochetesthemedia wasamendedwiththeantibacterialdrugsrifamycin,phosphomycin,and bromoethanesulfonate.dilution to extinctionenrichmentsfromwholegutcontents ofazootermopsisnevadensisworkerunder80%h2/20%co2(vol/vol)was performed.then,threesuccessivesingle colonypicksfromagardilutionseriesfor isolationwereundertaken(leadbetter&breznak1996,leadbetteretal.1999; Graber&Breznak2004;Graberetal.2004).Agardilutionseriesforisolationwere performedin10mlof4yacomedium(alsoin25mlbutylrubber stopperedbalch

41 1 30 tubes)solidifiedbyincorporating3%ultrapureagarose.sterile,de oxygenated needleswereusedtotransfersubsurface,well isolatedcoloniesfromonesuccessive isolationtoanother.allenrichmentandisolationcultureswereincubatedinthe dark,atroomtemperature,inahorizontalposition,andwithoutagitation.scalebar represents5µm. Thespirochetesuccessfullyisolatedshares100%16SrRNAsequenceidentitywith 1,andphylogeneticallyitsclosestculturedrelativesaretheseT.primitiastrains (AppendixItem1A 1).Given,however,thatthisisolatewasobtainedfromthea Zootermopsisnevadensisworkertermite,asopposedtostr.ZAS 1andZAS 2that wereobtainedfromz.angusticollis,thisisolatewasnamedt.primitiastr. ZNS 1. T.primitiastr.ZAS 2and99%16SrRNAsequenceidentitywithT.primitiastr.ZAS Mostcellsareapproximately0.2µmindiameterby3to15µmlong,withawave lengthorbodypitchofapproximately2µmsimilartostr.zas 1andZAS 2(Fig.1 7b).Fromtheirsuccessfulgrowthin4YAComediumwitha80%H2/20%CO2 (vol/vol)headspace,colony establishmentpreferenceclosetotheagar/headspace interfaceinagartubes,andconsumptionofheadspaceasindicatedbynegative pressure,str. ZNS 1 isveryprobablyanacetogen(atleastderivingacetatefromh2 +CO2)likestr.ZAS 1andZAS 2.T.primitiastr. ZNS 1 alsogrowswellat25 Cand aphof7.2(leadbetteretal.1999;graber&breznak2004;graberetal.2004). Identifying ZnD2Sec Toidentifythetermitehindgutmicrobialcommunitymemberencodingthe ZnD2Sec phylotyope,thephylotypethatisresponsibleforthemajority(40%)of theformatedehydrogenaseexpressioninthetermitehindgut,enrichmentsforthe organismthatencodethisphylotypewerepreparedthesameasenrichmentsfor acetogenicspirochetes,ontheassumptionthatthatbecausethisorganismis

42 1 31 responsibleforthemajorityofformatedehydrogenaseexpressionthatitisan acetogen(rosenthaletal.inreview).whileattemptstoisolatethetermitehindgut microbialcommunitymemberencodingthe ZnD2Sec phylotyopewere unsuccessful,inenrichments,uniformsmallcocciwereobserved,16srrnaanalysis confirmedthepresenceof ZnD2Sec inthesample,andqrt PCRconfirmedits relativeabundance.theseenrichmentswereusedastemplatesformicrofluidic digitalpcrtohaveabetterchanceattargetingbothfdh(fdhf)andbacterialssu rrnagiventheirrelativeabundanceintheenrichmentvs.gutcontents.over50% ofthesamplesthatco localized16srrnaandfdhfweredeltaproteobacteria.nonmatchestodeltaproteobacteriawereoftenunculturedorganisms. TreponemaprimitiaenzymaticdefensesagainstO2 stress Preliminaryworksuggeststhatculturesofthetermitehindgutspirochetes Treponemaprimitiamaintainedgrowthaftertheadditionofasmuchas0.5% (vol/vol)o2totheirheadspaceatmosphere,andexhibitedbothnad(p)h peroxidaseandnad(p)hoxidaseactivities(table1 3)(Graber&Breznak2004). Althoughneithercatalasenorsuperoxidedismutase,whichtogetherconstitutea systemtocombatreactiveoxygenspeciescommonamongaerobes,weredetected int.primitia(table1 3),otherenzymesthatscavengesuperoxide,hydrogen peroxide,andoxygenaboundinthecommonly studiedanaerobe,clostridium acetobutylicum,andmayprovideinsightintorelevantenzymesinthetermite system(graber&breznak2004;brioukhanov&netrusov2007;imlay2008a).

43 1 32 Table1 3:Treponemaprimitiastr.ZAS 2enzymaticdefensesagainstoxidative stress. Enzyme Activity a Genomic Evidence str.zas 1 str.zas 2 str.zas 1 str.zas 2 NADHoxidase + ++ Y NADPH + + oxidase NADH + + peroxidase NADPH + + peroxidase Catalase ND ND Superoxide ND ND dismutase afromgraber&breznak2004 +,lowactivity;++,highactivity;nd,notdetected;,noevidence;y,evidence For example, O2 can steal two electrons from a variety of reduced flavoenzymes, ultimatelygeneratingh2o2(brioukhanov&netrusov2007;imlay2008a).inturn, H2O2 can react with unincorporated intracellular iron to produce the powerful oxidant,ho,accordingtothefentonreaction: H2O2+Fe2 + HO +OH +Fe3 + InC.acetobutylicum,whenH2O2directlyoxidizesamononuclearironatomwithina peroxidestressregulator(perr),arepressorofperoxidestressresponses,the oxidizedperrproteinlosesthecapacitytobinddna.whereasperrwould normallybindtodnatorepressthesynthesisofproteinsthatsuppressfenton chemistry,theseproteinsareformedandthenhindertheconversionofh2o2toho (Fig.1 9).Further,rubrerythrinshavebeenassociatedwithbothperoxidaseand oxygen scavengingactivitieswithrubredoxinsservingaselectrondonorsinc.

44 1 33 acetobutylicum(fig.1 9).Inaddition,inlow oxygenenvironmentslow potential electroncarrierssuchasflavodoxins,rubredoxins,andferredoxinswilloftentimes reducerestingenzymes(brioukhanov&netrusov2007;imlay2008a). Fig.1 9:Proposedmechanismsforcombatingoxidativestressinthetermite hindgut.(1)molecularoxygencanstealtwoelectronsfromavarietyofreduced flavoenzymes,ultimatelygeneratingh2o2.whenh2o2directlyoxidizesa mononuclearironatomwithintheperoxidestressregulator(perr),theoxidized PerRproteinlosesthecapacitytobindDNA.WhereasPerRwouldnormallybindto DNAtorepressesthesynthesisofproteinsthatsuppressFentonchemistry,these proteinsareformedandhindertheconversionofh2o2toho.(2)inaddition, rubrerythrinshavebeenassociatedwithbothperoxidaseandoxygen scavenging activities,convertingh2o2ando2toh2o,respectively(dollaetal.2006; Brioukhanov&Netrusov2007;Rochaetal.2007;Imlay2008b;LeFournetal.2008; Sundetal.2008;Riebeetal.2009;Lakhaletal.2011;Xiaoetal.2011;Figueiredoet al.2012). AswithC.acetobutylicum,rubrerythrinandrubredoxininbothT.primitiastrains areoftentimesfoundwithinthesamegeneneighborhoods(fig.1 10a).So,too,are thegenesregulatingseveralflavodoxinsandferredoxinsintheseorganismsand

45 1 34 alsofoundincloseassociationwiththeaforementionedenzymes(fig.1 10b). Preliminaryobservations,therefore,suggestthatinadditiontoNAD(P)Hperoxidase andnad(p)hoxidase,otherenzymesimportantincombatingoxidativestressinthe termitehindgutmayincludeperrs,rubrerythrins,rubredoxins,flavodoxins,and ferredoxins(brioukhanov&netrusov2007;imlay2008b). Fig.1 10:GeneneighborhoodsofenzymesinvolvedinO2transformation. PredictedORFSinclude(a)rubrerythrin(red)andrubredoxin(darkblue)aswellas (b)peroxidestressregulator(green)andrubrerythrin(red)intreponemaprimitia str.zas 2. DISCUSSION: Yeastautolysateformediaandcultivation WhilegrowthratesoftheTreponemaisolatesonmediumwithRedStaryeast autolysatewere54to77hrs,growthratesoft.primitiastr.zas 2onmediumwith Fleischmann syeastautolysatemarkedlyimprovedto47hrs(table1 2).Growth ratesoft.primitiastr.zas 2onyeastautolysatepreparedfromdifferentbrands andpackagingofbaker syeastwerealsoquitedisparate(table1 2).Although yeastautolysatepreparedfromfreshbaker syeastisanintegralcomponentofthe 4YAComediumonwhichT.primitiastr.ZAS 1andZAS 2aswellasT.

46 1 35 azotonutriciumstr.zas 9grow,thatthereissucharangeofgrowthratesonyeast autolysatepreparedfromdifferentbrandsandpackagesofyeastisnoteworthy (Table1 2)(Leadbetteretal.1999).Theseresultssuggestthatonemustpaycareful attentionwhencultivatingorganismsinpureculturetoeachofthemedia componentsastheycanhaveconsiderableeffectsonculturegrowth.thisincludes payingattentiontotheirsource,howtheymayormaynotchange,theirage,and theirstorage,amongotherfactors. Moreover,whileanimprovementingrowthrateswasseenwithnewyeast autolysatepreparedfromjarsoffleischmann sbrandactivedryyeast,thetermite hindgutturnsoverevery24hours(bignell1984).consequently,ifinsituthe Treponemaisolatesweretodoubleevery47hrs,presumablytheywouldbelost fromthetermitesystem.theseobservations,therefore,hintthattreponemagrows withadoublingtimeunder24hrsinsituandthatcurrentcultureconditionsare suboptimal.thisdisparitycouldbearesultoftheisolatesevolvingastheyhave beenpassagedovertimetodoubleslowerand/orduetoalimitingfactorinthe media(seechapter4). Enrichmentsandisolation Methanogens Methanogensappearedtohavebeenenrichedforsuccessfullyasaprimerin enrichmentandisolationtechniques.thiswasbecausecoloniesinagardilution seriesculturesweregrowingclosetomethanogensubstrates,the80%h2/20%co2 (vol/vol)headspace,anduponpickingcoloniesandresuspendingtheminliquidfor

47 1 36 microscopicobservation,theydisplayedf420fluorescenceandmorphologies consistentwiththoseinliquidenrichments.additionally,becausetheagarmedia washomogenous,thatthesecoloniesdevelopedclosertotheheadspacewaslikely duetotheirpreferenceforh2andco2andnotaresultofanygradientofan inhibitoryfactorintheagarmedia.withadditionaltimeandattentionitwouldbe worthwhiletopursuemethanogenenrichments.thisisbecausetermite methanogensarecitedasasmallbutsignificantsourceoftheatmospherically relevanttracegasandtherehavebeenrelativelyfewstudiesonthesetermite symbionts(braumanetal.1992).moreover,previousmolecularanalyses,aswellas studiesinvolvingtheisolationandcultivationoftheseorganisms,haveshownthat themethanogensinhabitingthetermitegutaredistinctfromotherknown methanogenspecies(ohkumaetal.1999). Acetogenicspirochetes Aspirochete,Treponemaprimitiastr. ZNS 1, wasisolatedfromthezootermopsis nevadensistermitehindgut(fig.1 8b).Thisstrainexhibitspreliminaryevidenceof H2+CO2acetogenesis,ascoloniesofthestrainpreferentiallygrowattheagar/80% H2/20%CO2headspaceandnegativepressuregeneratedfromculturesgrowing under80%h2/20%co2isobserved(leadbetteretal.1999;graber&breznak 2004,Graberetal.2004).Similartothemethanogenagarcultures,becausetheagar mediawashomogenous,thatt.primitiastr. ZNS 1 coloniesdevelopedcloserto theheadspacewaslikelyduetotheirpreferenceforh2andco2andnotaresultof anygradientofaninhibitoryfactorintheagarmedia.

48 1 37 Identifying ZnD2Sec Effortstoisolatethetermitehindgutmicrobialcommunitymemberencodingthe ZnD2Sec phylotyope,wereunsuccessful.nevertheless,enrichmentcultureswere successfullyusedastemplatesformicrofluidicdigitalpcrandhelpedcorrelatethe ZnD2Sec phylotypewithadeltaproteobacteria andnotaspirocheteaspreviously thought(rosenthaletal.inreview).originally,itwasthoughtthat ZnD2Sec wasa spirochetebecausespirochetesareoneofthemostabundant,consistentlypresent prokaryotesintermitehindguts(breznak&brune1994;graber&breznak2004; Rosenthaletal.inreview).Moreover,itwasassumedthatbecausethisorganismis responsibleforthemajorityofformatedehydrogenaseexpressionthatitisan acetogen,andsincetwoofthethreespirochetesalreadyisolatedfromthetermite hindgutdemonstratebonafideh2+co2acetogenicactivities,itwasalsoassumed thisorganismisanacetogenicspirochete.nevertheless,multiplexmicrofluidic digitalpcrexperimentsconfirmedthatthetermitehindgutmicrobialcommunity memberencodingthe ZnD2Sec phylotypeisadeltaproteobacteria(rosenthalet al.inreview).thisfindingsupportsthatonemustremainopentotheunexpected (seechapter4).itisalsonoteworthythatthephylotypethatisresponsibleforthe second mostformatedehydrogenaseexpressioninthetermitehindgut(14%in contrastto40%for ZnD2Sec )isencodedbyafree livingspirochete(rosenthalet al.inreview). TreponemaprimitiaenzymaticdefensesagainstO2 stress Treponemaprimitiastr.ZAS 1andZAS 2lackhallmarkmechanismsfordealingwith oxidativestress,specificallycatalaseandsuperoxidedismutase(table1 3)(Graber

49 1 38 Breznak2004).Althoughtheydonothavecatalaseandsuperoxidedismutase,this doesnotnecessarilymeanthatstr.zas 1andZAS 2areespeciallysensitivetoO2. Likelyrather,theyhaveotherstrategiesandrelationshipswithO2intheir environment,whichisrelevanttoworkdiscussedinchapter3.possible mechanismsarealsolistedbelowandshouldbeinvestigatedfurther(appendix Table1A 2).

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55 1 44 CHAPTER1:APPENDIX Table1A 1:16SrRNAandfdhFmultiplexandsimplexprimerandprobedesigns. Table1A 2:EnzymaticdefensesagainstO2 stress. Item1A 1:Treponemaprimitiastr. ZNS 1 16SrRNAsequence.

56 1 45 Table1A 1:16SrRNAandfdhFmultiplexandsimplexprimerandprobedesigns. PrimerorProbe Sequence Target Experiments Prok27F 5 AGA GTTTGA TCCTGG CTCAG 3 Genbac16S rrna Isolate identification Prok1492R 5 TAC GGYTAC CTTGTT ACGACTT 3 Genbac16S rrna Isolate identification; Microfluidic digitalpcr onchip Prok357F 5 CTC CTACGG GAGGCA GCAG 3 Genbac16S rrna Microfluidic digitalpcr onchip GenBacHEX 1389Prb 5 HEX CTTGTA CACACC GCCCGTC 3BHQ1 3 Genbac16S rrna (probe) Microfluidic digitalpcr onchip FAM 1636Prb 5 ACTATG ACCGGC AATTGT CGCCTGTT 3 ZnD2Sec fdhf (probe) Microfluidic digitalpcr onchip ZNO1204F 5 AAC GAACAT GACGGC GTCTAC TCT 3 ZnD2Sec fdhf Tolookforin enrichments; Microfluidic digitalpcr onchip;chip wellpick check ZNO1792R 5 TCAGAC CCATAT CACGGC AAAGTT 3 ZnD2Sec fdhf Tolookforin enrichments; Microfluidic digitalpcr onchip;chip wellpick check Prok533F 5 GTGCCA GCMGCC GCGGTAA 3 Genbac16S rrna chipwellpick check Gen1100R: 5 AGG Genbac16S chipwellpick

57 1 46 GTTGCG CTCGTTG 3 rrna check

58 1 47 Table1A 2:PossibleenzymaticdefensesagainstO2 stress. Citation Enzymestoinvestigate Rochaetal.2007 glutathione/glutaredoxin;thioredoxin peroxidase Sundetal.2008 alkylhyperoxidereductase;cytochrome Cperoxidase/oxidase;ferritin;thiol peroxidasescavangase; oxygen induced starchutilization genes; global oxidativeresponse genes LeFournetal.2008 anyoxidoreductase;anyoxidase;any peroxidase Riebeetal.2009 rubrerythrin,rubredoxin,nadh: rubredoxinoxidoreductase Xiaoetal.2011 alkylhyperoxidereductase;pyridine nucleotide disulfidereductase;dna oxidativedamageprotectiveproteins (DNA binding);ribonucleotide reductase;nucleotidetriphosphate; pyrophosphohydrolase;polynucleotide phosphorylase,enolase Lakhaletal.2011 neelaredoxin Figueiredoetal.2012 bacterioferritin Dollaetal.2006 superoxidereductase;nigerythrin

59 1 48 Item1A 1:Treponemaprimitiastr. ZNS 1 16SrRNAsequence. TTACCTTGTTACGACTTCACCCTCCTCACTAAACGTACCTTCGACAGCGCGCTCCTTGCGG TTACGCTACCGGCTTCGGGTACCTCCAACTCGGATGGTGTGACGGGCGGTGTGTACAAGG CCCGGGAACGTATTCACCGCGCCATGCTGATGCGCGATTACTAGCGATTCCAACTTCATG AAGTCGGGTTTCAGACTTCAATCCGAACTACGGGCGGCTTTTTGCGCTTCGCTTTGACCT CGCGGTTTCGCGTCGCTTTGTACCGCCCATTGTAGCACGTGTGTAGCCCTGGACATAAGG GCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTTCCGCCAG AGTCCTCAGCGTTACCTGTTAGTAACTGGCAGTAGGGGTTGCGCTCGTTGCGGGACTTAA CCCAACACCTCACGGCACGAGCTGACGACAGCCATGCAGCACCTGTGATAGCGCGTATTG CTACGCTGATACATCTCTGCATCATTCACTACCATGTCAAACCCAGGTAAGGTTCCTCGC GTATCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGA GTTTCACCCTTGCGGGCATACTCCCCAGGCGGTGCACTTATCACGTTCGCTTCGGCACTG AGCCGCTTGGCCCAACACCTAGTGCACATCGTTTATAGTGCGGACTACCAGGGTATCTAA TCCTGTTTGCTCCCCGCACTTTCGCGCCTCAGCGTCAGTCATTGGCTAGTAGTTCGCCTTC GCCACCGGTGTTCTTCCGCATATCTACAGATTTCACCCCTACACGCGGAATTCCAACTAC CCCTCCATGA

60 2 1 CHAPTER2 ExploringgenomicevidenceofacetogenicdemethylationbyTreponema azotonutriciumstr.zas 9 ABSTRACT: UnliketheacetogenictermitehindgutspirocheteisolatesTreponemaprimitiastr. ZAS 1andZAS 2,theisolateT.azotonutriciumstr.ZAS 9doesnotdisplayacetogenic activityinvitro.moreover,t.azotonutriciumstr.zas 9lacksformate dehydrogenaseandmethylene tetrahydrofolatereductase,enzymesintegraltothe methylbranchoftheacetyl CoA(Wood Ljungdahl)pathwayofacetogenesisfound int.primitia.together,theseobservationssuggestthatthestrainisincapableof contributingtothetermitesystem sacetatepoolviaacetogenesis.nevertheless,t. azotonutriciumstr.zas 9 sgenomedoeshavetwocarbonmonoxidedehydrogenase homologsrepresentingtheotherbranchofthewood Ljungdahlpathway,the carboxylbranch,aswellas51genesannotatedaseithermethylasesor methyltransferaseswhich withavailablemethyl ormethoxylatedsubstrates can generatethesameendproductasthemethylbranchofthepathway.potentially, therefore,t.azotonutriciumstr.zas 9cancontributetoacetogenesisinthetermite hindgutbycouplingitsmethyl(transfer)aseandcarboxylbranchcapabilities.that thetermitehindgutisrepletewithwood derivedmeth(ox)ylatedcompoundsalso supportsthisnotion.totestthishypothesis,t.azotonutriciumstr.zas 9was grownonthemeth(ox)ylatedcompoundsdl methionine,dmso,methanol, methylamine,dimethylamine,trimethylamine,betaine,andtrimethoxybenzoate. Specifically,sinceacetateisthefuelforboththetermitehostanditsmicrobial

61 2 2 symbiontsalike,possibleincreasesingrowthrateandyieldoft.azotonutriciumstr. ZAS 9weremonitoredasaproxyformetabolismofthesemeth(ox)ylated compoundsintoacetate.despiteconvincinggenomicevidence,however,no significantlydifferentgrowthratesandyieldswereobservedinvitro.whilethereis currentlynoinvitroevidenceinsupportofacetogenicdemethylationbyt. azotonutriciumstr.zas 9,thisdoesnotmeanthatthisfunctionisnotoccurringin situandinconjunctionwithothermembersofthetermitehindgutmicrobial community.therefore,thishypothesiscouldbere evaluatedusingacombination ofdiversemicrobialecologyresearchtechniquesdiscussedhere. INTRODUCTION: Acetateisanimportantmetaboliteinthetermite hindgutmicrobial communitymutualism Hungatewasthefirsttodetectacetateintermitehindguts,specificallyinthe hindgutsofthephylogenetically lower termiteszootermopsissp.and Reticulitermesclaripennis(1939,1943).Decadeslater,ithasbecomewellestablishedthatacetateisthe biofuel ofthetermitehostitselfaswellasits mutualistichindgutmicrobialcommunity(odelson&breznak1983;breznak& Switzer1986;Breznak1994;Leadbetteretal.1999;Tholen&Brune2000;Graber& Breznak2004;Graberetal.2004).Inparticular,acetateservesastheprimary oxidizableenergysourceandaprecursorofaminoacids,hydrocarbons,and terpenesinthetermitesystem(blomquistetal.1979;mauldin1982;odelson& Breznak1983;Breznak&Switzer1986;Braumanetal.1992;Breznak&Brune 1994).InReticulitermsflavipes,forexample,acetategeneratedbythehindgut

62 2 3 microbiotasupports77to100%oftherespiratoryrequirementoftheinsectsystem (Mauldin1982;Breznak&Switzer1986). Inthewood feeding,phylogenetically lower termites,symbioticcellulolytic hindgutprotozoaareknowntohydrolyzewoodpolysaccharidesandfermentthe resultingsugarmonomersintoacetate,carbondioxide,andhydrogen(pleasesee Chapter1Fig1 2)(Hungate1955;Yamin1980;Odelson&Breznak1983;Odelson& Breznak1985a;Odelson&Breznak1985b;Breznak&Brune1994).Odelsonand Breznak(1983)hypothesizedthatadditionalacetateisgeneratedfromtheH2+CO2 fermentationbyproducts,andbreznakandswitzer(1986)validatedh2+co2 acetogenicactivityinthetermitehindgut.specifically,acetogenicbacteriaare responsiblefortransformingthish2+co2intoadditionalacetate(pleasesee Chapter1Fig1 2)(Breznak&Switzer1986;Braumanetal.1992;Leadbetteretal. 1999;Pester&Brune2007).Thisbacteriamediatedsynthesisofacetatefrom fermentation generatedh2+co2issignificantandcouldaccountforupto approximately1/3ofalltheacetateproducedduringthehindgutfermentation (Breznak&Switzer1986). Acetogensisolatedfromthetermitehindgut SeveralH2+CO2acetogenicbacteriahavebeenisolatedfromthehindgutsof nutritionallydiversetermites.reflectiveoftheimportanceofh2+co2acetogenesis totermitenutrition,theseorganismsarediverseandincludesporomusatermitida obtainedfromthewood feeding higher termitenasutitermesnigriceps(breznaket al.1988),acetonemalongumfromthedrywood feeding lower termite

63 2 4 Pterotermesoccidentis(Kane&Breznak1991),Clostridiummayombeifromthe Africansoil feeding higher termitecubitermesspeciosus(kaneetal.1991),and Treponemaprimitiastr.ZAS 1andZAS 2fromZootermopsisangusticollis (Leadbetteretal.1999). WhileitisknownthattheseorganismsperformH2+CO2acetogenesisinvitro,there isnotagoodunderstandingoftheircontributiontoacetogenesisand,inturn,to nutritionandenergyinthetermitehindgut(brune2006).leadbetter s enlighteningisolationof,andworkwith,t.primitiastr.zas 1andZAS 2illustrated thatspirochetes prominentmembersoftermitehindgutmicrobialcommunities couldbesignificantcontributorstothetermitesystem sacetatepoolgiventheir numbersandacetogeniccapabilities(1999).inadditiontoh2+co2acetogenesis, botht.primitiastrainswereabletofermenthomoacetogenicallyvarioushexoses, pentoses,anddisaccharides(graber&breznak2004).t.primitiastr.zas 2was foundtoalsoutilizemethoxylatedaromaticcompounds(graber&breznak2004). Homoacetogenicgrowthonmeth(ox)ylatedcompounds Inobservingenrichmentculturesfromsewagesludge,Fischerwasthefirstto reporttheformationofacetatefromh2+co2(1932).thenwieringareportedthe firstbacteriumtoderiveenergyforgrowthbyacetatesynthesisfromh2+co2, Clostridiumaceticum(1936).MostacetogenicbacteriaderiveacetatefromH2+CO2 withh2servingastheelectrondonorforco2reductiontoacetate(diekert1992). Specifically,themethylgroupofacetateisformedfromCO2viaformateandthe carboxylgroupisderivedfromco,synthesizedfromo2bycarbonmonoxide

64 2 5 dehydrogenase(diekert1992).nevertheless,acetogensareaverymetabolically versatilegroupofanaerobicbacteria(diekert1992;schink1995).mostareableto growonavarietyofdifferentsubstratesincludingsugars,c 1compounds, methylatedcompounds,non aromaticoraromaticmethoxylatedcompounds,and alcohols(schink1995). Acetogensthatgrowwithmeth(ox)ylatedcompoundsareknownas demethylating acetogens andincludebutyribacteriummethylotrophicum(lynd&zeikus1983), Eubacteriumlimosum(Genthneretal.1981),Acetobacteriumwoodii(Bache& Pfennig1981),Sporomusasp.(Breznak1988),Clostridiumformicoaceticum,and Acetobacteriumcarbinolicum(Diekert1992).Commonlyusedmethylsubstratesare methanol(zeikusetal.1980),methoxylatedaromaticcompoundssuchasvanillate orsyringate(bache&pfennig1981),andmethylchloride(trauneckeretal.1991). Theseandsimilarcompoundsmaybeformedasintermediatesofwood decompositioninthetermitehindgutandofferanalternativemethodofgenerating acetate(bache&pfennig1981). Experimentalmotivation Twotermitehindgutspirocheteisolates,Treponemaprimitiastr.ZAS 1andZAS 2, arebonafideh2+co2acetogens(leadbetteretal.1999).inparticular, acetogenesisfromh2+ 14 C labelledco2wasfoundtosupportmostofthegrowthof T.primitiastr.ZAS 1andZAS 2inaccordancewithacetyl CoA(Wood Ljungdahl) stoichiometries(leadbetteretal.1999).bothstrainsalsodemonstratedtheuseof otherorganicsubstrateshomoacetogenically(graber&breznak2004).

65 2 6 Athirdspirocheteisolatefromthesametermitespecieshindgut,T.azotonutricium str.zas 9,however,doesnotdisplayacetogenicphysiologyinpureculture(Graber etal.2004).afterfirstbeingobtainedinpureculture,thegenomesofthethree Treponemaspecieshavebecomeavailableforanalysisandcomparison(Rosenthal etal.2011;balloretal.2012).althoughthephysiologyisnotpresentinvitro,t. azotonutricium sgenomeisnotcompletelydevoidofwood Ljungdahlpathway homologs(rosenthaletal.2011;balloretal.2012). Asbackground,theacetyl CoA(Wood Ljungdahl)pathwayofacetogenesisconsists ofacarboxylbranchandamethylbranch(fig.2 1)(Diekert1992;Drake1994; Ragsdale1997).Inthecarboxylbranch,carbonmonoxidedehydrogenasereduces CO2toCOformingthecarboxylgroupofacetate(Fig.2 1).Inthemethylbranch, CO2isreducedtoformate,whichissubsequentlyboundtotetrahydrofolate generatingformyl tetrahydrofolate(fig.2 1).Thisformyl tetrahydrofolateisthen reducedtomethyl H4folateviamethenyl andmethylene H4folate(Fig.2 1).The resultingmethylgroupisnexttransferredtoacorrinoidproteinthatbecomesthe methylgroupofacetate(fig.2 1)(Ljungdahletal.1966;Diekert1992;Drake1994; Ragsdale1997). T.azotonutriciumstr.ZAS 9hasgeneswiththerequisitePfamdomainssuggesting functionalityforthecarboxylbranchofacetogenesis(representedbycarbon monoxidedehydrogenase,althoughinitialstudiesdidnotdetectactivityforthis enzyme;graber&breznak2004)andhomologsalsowithrequisitepfamsforfourof thesixenzymesresponsibleforthemethylbranchofacetogenesis(fig.2 1)

66 2 7 (Rosenthaletal.2011;Balloretal.2012).Specifically,T.azotonutriciumstr.ZAS 9 hasallofthegenesforthewood LjungdahlpathwayofacetogenesisthatT.primitia str.zas 1andZAS 2have,withtheexceptionofformatedehydrogenaseand methylene tetrahydrofolatereductase(fig.2 1)(Rosenthaletal.2011;Balloretal. 2012). Fig.2 1:Acetyl CoA(Wood Ljungdahl)pathwayforCO2 reductiveacetogenesis. THF,tetrahydrofolate;Acetyl P,acetyl phosphate.pathwayenzymeswithhomologs intreponemaazotonutriciumstr.zas 9 sgenomearedarkwhereaspathway enzymeswithouthomologsint.azotonutriciumstr.zas 9 sgenomearelight. Dashedlineindicatesintermediatecanbeinsteadutilizedforbiosynthetic processes.(figurecourtesyoftheleadbetterlab).

67 2 8 Formatedehydrogenaseisresponsibleforthefirststepofthemethylbranchofthe Wood Ljungdahlpathwayfortheformationofthemethylgroupofacetate(Fig.2 1) (Diekert1992).ItcatalyzesthereductionofCO2toformateandisanoxygen sensitiveenzyme(fig.2 1)(Diekert1992).Giventhatthetermitehindgutis actuallyanenvironmentwithvaryinglevelsofoxygen,itisplausiblethatt. azotonutriciumstr.zas 9haslostthisenzymeinfavorofaroutetogenerateacetate thatcanwithstandanenvironmentofvaryingoxygenlevels(bruneetal.1995). andmethylene tetrahydrofolatereductasemeansthatt.azotonutriciumstr.zas 9 cannotgeneratethemethylgroupofacetateviathismethylbranch,t. azotonutriciumstr.zas 9 sgenomecontains51annotatedmethyl(transfer)ases (AppendixTable2A 1).Itispossible,therefore,thatT.azotonutriciumstr.ZAS 9 candirectlyshuntmethylgroupsfrommeth(ox)ylatedcompoundsinthetermite hindgutenvironmentintothewood Ljungdahlpathwaytocontributetothemethyl groupofacetate,bypassingthemajorityofthemethylbranch(fig.2 1)(Ljungdahl etal.1966;diekert1992;drake1994;ragsdale1997).althought.azotonutricium T.azotonutriciumstr.ZAS 9lacksanotherkeymethylbranchenzyme,methylenetetrahydrofolatereductase(Fig.2 1).Whilemissingbothformatedehydrogenase str.zas 9doesderiveacetateaswellasethanol,H2,andCO2fromnonhomoacetogenicfermentationofcarbohydrates,thisabilitywouldallowthestrain tocontributeevenmoretotermitenutritionviaacetogenesisfromdemethylationof organiccompounds.totestthishypothesisliquidculturesoft.azotonutriciumstr. ZAS 9weregrownwithavarietyofmeth(ox)ylatedcompounds.Growthratesand yieldsweremeasuredasaproxyforacetateproduction.

68 2 9 MATERIALSANDMETHODS: Mediaandcultivation RoutineinvitrogrowthandmaintenanceofTreponemaazotonutriciumstr.ZAS 9in 4YAColiquidmediumunderaheadspaceof80%N2/20%CO2wascarriedoutas previouslydescribed(pleaseseechapter1materialsandmethods)(graberetal. 2004).ForexaminationofgrowthofT.azotonutriciumstr.ZAS 9onseveral meth(ox)ylatedcompounds,thegrowthmediumwasmaintainedat4yacoor reformulatedto1yaco(yeastautolysateforyacomediapreparedfromjarsof Fleischmann sbrandyeast,pleaseseechapter1),andmaltosewaseitherincreased to40mm,retainedat20mm,decreasedto5mm,2.5mm,or1mmfinalconcentration, oromitted.insomeinstances100%h2wasaddedsterilytothecultureheadspace of80%n2/20%co2(toachieveafinalconcentrationof0.5%h2,vol/vol)priortoor atthetimeofinoculation.cultureswereincubatedinthedark,atroom temperature,inahorizontalposition,andwithoutagitation. Growthinthepresenceofmeth(ox)ylatedcompounds Individual50mMstocksolutionsofDL methionine,dmso,methanol,methylamine, dimethylamine,trimethylamine,betaine,andtrimethoxybenzoatewereprepared sterily,undern2,andstoredinthedark(table2 1).Asneeded,stocksolutions wereneutralizedwithnaohduringthepreparations. Toinvestigatethepotentialtoxicityofthesemeth(ox)ylatedcompoundson Treponemaazotonutriciumstr.ZAS 9,initialstudiescomparedgrowthofthestrain oneachsubstrateat1mm,5mm,or10mmfinalconcentration.controlswereliquid

69 2 10 culturesofthesameexperimentalconditionsexceptwithwaterinsteadof meth(ox)ylatedsubstrate.asaresultofpreliminarystudies,culturesoft. azotonutriciumstr.zas 9werescreenedformetabolismofmeth(ox)ylated compoundsatafinalconcentrationof5mm.changesinopticaldensityofgrowing cultureswasaproxyformeth(ox)ylatedcompoundutilizationandweremeasured at600nmwithaspectronic20colorimeter. Bioinformatics ForidentificationofmethylasesandmethyltransferasesinTreponema azotonutriciumstr.zas 9 sgenome,datawereobtainedfromrast(azizetal. 2008),JGIIMG/M(Markowitzetal.2006),andNCBIBLAST(Geeretal.2010). RESULTS: Treponemaazotonutriciumstr.ZAS 9hasputativemethyl(transfer)asesin neighborhoodsofacetyl CoA(Wood Ljungdahl)pathwayhomologs Severalmethyl(transfer)asesareinthegeneneighborhoodsofWood Ljungdahl pathwayhomologsinthegenomeoft.azotonutriciumstr.zas 9,andthis arrangementmighthaveimplicationstofunctionality(fig.2 2).Thislocalization supportsthehypothesisthatt.azotonutriciumstr.zas 9canbypassthemethyl branchofthewood Ljungdahlpathwayofacetogenesisand,instead,directly transfermethylgroupsfromwood derivedmeth(ox)ylatedcompoundsinthe termitehindgutdirectlytocarboxylicacidgeneratedfromthecarboxylbranchof

70 2 11 thepathwaytoformacetate. Fig.2 2:Treponemaazotonutriciumstr.ZAS 9geneneighborhoodswithboth putativemethyl(transfer)asesandhomologsoftheacetyl CoA(Wood Ljungdahl) pathwayofacetogenesis.relevantgenesareinblack,andunrelatedorhypothetical proteinsareingrey.thf,tetrahydrofolate;met,methionine;transp.,transporter; bind.,binding;(l),largesubunit;(s),smallsubunit;codh,carbonmonoxide dehydrogenase;cys,cysteine;methyltransf.,methyltransferase;vit.,vitamin;fam., family.

71 2 12 Treponemaazotonutriciumstr.ZAS 9growthonmeth(ox)ylatedcompoundsis inconclusive Asapreliminaryscreenofpotentialtoxicityofmeth(ox)ylatedcompoundson Treponemaazotonutriciumstr.ZAS 9,growthofthestrainwasevaluatedoneach substrateat1mm,5mm,or10mmfinalconcentrationin1yacomediumwith20mm maltose(datanotshown).growthwassuccessfulandgrowthyieldsweregreatest oncompoundsprovidedat5mmconcentrationcomparedto1and10mm(datanot shown).fromthispointon,substrateswereaddedtoculturesto5mmfinal concentrationforgrowthexperiments. Next,asafirstpassatcomparisonofgrowthofT.azotonutriciumstr.ZAS 9on5mM ofeachmeth(ox)ylatedsubstrate,liquidculturesofthestrainwerepreparedin duplicatein1yacomediumwith20mmmaltose(fig.2 3a;AppendixFig.2A 1).A smallbutsignificantincreaseingrowthyieldwasobservedint.azotonutriciumstr. ZAS 9culturesgrownwithDMSO,betaine,andtrimethoxybenzoaterelativeto growthonothersubstratesorjustwaterasacontrol(fig.2 3a;AppendixFig.2A 1, Table2A 2).Nodiscernabledifferencesingrowthratewereobserved(Fig.2 3a; AppendixFig.2A 1). Asaresultofthisinitialcomparison,betaine,DMSO,andtrimethoxybenzoate appearedtobepromisingcompoundsforfurthertesting,butsignificantincreasesin growthyieldonthesesubstratesrelativetocontrolsweresmall(fig.2 3a;Appendix Fig.2A 1,Table2A 2).Therefore,growthonalloftheaforementioned meth(ox)ylatedcompoundswasre evaluatedat5mmconcentrationandwithor without5mmmaltose.theamountofmaltoseaddedtoexperimentalcultureswas

72 2 13 reducedontheassumptionthat20mmmaltose,asexaminedearlier,providedtoo muchsubstrateforgrowth.perhaps,t.azotonutriciumstr.zas 9waspreferentially utilizingmaltoseinsteadofthemeth(ox)ylatedcompounds.therefore,byreducing maltoseto5mmoromittingit,itwassupposedthatstr.zas 9wouldmetabolizethe meth(ox)ylatedcompoundsandmorestrikinggrowthyielddifferenceswouldbe seen.thistime, sparking amountsof100%h2werealsoaddedtothe80% N2/20%CO2headspaceincaseH2hadbeenalimitingfactorintheprevious experiment.asmallbutsignificantincreaseingrowthyieldwasobservedint. azotonutriciumstr.zas 9culturesgrownwithmethylamine,dimethylamine,and trimethoxybenzoaterelativetogrowthonothersubstratesorjustwaterasacontrol (Fig.2 3b,c,andd;AppendixFig.2A 2a,b,c,andd,Table2A 3,Table2A 4).No discernabledifferencesingrowthratewereseen(fig.2 3b,c,andd;AppendixFig. 2A 2a,b,c,andd). Becausethemostsignificantlydifferentincreaseingrowthyieldrelativetocontrols wasobservedwithgrowthondimethylamine,dimethylaminewasexaminedwith variousyaco,maltose,andh2regimesinanefforttofindthecombinationofyaco, maltose,andh2thatwould kickstart growthandpromptoptimalutilizationofthe meth(ox)ylatedcompound.theincreasesingrowthyieldorratewith dimethylamine,however,werenotsignificantdespiteevaluatingallofthese parameters(fig.2 3e;AppendixFig.2A 3aandb).Theresultsobtaineddidnot warrantfurtherinvestigationusingtheseapproaches.

73 2 14 a b c d D!#("!#'" +,-./012,"3" +,-./012,"4" +,-./012,"5" "3" "4" "5"!#&"!#%" """"9: )!!7;"!#$"!"!"#$%&'()*%!" $!!" %!!" &!!" '!!" (!!" )!!" *!!" (#&" (#%" (#$" e ("!#'"!#&"!#%"!#$"!" 78 &!!59" :-9*";<6=>"!" $!!" %!!" &!!" '!!" (!!!" ($!!" (%!!" )*+,-./0*"1" )*+,-./0*"2" )*+,-./0*"3" ,"1" ,"2" ,"3"

74 2 15 Fig.2 3:GrowthofTreponemaazotonutriciumstr.ZAS 9withvarious meth(ox)ylatedcompounds.smallbutsignificantincreasesingrowthyieldwere observedfrom1yacoliquidculturesoft.azotonutriciumstr.zas 9undera headspaceof80%n2/20%co2growingwith(a)5mmdmso,betaine,and trimethoxybenzoate(tmb)andsupplementedwith20mmmaltose(initial experiment)and(b)5mmmethylamine,(c)5mmdimethylamine,and(d)5mm trimethoxybenzoateallsupplementedwith5mmmaltoseand100%h2toachievea finalconcentrationof0.5%vol/vol(allsecondexperimentresults)comparedto controlswithwaterinsteadofthemeth(ox)ylatedcompounds.inconclusive increasesingrowthyieldwereobservedfrom(e)liquidculturesoft. azotonutriciumstr.zas 9growingin4YAComediumwith5mMdimethylamine, 40mMmaltose,and100%H2toachieveafinalconcentrationof0.5%vol/vol comparedtocontrolswithwaterinsteadofthemeth(ox)ylatedcompound.all cultureswereincubatedinthedark,atroomtemperature,inahorizontalposition, andwithoutagitation.changesinopticaldensityofgrowingcultureswasaproxy formeth(ox)ylatedcompoundutilizationandweremeasuredat600nmwitha Spectronic20colorimeter.Nodiscernabledifferencesingrowthratewereseen.

75 2 16

76 2 17 DISCUSSION: Treponemaazotonutriciumstr.ZAS 9growthonmeth(ox)ylatedcompoundsis inconclusive NosignificantdifferencesingrowthratesofliquidculturesofTreponema azotonutriciumstr.zas 9withmeth(ox)ylatedcompoundsrelativetocontrolswere seen(fig.2 3a,b,c,d,ande;AppendixFig.2A 1,Fig2A 2a,b,c,andd,Fig.2A 3a andb).anysignificantgrowthyieldincreasesinexperimentalculturescomparedto controlsweresmallandnotreproducibleamongthedifferentmeth(ox)ylated substratestested(fig.2 3a,b,c,d,ande;AppendixFig.2A 1,Table2A 2,Fig2A 2a, b,c,andd,table2a 3,Table2A 4,Fig.2A 3aandb).Specifically,in1YAColiquid mediumunderan80%n2/20%co2headspaceandsupplementedwith20mm maltose,culturesoft.azotonutriciumstr.zas 9displayedsignificantbutrelatively smallincreasesingrowthyieldwith5mmdmso,betaine,ortrimethoxybenzoate (TMB)(Fig.2 3a,b,c,d,ande;AppendixFig.2A 1,Table2A 2).Ontheotherhand, in1yacoliquidmediumunderan80%n2/20%co2headspacewith100%h2added toachieve0.5%vol/volfinalconcentration,andsupplementedwith5mmmaltose ornomaltose,culturesoft.azotonutriciumstr.zas 9displayedsignificantbut relativelysmallincreasesingrowthyieldwith5mmmethylamine,dimethylamine, ortmb(fig.2 3b,c,andd;AppendixFig.2A 2a,b,c,andd,Table2A 3,Table2A 4). Althoughfromtheseexperimentsdimethylamineappearedtobethemost promisingsubstrate,experimentsfocusingondimethylaminewithvariousregimes ofyaco,maltose,andh2inanefforttoobservegreaterdifferencesingrowthyield betweenexperimentalandcontrolcultureswereinconclusive(fig.2 3e;Appendix Fig.2A 3aandb).

77 2 18 Futurework: AnalyzeTreponemaazotonutriciumstr.ZAS 9methyl(transfer)asesgenesand geneneighborhoods First,whilemethyl(transfer)aseswereobservedinthesamegeneneighborhoodsas homologsoftheacetyl CoA(Wood Ljungdahl)pathwayofacetogenesis,itwouldbe worthwhiletoexaminethoseneighborhoodsforevidenceofpromoters,operons, andregulationstogaugefunction.further,asimpleexercisetoalsoevaluate functionwouldbetolookforpfamdomainsandkeyresiduesknowntobe importantforfunctionalityinthesegeneandproteinsequences.thepresenceof keypfamdomainsandaminoacidresiduescouldsupportthehypothesisof acetogenicdemethylationbyt.azotonutriciumstr.zas 9.TheabsenceofkeyPfam domainsandaminoacidresidueswouldnotnecessarilyruleoutthishypothesis,but couldre directstudieskeepingthesedifferencesinmind. Decreaseexperimentalconcentrationofmeth(ox)ylatedsubstrate Thatdimethylaminewasthesubstratethatledtothemostpromisingpossible increasesint.azotonutriciumstr.zas 9growthyieldcorroboratesearlierstudies demonstratingthatt.primitiastr.zas 2isabletousemethoxylatedaromatic compounds,suchastmb,asenergysources(graber&breznak2004).similarto theworkwitht.azotonutriciumstr.zas 9presentedherein,T.primitiastr.ZAS 2 didnotgrowonmethanol(graber&breznak2004).intheworkwitht.primitia str.zas 2,however,meth(ox)ylatedcompoundsweresuppliedatconcentrations lessthanorequalto2.5mmbecausehigherconcentrationsinhibitedgrowth (Graber&Breznak2004).Perhapstestinggrowthwith5mMofmeth(ox)ylated

78 2 19 aromaticcompoundsherewasinhibitory.themaximumconcentrationof meth(ox)ylatedsubstratesthatdidnotleadtoinhibitoryeffectswaschosenfor experimentalevaluationundertheassumptionthatitwouldleadtomostproduct formationandobservabledifferencesbetweenexperimentalandcontrolcultures. However,perhapsgrowthwasnotvisiblyinhibitedby5mMconcentrationsbut compoundutilizationwasbecauseenzymesubstratebindingsiteswereoverloaded.inthefutureonecouldtestarangeofconcentrationsbetweenthe1mmand 5mMconcentrationsexaminedhereandsimultaneouslymeasurehowmuchofthe meth(ox)ylatedisused.theexperimentcouldthenberepeatedusingthat particularconcentrationinordertonotoverwhelmthesystem.anotherpotential experimentwouldbetore evaluatethishypothesisafter priming T. azotonutriciumstr.zas 9thathasbeensub culturedawayfromitsoriginal environmentforoveradecadewithmeth(ox)ylatedcompoundsto kickstart this putativemetabolism. Directlymeasuremeth(ox)ylatedcompoundusageandacetateproduction Thatsignificantbutsmallboostsingrowthyieldwereobservedsuggeststhat,under theexperimentalconditionsexaminedherein,anypossibleacetategenerationbyt. azotonutriciumstr.zas 9viatheproposedacetogenicdemethylationmechanism doesnotresultinanincreaseingrowthrateandyieldsuchascanbedetectedby opticaldensitymeasurements.whileitisstillundeterminedwhetherornott. azotonutriciumstr.zas 9cangenerateacetogenesisviaacetogenicdemethylation, perhapsdifferentexperimentalapproachessuchasradio labelingandtracingofthe methylgroupsoftheexaminedmeth(ox)ylatedcompounds,orgc MS,ICS,orLC MS

79 2 20 measurementsofculturemediaforacetateproductionovertimeofculturegrowth, willprovidemoreconclusiveanswers. Feedingexperimentsandco culturing EarlierandsubsequentbioinformaticandgenomicworkwiththeT.primitiametapathwayhomologsalsoindicatesthatgrowthyieldincreasesarenotnecessarilythe bestproxyforanacetate yieldingfunction(pleaseseechapter3).thismaybe becauseinsitu,perhaps,thisacetateisnotusedbythegeneratingorganism,butis insteadgiventoanothermemberofthehindgutmicrobialcommunity.forexample, ithasbeensuggestedthatrelatedtotheirsymbioticrelationshipswiththetermite gutmicrobialcommunityandthetermitehostitself,t.primitiastr.zas 1andZAS 2 andt.azotonutriciumstr.zas 9donotconvertsubstratestoproductsforrapid growthandefficientgenerationofcellmaterial(graber&breznak1994;graberet al.1994).rather,theygivetheacetatetheyproducetotherestofthehindgut microbialcommunityandtheirhost,anddisplaylimitedproductionofbiomass (Graber&Breznak1994;Graberetal.1994).Thistransferofacetatemaybeableto bemeasuredbyconductingfeedingexperimentswherebycell freet. azotonutriciumstr.zas 9culturefluidisfedtootherculturesthatwould demonstrateamarkedincreaseingrowthrateandyieldfromacetate.successof co cultureexperimentswitht.azotonutriciumandthisorganismcouldalsobe informative(pleaseseechapters2and3)(rosenthaletal.2011). Whatisknownecologicallyaboutthetermitehindgutecosystemsuggeststhatthe abilitytoperformacetogenicdemethylationwouldnotonlycontributetothe

80 2 21 acetatepoolinthetermitesystem,butmayalsobeacompetitivestrategyina complexecosystem.becausehomoacetogensarespecialistsinc1metabolismand competewithmethanogensandsulfatereducers,methylgroupsofmeth(ox)ylated carboncompoundsareafoodsourcethatisrelativelylesscompetedfor(schink 1995).Moreover,ligninisabundantinthetermitehindgutandligninderivatives havebeenpostulatedtoactasmethyldonorsformethyl groupformationby homoacetogens(schink1995).inlightofsubsequentworkwiththet.primitia meta cleavagepathway,ift.azotonutriciumutilizesthemethylgroupsofligninderivedaromatics,thiscouldhelppreparethesesubstratesforthet.primitia catechol2,3 dioxygenases(pleaseseechapter3).thishypothesiscouldbefurther testedwitht.azotonutriciumandt.primitiaco cultureexperiments(perrosenthal etal.2011).iffurtherinvitroanalysesstilldonotdemonstrateacetogenic demethylationbyt.azotonutriciumstr.zas 9,theculturemediaconditionsshould bere examined. Enzymeactivityassaysandexpressionwork Lastly,itmightbeworthwhiletocloneandexpressthesegenesinanexpression vectorsuchase.colitoevaluateenzymefunctionalityandactivitywithoutvariables inthet.azotonutriciumculturesthatmaybenegativelyimpactingthese genes /enzymes functions.thesegenescanbeexpressedandevaluated individuallyortogetherandenzymeactivityassayscouldbperformed.qrt PCR withprimersdesignedtotargetrelevantgenescouldalsohintatexpressionorlack thereof.

81 2 22 IfwithadditionalworkthehypothesisthatT.azotonutriciumstr.ZAS 9isableto bypassthemethylbranchofthewood Ljungdahlpathwayandgenerateacetate withmethylgroupsittakesfromwoodisnotsupported,atleasttheseexperiments demonstratethatanorganism,evenwithacompletesetofgenesrepresentinga particularfunction,doesnotnecessarilyperformthatfunction.

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83 2 24 FischerF,LieskeR,WinzerK(1932)BiologischeGasreaktionen.II:ÜberdieBildung vonessigsäurebeiderbiologischenumsetzungvonkohlenoxydundkohlensäure mitwasserstoffzumethan.biochemischezeitschrift,245,2 12. GeerLY,Marchler BauerA,GeerRCetal.(2010)TheNCBIBioSystemsdatabase. NucleicAcidsResearch,38,D492 D496. GenthnerBRS,DavisCL,BryantMP(1981)Featuresofrumenandsewagesludge strainsofeubacteriumlimosum,amethanolandh2 CO2 utilizingspecies.applied andenvironmentalmicrobiology,42, GraberJR,BreznakJA(2004)PhysiologyandnutritionofTreponemaprimitia,an H2/CO2 acetogenicspirochetefromtermitehindguts.appliedandenvironmental Microbiology,70, GraberJR,LeadbetterJR,BreznakJA(2004)DescriptionofTreponema azotonutriciumsp.nov.andtreponemaprimitiasp.nov.,thefirstspirochetes isolatedfromtermiteguts.appliedandenvironmentalmicrobiology,70, HungateRE(1939)ExperimentsonthenutritionofZootermopsis.III.Theanaerobic carbohydratedissimilationbytheintestinalprotozoa.ecology,20, HungateRE(1943)Quantitativeanalysesonthecellulosefermentationbytermite protozoa.annalsoftheentomologicalsocietyofamerica,36, HungateR(1955)MutualisticIntestinalProtozoa.In:BiochemistryandPhysiologyof Protozoa(edsHunterS,LwoffA),pp AcademicPress,NewYork. KaneMD,BraumanA,BreznakJA(1991)Clostridiummayombeisp.nov.,anH2/CO2 acetogenicbacteriumfromthegutoftheafricansoil feedingtermite,cubitermes speciosus.archivesofmicrobiology,156, KaneMD,BreznakJA(1991)Acetonemalongumgen.nov.sp.nov.,anH2/CO2 acetogenicbacteriumfromthetermite,pterotermesoccidentis.archivesof Microbiology,156, LeadbetterJR,SchmidtTM,GraberJR,BreznakJA(1999)AcetogenesisfromH2plus CO2byspirochetesfromtermiteguts.Science,283, LjungdahlLG,IrionE,WoodHG(1966)Roleofcorrinoidsinthetotalsynthesisof acetatefromco2byclostridiumthermoaceticum.federationofamericansocieties forexperimentalbiologyjournal,25, Lynd LH, Zeikus JG (1983) Metabolism of H2 CO2, methanol, and glucose by Butyribacterium methylotrophicum.journalofbacteriology,153,

84 2 25 MarkowitzVM,KorzeniewskiF,PalaniappanKetal.(2006)Theintegrated microbialgenomes(img)system.nucleicacidsresearch,34,d344 D348. MauldinJK(1982)Lipidsynthesisfrom[ 14 C] acetatebytwosubterraneantermites, ReticulitermesflavipesandCoptotermesformosanus.InsectBiochemistry,12, OdelsonDA,BreznakJA(1983)Volatilefattyacidproductionbythehindgut microbiotaofxylophagoustermites.appliedandenvironmentalmicrobiology,45, Odelson DA, Breznak JA (1985a) Nutrition and growth characteristics of Trichomitopsis termopsidis, a cellulolytic protozoan from termites. Applied and EnvironmentalMicrobiology,49, OdelsonDA,BreznakJA(1985b)Cellulaseandotherpolymer hydrolyzingactivities oftrichomitopsistermopsidis,asymbioticprotozoanfromtermites.appliedand EnvironmentalMicrobiology,49, PesterM,BruneA(2007)Hydrogenisthecentralfreeintermediateduring lignocellulosedegradationbytermitegutsymbionts.ismejournal,1, RagsdaleSW(1997)TheeasternandwesternbranchesoftheWood/Ljungdahl pathway:howtheeastandwestwerewon.biofactors,6,3 11. RosenthalAZ,MatsonEG,EldarA,LeadbetterJR(2011)RNA seqreveals cooperativemetabolicinteractionsbetweentwotermite gutspirochetespeciesin co culture.ismejournal,5, SchinkB(1995)Diversity,Ecology,andIsolationofAcetogenicBacteria.In: Acetogenesis(edDrakeHL),pp Chapman&Hall,NewYork. TholenA,BruneA(2000)Impactofoxygenonmetabolicfluxesandinsituratesof reductiveacetogenesisinthehindgutofthewood feedingtermitereticulitermes flavipes.environmentalmicrobiology,2, TrauneckerJ,PreussA,DiekertG,PreussA(1991)Isolationandcharacterizationof amethyl chlorideutilizing,strictlyanaerobicbacterium.archivesofmicrobiology, 156, WieringaKT(1940)Theformationofaceticacidfromcarbondioxideandhydrogen byanaerobicspore formingbacteria.antonievanleeuwenhoek,6, YaminMA(1980)CellulosemetabolismbythetermiteflagellateTrichomitopsis termopsidis.appliedandenvironmentalmicrobiology,39,

85 2 26 CHAPTER2:APPENDIX Table2A 1:Genesannotatedasmethyl(transfer)asesinthegenomeofTreponema azotonutriciumstr.zas 9. Fig.2A 1:GrowthofTreponemaazotonutriciumstr.ZAS 9in1YAColiquidmedium underaheadspaceof80%n2/20%co2with5mmvariousmeth(ox)ylated compounds(specifiedongraphs)supplementedwith20mmmaltose. Table2A 2:Student st testp valueresults(correspondingtofig.2a 1). Fig2A 2a,b,c,andd:GrowthofTreponemaazotonutriciumstr.ZAS 9in1YACo liquidmediumunderaheadspaceof80%n2/20%co2supplementedwith100%h2 toachieveafinalconcentrationof0.5%vol/voland5mmvariousmeth(ox)ylated compounds.culturesrepresentedincanddalsohave5mmmaltose. Table2A 3:Student st testp valueresults(correspondingtofig.2a 2CandD). Table2A 4:AveragehighestOD600nmobtainedoftriplicatecultures. Fig2A 3aandb:GrowthofTreponemaazotonutriciumstr.ZAS 9in1YACo(a)or 4YACo(b)liquidmediumunderaheadspaceof80%N2/20%CO2and5mM dimethylaminewith100%h2toachieveafinalconcentrationof0.5%vol/voland differentmaltoseadditions(specifiedongraphs). Table2A 2:Genesannotatedasmethyl(transfer)asesinthegenomeofTreponema azotonutriciumstr.zas 9.

86 2 27 Table2A 1:Genesannotatedasmethyl(transfer)asesinthegenomeofTreponema azotonutriciumstr.zas 9. Accession# Annotation YP_ SrRNA(guanine N 2 )methyltransferaserlmlec ) YP_ RibosomalRNAsmallsubunit methyltransferasef(ec ) YP_ SrRNA(Uracil 5 )methyltransferaseruma(ec ) YP_ trna(guanine46 N7 ) methyltransferase(ec ) YP_ prophagelambdaso,dnamodification methyltransferase,putative YP_ RibosomalRNAsmallsubunit methyltransferased(ec ) YP_ SrRNAmethyltransferaseand Florfenicol/chloramphenicol resistanceprotein/radicalsamfamily enzyme,upf0063family YP_ Methyltransferase/methylase YP_ S adenosyl methyltransferasemraw (EC ) YP_ COG1092familypredictedtRNA methylase YP_ tetrapyrrolemethylasefamily protein/mazgfamilyprotein YP_ Methylcobalamin:coenzymeM methyltransferase,methylaminespecific YP_ Methylcobalamin:coenzymeM methyltransferase,methylaminespecific YP_ Dimethylaminemethyltransferase corrinoidprotein YP_ Methylcobalamin:coenzymeM methyltransferase,methylaminespecific YP_ PredictedN6 adenine specificrna methylasecontainingthumpdomain YP_ methyltetrahydrofolate homocysteinemethyltransferase(ec ) YP_ methyltetrahydrofolate:corrinoid

87 2 28 iron sulfurproteinmethyltransferase YP_ methyltetrahydrofolate homocysteinemethyltransferase(ec ) YP_ Corrinoidmethyltransferaseprotein YP_ Uroporphyrinogen III methyltransferase(ec ) /Uroporphyrinogen IIIsynthase(EC ) YP_ PutativeRNA2' O ribose methyltransferasemtfa(ec ) YP_ Tetrapyrrole(Corrin Porphyrin) methylasefamilyproteinupf0011 YP_ TRNA/rRNAmethyltransferase YP_ hypotheticaltrna/rrna methyltransferaseyfif[ec: ] YP_ Methyltransferase(EC );N6 adenine specificdnamethylase YP_ rrnamethylases YP_ Serinehydroxymethyltransferase(EC ) YP_ Chemotaxisproteinmethyltransferase CheR(EC ) YP_ methyltransferase YP_ methylated DNA protein cysteine methyltransferase relatedprotein YP_ Methylcobalamin:coenzymeM methyltransferase,methanol specific YP_ Methylcobalamin:coenzymeM methyltransferase,methanol specific YP_ Methylcobalamin:coenzymeM methyltransferase,methanol specific YP_ RNAmethyltransferase,TrmHfamily YP_ DNAmodificationmethylase(Adeninespecificmethyltransferase)(EC ) YP_ Methyl directedrepairdnaadenine methylase(ec ) YP_ TypeIrestriction modificationsystem, DNA methyltransferasesubunitm(ec )

88 2 29 YP_ TypeIrestriction modificationsystem, DNA methyltransferasesubunitm(ec ) YP_ trna(guanine37 N1)methyltransferase(EC ) YP_ methyltetrahydrofolate homocysteinemethyltransferase(ec ) YP_ MethyltransferasegidB(EC2.1.. ) YP_ HEN1C terminaldomain;doublestrandedrna3' methylase YP_ Methylaseofpolypeptidechainrelease factors YP_ trna:cm32/um32methyltransferase YP_ Smallribosomalsubunit16SrRNA methyltransferase##u1498 specific ine.coli YP_ Aminomethyltransferase(glycine cleavagesystemtprotein)(ec ) YP_ trna(5 methylaminomethyl 2 thiouridylate) methyltransferase(ec ) YP_ TypeIrestriction modificationsystem, DNA methyltransferasesubunitm(ec ) YP_ GlycineN methyltransferase(ec ) YP_ Methylated DNA protein cysteine methyltransferase(ec )

89 2 30

90 2 31 Fig.2A 1:GrowthofTreponemaazotonutriciumstr.ZAS 9in1YAColiquidmedium underaheadspaceof80%n2/20%co2with5mmvariousmeth(ox)ylated compounds(specifiedongraphs)supplementedwith20mmmaltose.controlshave waterinsteadofthemeth(ox)ylatedcompounds.smallbutsignificantincreasesin growthyieldwereobservedfromculturesoft.azotonutriciumstr.zas 9growing with5mmdmso,betaine,ortrimethoxybenzoate(tmb).thesecultureswere incubatedinthedark,atroomtemperature,inahorizontalposition,andwithout agitation.changesinopticaldensityofgrowingcultureswasaproxyfor meth(ox)ylatedcompoundutilizationandweremeasuredat600nmwitha Spectronic20colorimeter.

91 2 32 Table2A 2:Student st testp valueresults Meth(ox)ylatedCompound P Value Betaine DMSO Methanol 0.37 D LMethionine 0.57 Dimethylamine 0.52 Methylamine 0.11 Trimethylamine 0.29 TMB ResultscorrespondtoFig.2A 1. P valuecompareshighestgrowthyield(measuredod600nm)achievedbytreponema azotonutriciumstr.zas 9grownwith5mMofvariousmeth(ox)ylatedcompounds, comparedtohighestgrowthyieldachievedbycontrolswithwaterinlieuof meth(ox)ylatedsubstrate.specifically,themeansofreplicatedatarepresenting eachgrouparecompared.allcultureswerein1yacoliquidmediumsupplemented with20mmmaltoseunderan80%n2/20%co2headspace. 95%confidencelevelwasused,thereforeaP value<0.05signifiesthatthemeans ofthedatafromeachmeth(ox)ylatedtreatmentreplicateissignificantlydifferent fromthatofthecontrols(bold).

92 2 33 a

93 2 34 b

94 2 35 c

95 2 36 d

96 2 37 Fig.2A 2:GrowthofTreponemaazotonutriciumstr.ZAS 9in1YAColiquidmedium underaheadspaceof80%n2/20%co2supplementedwith100%h2toachievea finalconcentrationof0.5%vol/voland5mmvariousmeth(ox)ylatedcompounds (specifiedongraphs).controlshavewaterinsteadofthemeth(ox)ylated compounds.someculturesalsohave5mmmaltose(candd).aandcgraphsare thelogarithmicplotsofbanddgraphs,respectively.smallbutsignificantincreases ingrowthyieldwereobservedfromculturesoft.azotonutriciumstr.zas 9growing with5mmmethylamine,dimethylamine,ortrimethoxybenzoate(tmb)andwith 5mMmaltose.Thesecultureswereincubatedinthedark,atroomtemperature,ina horizontalposition,andwithoutagitation.changesinopticaldensityofgrowing cultureswasaproxyformeth(ox)ylatedcompoundutilizationandweremeasured at600nmwithaspectronic20colorimeter.

97 2 38 Table2A 3:Student st testp valueresults Meth(ox)ylatedCompound P Value Betaine 0.65 DMSO 0.45 Methanol 0.89 D LMethionine < Methylamine Dimethylamine Trimethylamine 0.23 TMB ResultscorrespondtoFig.2A 2candd. P valuecompareshighestgrowthyield(measuredod600nm)achievedbytreponema azotonutriciumstr.zas 9grownwith5mMofvariousmeth(ox)ylatedcompounds, comparedtohighestgrowthyieldachievedbycontrolswithwaterinlieuof meth(ox)ylatedsubstrate.specifically,themeansofreplicatedatarepresenting eachgrouparecompared.allcultureswerein1yacoliquidmediumsupplemented with5mmmaltoseunderan80%n2/20%co2headspacetowhich100%h2was addedto0.5%vol/volfinalconcentration. 95%confidencelevelwasused,thereforeaP value<0.05signifiesthatthemeans ofthedatafromeachmeth(ox)ylatedtreatmentreplicateissignificantlydifferent fromthatofthecontrols(bold).

98 2 39 Table2A 4:AveragehighestOD600nmobtainedoftriplicatecultures Sample HighestOD600nmObtained 4YaCo,40mMMaltose YaCo,40mMMaltose(w/H2) YaCo,20mMMaltose YaCo,20mMMaltose(w/H2) YaCo,5mMMaltose YaCo,5mMMaltose(w/H2) YaCo YaCo(w/H2) AllofthefollowinghaveH2: 1Yaco,5mMBetaine YaCo,5mMBetaine,5mMMaltose YaCo,5mMDMSO YaCo,5mMDMSO,5mMMaltose YaCo,5mMMethanol YaCo,5mMMethanol,5mMMaltose YaCo,5mMD LMethionine YaCo,5mMD LMethionine,5mM Maltose 1YaCo,5mMMethylamine Yaco,5mMMethylamine,5mMMaltose YaCo,5mMDimethylamine YaCo,5mMDimethylamine,5mM Maltose 1YaCo,5mMTrimethylamine YaCo,5mMTrimethylamine,5mM Maltose 1YaCo,5mMTMB YaCo,5mMTMB,5mMMaltose 0.424

99 2 40 a

100 2 41 b

101 2 42 Fig.2A 3:GrowthofTreponemaazotonutriciumstr.ZAS 9in(a)1YACoor(b) 4YAColiquidmediumunderaheadspaceof80%N2/20%CO2supplementedwith 100%H2toachieveafinalconcentrationof0.5%vol/volanddimthylaminewith differentmaltoseadditions(specifiedongraphs).controlshavewaterinsteadof themeth(ox)ylatedcompounds.thesecultureswereincubatedinthedark,atroom temperature,inahorizontalposition,andwithoutagitation.changesinoptical densityofgrowingcultureswasaproxyformeth(ox)ylatedcompoundutilization andweremeasuredat600nmwithaspectronic20colorimeter.

102 3 1 CHAPTER3 Catechol2,3 dioxygenaseandothermeta cleavagecatabolicpathway genesinthe anaerobic termitegutspirochetetreponemaprimitia ABSTRACT: Catechol2,3 dioxygenaseswereobservedinthegenomesofstrainsofthesymbiotic termitehindgut anaerobe, Treponemaprimitia.Bioinformaticsconfirmedthe presenceofacassetteencodinggenes,pfamdomains,andexpectedkeyresidues forenzymesassociatedwithacompletearomaticmeta cleavagepathwaytypicalto aerobicpseudomonads.curiously,transcriptsforeachgenewereevenobserved expressedinstrictlyanaerobicculturesoft.primitia.phylogeneticanalysessuggest thatthedioxygenaseandseveralotheressentialgenesofthemeta pathwaywere acquiredbyt.primitiafromanalphaproteobacteriuminthedistantpast,to augmentseveralgenesacquiredfromanaerobicfirmicutesthatdonotdirectly catabolizearomaticsbutcancontributetofinalmeta pathwaysteps.toexamineif thegeneswerefunctional,culturesoft.primitiawereadaptedtogrowin unreducedmediacontainingamicrooxicheadspace.theadditionofcatechol resultedinthetransientaccumulationoftraceamountsofayellowintermediate havingthespectrophotometriccharacteristicsoftheexpectedringcleavage product,hydroxymuconicsemialdehyde.thisisthefirstevidenceforaromaticring cleavageinthephylum(division)spirochetes.catechol2,3 dioxygenasemetacleavagepathwaysarenotfoundwidespreadacrossthebacteriallineofdescent, previouslyhavingbeenobserved,viaphysiologyorgenomics,restrictedtoselect speciesrepresentingonlythreeofthemajorbacterialphyla(divisions):

103 3 2 Proteobacteria,Firmicutes,andActinobacteria.However,T.primitiadidnot consumecatecholtoexhaustionnorexhibitanymarkedstimulationsingrowthor acetateyield.nevertheless,theresultssuggestthatthepotentialforo2 dependent INTRODUCTION: Manytermitesthriveononeoftheplanet smostabundantformsofbiomass,wood, comprisedprimarilyofcellulose,hemicellulose,andlignin(breznak&brune1994). Thepresenceandmanyrolesofthecomplex,obligate,andnutritionally mutualistic symbioticmicrobialcommunitiesintermitegutsduringthemetabolicprocessingof lignocellulosehaslongbeenestablished(leidy1877;cleveland1926;hungate 1955;Yamin&Trager1979;Yamin1980).Inthewood feeding,phylogenetically lower termites,symbiotichindgutprotozoaareknowntohydrolyzewood polysaccharidesandfermenttheresultingsugarmonomersintoacetate,carbon dioxide,andhydrogen(hungate1955;yamin1980;odelson&breznak1983; Odelson&Breznak1985a;Odelson&Breznak1985b;Breznak&Brune1994). HomoacetogenicbacteriathentransformtheH2+CO2intoadditionalacetate (Breznak&Switzer1986;Braumanetal.1992;Leadbetteretal.1999;Pester& Brune2007).Polysaccharide derivedacetateservesastheoxidizableenergysource yetnon respiratorymetabolismsofplant derivedandotheraromaticsshouldbereevaluatedintermitehindgutcommunities. aswellasaprecursorofaminoacids,hydrocarbons,andterpenesinthewoodfeedinghostinsect(blomquistetal.1979;mauldin1982;odelson&breznak1983; Breznak&Switzer1986;Braumanetal.1992).Together,termitesandtheirgut

104 3 3 microbiotaareprimeexamplesoftinyandnaturalbioreactorsoperativeona massivescaleinmanyterrestrialenvironments(brune1998a). Asreviewedpreviously(Breznak&Brune1994),thefateofthemostinaccessible componentoflignocelluloseinthedietofwoodfeedingtermites,lignin,hasbeen andremainscomparativelyunclearandoftencontroversial(butler&buckerfield 1979;Cookson1987;Pastietal.1990;Breznak&Brune1994;Kuhnigketal.1994; Scharf&Tartar2008;Tartaretal.2009;Scharf&Boucias2010;Sethietal.2013). ReliablymeasuringtheoxidationofC 14 labelledligninsbefore,during,andafter passagethroughthetermiteguttracthasbeenchallenging(butler&buckerfield 1979;Cookson1987;Pastietal.1990;Breznak&Brune1994;Kuhnigketal.1994; Bruneetal.1995a).Inaddition,studieshavehypothesizedthatO2isanecessarycosubstrateforthecompletedegradationofligninaromaticmonomers(Bruneetal. 1995a).Studiesoverthepasttwodecadeshaverevealedthatthehindgutsof termitescontainvariouslevelsofoxygenintheperiphery,andthatonlythecentral portionsofthehindgutaretotallyanoxic(bruneetal.1995b;brune1998a;kappler &Brune1999;Kappler&Brune2002;Zimmer&Brune2005;Pester&Brune 2007).Cultivationandgene basedresultshaverevealedthatmicroorganismswith varyingdegreesofoxygentoleranceandoxygenmetabolismsareoperativeasbona fidemembersoftermitegutcommunities(leadbetter&breznak1996;shimaetal. 1999;Shimaetal.2001;Boga&Brune2003;Bogaetal.2003;Tholenetal.2007; Wertz&Breznak2007a;Wertz&Breznak2007b;Wertzetal.2012).

105 3 4 Inanefforttogleanmoreaboutrolesofthegutmicrobiotainthesymbiosis,the genomesofthreestrainsofsugarandh2metabolizingtermitegutsymbiotic spirochetesandthepartialtermitegutmetagenome,allpreviouslydescribed,were compared(warneckeetal.2007;rosenthaletal.2011;balloretal.2012).many genesofinterestinthemetagenomicdatasetwererepresentedinthegenomesof theisolates,confirminganotherleveloftheirvalidityasmodelsforinvitrostudies (Warneckeetal.2007;Rosenthaletal.2011;Balloretal.2012).Perhapsmore surprising,though,werenumerousexamplesofgenespresentintheisolatesthat werenotrepresentedinthelarger(albeitnotexhaustive)metagenomicdataset. Here,asubsetofsuchgenesthathaverelevancetooxygenandaromaticmetabolism inthetermitehindgutfromtwostrainsoftreponemaprimitiaaredescribed. MATERIALSANDMETHODS: Mediaandcultivation RoutineinvitrogrowthandmaintenanceofTreponemaprimitiastr.ZAS 1andZAS 2in2YAComediumwasaspreviouslydescribed(Leadbetteretal.1999;Graber& Breznak2004;Graberetal.2004).ForexaminationofthemicrooxicgrowthofT. primitiastr.zas 1andZAS 2,thegrowthmediumwasreformulatedtocontainno dithiothreitol(dtt)orotherreducingagent,noresazurin(aredoxindicator),and nomaltose(unlessusedasasubstrateforpositivecontrols).theheadspacewas 80%N2/20%CO2.Toserum stopperedculturetubesofthisunreducedmedium, roomairwasinjectedthrougha0.2µmfilterintothecultureheadspace(toachieve afinalconcentrationof0.5%o2,vol/vol)priortooratthetimeofinoculation.

106 3 5 Thesecultureswereincubatedat25 Cinahorizontalpositionwithoutagitation. BecauseT.primitiastr.ZAS 1grewmoresuccessfullythanT.primitiastr.ZAS 2 undertheseconditions,t.primitiastr.zas 1wasusedforfurtherliquidculture physiologicalexperimentswiththismicroorxicregime. Itisnoteworthythatthe YACo mediaformulationcontainscofactorsrequiredof allthemeta cleavagepathwayenzymes(catechol2,3 dioxygenaserequiresfe 2+ ;2 oxopent 4 enoatehydrataserequiresmn 2+ ormg 2+ (Izumietal.2007);4 hydroxy 2 oxopentanoatealdolaserquiresmn 2+ (Leietal.2008);acetaldehyde dehydrogenaserequirescoashandnad + (Leietal.2008)). Growthinthepresenceofaromaticcompounds Individual50mMstocksolutionsofcatechol,guaiacol,veratrole, protocatechualdehyde,protocatechuicacid,vanillicacid,gallicacid, homoprotocatechuicacid,homoveratricacid,hydrocaffeicacid,caffeicacid,and ferulicacidwerepreparedundern2andstoredinthedark.asneeded,stock solutionswereneutralizedwithnaohduringthepreparations. LiquidculturesofT.primitiastr.ZAS 1(preparedasdescribedabovefor examinationofmicrooxicgrowthandwith0.5mmaromaticcompound)were screenedforincreasesingrowthrateandyieldasaproxyforacetategeneration frommeta cleavagepathwaymetabolismofaromaticcompounds.changesin opticaldensityofgrowingculturesweremeasuredat600nmwithaspectronic20 colorimeter.uv/visabsorbancespectrawereobtainedusingacarywinuv Spectrophotometer.

107 3 6 Fortheexaminationofgrowthinoxygengradients,1mLofmolten3%agarose containing10mmcatechol,protocatechuicacid,hydrocaffeicacid,orcaffeicacid(or waterasanegativecontrol)wasdispensedinto25mlbalchtubes.afterhardening, thisplugwasoverlaidwith10mlofamoltenagarosemedium.themediumused wastheunreduced2yacomediumsupplementedwithlowmeltingpointagaroseto afinalconcentrationof0.8%,andresazurinasanoxygenandredoxindicator.upon agaroseadditionstothebalchtubes,carewastakentoensureresidualagarosedid notsticktothewallsofthetubesothatthelevelofagaroseinthetubeswas consistentamongthetubes.oncetheagarosehardened,steriletuberculinsyringes with6in.,18ga.needleswereusedtoinoculateaconsistentvolume(400µl)oft. primitiacells(orwaterasacontrol)alongtheverticallengthoftheagarose.room airwastheninjectedthrougha0.2µmfilterintothecultureheadspacetoachievea headspaceconcentrationof4%o2,70%n2,10%co2,vol/vol.cultureswere cultivatedat25 Cinaverticalposition.T.primitiastr.ZAS 2wasusedforthese initialgradienttubeexperimentsinadditiontot.primitiastr.zas 1toseeifitcould establishitselfinanoptimalo2/aromaticgradientandgrowmoresuccessfullythan inliquidmedia.still,however,t.primitiastr.zas 1performedbetterunderthese conditionsandwasusedforsubsequentgradienttubeexperiments. Enrichments Enrichmentculturestoobtainafresh,O2/aromatic metabolizingisolatefromthe termitehindgutenvironmentwerepreparedfromthewholegutcontentsofa Zootermopsisnevadensisworkertermite.Enrichmentculturemediawasprepared

108 3 7 asdescribedaboveforexaminationofmicrooxicgrowth,alsowith0.5mmaromatic compound(seechapter1forenrichmentandisolationprotocol).enrichment cultureswithferulicacidorvanillicaciddidtransientlyturnvariousshadesof yellowforthefirsttwosetsoftransfers,butnotafter,suggestivethatatsomeearly pointinenrichmentworko2/aromatic metabolizingorganismsmighthavebeen presentintheseenrichments.thisbodespromisingforfutureattemptsat enrichmentandisolation. Bioinformatics Sequenceswereobtainedfromavarietyofsources:JGIIMG/M(Markowitzetal. 2006);NCBIBLAST(Geeretal.2010);SangerPfam(Puntaetal.2012);CAMERA (Sunetal.2011);andJCVICMR(Davidsenetal.2010)tocollectascompleteofa datasetaspossible.fortheconstructionofphylogenetictrees,initiallyindividual datasetsrangingfromapproximately500to1500topblastsequencehits correspondingtoeachofthetreponemaprimitiameta cleavagepathwayproteins, orpfamdomains,wereassembled.datasetswerecollectedbylookingfornatural cut offsinsequenceidentityandsimilarityinblastresults,insteadofchoosingan arbitrarynumberofthetopblasthits,astonotmisspotentiallyrelevant sequences. ForeachproteinorPfamCLUSTALW,MAFFT,DIALIGN,T Coffee,andMUSCLE alignmentsweregeneratedonthemobyleserverandcompared(néronetal.2009). Consistently,MUSCLEgeneratedthebestalignmentsandthereforesequenceswere unambiguouslyalignedusingthisprogram(fig.3 3b).Conservativefilterswere

109 3 8 usedtodisregardcolumnsofdatacontainingsequencegapsorambiguous alignments.afterinitialanalyses,phylogramsusingfewerandmorerelevant sequencesfromtheoriginallargedatasetswerepreparedusingpackageswithin ARBv.5.2(Ludwigetal.2004)andMr.Bayesv.3.2usingmixed modelsettings (Huelsenbeck&Ronquist2001;Ronquist&Huelsenbeck2003).Additionaldetails oftreeconstructionarefoundinthetreelegends.accessionnumbersarereported inappendix(appendixtable3a 1). Putativepromoterregionswithinoradjacenttothemeta cleavagepathwaygene neighborhoodsweredeterminedusingvirtualfootprintv.3.0(münchetal.2005). Forexaminationofthegenesusingselectionpressuremodels,MEGAv.5.05 (Tamuraetal.2007)wasused.Theseveralmodelstestedincludedapositive selectionhypothesis(ds>dn)viaacodon basedz test:ng86(nei Gojobori methodwithjukes Cantornucleotidesubstitutionmodel);modifiedNG86(modified Nei GojoborimethodswiththeJukes Cantornucleotidesubstitutionmodel);LWL85 (Li Wu Loumethod);PBL85(Pamilo Bianchi Limethod);andKumar(Kumar method).resultsarereportedastheoverallaverageofpair wiseanalysesofthe followingnumberofunambiguouscodonpositionswithoutgapsforeachgene: ferredoxin likepeptide,73;catechol2,3 dioxygenase,291;2 hydroxymuconic semialdehydehydrolase,264;2 oxopent 4 enoatehydratase,260;4 hydroxy 2 oxopentanoatealdolase,313;andacetaldehydedehydrogenase,261.allanalyses wererunwith1000bootstrapreplicates.zmustbelessthanzero(ds>dn)ina statisticallysignificantmannertoindicatepositiveselection. ND denotescasesin whichitwasnotpossibletoestimateevolutionarydistances.

110 3 9 Forexaminationofkeyresiduesandotherfeaturesoftheputativemeta cleavage pathwayproteinsint.primitiastr.zas 1andZAS 2,aswellasinotherorganisms, datawereobtainedfromrast(azizetal.2008),meta Cyc(Caspietal.2012),and KEGG(Kanehisa&Goto2000),inadditiontothedatabasesnotedabove.These analyses,coupledwithextensiveliteraturereview,werealsousedtodetermine whereacrossthebacteriallineofdescentcatechol2,3 dioxygenasemeta cleavage pathwaysarefound. Forexaminationofrelevantgeneexpressionunderroutinecultivationconditions, dataobtainedinapreviousstudywasre analyzed(rosenthaletal.2011).first, expressiondatafromallt.primitiastr.zas 2geneswerecomparedtogaugethe rangeofreads/kbmeasurements(appendixtable3a 2)(Rosenthaletal.2011). Next,expressiondataforeachmeta cleavagepathwaygenewasevaluatedrelative toothergenesincludingthoserepresentinghousekeepingandotherandmetabolic functions(appendixtable3a 3). Enzymeactivityassays Activityassaysforcatechol2,3 dioxygenasewereconductedwith0.5mmcatechol, 50mMpotassiumphosphatebuffer,and10µLcrudecelllysateinafinalvolumeof 1mLatroomtemperatureinthedark.Catecholwasaddedtobeginreactionand reactionwasexposedtoroomairandinvertedforo2exposure.althoughmany iterationsofassayswereperformed,noneyieldedresults.iterationswerea combinationof:

111 3 10 AddingcatecholandO2toculturesatvarioustimepointspriortotheassayto allowculturestobegintoexpresscatechol2,3 dioxygenaseinanticipationof assay NotaddingcatecholorO2untilthetimeoftheenzymeassaysothat maximumactivityhasnotalreadyoccurredpriortoassay Preparingcrudelysatefrom100xconcentratedexponentialphase Treponemaprimitiastr.ZAS 1cultures DisruptingT.primitiastr.ZAS 1cellsviaCellLytic(microscopecheck confirmedapproximately50%ofcellsdisrupted) DisruptingT.primitiastr.ZAS 1cellsviasonnication(microscopecheck confirmedapproximately80%ofcellsdisrupted) DisruptingT.primitiastr.ZAS 1cellsviamicrosonnication(onice) (microscopecheckconfirmedapproximately80%ofcellsdisrupted) PreparingcrudecelllysateunderN2 Adding3µLof2.1g/100mLFeS04toreactionmixtoensureenoughFe 2+ cofactorwasavailable AnappropriatepositivecontrolwouldbePseudomonasputidaandanappropriate negativecontrolwouldbet.azotonutriciumstr.zas 9,butthesewerenotexamined here(kitaetal.1999). RESULTS: ReciprocalBLASTrevealedcatechol2,3 dioxygenaseintreponemaprimitia Tocomparethemetabolicpotentialsofphylogenetically higher and lower termitehindgutmicrobesusingavailabledatasets,reciprocalblastanalyseswere conductedbetweengenomesfromthree lower termitehindgutisolates, Treponemaprimitiastr.ZAS 1(3.8Mb)andZAS 2(4.1Mb),andT.azotonutricium str.zas 9(3.9Mb)(Leadbetteretal.1999;Graber&Breznak2004;Graberetal. 2004;Rosenthaletal.2011;Balloretal.2012),andtheP3hindgutregion

112 3 11 metagenomefromthe higher termite,nasutitermessp.(62mb)(warneckeetal. 2007).Specifically,genespresentinanyofthethree lower termitehindgut isolates genomes,butnotinthe higher termitehindgutmetagenome,andvice versa,wereidentified.21%,20%,and17%ofthet.primitiastr.zas 1,T.primitia str.zas 2,andT.azotonutriciumstr.ZAS 9genomes,respectively,hadnoorthologs inthemetagenomicdataset(seediscussionforwhymetagenomedatasetdoesnot representfull coverageof higher termitesample)(appendixtable3a 4).Among thosenon orthologs,aputative catechol2,3 dioxygenase genewasfoundineach ofthet.primitiastr.zas 1andZAS 2genomes,butnotintheT.azotonutriciumstr. ZAS 9genome(AppendixTable3A 4).Nocatechol2,3 dioxygenasehomologswere foundinthe higher termitemetagenomeeither,thuscorroboratingearlier analyses(appendixtable3a 4)(Warneckeetal.2007).Becausecatechol2,3 dioxygenaseisakeyenzymeofthemeta cleavagepathwayofmanyaerobicbacteria andbothstrainsareconsideredanaerobes andpreliminarygenefunction assignmentsareoftenpronetoerror amoreindepthevaluationofthegenome andmetabolicpotentialsforo2 dependentaromaticmetabolismbyt.primitiawas initiated. TreponemaprimitiastrainsZAS 1andZAS 2havegenesrepresenting completecatechol2,3 dioxygenasemeta cleavagepathways Asbackground,duringthemetabolismofmanyaromaticcompoundsnumerous preparativeor upper pathwaysgenerate(di)hydroxylatedintermediatesfroma widerrangeofstructurallydiversearomaticcompounds.then, lower pathways transformtheresultingaromaticsintonon aromatic,centralcellmetabolites

113 3 12 (Viggianietal.2004;Sianietal.2006).Catechol2,3 dioxygenasesareoften employedtocatalyzethekeyringcleavagestepinsuch lower pathwaysof monoaromaticmetabolism(fig.3 1). Fig.3 1:Catechol2,3 dioxygenase basedmeta cleavagepathwayofaromatic metabolismandshunts.treponemaprimitiastr.zas 1andZAS 2havegenesfora completemeta cleavagepathwayrepresentedbysteps1,2,3,4,and5(black).two alternativeroutes(grey)are,shunta:steps1,6,7,8,3,4,and5(skipsstep2)shunt B:steps1,6,7,9,4,and5(skipssteps2and3).SubstratesandproductsareA: catechol;b:2 hydroxymuconicsemialdehyde;c:2 oxopent 4 enoate;d:4 hydroxy 2 oxopentanoate,e:2 hydroxymuconicacid,andf:γ oxalocrotonate.centralcell metaboliteproductsareoutlinedinboxes.enzymesrepresentedare:1,catechol 2,3 dioxygenase;2,2 hydroxymuconicsemialdehydehydrolase;3,2 oxopent 4 enoatehydratase;4,4 hydroxy 2 oxopentanoatealdolase;5,acetaldehyde dehydrogenase;6,2 hydroxymuconicsemialdehydedehydrogenase;7,4 oxalocrotonatetautomerase;8,4 oxalocrotonatedecarboxylase;and9,4 oxalocrotonatecarboxy lyase. Catechol2,3 dioxygenasesareclassifiedasextradioldioxygenases,thusthe lower pathwaystheyinitiateareoftenreferredtoas meta cleavage pathways(bugg& Winfield1998;Vaillancourtetal.2006).Catechol2,3 dioxygenases,therefore,

114 3 13 typicallyfunctioninconjunctionwithasuiteofuptoeightothermeta pathway enzymesincluding:2 hydroxymuconicsemialdehydehydrolase(pf00561,step2); 2 oxopent 4 enoatehydratase(pf01557, step3);4 hydroxy 2 oxopentanoate aldolase(pf00682andpf07836,step4);acetaldehydedehydrogenase(pf01118 andpf09290,step5);2 hydroxymuconicsemialdehydedehydrogenase(pf00171, step6);4 oxalocrotonatetautomerase(pf01361,step7);4 oxalocrotonate decarboxylase(pf01557,step8);and4 oxalocrotonatecarboxy lyase(pf01557, step9)(table3 1,Fig.3 1)(Harayamaetal.1987;Harayama&Rekik1990; Furukawaetal.1993;Suenagaetal.2007).Anassociatedferredoxin likepeptide (PF00111,step1')playsanindirectpathwayrole,reactivatingO2 inactivated catechol2,3 dioxygenasebyreducingitsoxidizedironcofactor(table3 1,Fig.3 1) (Polissi&Harayama1993;Hugoetal.2000).Catechol2,3 dioxygenaseinitiated meta cleavagepathwaysgenerallyproceedviaoneroute(fig.3 1,black,steps:1,2, 3,4,and5),buttwoalternativeshuntsareknown(Fig.3 1,grey,ShuntA:steps1,6, 7,8,3,4,and5,ShuntB:steps1,6,7,9,4,and5)(Khajamohiddinetal.2008).Inan efforttodeterminewhethertheannotationofgenesintreponemaprimitiastr.zas 1andZAS 2asbeing catechol2,3 dioxygenases isvalid,theirgeneneighborhoods (andtherestoftheirgenomes)wereanalyzedforthepresenceofotherkeygenes formeta cleavagepathways.

115 3 14 Table 3-1: Treponema primitia str. ZAS-1 and ZAS-2 meta-cleavage pathway genes and pfam features Present in Protein Size (a.a.) in Pfam Residues in Step Gene Annotation str. str. str. str. Pfam(s) Represented (PF#, family, domain) str. str. ZAS-1 ZAS-2 ZAS-1 ZAS-2 ZAS-1 ZAS-2 1' ferredoxin-like peptide PF00111, Fer2, [2Fe-2S] cluster binding catechol 2,3-dioxygenase PF00903, Glyoxalase, glyoxalase/bleomycin resistance protein/dioxygenase PF00903, Glyoxalase, glyoxalase/bleomycin resistance protein/dioxygenase hydroxymuconic semialdehyde hydrolase PF00561, Abhydrolase 1, α/β hydrolase fold oxopent-4-enoate hydratase PF01557, FAA hydrolase, fumarylacetoacetate hydrolase hydroxy-2-oxopentanoate aldolase PF00682, HMGL-like, HMGL-like PF07836, DmpG comm., DmpG-like communication acetaldehyde dehydrogenase PF01118, Semialdehyde dh, semialdehyde dehydrogenase NAD binding hydroxymuconic semialdehyde dehydrogenase - - PF00171 Aldedh, aldehyde dehydrogenase family 7 4-oxalocrotonate tautomerase - - PF01361 Tautomerase, tautomerase enzyme PF09290, Acetdehyd dimer, prokaryotic acetaldehyde dehydrogenase dimerisation oxalocrotonate decarboxylase - - PF01557 FAA hydrolase, fumarylacetoacetate hydrolase 9 4-oxalocrotonate carboxy-lyase - - PF01557 FAA hydrolase, fumarylacetoacetate hydrolase InbothT.primitiastr.ZAS 1andZAS 2,directlyupstreamoftheputative catechol 2,3 dioxygenase arefourotherputativemeta cleavagepathwaygenes(fig.3 2). Collectively,inordertheyare:2 hydroxymuconicsemialdehydehydrolase;2 oxopent 4 enotehydratase;acetaldehydedehydrogenase;4 hydroxy 2 oxopentanoatealdolase;ferredoxin likepeptide;andcatechol2,3 dioxygenase(fig. 3 2).Thus,T.primitiastr.ZAS 1andZAS 2havegenesrepresentingallkey enzymaticstepsknownforthebetterstudiedmeta pathwayroute,butnotfor eitherofthealternativeshunts(fig.3 1inblack). PfamsandconservedresiduesinTreponemaprimitiameta pathwaygenes suggestfunctionality The2 hydroxymuconicsemialdehydehydrolase;2 oxopent 4 enotehydratase; acetaldehydedehydrogenase;4 hydroxy 2 oxopentanoatealdolase;ferredoxin like peptide;andcatechol2,3 dioxygenaseintreponemaprimitiastr.zas 1andZAS 2,

116 3 15 areconsistentinsizewiththeirequivalent,reported,functionalmeta cleavage pathwaygenes,andeachgenes correspondingproteinhastheexpectedpfam (proteinfamily)domainsandconservedaminoacidresiduessuggestiveofmetapathwayfunctionality(table3 1;AppendixFig.3A 1)(Díaz&Timmis1995;Kitaet al.1999;nardini&dijkstra1999;holmquist2000;nandhagopaletal.2001;reaet al.2005;izumietal.2007;leietal.2008). TheT.primitiastr.ZAS 1ferredoxin likepeptidehasonefamilyfer2,[2fe 2S] clusterbindingdomain(pf00111)spanning73residues(13to85)ofthe98amino acid longproteinconsistentwithotherstudiedmeta pathwayferredoxins(table3 1;AppendixFig.3A 1)(Azizetal.2008;Puntaetal.2012).LikewisetheT.primitia str.zas 2ferredoxin likepeptidehasonefamilyfer2,[2fe 2S]clusterbinding domain(pf00111)spanning72residues(14to85)ofthe94aminoacid long protein(table3 1;AppendixFig.3A 1)(Azizetal.2008;Puntaetal.2012).TheT. primitiastr.zas 1ferredoxin likepeptidehasanactivesitecomprisedofval 36, Ser 37,Gly 43,Gly 46,Ala 47,andLeu 80,andaniron bindingsitethatalso contributestoactivitycomprisedofcys 40,Cys 45,Cys 48,andCys 81(Appendix Fig.3A 1)(Azizetal.2008).TheT.primitiastr.ZAS 2ferredoxin likepeptidehas thesamefeaturesexceptforathr 37insteadofaSer 37intheactivesite (AppendixFig.3A 1)(Azizetal.2008).Thefourcysteineresiduesarehighly conservedamongcatechol2,3 dioxygenase associatedferredoxins,andhavea conservedrolealsoasligandsforthe[2fe 2S]cluster(Hugoetal.2000).

117 3 16 Next,boththeT.primitiastr.ZAS 1andZAS 2catechol2,3 dioxygenasesare308 aminoacidsinlength,consistentwiththeresiduespanofpreviouslyreported catechol2,3 dioxygenases(table3 1;AppendixFig.3A 1)(Kitaetal.1999;Viggiani etal2004).alsolikeknowncatechol2,3 dioxygenases,thet.primitiastr.zas 1 andzas 2geneseachcontainanN andc terminaldomainthatbothrepresentthe glyoxalase/bleomycinresistanceprotein/dioxygenasesuperfamilyofproteins (PF00903)(Table3 1;AppendixFig.3A 1).Thesizesofeachofthosedomainsin boththet.primitiastr.zas 1andZAS 2genealsocorrelatewithpublishedlengths (Table3 1;AppendixFig.3A 1). Previousprimarystructureanalysisofnearly40diversecatechol2,3 dioxygenase sequencessuggeststhatconservedresiduesincludethreemetalligands,his 146, His 210,andGlu 260,andthreeadditionalactivesiteresidues,His 195,His 241, andtyr 250.Theotherstrictly conservedresidues,gly 28,Leu 165,andPro 254, areremotefromtheactivesite,andthusarelikelytoplaystructuralorfoldingroles (Suenagaetal.2009).TheseconservedresiduesareallfoundintheT.primitiastr. ZAS 1andZAS 2catechol2,3 dioxygenase,andlikeotherwell studiedcatechol2,3 dioxygenasesthec terminaldomainofthet.primitiastr.zas 1andZAS 2gene appearstocontaintheactivesite(appendixfig.3a 1). Regardingthe2 hydroxymuconicsemialdehydehydrolase,boththet.primitia2 hydroxymuconicsemialdehydehydrolaseshaveonefamilyabhydrolase1,α/β hydrolasefolddomain(pf00561)spanning235residues(32to266)ofthe274

118 3 17 amino acidlongproteinslikeotherwell studied2 hydroxymuconicsemialdehyde hydrolases(table3 1;AppendixFig.3A 1)(Azizetal.2008;Puntaetal.2012).In addition,boththet.primitia2 hydroxymuconicsemialdehydehydrolaseseachhave anactivesitecomprisedofser 105,Asp 226,andHis 254(AppendixFig.3A 1) (Azizetal.2008).Thesethreeresiduesreflectthecatalytictriadconfiguration, typicallynucleophile acid histidineorser 107,Asp 228,andHis 256,conserved amongotherfunctionalα/βhydrolasefold2 hydroxymuconicsemialdehyde hydrolases(díaz&timmis1995;nandhagopaletal.2001).further,a nucleophilic elbow consensussequence,sm X Nu X Sm Sm,typicallysurroundstheSer 107 nucleophileinα/β hydrolasefoldenzymeswithsmrepresentingsmallaminoacids (generallyglycine)andxrepresentinganyaminoacid(díaz&timmis1995).this nucleophilicelbow inpseudomonasputida,isgly 105,Asn 106,Ser 107,Phe 108, Gly 109,andGly 110(Díaz&Timmis1995).Similarly,boththeT.primitia2 hydroxymuconicsemialdehydehydrolaseshavea nucleophilicelbow ofgly 103, Asn 104,Ser 105,Phe 106,Gly 107,andGly 108(AppendixFig.3A 1). Moreover,aconservedmotifamongmeta cleavageproducthydrolasesishis 35, Gly 36,X 37,Gly 38,Pro 39,andGly 40withXrepresentingasmallaminoacidand bothhis 35andGly 36involvedintheformationofanoxyanionholecriticalto catalyticfunction(nandhagopaletal.2001).indeedboththet.primitia2 hydroxymuconicsemialdehydehydrolaseshavethemotifhis 34,Gly 35,Ser 36, Gly 37,Pro 38,andGly 39withthecatalyticHis 34andGly 35residues(Appendix Fig.3A 1).Otherconservedfeaturesamongmeta cleavageproducthydrolasesare

119 3 18 locatedinthebottomofthesubstrate bindingpocket.theseareasn 46,Asn 109, andgln 266(Nandhagopaletal.2001).Correspondingly,theT.primitiastr.ZAS 1 2 hydroxymuconicsemialdehydehydrolasehasresiduesasn 47,Asn 104,andGln 262,andtheT.primitiastr.ZAS 22 hydroxymuconicsemialdehydehydrolasehas residuesasn 45,Asn 104,andGln 257(AppendixFig.3A 1). Next,theT.primitiastr.ZAS 12 oxopent 4 enoatehydratasehasonefamilyfaa hydrolase,fumarylacetoacetatehydrolasedomain(pf01557)spanning199 residues(61to259)ofthe260aminoacid longproteinasreportedforother2 oxopent 4 enoatehydratases(table3 1;AppendixFig.3A 1)(Azizetal.2008; Puntaetal.2012).TheT.primitiastr.ZAS 22 oxopent 4 enoatehydratasehasone FamilyFAAhydrolase,fumarylacetoacetatehydrolasedomain(PF01557)spanning 200residues(60to259)ofthe260aminoacid longprotein(table3 1;Appendix Fig.3A 1)(Azizetal.2008;Puntaetal.2012).Threeconservedresiduesofthese hydratases,glu 106,Glu 108,andGlu 139,coordinatethemetalion(Izumietal. 2007).BoththeT.primitia2 oxopent 4 enoatehydrataseshavethesethree conservedresidues,representedbyglu 104,Glu 106,andGlu 137(AppendixFig. 3A 1)(Azizetal.2008).Otherconservedresiduesofmeta cleavagepathway2 oxopent 4 enoatehydratasesarelys 61,Leu 64,Asp 79,andAsn 168that comprisetheactivesite(izumietal.2007).int.primitiatheseconservedactivesite residuesarelys 60,Leu 63,Asp 78,andAsn 168instr.ZAS 1andAsn 157instr. ZAS 2(AppendixFig.3A 1)(Azizetal.2008).

120 3 19 TheT.primitia4 hydroxy 2 oxopentanoatealdolaseseachhaveonehmgl like familydomain(pf00682)spanning232residues(19to250instr.zas 1and14 245instr.ZAS 2)andoneFamilyDmpGcomm.,DmpG likecommunicationdomain (PF07836)spanning66residues( instr.ZAS 1and instr.ZAS 2) ofthe340and335aminoacids longproteinsint.primitiastr.zas 1andZAS 2, respectivelylikeotherresearched4 hydroxy 2 oxopentanoatealdolases(table3 1; AppendixFig.3A 1)(Azizetal.2008;Puntaetal.2012). Conservedmetalionligandsof4 hydroxy 2 oxopentanoatealdolasesareasp 18, His 200,andHis 202.Accordingly,T.primitiastr.ZAS 1 s4 hydroxy 2 oxopentanoatealdolasehasconservedmetalionligandsatasp 20,His 200,and His 202whereasT.primitiastr.ZAS 2 s4 hydroxy 2 oxopentanoatealdolasehas conservedmetalionligandsatasp 15,His 195,andHis 197(AppendixFig.3A 1) (Azizetal.2008).Theseconservedmetalionligandsinteractwiththeconserved activesiteresiduesarg 17,His 21,Gly 52,andTyr 291whicharerepresentedby Arg 19,His 23,Gly 54,andTyr 291inT.primitiastr.ZAS 1,andArg 14,His 18,Gly 54,andTyr 286instr.ZAS 2(AppendixFig.3A 1)(Azizetal.2008). TheT.primitiaacetaldehydedehydrogenaseseachhaveoneFamilySemialdehyde dh/semialdehydedehydrogenase,nadbindingdomain(pf01118)spanning110 residues(7 116instr.ZAS 1and5 114instr.ZAS 2)andoneFamilyAcetdehyd dimmer,prokaryoticacetaldehydedehydrogenasedimerisationdomain(pf09290) spanning137residues( instr.ZAS 1and instr.ZAS 2)ofthe291 and288aminoacid longproteinsint.primitiastr.zas 1andZAS 2,respectively

121 3 20 consistentwithpreviouslyreportedacetaldehydedehydrogenases(table3 1; AppendixFig.3A 1)(Azizetal.2008;Puntaetal.2012).Theactivesiteof acetaldehydedehydrogenasesislocatedbetweenitsnad+ bindingand dimerizationdomainsandhasaconservedresidue,cys 132.LikewiseintheT. primitiaacetaldehydedehydrogenasethisresidueiscys 126(str.ZAS 1)andCys 124(str.ZAS 2)(AppendixFig.3A 1).

122 3 21

123 3 22 Fig.3 2:Geneneighborhoodsrepresentingcompletecatechol2,3 dioxygenasebasedmeta cleavagepathways.inparenthesesnearthenamesoforganismswith genesrepresentingcompletemeta cleavagepathways, M, A, and/or B, representshavinggenesforthemainpathway,shunta,orshuntb,respectively. Ferredoxin likepeptide(step1,red),catechol2,3 dioxygenase(step1,orange),2 hydroxymuconicsemialdehydehydrolase(step2,lightblue),2 oxopent 4 enoate hydratase(step3,darkgreen),4 hydroxy 2 oxopentanoatealdolase(step4,dark blue),acetaldehydedehydrogenase(step5,purple),2 hydroxymuconic semialdehydedehydrogenase(step6,lightgreen),4 oxalocrotonatetautomerase (step7,yellow),4 oxalocrotonatedecarboxylase(step8,pink),putativesregulatory genes(darkgrey),andhypotheticalproteins(lightgrey)aredepicted.regulatory familiesarenotedinblueprintwhenknown. TheT.primitiastr.ZAS 1andZAS 2meta cleavagepathwaygeneneighborhoods arearrangedsimilarlybetweentheirrespectivegenomes(fig.3 2).Incontrast, theirarrangementisdissimilarfromtheorderofthemeta cleavagepathway dioxygenasemeta cleavagepathwaygeneneighborhoodsinotherorganisms enzymaticstepstheyrepresent(fig.3 1and3 2).Relativetocatechol2,3 possessingthispathway,thet.primitiastrains genearrangementisdistinct(fig.3 2). Noevidencefor upper preparatorypathwaygenesintreponemaprimitia Manymeta cleavagepathway containingorganismsperformpreparatoryreactions beforeproceedingtocleavearomaticsubstrates,andoftenhavegenesfor monooxygenasesanddioxygenases.theseareoftenfounddirectlyupstream, downstream,oramongthemeta cleavagepathway encodinggenecluster. Nevertheless,noevidenceforpreparatoryreactionswasobservedinthegenomes ofthetreponemaprimitiaisolates.

124 3 23 EvolutionaryoriginsoftheTreponemaprimitiameta cleavagepathway Inordertolearnmoreabouttheevolutionaryoriginsandhistoryofthepathway genesintreponemaprimitia,aphylogeneticanalysiswasperformedoneachby comparingeachgeneinthepathwaytodatabasesoftheirrespectivehomologs.the resultsofthatanalysissuggestthatthegenesforthepathwaylikelyoriginatefrom atleasttwo,ifnotthreesources:analphaproteobacterium,amemberofthe clostridiales,andpossiblyamemberofthebacillales(fig.3 3,3 4,and3 5; AppendixFig3A 2,3A 3,and3A 4).However,genetransfereventsseemtohave occurredinthedistantpast,inasmuchasrelativelylongbranchlengthsare observedforeacht.primitiahomolog,withameliorationofthegcandcodonusage.

125 3 24 a b Fig.3 3:(a)PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 catechol2,3 dioxygenase(pf00903,step1).bayesianproteinphylogeneticanalysis (90treesfrom36,000generations;PSRF=0.999;averagestandarddeviationof splitfrequencies= )isbasedon297unambiguouslyalignedaminoacid

126 3 25 positionsofa308aminoacid longprotein.bayesvalues(whengreaterthan50)are reportedabovethenodes.phylipprotparsmaximumparsimonysupportafter analysisof1000bootstraps(whengreaterthan50%)isreportedbelownodes. Shadedcircles( )indicatenodessupportedbybothmaximumparsimonyand Fitchdistancematrixmethods.Opencircles( )indicatenodessupportedbyone ofthosemethods.diamonds( )indicateorganismswithgenesforacomplete catechol2,3 dioxygenase basedmeta cleavagepathway.scalebarindicates distancedepictedas0.1aminoacidchangesperalignmentposition.accession numbersarereportedinappendix(appendixtable3a 1).(b)ExampleofMUSCLE alignmentsusedtocreatetrees.foreachproteinorpfamclustalw,mafft, DIALIGN,T Coffee,andMUSCLEalignmentsweregeneratedontheMobyleServer andcompared(néronetal.2009).consistently,musclegeneratedthebest alignmentsandthereforesequenceswereunambiguouslyalignedusingthis program.conservativefilterswereusedtodisregardcolumnsofdatacontaining sequencegapsorambiguousalignments.

127 3 26 Fig.3 4:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 2 hydroxymuconicsemialdehydehydrolase(pf00561,step2).bayesianprotein phylogeneticanalysis(2540treesfrom1,016,000generations;psrf=1.000; averagestandarddeviationofsplitfrequencies= )isbasedon222 unambiguouslyalignedaminoacidpositionsofa274aminoacid longprotein. Bayesvalues(whengreaterthan50)arereportedabovethenodes.Phylip PROTPARSmaximumparsimonysupportafteranalysisof1000bootstraps(when greaterthan50%)isreportedbelownodes.shadedcircles( )indicatenodes supportedbybothmaximumparsimonyandfitchdistancematrixmethods.open circles( )indicatenodessupportedbyoneofthosemethods.diamonds( ) indicateorganismswithgenesforacompletecatechol2,3 dioxygenase basedmetacleavagepathway.scalebarindicatesdistancedepictedas0.1aminoacidchanges peralignmentposition.accessionnumbersarereportedinappendix(appendix Table3A 1).

128 3 27 Fig.3 5:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 22 oxopent 4 enoatehydratase(pf01557,step3).bayesianproteinphylogenetic analysis(313treesfrom125,000generations;psrf=1.000;averagestandard deviationofsplitfrequencies= )isbasedon243unambiguouslyaligned aminoacidpositionsofa260aminoacid longprotein.bayesvalues(whengreater than50)arereportedabovethenodes.phylipprotparsmaximumparsimony supportafteranalysisof1000bootstraps(whengreaterthan50%)isreported belownodes.shadedcircles( )indicatenodessupportedbybothmaximum parsimonyandfitchdistancematrixmethods.opencircles( )indicatenodes supportedbyoneofthosemethods.diamonds( )indicateorganismswithgenes foracompletecatechol2,3 dioxygenase basedmeta cleavagepathway.scalebar indicatesdistancedepictedas0.1aminoacidchangesperalignmentposition. AccessionnumbersarereportedinAppendix(AppendixTable3A 1).

129 3 28 Thecatechol2,3 dioxygenasesfromthetwot.primitiastrainsareeachother s dioxygenasesfromα,β,andγ proteobacteria,aswellasseveralenvironmental sequences(fig.3 3).Specifically,themajorityoftheclosest culturedrelativesare alphaproteobacteria,specifically sphingomonads (Fig.3 3).Similarly,theshort ferredoxin likepeptidesfromthet.primitiastrainsaffiliatephylogeneticallywith thoseassociatedwithmeta cleavagepathwaysindiverseproteobacteria(appendix closestrelative,anddemonstrateastrongaffiliationwithhomologsofcatechol2,3 Fig.3A 2).Theproteinthatcatalyzesthesecondstepinthepathway,2 hydroxymuconicsemialdehydehydrolase,alsosupportsaproteobacterialorigin (Fig.3 4). Incontrast,theproteinsfortheremainderofthestepsinthepathway,2 oxopent 4 enoatehydratase,4 hydroxy 2 oxopentanoatealdolase,andacetaldehyde dehydrogenase,demonstratestrongaffiliationswiththosefromfirmicutes(fig.3 5; AppendixFig3A 3and3A 4,respectively).Thehydrataseisrelatedtoother hydratasesfromaerobicspeciesofthebacillales,whereasthealdolaseand acetaldehydedehydrogenaseaffiliatephylogenticallywithhomologsthatareoften foundwidespreadacrossmanyanaerobicclostridiales,includingmany homoacetogens(fig.3 5;AppendixFig.3A 3and3A 4,respectively),noneofwhich encodetheenzymescatalyzingthekeyearlystepsinthepathway.extensive phylogeneticanalysesoneachofthespecificpfam(proteinfamily)domainsor modulespresentwithincatechol2,3 dioxygenase,4 hydroxy 2 oxopentanoate aldolase,andacetaldehydedehydrogenaseyieldedresultsconsistentwithallofthe

130 3 29 aboveconclusions(appendixfig.3a 5,3A 6,3A 7,3A 8,3A 9,3A 10,3A 11,3A 12, 3A 13,and3A 14). Selectionpressureandexpressionanalyses NoneofthepathwayrelatedORFsintheTreponemaprimitiastrainsappeartobe pseudogenes,andallappeartoencodefunctionalenzymes.inordertoexamine howimportantthesegenesmaybeovermorerecentevolutionarytime,selection pressureanalyseswereperformedtotestthehypothesisthatthesegenesmight demonstratesignsofpositiveselection.allofthepathwaygeneswiththeexception of2 hydroxymuconicsemialdehydehydrolaseappeartobeunderpositiveselection, astheirdeterminedzvaluesweresignificantlylessthanzero(i.e.thenumbersof synonymouschanges(ds)ineachweresignificantlygreaterthanthenumberof nonsynonymouschanges(dn))(table3 2).Curiously,the2 hydroxymuconic semialdehydehydrolasegenesfromthetwospirocheteseachappeartobeunder negativeselectionpressure(table3 2).

131 3 30 Table 3-2: Selection pressure analysis of Treponema primitia str. ZAS-1 and ZAS-2 meta-cleavage pathway genes Gene Method Z P-value Selection Pressure ferredoxin-like peptide NG Modified NG LWL PBL Kumar catechol 2,3-dioxygenase NG Modified NG LWL PBL Kumar hydroxymuconic semialdehyde hydrolase NG Modified NG LWL PBL Kumar oxopent-4-enoate hydratase NG Modified NG LWL PBL Kumar hydroxy-2-oxopentanoate aldolase NG Modified NG LWL PBL Kumar acetaldehyde dehydrogenase NG Modified NG86 NA NA + LWL PBL Kumar Inare analysisofdatafromapublishedtranscriptomicsstudy(rosenthaletal. 2011),theexpressionofgenesinthepathwaywereevaluatedforT.primitiastr. ZAS 2grownunderanaerobic,homacetogenicgrowthconditionsasapure culture orasco culturewitht.azotonutriciumstr.zas 9(AppendixTable3A 3).Although bothpure andco culturesoft.primitiastr.zas 2hadbeengrowninananoxic, DTT reducedmediumwithoutaddedaromaticsubstratesoro2,allofthemeta

132 3 31 cleavagepathwaygeneswereobservedasbeingexpressed(appendixtable3a 2 andtable3a 3).Catechol2,3 dioxygenaseexpressionwasatahigherlevelthanthe othermeta cleavagepathwaygenes,andatalevelcomparabletothatofakeygene inthisspecies anaerobicmetabolism formatedehydrogenase(appendixtable3a 2andTable3A 3).Comparisonsofexpressionlevelsofthegenesinthepathway withotherreferencegenesarepresentedinthesupplementarymaterials(appendix Table3A 2andTable3A 3). Regulation Expressionofthemeta cleavagepathwaygenesappearstoberegulatedbyagntr familytranscriptonregulatorupstreamofthe2 hydroxymuconicsemialdehyde hydrolase andthusallgenesrepresentingthemeta cleavagepathway inboth Treponemaprimitiastr.ZAS 1andZAS 2(AppendixFig.3A 15).TheGntRfamilyof transcriptionalregulatorsincludesmorethan1,000membersandisdistributed amongdiversebacterialgroupsandbiologicalprocesses(rigalietal.2002;hillerich &Westpheling2006).Thisfamilywasnamesaftertherepressorofthegluconate operoninbacillussubtilis(haydon&guest1991).gntrfamilymembersareknown toalsoregulatethedegradationofaromaticcompounds,functioningas transcriptionalrepressorsintheabsenceofpathwaysubstratessuchasaromatics (Tropel&vanderMeer2004).Otherregulatoryfamiliesinvolvedinaromatic degradationpathwaysincludelysr,iclr,arac,xyl,tetr,andxylrs,andarefound nearcatechol2,3 dioxygenase basedmeta cleavagepathwayneighborhoodsin otherorganismsexaminedhere(tropel&vandermeer2004).preliminary phylogeneticanalysessuggestthet.primitiagntrs closestculturedrelativeisa

133 3 32 memberofthephylumspirochaetes(appendixfig.3a 16andFig.3A 17).Other closeculturedrelativesarerepresentativesofthephylumfirmicutes(appendixfig. 3A 16andFig.3A 17). InadditiontoaGntRfamilytranscriptionalregulator,twopromoterregionsmight belocatedwithinthet.primitiastr.zas 1andZAS 2geneneighborhoodsupstream ofthegntrandbeforetheacetaldehydedehydrogenaseineach(appendixfig.3a 15).Also,althoughselenocysteineinsertionsequence(SECIS)elements that recodein frameugacodonswhichnormallyfunctionasstopsignalstoserveas selenocysteinecodons arefoundint.primitiaformatedehydrogenases(fdh), nonewerefoundinthet.primitiameta cleavagepathwaygenesorgene neighborhoods(zhangetal.2011). Metabolismofcatecholundermicrooxicconditions NeitherofthegenomesoftheTreponemaprimitiastrainshaveanyobviousgenes foroxygenrespiration,forcytochrome associatedproteinsormetabolism,orfor electrontransportphosphorylation.therefore,anyfunctionofthemeta cleavage pathwaygenesint.primitiamustbeverydifferentfromallofthecanonicalaerobes thattypicallyencodethesepathwaysandwhichmineralizetheringcarbontoco2 duringoxidativephosphorylation.however,anearlierstudybygraberand BreznakdocumentedthatT.primitiacultureshaveanumberofenzymeactivities relevanttoo2detoxification,andthatthisspeciescanconsumelowamountsofo2 (2004).Inculturesspikedwithair,growthceaseduntiltheoxygenhadbeenfully consumed,whereupongrowthre initiated(graber&breznak2004).thoseresults

134 3-33 raise the possibility that aromatic metabolism might be used by the spirochete as an accessory mechanism to consume low amounts of oxygen, and possibly to generate intermediates that can feed into its (or the surrounding microbial community s) oxygen independent energy metabolism. Here, culture conditions were modified allowing the observation of good growth and successive serial transfer of T. primitia str. ZAS- 1 cultures in an unreduced medium under a 0.5% O2 vol/vol headspace. Such media was competent for culturing this strain if the cultures were incubated statically. Other than occasionally exhibiting longer initial lag phases, growth progressed at the same rates and yields as those observed in reduced, anoxic media (Fig. 3-6). Fig. 3-6: Microoxic growth of Treponema primitia str. ZAS- 1. T. primitia str. ZAS- 1 grew in 5mL of 2YACo, DTT- free media, with an 80% N2/20% CO2 headspace to

135 3 34 whichairwasaddedsterilelytoachieve0.5%vol/volo2(red)and5mlof4yaco, DTT reducedmediawithan80%h2/20%co2headspace(blue).bothtreatment typesweresupplementedwithavitaminmixture(12 vitamin,vitaminb12,and folinicacidsolutions,graber&breznak,2004).cultureswerecontainedin25ml butylrubber stopperedbalchtunesandincubatedat25 Chorizontallyandstill. Firstthreedaysofgrowthareshown. Stoichiometricpredictionsofcatecholconversiontoacetateviathismeta cleavage pathwaysuggestanincreaseinacetateyieldthanwhatisobtainedfromh2+co2 acetogenesis: H2+CO2Acetogenesis: 4H2+2CO2 CH3OOH+2H2O Catechol FormicAcid+PyruvicAcid+AceticAcid: C6H6O2+O2+3H2O H2CO2+C3H3O3H+CH3COOH+H2(+ATP) FormicAcid+PyruvicAcid AceticAcid: H2CO2+C3H3O3H+2[2e +2H + ] 2CH3COOH+H2O(+2ATP) Catechol AceticAcid: C6H6O2+O2+2H2O+2[2e +2H + ] 3CH3COOH+H2(+3ATP) Therefore,potentialincreasesingrowthyieldsofculturestowhichcatechol like aromaticsando2wereaddedwereevaluatedasaproxyforacetateproductionvia thismeta cleavagepathway.whent.primitiastr.zas 1culturesweretransferred intothismicrooxicmediumamendedwithcatecholtoafinalconcentrationof 0.5mM,nosignificantstimulationsofgrowthororganicacidyieldswereobserved, andthemajorityofthesubstratewentunreactedovertime.however,theculture fluidstransientlyturnedadistinctlemon yellowcolorafterseveraldaysofgrowth, havingreachedanod600nmbetween0.2and0.3(table3 3;AppendixFig.3A 18and Fig.3A 19).Analysisofculturefluidstowhich0.5mMaromaticsubstrateand0.5%

136 3 35 vol/volo2wereaddedrevealedthatthecatechol dependentyellowmaterialhadan absorbancemaximumat375nm(table3 3;AppendixFig.3A 19),consistentwith hydroxymuconicsemialdehyde.thispeakwasnotseeninculturestowhichonly 19).Culturestransferredintomediacontaining0.5mMguaiacolalsogeneratedthis sameyellowintermediate(table3 3).Thus,thedioxygenaseinthisspirochete appearstobefunctionaloncatecholandguaiacol.nogenerationofobvious intermediateswasobservedwhenanyofthefollowingcompoundsweretested: veratrole,protocatechualdehyde,protocatechuicacid,vanillicacid,gallicacid, homoprotocatechuicacid,homoveratricacid,hydrocaffeicacid,caffeicacid,or ferulicacid. Asaback of the envelopecalculation,that0.5mmcatecholaddedtothecultures examinedbyuv/vismeansthattherewere275µmolesofcatecholineach5ml thepresenceoftheexpecteddioxygenase mediatedringcleavageproduct,2 0.5mMaromaticsubstrateoronly0.5%vol/volO2wasadded(AppendixFig.3A culture.therefore,ifcatecholgeneratestheringcleavageproduct,2 hydroxymuconicsemialdehyde,inaratioof1:1accordingtothesoichiometry above,thenonewoudexpectallofthiscatecholtobeconvertedto275µmolesof ringcleavageproduct.nevertheless,approximately22µmolesoftheringcleavage productwereformed(ε=36,000lmol 1 cm 1 andculturesamplediluted1:100for UV/Visspecreadings)indicatingroughly10%oftheoriginalcatecholinputtothe cutureswastransformedintotheringcleavageproduct(appendixfig.3a 19).

137 3 36 Table 3-3: Monoaromatic compound cleavage by Treponema primitia str. ZAS-1 Monoaromatic Compound Abs. Max (nm) Ring Cleavage Product Abs. Max (nm) Ring Cleavage catechol (1,2-dihydroxybenzene) guaiacol (2-methoxyphenol) Growth on veratrole, protocatechualdehyde, protocatechuic acid, vanillic acid, gallic acid, homoprotocatechuic acid, homoveratric acid, hydrocaffeic acid, caffeic acid, and ferulic acid was tested, but no evidence of ring cleavage was detected. AsanalternativetogrowingT.primitiainaliquidmediaunderadefinedmicrooxic headspace,cultureswerealsoinitiatedintubescontaininganagarose solidified mediaunderamicrooxicheadspaceinordertoestablishoxygengradientcultures. UndertheseconditionsitbecameclearthatbothstrainsofT.primitiainfluencethe extentoftheinwarddiffusionofoxygenintothemedia,withorwithoutanaromatic substratepresent.moreover,thedistributionofthegrowthintheanaerobic portionsoftheagarwasnotuniform.rather,cellsaccumulatedupwardstowards theoxicanoxicinterfaceasvisualizedbythetransitionoftheredoxindicatordye resazurinfrompinkincolortoclear.becausethebalanceofthemicrooxic headspacewasn2/co2,theresultssuggestthatthepresenceofoxygenstimulates theyieldofelectrondonorspresentinthemedia(yeastautolysateand,when present,utilizablearomaticsubstrates).inseveralcases,theadditionofcertain aromaticsubstrates(catechol,protocatechuicacid,hydrocaffeicacid,andcaffeic acid)furtherstimulatedtheabilityoft.primitiatoimpactthedegreeofoxygen

138 3 37 penetrationintotheagar,suggestingthatthesecompoundsarebeingmetabolized inoxygensinkreactions(table3 4).Theimpactofthepresenceorabsenceof aromaticsand/orviablecellsonoxygenpenetrationissummarizedintable3 4and Fig.3 7aswellasAppendixFig.3A 20aandband3A 21aandb.Theseanalyses provideadditionalsupportthatt.primitiastr.zas 1isperhapsthebettercandidate ofthetwostrainsforfuturestudiesonoxygenandaromaticmetabolismsinthis anaerobic species(appendixfig.3a 20aandb). Table 3-4: Average depth of O 2 penetration in Treponema primitia str. ZAS-1 and ZAS-2 O 2 /aromatic gradient cultures Aromatic Substrate str. ZAS-1 Ave. +/- Std. Error Depth (mm) str. ZAS-2 Ave. +/- Std. Error catechol 5 +/-1 ab 20 +/- 1 protocatechuic acid 6 +/-3 ab 24 +/- 1 hydrocaffeic acid 6 +/- 1 ab 13 +/- 1 a caffeic Acid 7 +/- 2 b 18 +/-1 H 2 O 9 +/- 2 b 19 +/- 1 a Significantly different from corresponding H 2 O control as determined by Student's t-test p-value calculations at 95% confidence. b Significantly different from corresponding T. primitia str. ZAS-2 cultures as determined by Student's t-test p-value calculations at 95% confidence. After 7 days of incubation, distance of O 2 penetration into semi-solid agar culture media determined by resazurin front. Data is average of triplicate cultures. For comparison, O 2 reached the bottom of cell-free control tubes (22mm) by day 3 (Fig. 3-6a).

139 3 38 A B Fig.3 7:Treponemaprimitiastr.ZAS 1O2/catecholgradientculturesandcontrols. OxygengradienttubesunderaheadspaceofN2/CO2/O2(70:10:4)after7daysof incubation.(a)fromlefttoright:uninoculatedo2andcatecholcontrol; uninoculatedo2 onlycontrol;inoculatedo2 onlycontrol;andinoculatedo2and catechol.(b)left:closerviewofinoculatedo2 onlycontrol;right:closerviewof inoculatedo2andcatechol.arrowsindicatetheredoxtransitionzoneas representedbyresazurindecolorization.bracketshighlightaccumulationsofcell density.mediaandcultivationconditionsaredetailedinthematerialsandmethods. Bar=15mm. WithacatecholgradientonlyandnoO2available,T.primitiastr.ZAS 1culturesdo notdisplayrobustgrowth.rather,theyappearliketheirunoculatedcontrol (AppendixFig.3A 21a). DISCUSSION: TreponemaprimitiastrainsZAS 1andZAS 2havegenesrepresenting completecatechol2,3 dioxygenasemeta cleavagepathways ThatreciprocalBLASTanalysesconductedbetweengenomesfromthree lower termitehindgutisolates,treponemaprimitiastr.zas 1andZAS 2,andT. azotonutriciumstr.zas 9(Leadbetteretal.1999;Graber&Breznak2004;Graberet al.2004;rosenthaletal.2011;balloretal.2012),andthep3hindgutregion

140 3 39 metagenomefromthe higher termite,nasutitermessp.(warneckeetal.2007) revealedaputative catechol2,3 dioxygenase geneineachofthet.primitiastr. ZAS 1andZAS 2genomes,butnotintheT.azotonutriciumstr.ZAS 9genomenor highertermitemetagenome,wasunexpectedbuthelpsreconcileo2inthetermite hindgut,strategiesof anaerobic organismstodealwitho2 relatedstress,ando2 requiringmetabolismsrelatedtolignocellulosedegradation. Itshouldalsobenotedthatthe higher termitemetagenomecoveragewasless than1xtheentirehindgutpopulation(warneckeetal.2007).likelycoveragewas morethan1xforthemostabundantspeciesbutlessthan1xforlowerabundance members.becausethetermiteguthasaconstantlychangingcompositionof membersaswellascommunitymembersinrelativelylowabundance,itwas difficulttomakesureareliable coverage isobtainedsincean a priori knowledge ofwhatisinthegut whenthisgenomewasgenerated wasnotavailable. Nevertheless,currenttechnologycouldaquirealargerdataset,andwiththis higher termitemetagenomicdatasetasareference,anothersimilarprojectto obtainmorecoveragecouldbeconducted. Genomichintsofmeta pathwaysubstratepreference KeyresiduesoftheT.primitiameta cleavagepathwaygenesprovidehintsabout theirfunctions(díaz&timmis1995;kitaetal.1999;nardini&dijkstra1999; Holmquist2000;Nandhagopaletal.2001;Reaetal.2005;Izumietal.2007;Leiet al.2008).forexample,inpseudomonasputidastr.mt 2,catecholandO2are thoughttoentertheactivesitethroughachannelcomprisedmainlyoflarge

141 3 40 Moreover,thefactthatT.primitiahasgenesrepresentingone,butnottheother, meta pathwayrouteshasbearingonunderstandingthearomaticsubstratesforthe pathway.paststudieshaveindicatedthatthe2 hydroxymuconicsemialdehyde hydrolase(step2),forinstance,prefersketonegroup containingringcleavage products,incomparisontothe2 hydroxymuconicsemialdehydedehydrogenase (step6)thatisactiveonlyonaldehydegroup containingringcleavageproducts residues:his 153,His 199,Tyr 255,andPhe 302.Theserelativelybulkyresidues makethechannelisnarrowerthanthatofotherdioxygenases,consistentwiththe factthatcatecholisarelativelysmallaromaticsubstrate(kitaetal.1999). Similarly,thesebulkyresiduesarerepresentedintheT.primitiacatechol2,3 dioxygenases(his 154,His 200,Tyr 256,Phe 303)suggestingtheseenzymesalso prefersmallersubstratessuchascatechol. (Fig.3 1)(Khajamohiddinetal.2006).ThissuggeststhatT.primitiacatechol2,3 dioxygenasesprefersubstrateswithmeta cleavageproductsthatcontainketone groups. TheT.primitiameta cleavagepathwaygeneneighborhoodarrangementisdistinct fromotherorganismspossessingthispathway,whichisalsouniqueasthereare onlyafewconservedgeneneighborhoodarrangementsamongthemeta cleavage pathwaycontainingorganisms(fig.3 2)(Suenagaetal.2009). Noevidencefor upper preparatorypathwaygenesintreponemaprimitia Inadditiontotheuniquearrangementofmeta pathwaygenes,thereisnoevidence ofgenesrepresenting upper preparatorypathwaysintreponemaprimitia,

142 3 41 commonlyfoundinotherorganismspossessingmeta pathwayhomologs(suenaga etal.2009).thissuggeststhatonlyalimitednumberofcandidatearomatic substratesmightserveaspossiblesubstrates.alternatively,giventhatt.primitia str.zas 1andZAS 2naturallyarepartofacomplexmicrobialecosystem,perhaps anothercommunitymemberhas upper pathwaycapabilitiesandprovides lower pathwaysubstratestot.primitia.onewaytotestwhetherornototherorganisms provide upper pathwayproductstot.primitiatofueltheir lower pathway metabolism,wouldbetofeedt.primitiastr.zas 1andZAS 2cell freeculturefluid fromanorganismknowntoperformthe upper pathwayinlieuofaromatic like substratesandfilter sterilizedtermitehindgutfluid(whichwouldrequirealotof termites)and/orcell freeenrichmentculturefluidthatwaspreparedtoselectfor organismswith upper pathwaycapabilities.azotobactervinelandii, Novosphingobiumaromaticivorans,Novosphingobiumsp.,Methylocellasilvestris, Sphingobiumjaponicum,Azoarcussp.,Pseudomonasputida,Thauerasp., Dechloromonasaromatica,andMethylibiumpetroleiphilumareknowntoperform upper pathways.distinctmeta cleavageproductscouldbedetected,forexample, colorimetricallyandwithuv/vis. EvolutionaryoriginsoftheTreponemaprimitiameta cleavagepathway Interestingly,inlightofthefactthatthemajorityoftheclosest culturedrelatives phylogeneticallyassociatingwiththetreponemaprimitiacatechol2,3 dioxygenases are Sphingomonad alphaproteobacteria,examinationofgeneneighborhoodsand genomesinthedatabasesrevealsthatcatechol2,3 dioxygenasesandotherkey pathwaygenesinthese sphingomonads aretypicallyfoundonplasmidsandother

143 3 42 mobilegeneticelements,andthisispossiblyhowcatechol2,3 dioxygenasewas acquiredbyt.primitia(harayama&rekik1990;stillwelletal.1995)(fig.3 3). Becausephylogeneticanalysessuggestthatthegenesforthepathwaylikely originatefromatleasttwo,ifnotthreesources analphaproteobacterium,a memberoftheclostridiales,andpossiblyamemberofthebacillales itisplausible dependentutilizationofaromaticcompounds,wasaugmentedwithgenesacquired bylateraltransfer. Moreover,analysesofthegeneandgenomedatabasesindicatethatevidencefor meta cleavagepathwayshaspreviouslybeenrestrictedtospeciesrepresenting threebacterialphyla:proteobacteria,firmicutes,andactinobacteria.thus,the currentresultsextendthedistributionofthegenesforthispathwaytoafourth phylum,thespirochetes.theavailabledatasuggestsmeta cleavagepathwaysare absentinallotherspirochetesthathavebeenstudied. Selectionpressureandexpressionanalyses ThatnoneofthepathwayrelatedORFsintheTreponemaprimitiastrainsappearto bepseudogenes,andallappeartoencodefunctionalenzymes,andallofthe pathwaygeneswiththeexceptionof2 hydroxymuconicsemialdehydehydrolase thatapathwayfoundinmanyanaerobes,butthatdoesnotinvolvetheo2 appeartobeunderpositiveselection,suggeststhatthemajorityofthemetacleavagepathwayhavebeenusedovertimeandhaveprovidedbenefittot.primitia (Table3 2).

144 3 43 Further,thatallofthemeta pathwaygenesareexpressedevenaftercultivation withouto2oraromaticsubstratesincetheywereisolatedoveradecadeago suggeststhemeta pathwaygenesareconstitutivelyexpressed(appendixtable3a 2andTable3A 3).Nevertheless,perhapstracelevelsofO2areinthemedia,O2isa metabolicby productgeneratedinthemedia,andaromaticcompoundsare availableintheundefinedyeastautolysateleadingtothemeta pathwaygenesbeing expressed.ifthiswerethecase,however,thedistinctringcleavageyellowmay havebeenseenwhichhasnotbeenobservedbeforethisexperiment. Metabolismofcatecholundermicrooxicconditions Itisconcludedthatbotharomaticsubstrateandoxygenareneededtobeaddedto culturestoobserveringcleavageproducts.thisisnotaphenomenonobservedin culturestowhicharomaticsubstrateisaddedalone(appendixfig.3a 19).Further, theringcleavageproductpeakat375nmisonlyobservedwhenboth0.5mm aromaticsubstrateand0.5%vol/volo2isaddedtocultures.thispeakwasnotseen inculturestowhicheither0.5mmaromaticsubstrateor0.5%vol/volo2wasadded (AppendixFig.3A 19). InliquidcultureofT.primitiastr.ZAS 1withcatechol,thefactthatthemetacleavagefirstproductwasdetectedcolorimetricallyandwithUV/Visbutwas transientsuggeststhattheintermediateisbeingmetabolizedfurtherbytheother meta cleavagepathwayenzymes.theaccumulationoftheexpectedfinalproducts wasnotmeasuredastheywouldbeatthelimitofdetection,takingintoaccountthe smallamountsofthesubstratemetabolized.

145 3 44 WithacatecholgradientonlyandnoO2available,T.primitiastr.ZAS 1culturesdo notdisplayrobustgrowth.rather,theyappearliketheirunoculatedcontrol (AppendixFig.3A 21a).Thiscouldbebecausethesearomaticcompoundsare inhibitorytot.primitiaorthatinthismediathatlacksacarbonsourceotherthan thearomaticsubstrate(exceptwhatmybeminimallyprovidedinthe2yaco)t. primitiastarvesbecauseitcannotmetabolizethearomaticsubstrate.however, whenwaterisprovidedasacontrolsubstrateandnoo2isavailablegrowth(albeit small)isstillseen(appendixfig.3a 21b).Thereforeitislikelythesearomatic compoundsareinhibitoryandperhapsespeciallysowhencellsarestarvedtosome degree. ThistrendcouldmeanthatincultureswithbotharomaticandO2gradientsinwhich lesso2penetrationisseen(table3 4,Fig.3 7)thatcellsarepreferentiallygrowing towardstheheadspacetomoveawayfromthearomatic.decreasingthe concentrationofaromaticinthegradienttubeandgeneratingaromaticgradient onlycultureswithrobustgrowththroughoutcouldestablishanaromatic concentrationregimetore investigatetheseo2andaromaticgradienttubes. Perhaps,however,withO2available,T.primitiacancleavetheinhibitoryaromatic substratesandgeneratepresumablylesstoxicringcleavageproducts.thiscouldbe whygrowthisseeninaromaticgradienttubestowhicho2isadded.otherworkin thisstudyverifiescatecholringcleavagebyt.primitiastr.zas 1,andthisability couldbeastrategytodetoxifythehindgutenvironmentoflignocellulose derived aromaticmonomers(bruneetal.1995a).

146 3 45 Conclusions Thegenomesoftwostrainsofthe anaerobic spirochetet.primitiabothcontainall genesforacompleteandpossiblyfunctionalcatechol2,3 dioxygenasemetacleavagepathway.moreover,t.primitiastr.zas 1performsatleastthefirst enzymaticstepofthemeta pathwayinvitro,utilizingo2tocleavetheringof catecholtotheexpecteddegradationintermediate.thedietofwood feeding termitesisrichinaromatics,albeitinaformthatisrecalcitranttodegradation(the aromaticpolymerlignin).theseresultssuggestthatanymonomericaromatics possiblyliberatedbyinsectandmicrobialmetabolisminthegutmightbecome substratesinnon canonicaloxygenco utilizingreactions.indeed,manypaststudies havefocusedonaromaticdegradationactivitiesandculturestrategiesunderstrictly aerobicoranaerobicconditions(brune1998b;lovely2000;gibson&harwood 2002;Fuchsetal.2011),conditionsthatmighthaveoverlookedtheactivitiesof oxygendependentreactionsthatdonotresultintherespiratorymineralizationof theringcarbon.inthefuture,itwilllikelybeimportanttore examineanypotential foraromaticmetabolismintermitehindgutcommunitieswiththisinmind. Futurework AlthoughwedonotseeanimprovementofgrowthbytheTreponemaprimitia strainsculturedwitharomaticando2,thisdoesnotnecessarilyindicatethatthey arenotgeneratingacetatewiththesetwosubstratesviathemeta cleavagepathway. Rather,itcanbeconcludedthatanypotentialacetateproductionisnotresultingin anincreaseingrowthratesoryield(graber&breznak2004;graberetal.2004).

147 3 46 AnalyticalMethods Ifappropriateandsensitiveprotocolscanbeestablished,acombinationofgas chromatography,liquidchromatography massspectrometry,andion chromatographycouldbeusedtomeasureo2drawdownbyt.primitiastr.zas 1 Therefore,inthefutureothertechniquescouldbepursuedtoevaluatemetacleavagepathwaycapabilitiesbyT.primitia. chromatographycouldbeemployedtoevaluatedifferentaspectsofputativemetacleavagepathwayfunctionalityintreponemaprimitiastr.zas 1.First,gas cultures,indicativeofthefirststepofthemeta pathwayperformedbycatechol2,3 dioxygenase,towhichbothanaromaticsubstrateando2havebeenadded. Moreover,liquidchromatography massspectrometrycouldpotentiallydetect substrates,intermediates,andproductsalongthemeta cleavagepathwayroute.to evaluatetheformationofthemeta cleavagepathwayendproduct,acetate,ion chromatographycouldbeutilized.tracingradio labeledmethylgroupsinto possibleacetategeneratedbythestrainisanothermore directmethod. Revisemediadesign Similarly,whiletheconcentrationsofaromaticsubstratesandO2addedtocultures thusfardonotappeartobeinhibitorytogrowth,theycouldstillbeinhibitoryto enzymefunctionseeingasnotallaromaticaddedtotheculturesisutilizedinthis set up(andthedrawdownofo2incultureheadspaceisunknown).therefore,a decreaseinaromaticando2substrateswouldbeagoodwaytostartamending mediadesign.

148 3 47 Feedingexperimentsandco culturing OnewaytotestiftheTreponemastrainsaregeneratingacetatewhichisusedby otherorganismswouldbetoprepare feeding experimentsandlookformarked increasesingrowthrateandyieldofthe fed organism.forinstance,insteadof acetateasasubstrate,cell freetreponemaculturefluidcouldbeprovidedtoother cultures organismsknowntometabolizeacetateand/orotherorganismsisolated fromthetermitehindgutenvironment.co culturesbetweenthetreponemastrains andtheseorganismscouldalsobeinformativeifsuccessful(rosenthaletal.2011). Moleculartechniques Underthese,andnormalcultureconditions,qRT PCRofrelevantgenes(Appendix Methods3A 1)andcloning/expression/enzymeactivityassayworkcouldalso measureactivity,expression,orlackthereofoftheserelevantmeta cleavage pathwaygenes.genome widetranscriptomicscouldleadtoabetterunderstandof thetreponemaprimitiastrains relationshipwitho2. Visualization AZootermopsisnevadensisworkertermitehindgutwassuccessfullysectioned. Protozoawereprominent,includingStreblomastixstrixisanoxymonadsymbiontof thetermitezootermopsisangusticoli(appendixfig.3a 22b)(Leander&Keeling 2004).Whileartifactscanbeinducedduringthefixation,dehydration,and embeddingprotocols,hcr FISHtomapmRNAexpressioncouldco localizethe Treponema16SrRNAgenewithrelevantmeta cleavagepathwaygenes.dueto peristalticmovementofthegut,membersofthegutmicrobialcommunityarelikely

149 3 48 constantlybeingdisorientedandre establishingthemselvesinpreferential locations.whileasingletime pointsectionmaynotcapturet.primitia sideal location,co localizing16srrnagenewithrelevantmeta cleavagepathwaygenesin situcouldprovideusefulinformation.

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158 3 57 CHAPTER3:APPENDIX Table3A 1:GenBankaccessionnumbersandreferencesoforganismsandproteins presentedintextandfigures. Table3A 2:GeneexpressionpatternsofTreponemaprimitiastr.ZAS 2inmonocultureorco culturewitht.azotonutriciumstr.zas 9. Table3A 3:ExpressionofTreponemaprimitiastr.ZAS 2genes. Table3A 4:GenesuniquetoTreponemagenomesrelativetometagenomeof Nasutitermessp.P3hindgutregion. Fig.3A 1:PFAMdomainsandconservedfunctionalresiduesandsequencemotifsin Treponemaprimitiastr.ZAS 1andZAS 2meta cleavagepathwayproteins. Fig.3A 2:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 ferredoxin likepeptide(pf00111,step1 ). Fig.3A 3:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 24 hydroxy 2 oxopentanoatealdolase(pf00682andpf07836,step4). Fig.3A 4:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 acetaldehydedehydrogenase(pf01118andpf09290,step5). Fig.3A 5:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2NterminalDomain(PF00903). Fig.3A 6:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2NterminalDomain(PF00903)withextra domainregion. Fig.3A 7:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2NterminalDomain(PF00903)withintra domainregion. Fig.3A 8:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2CterminalDomain(PF00903). Fig.3A 9:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2CterminalDomain(PF00903)withextra domainregion. Fig.3A 10:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2CterminalDomain(PF00903)withintra domainregion. Fig.3A 11:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 24 hydroxy 2 oxopentanoatealdolasehmgl likedomain(pf00682).

159 3 58 Fig.3A 12:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 24 hydroxy 2 oxopentanoatealdolasedmpg likecommunicationdomain(pf07836). Fig.3A 13:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 acetaldehydedehydrogenase,nadbindingdomain(pf01118). Fig.3A 14:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 acetaldehydedehydrogenasedimerisationdomain(pf09290). Fig.3A 15:TranscriptionalregulatoryelementswithintheTreponemaprimitiastr. ZAS 1andZAS 2meta cleavagepathwaygeneneighborhoods. Fig3A 16:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2metapathwayassociatedGntRfamilytranscriptionalregulator(PF00392). Fig.3A 17:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2metapathwayassociatedGntRfamilytranscriptionalregulator,Bacterialregulatory proteinsgntrfamilydomain(pf00392). Fig.3A 18:YellowTreponemaprimitiastr.ZAS 1cultures. Fig.3A 19:AbsorbancespectraofTreponemaprimitiastr.ZAS 1cultures. Fig.3A 20:Treponemaprimitiastr.ZAS 1andZAS 2O2/catecholgradientcultures andcontrols.(a)uninoculatedcatecholando2controlandtriplicatesof O2/catecholgradienttubesinoculatedwithofT.primitiastr.ZAS 2(b)O2/catechol gradienttubesinoculatedwithoft.primitiastr.zas 2comparedtoT.primitiastr. ZAS 1. Fig.3A 21:Treponemaprimitiastr.ZAS 1(a)catecholgradientculturesand controlsand(b)watergradientculturesandcontrols. Method3A 1:VisualizingTreponemaprimitiaintermitehindgut. Method3A 2:qRT PCRofeachmeta cleavagepathwaygene.

160 3 59 Table 3A-1: GenBank accession numbers and references of organisms and proteins presented in text and figures Organism Accession Figure Organisms Name Number Reference(s) Keil et al. 1985; Keil et al. 1987a; Keil et al. 1987b; Osborne et al. 1988; Assinder et al. 1992; Assinder et al. 1993; Gallegos et al. 1997; Sentchilo et al. 2000; Tsuda and Genka 2001; Yano et al. 2007; Miyakoshi et al Fig. 3-2 Pseudomonas putida AB Fig. 3-2 Treponema primitia str. ZAS-2 CP Rosenthal et al Fig. 3-2 Treponema primitia str. ZAS-1 CP Ballor et al Fig. 3-2 Novosphingobium aromaticivorans AF Romine et al Fig. 3-2 Novosphingobium sp. FR D'Argenio et al Fig. 3-2 Methylocella silvestris CP Chen et al Fig. 3-2 Sphingobium japonicum AP Nagata et al Fig. 3-2 Azoarcus sp. AM Krause et al Fig. 3-2 Thauera sp. CP NA Fig. 3-2 Dechloromonas aromatica CP NA Fig. 3-2 Azotobacter vinelandii CP Setubal et al Fig. 3-2 Methylibium petroleiphilum CP Kane et al Catechol 2,3- dioxygenase Accession Number Fig. 3-3 Novosphingobium aromaticivorans NP_ Romine et al.1999 Fig. 3-3 Sphingomonas agrestis AAB03075 Yrjala et al.1994 Fig. 3-3 Rhizobium sp. ABF82226 NA Fig. 3-3 Beijerinckia sp. B57264 Kim and Zylstra 1995 Fig. 3-3 Sphingomonas sp. AAM14600 NA Fig. 3-3 Sphingomonas sp. ADK27485 NA Fig. 3-3 Novosphingobium pentaromativorans ZP_ NA Fig. 3-3 Novosphingobium sp. YP_ D'Argenio et al Fig. 3-3 Sphingomonas sp. AAD11448 NA Fig. 3-3 Sphingomonas sp. AAD11452 NA Fig. 3-3 Treponema primitia str. ZAS-1 ZP_ Ballor et al Fig. 3-3 Treponema primitia str. ZAS-2 YP_ Rosenthal et al Fig. 3-3 lake sediment Fig. 3-3 activated sludge BAH89314 Suenaga et al. 2007; Suenaga et al Fig. 3-3 activated sludge BAH90343 Suenaga et al. 2007; Suenaga et al Fig. 3-3 Methylocella silvestris YP_ Chen et al Fig. 3-3 Sphingobium japonicum YP_ Nagata et al. 2010

161 3 60 Fig. 3-3 Pseudomonas putida BAB62050 NA Fig. 3-3 Thauera sp. YP_ NA Fig. 3-3 Dechloromonas aromatica YP_ NA Fig. 3-3 Azotobacter vinelandii YP_ Setubal et al Fig. 3-3 Pseudomonas putida YP_ Keil et al. 1985; Keil et al. 1987a; Keil et al. 1987b; Osborne et al. 1988; Assinder et al. 1992; Assinder et al. 1993; Gallegos et al. 1997; Sentchilo et al. 2000; Tsuda and Genka 2001; Yano et al. 2007; Miyakoshi et al Fig. 3-3 Alcaligenes xylosoxydans Fig. 3-3 Marinobacter algicola ZP_ NA Fig. 3-3 whale fall rib bone Fig. 3-3 Marinobacterium stanieri ZP_ NA Fig. 3-3 Marinobacterium stanieri ZP_ NA Fig. 3-3 marine gamma proteobacterium HTCC2207 ZP_ NA Fig. 3-3 sludge Fig. 3-3 Novosphingobium nitrogenifigens ZP_ NA Fig. 3-3 Cupriavidus necator Fig. 3-3 Methyloversatilis universalis ZP_ NA Fig. 3-3 Methylibium petroleiphilum YP_ Kane et al hydroxymuconic hydrolase Accession Number Letek et al. 2008; Letek et al Fig. 3-4 Rhodococcus equi YP_ Fig. 3-4 Intrasporangium calvum YP_ Del Rio et al Fig. 3-4 Kyripidia tusciae YP_ NA Fig. 3-4 Solibacillus silvestris YP_ Morohoshi et al Fig. 3-4 Bacillus tusciae YP_ NA Fig. 3-4 Novosphingobium aromaticivorans NP_ Romine et al Fig. 3-4 Novosphingobium pentaromativorans ZP_ NA Fig. 3-4 Novosphingobium sp. YP_ D'Argenio et al Fig. 3-4 whale fall rib bone Fig. 3-4 Lysobacter sp. BAH80176 NA Fig. 3-4 Treponema primitia str. ZAS-1 ZP_ Ballor et al Fig. 3-4 Treponema primitia str. ZAS-2 YP_ Rosenthal et al Fig. 3-4 lake sediment Fig. 3-4 Arcobacter sp. YP_ Toh et al Fig. 3-4 Leptothrix cholodnii YP_ NA Fig. 3-4 Delftia sp. ZP_ NA Fig. 3-4 Alcaligenes faecalis ABV30923 NA

162 3 61 Fig. 3-4 Pseudomonas reinekei ABH07023 Nikodem et al. 2003; Camara et al. 2007a; Camara et al. 2007b Fig. 3-4 Dechloromonas aromatica YP_ NA Fig. 3-4 Azoarcus sp. YP_ Krause et al Fig. 3-4 Acidovorax sp. YP_ NA Fig. 3-4 activated sludge BAH89584 Suenaga et al., 2007; Suenaga et al., 2009 Fig. 3-4 Methylibium petroleiphilum YP_ Kane et al Fig. 3-4 Thauera sp. YP_ NA Fig. 3-4 Pseudomonas putida YP_ Keil et al. 1985; Keil et al. 1987a; Keil et al. 1987b; Osborne et al. 1988; Assinder et al. 1992; Assinder et al. 1993; Gallegos et al. 1997; Sentchilo et al. 2000; Tsuda and Genka 2001; Yano et al. 2007; Miyakoshi et al Fig. 3-4 Methyloversatilis universalis ZP_ NA Fig. 3-4 activated sludge BAH90404 Suenaga et al., 2007; Suenaga et al., 2009 Fig. 3-4 Novosphingobium nitrogenifigens ZP_ NA Fig. 3-4 sludge Fig. 3-4 Thiothrix nivea WP_ NA Fig. 3-4 Rhodococcus jostii YP_ McLeod et al Fig. 3-4 Rhodococcus erythropolis NP_ Stecker et al oxopent-4-enoate Hydratase Accession Number Fig. 3-5 Azoarcus sp. YP_ Krause et al Fig. 3-5 activated sludge BAH89311 Suenaga et al., 2007; Suenaga et al., 2009 Fig. 3-5 Novosphingobium pentaromativorans ZP_ NA Fig. 3-5 Methylocella silvestris YP_ Chen et al Fig. 3-5 Sphingobium japonicum YP_ Nagata et al Fig. 3-5 Azotobacter vinelandii YP_ Setubal et al Fig. 3-5 Methylibium petroleiphilum YP_ Kane et al Fig. 3-5 Burkholderia sp. Fig. 3-5 Arcobacter sp. YP_ Toh et al Fig. 3-5 Marinobacter manganoxydans ZP_ NA Fig. 3-5 activated sludge BAH89583 Suenaga et al., 2007; Suenaga et al., 2009 Fig. 3-5 Pseudomonas putida YP_ Keil et al. 1985; Keil et al. 1987a; Keil et al. 1987b; Osborne et al. 1988; Assinder et al. 1992;

163 3 62 Assinder et al. 1993; Gallegos et al. 1997; Sentchilo et al. 2000; Tsuda and Genka 2001; Yano et al. 2007; Miyakoshi et al Fig. 3-5 Methyloversatilis universalis ZP_ NA Fig. 3-5 Dechloromonas aromatica YP_ NA Fig. 3-5 Ralstonia eutropha YP_ Pohlmann et al Fig. 3-5 Geobacillus sp. YP_ NA Fig. 3-5 Carboxydothermus hydrogenoformans YP_ Wu et al., 2005 Fig. 3-5 Thermosinus carboxydivorans ZP_ NA Kosaka et al., 2006; Kosaka et al., 2008 Fig. 3-5 Pelotomaculum thermopropionicum YP_ Fig. 3-5 Desulfotomaculum nigrificans ZP_ NA Fig. 3-5 Moorella thermoacetica YP_ Pierce et al., 2008 Fig. 3-5 Flavonifractor plautii ZP_ NA Fig. 3-5 Acetonema longum ZP_ NA Fig. 3-5 Bacillus cereus ZP_ Fig. 3-5 Treponema primitia str. ZAS-1 ZP_ Ballor et al Fig. 3-5 Treponema primitia str. ZAS-2 YP_ Rosenthal et al Fig. 3-5 Solibacillus silvestris YP_ Morohoshi et al., 2012 Fig. 3-5 Brevibacillus brevis YP_ NA Fig. 3-5 Paenibacillus sp. BAH79101 Kasai et al., 2009 Fig. 3-5 Geobacillus stearothermophilus AAZ76889 NA Fig. 3-5 Sulfobacillus acidophilus YP_ NA Fig. 3-5 Halobacterium sp. ZP_ NA Fig. 3-5 Alicyclobacillus acidocaldarius YP_ Mavromatis et al., 2010 Fig. 3-5 Thermomicrobium roseum YP_ Wu et al., 2009 Fig. 3-5 Salmonella bongori YP_ NA Fig. 3-5 Intrasporangium calvum YP_ Del Rio et al., 2010

164 3 63 Table 3A-2: Gene expression patterns of Treponema primitia str. ZAS-2 in mono-culture or co-culture with T. azotonutricium str. ZAS-9 Cohort Expression Range Mono-Culture Co-Culture (reads/kb) No. genes % No. genes % 1 a b c > Analysis complements Table 3A-3. Analysis based on expression dataset from Rosenthal et al a In addition to having 0 reads/kb, all genes in Cohort 1 have 0 expression. b Cohort includes meta-pathway 2-hydroxymuconic semialdehyde hydrolase, 2-oxopent-4-enoate hydratase, 4- hydroxy-2-oxopentanoate aldolase, acetaldehyde dehydrogeanse, and the associated ferredoxin-like peptide. c Cohort includes meta-pathway catechol 2,3-dioxygenase.

165 3 64 Table 3A-3: Expression of Treponema primitia str. ZAS-2 genes Gene Pure-Culture Expression Co-Culture Expression (reads/kb) (reads/kb) Meta-pathway Ferredoxin-like peptide a catechol 2,3-dioxygenase b hydoxymuconic semialdehyde hydrolase a oxopent-4-enoate hydratase a hydroxy-2-oxopentanoate aldolase a acetaldehyde dehydrogenase a House-keeping ATP-dependent Clp protease ATP-binding subunit ClpX Metallo-beta-lactamase family protein, RNA-specific RNA polymerase sigma factor RpoD RNA polymerase sigma factor RpoD RNA polymerase sigma factor RpoD RNA polymerase sigma factor RpoD RNA polymerase sigma factor RpoD Acetogenesis Formate dehydrogenase chain D 62 2 Formate dehydrogenase chain D Formate dehydrogenase; cysteine-containing variant Formate dehydrogenase; selenocysteine-containing variant Carbon monoxide dehydrogenase CooS subunit Formate-tetrahydrofolate ligase (FTHFS) Glycolysis Hexokinase Glucose-6-phosphate isomerase phosphofructokinase Fructose-bisphosphate aldolase class II Triosephosphate isomerase NAD-dependent glyceraldehyde-3-phosphate dehydrogenase NAD-dependent glyceraldehyde-3-phosphate dehydrogenase ,3-bisphosphoglycerate-independent phosphoglycerate mutase Enolase Pyruvate kinase Misc. acetate kinase Fe-S cluster containing hydrogenase components Periplasmic [Fe] hydrogenas large subunit Expression data from Rosenthal et al a Belong to Cohort 3 (Table 3A-2). b Belongs to Cohort 4 (Table 3A-2).

166 3 65 Table 3A-4: Genes unique to Treponema genomes relative to metagenome of Nasutitermes sp. P3 hindgut region Treponema primitia str. ZAS-1 DCMPdeaminase UncharacterizedproteinTP_0813 SignalpeptidaseI(EC ) Probablesignalpeptideprotein UncharacterizedproteinTP_0181 Xyloseisomerase(EC ) transcriptionalregulator,aracfamily oxidoreductase,aldo>ketoreductasefamily anti-sigmaffactorantagonist(spoiiaa-2); HPrkinase>phosphorylase(EC )(EC Aspartyl-tRNA(Asn)amidotransferasesubunitB RiboseABCtransportsystem,periplasmic RiboseABCtransportsystem,ATP-binding RiboseABCtransportsystem,permeaseprotein Biofilm-associatedprotein HydroxymethylpyrimidineABCtransporter,ATPase HydroxymethylpyrimidineABCtransporter, CelldivisionproteinmraZ S-adenosyl-methyltransferasemraW(EC ) Rossmannfoldnucleotide-bindingproteinSmf Ribosome-bindingfactorA sensoryboxhistidinekinase>response PhnBprotein;putativeDNAbinding Probableextracellularnuclease Nitrogenaseironprotein(EC ) Single-strandedDNA-bindingprotein Threonyl-tRNAsynthetase(EC ) ABCtransporter,ATP-bindingprotein ABCtransporter,permeaseprotein Two-componentresponseregulator RiboseABCtransportsystem,periplasmic RiboseABCtransportsystem,permeaseprotein Riboseoperonrepressor Esterase>lipase Transcriptionalregulator,MarRfamily Cellsurfaceprotein Streptococcalhemagglutininprotein Cystathioninebeta-lyase(EC ) Sensoryboxhistidinekinase>response Aspartatecarbamoyltransferase(EC ) Putativesugartransporter Nitrogenasemolybdenum-cofactorsynthesis Nitrogenasevanadium-cofactorsynthesisprotein ABC-typenitrate>sulfonate>bicarbonate ABC-typenitrate>sulfonate>bicarbonate TaurinetransportATP-bindingproteintauB Cysteinesynthase(EC )

167 3 66 Nitrogenasevanadium-cofactorsynthesisprotein Nitrogenase(molybdenum-iron)alphachain(EC Transketolase,C-terminalsection(EC ) Transketolase,N-terminalsection(EC ) L-idonate5-dehydrogenase(EC )> Melibioseoperonregulatoryprotein RiboseABCtransportsystem,ATP-binding RiboseABCtransportsystem,permeaseprotein Methyl-acceptingchemotaxisprotein dtdp-rhamnosyltransferaserfbf(ec ) O-antigenpolymerase Mannosyltransferase(EC ) UDP-glucose4-epimerase(EC ) MaltoseO-acetyltransferase(EC ) O-antigenflippaseWzx Beta-1,3-glucosyltransferase UDP-glucose4-epimerase(EC ) dtdp-4-dehydrorhamnose3,5-epimerase(ec Exopolysaccharideproductionprotein capsularpolysaccharidebiosynthesisprotein Glycosyltransferase Beta-1,3-glucosyltransferase Beta-1,3-glucosyltransferase oxidoreductaseofaldo>ketoreductasefamily, 3-oxoacyl-acyl-carrier-protein]synthase, 3-oxoacyl-acyl-carrier-protein]reductase 3-oxoacyl-ACP]reductase 3-oxoacyl-acyl-carrier-protein]synthase, 3-oxoacyl-acyl-carrier-protein]synthase, RelatedtoF420H2-dehydrogenase,betasubunit dtdp-glucose4,6-dehydratase(ec ) Cytosine>purine>uracil>thiamine>allantoin PutativeDNAprimase>helicase Tyrosine-proteinkinasewzc(EC ) PolysialicacidtransportproteinkpsD SugarABCtransporter,periplasmic Maltose>maltodextrinABCtransporter,permease Maltose>maltodextrinABCtransporter,permease DNA-bindingtranscriptionalregulator Kef-typeK+transportsystems(NAD-binding HITfamilyprotein NAD(P)-dependentglyceraldehyde3-phosphate Phosphoglyceratekinase(EC ) Inososedehydratase(EC ) Epi-inositolhydrolase 5-keto-2-deoxygluconokinaseB(EC ) SugarABCtransporter,periplasmic RiboseABCtransportsystem,ATP-binding RiboseABCtransportsystem,permeaseprotein

168 3 67 IolIprotein Branched-chainaminoacidABCtransporter, High-affinitybranched-chainaminoacid Branched-chainaminoacidtransportsystem Branched-chainaminoacidtransportATP-binding Branched-chainaminoacidtransportATP-binding Branched-chainaminoacidtransportATP-binding Branched-chainaminoacidtransportATP-binding Branched-chainaminoacidtransportATP-binding High-affinitybranched-chainaminoacid Branched-chainaminoacidtransportsystem Branched-chainaminoacidABCtransporter, diguanylatecyclase(ggdefdomain)withpas>pac Membranespanningprotein NitrogenregulatoryproteinP-II Ornithinedecarboxylase(EC )> UPF0118membraneproteinBB_0006 methyl-acceptingchemotaxisprotein methyl-acceptingchemotaxisprotein OligopeptidetransportATP-bindingproteinoppF OligopeptidetransportATP-bindingproteinoppD Oligopeptidetransportsystempermeaseprotein archaealatpase,fusedtoc-terminalduf234 AnaerobicdimethylsulfoxidereductasechainA O-acetylhomoserinesulfhydrylase(EC ) Putativeamino-acidtransporterperiplasmic MethionineABCtransporterATP-bindingprotein D-galactose-bindingperiplasmicprotein GalactosidetransportATP-bindingproteinmglA Galactosidetransportsystempermeaseprotein CircadianinputkinaseA Two-componentsystemsensorprotein Ureacarboxylase-relatedABCtransporter, NitrogenregulatoryproteinP-II NitrogenregulatoryproteinP-II Ureacarboxylase(EC ) sensoryboxhistidinekinase>response Acylcarrierprotein BchE>P-methylasefamilyprotein Xaa-Proaminopeptidase(EC ) Uncharacterizedproteininarchaea TVG protein Cellsurfaceprotein Antiholin-likeproteinLrgA lrga-associatedmembraneproteinlrgb MultiplesugarABCtransporter, MultiplesugarABCtransporter, MultiplesugarABCtransporter, MaltoseoperontranscriptionalrepressorMalR,

169 3 68 OmpAfamilyprotein 3-oxoacyl-(acyl-carrier-protein)synthase Carbondioxideconcentratingmechanismprotein Propanediolutilizationpolyhedralbodyprotein Propanediolutilizationpolyhedralbodyprotein classiialdolase>adducindomainprotein L-fuculokinase(EC ) RiboseABCtransportsystem,periplasmic RiboseABCtransportsystem,ATP-binding RiboseABCtransportsystem,permeaseprotein Redox-sensitivetranscriptionalregulator D-tagatose3-epimerase(EC ) Putativeoxidoreductase IolIprotein MultiplesugarABCtransporter, MultiplesugarABCtransporter, Probablehemagglutinin>hemolysin-related putativednaprimase>helicase Regulatorofpolyketidesynthaseexpression Hydantoinracemase(EC ) Catalyzesthecleavageof FlagellinproteinflaA Two-componentsystemsensorprotein CTP:Inositol-1-phosphatecytidylyltransferase CapsularpolysaccharidesynthesisenzymecpsI, Innermembraneprotein Innermembraneprotein putativebifunctionalpolymerase Alpha-1,4-N-acetylgalactosaminetransferase UDP-glucosedehydrogenase(EC ) Bll3360protein Beta-1,3-glucosyltransferase Phospholipid-lipopolysaccharideABC MoxR-likeATPases Benzoyl-CoAreductasesubunitBadG(EC FormatedehydrogenasechainD(EC ) FormatedehydrogenaseH(EC ) Tropomodulin1 DNA-damage-inducibleproteinJ,putative Smallribosomalsubunit16SrRNA anti-sigmaffactorantagonist(spoiiaa-2); PhageshockproteinA FOG:CheY-likereceiver Transcriptionalregulator,luxRfamily, Glycerol-3-phosphateABCtransporter, Methyl-acceptingchemotaxisprotein TypeIIIrestriction-modificationenzyme ATPasefamilyprotein RadicalSAMdomainprotein

170 3 69 Mlr4706protein Sensorytransductionhistidinekinase Antibioticresistanceprotein GMPsynthaseglutamine-hydrolyzing](EC Nitrogenase(iron-iron)betachain(EC Nitrogenase(iron-iron)deltachain(EC Nitrogenase(iron-iron)alphachain(EC NitrogenregulatoryproteinP-II NitrogenregulatoryproteinP-II Nitrogenaseironprotein(EC ) L-threonine3-O-phosphatedecarboxylase(EC NAD-dependentproteindeacetylaseofSIR2 SAPDNA-bindingdomain-containingprotein Transcriptionalregulator,DeoRfamily Ribose5-phosphateisomeraseB(EC ) Tributyrinesterase RiboseABCtransportsystem,permeaseprotein Oxidoreductase,shortchain PutativeROK-familytranscriptionalregulator Nitroreductase Propionatecatabolismoperonregulatoryprotein Phosphopantothenoylcysteinesynthetase(EC ENDO-TYPE6-AMINOHEXANOATEOLIGOMERHYDROLASE HcptranscriptionalregulatorHcpR(Crp>Fnr Sensorproteinofzincsigma-54-dependent Type4fimbriaeexpressionregulatoryprotein Long-chain-fatty-acid--CoAligase(EC ) Peptidoglycan-bindingLysM Signaltransductionhistidinekinase Integrase TnpY TPRrepeatprecursor CatabolitecontrolproteinA RiboseABCtransportsystem,ATP-binding RiboseABCtransportsystem,permeaseprotein RiboseABCtransportsystem,permeaseprotein RiboseABCtransporter,periplasmic Mll7147protein RiboseABCtransportsystem,periplasmic RiboseABCtransportsystem,ATP-binding RiboseABCtransportsystem,permeaseprotein Uroporphyrinogen-IIIdecarboxylase RiboseABCtransportsystem,permeaseprotein RiboseABCtransporter,periplasmic RiboseABCtransportsystem,ATP-binding Putativeexportedproteinprecursor Sodium-dependentphosphatetransporter Xylulosekinase(EC ) Hemerythrin-likeproteinMJ0747

171 3 70 Putativelipoprotein Outermembranelipoproteinomp16precursor Sensoryboxhistidinekinase Two-componentresponseregulator LSUribosomalproteinL32p Transcriptionalregulator,GntRfamily Dihydroxy-aciddehydratase(EC ) Nitroreductase methyl-acceptingchemotaxisprotein EnergyconservinghydrogenaseEhb Hydrolase(HADsuperfamily) Iron-sulfurclusterregulatorIscR Cystathioninebeta-lyase(EC ) Transcriptionalregulator,Cro>CIfamily Phospholipid-lipopolysaccharideABC GalactosideO-acetyltransferase(EC ) Carbamoyl-phosphatesynthaselargechain(EC Taurinetransportersubstrate-bindingprotein ABC-typenitrate>sulfonate>bicarbonate TaurinetransportsystempermeaseproteintauC TaurinetransportATP-bindingproteintauB UncharacterizedproteinRv1507c>MT1555 Glycosyltransferase Beta-1,3-galactosyltransferase> CDP-ribitol:poly(ribitolphosphate)ribitol Glycosyltransferasedomainprotein membraneprotein,putative Choline-phosphatecytidylyltransferase CholinepermeaseLicB Beta-1,3-glucosyltransferase Acetyltransferase,CYSE>LACA>LPXA>NODLfamily O-antigenpolymerase O-antigenpolymerase Glycosyltransferase(EC ) Succinoglycanbiosynthesisprotein UDP-glucose4-epimerase(EC ) Glycosyltransferase,group1familyprotein Colanicacidbiosynthesisglycosyltransferase Putativetelluriumresistanceprotein Two-componentresponseregulator-likeprotein serine>threoninekinase serine>threoninekinase Leaprotein-soybean Probabletwo-componentsensor,nearpolyamine sensoryboxhistidinekinase>response cellwallsurfaceanchorfamilyprotein twitchingmotilityproteinpilh Transcriptionalregulator ATP-dependenthelicaseHrpB

172 3 71 putativetransposase Cellsurfaceprotein InterPro:FibronectintypeIIIdomain TonB-dependentreceptor 5'-nucleotidase(EC ) Neopullulanase(EC ) GlutathioneS-transferasedomainprotein Cystathioninegamma-lyase(EC ) ABCtransporter,substrate-bindingprotein ABCtransporter,substrate-bindingprotein Catalyzesthecleavageof TetRfamilytranscriptionalregulatorprobably GlutamatesynthaseNADPH]smallchain(EC RegulatoryproteinrecX Sensoryboxhistidinekinase>response FOG:CheY-likereceiver Helix-turn-helixprotein,CopG Transcriptionalactivatorofmaltoseregulon, Corrinoidmethyltransferaseprotein Transposase,mutatorfamily Sensoryboxhistidinekinase>response Responseregulator>phosphatase Na+drivenmultidrugeffluxpump Transcriptionalregulator,MarRfamily Flagellarhook-lengthcontrolproteinfliK MembranemucinMUC17 N-acyl-D-amino-aciddeacylasefamilyprotein Transcriptionalregulator,DeoRfamily ProteininvolvedincatabolismofexternalDNA DHHfamily>DHHA1domainprotein Lactoylglutathionelyase(EC ) Chloridechannelprotein Mg-protoporphyrinIXmonomethylesteroxidative OligoendopeptidaseF CorecomponentFbpofpredictedfolateECF ATPasecomponentofgeneralenergizingmodule Transmembranecomponentofgeneralenergizing bacterialseryl-trnasynthetaserelated NADbindingoxidoreductase 2-hydroxy-6-oxohepta-2,4-dienoatehydrolase Transcriptionalregulator,TetRfamily TypeIrestriction-modificationsystem, TypeIrestriction-modificationsystem, putativetwo-componentsystemsensorhistidine Adenosylcobinamide-phosphate Nitrogenasemolybdenum-cofactorsynthesis Nitrogenaseironprotein(EC ) RibonucleasePproteincomponent(EC ) DNA-bindingdomainofModE

173 3 72 D-tagatose3-epimerase(EC ) Formiminotetrahydrofolatecyclodeaminase(EC Lead,cadmium,zincandmercurytransporting Acetylornithine aminoacidabctransporter,atp-binding aminoacidabctransporter,aminoacid-binding AminoacidABCtransporter,permeaseprotein AminoacidABCtransporter,permeaseprotein Methylcobalamin:coenzymeMmethyltransferase, Beta-phosphoglucomutase(EC ) Maltosephosphorylase(EC )>Trehalose MultiplesugarABCtransporter, SOS-responsetranscriptionalrepressors Fe-Soxidoreductase Regulatorofpolyketidesynthaseexpression VrlP SuperfamilyIIDNA>RNAhelicases,SNF2family ATP-dependentRNAhelicase VrlK VrlJ ATPase transposase,mutatorfamily FOG:CheY-likereceiver Sensoryboxhistidinekinase>response MutTdomainprotein-like AbortiveinfectionproteinAbiGII abortiveinfectionproteinabigi Uroporphyrinogen-IIIdecarboxylase LysRfamilyregulatoryproteinCidR Ferricsiderophoretransportsystem, MolybdenumtransportATP-bindingproteinmodC ABC-TYPEIRON(III)TRANSPORTSYSTEM Iron(III)dicitratetransportsystempermease IronABCtransporter,solute-bindingprotein Methlytransferase,UbiE>COQ5family Ni2+-bindingGTPaseinvolvedinregulationof ABCtransporter,ATP-bindingprotein Spermidine>putrescine-bindingprotein Glucitoloperonrepressor Transketolase,C-terminalsection(EC ) Maltose>maltodextrinABCtransporter,substrate MultiplesugarABCtransporter, Glycerolkinase(EC ) Serine>threonineproteinkinase Beta-1,3-glucosyltransferase Adenylatekinase(EC ) MultiplesugarABCtransporter, SN-glycerol-3-phosphatetransportsystem MultiplesugarABCtransporter,

174 3 73 Ribose5-phosphateisomeraseB(EC ) Helix-turn-helixmotif Xylulosekinase(EC ) MultiplesugarABCtransporter, N-Acetyl-D-glucosamineABCtransportsystem, L-fucosemutarotase RiboseABCtransportsystem,permeaseprotein RiboseABCtransportsystem,ATP-binding RiboseABCtransportsystem,periplasmic Glucokinase(EC ) PossibleD-erythrulose4-phosphate Galactitol-1-phosphate5-dehydrogenase(EC Ribulose-phosphate3-epimerase(EC ) Mo>Fe-nitrogenase-specifictranscriptional ATPase Thiolperoxidase,Tpx-type(EC ) Predictedmolybdate-responsiveregulatorYvgK HuntingtoninteractingproteinHYPE Probablemultipleantibioticresistanceprotein Purinenucleotidesynthesisrepressor RiboseABCtransportsystem,periplasmic RiboseABCtransportsystem,ATP-binding RiboseABCtransportsystem,permeaseprotein DNA-damage-inducibleproteinJ Metaltransporter,ZIPfamily L-rhamnosemutarotase RiboseABCtransportsystem,ATP-binding RiboseABCtransportsystem,permeaseprotein RiboseABCtransportsystem,permeaseprotein Transcriptionalregulator,Cro>CIfamily XyloserepressorXylR(ROKfamily) RiboseABCtransportsystem,periplasmic RiboseABCtransportsystem,ATP-binding Sorbitoldehydrogenase(EC ) L-xylulose>3-keto-L-gulonatekinase(EC L-xylulose5-phosphate3-epimerase(EC ) Ribosomallargesubunitpseudouridinesynthase RiboseABCtransportsystem,periplasmic RiboseABCtransportsystem,permeaseprotein RiboseABCtransportsystem,ATP-binding Leaderpeptidase(Prepilinpeptidase)(EC glycosylhydrolase Hydrolase(HADsuperfamily) L-2-haloalkanoicaciddehalogenase Putativehigh-affinityironpermease Periplasmicproteinp19involvedin probableintegralmembraneproteincj1660 SimilartoABCtransporter:egYBJZ_ECOLI Putativemembraneprotein

175 3 74 PutativeABCtransportsystemATP-binding Putativepheromoneprecursorlipoprotein Signaltransductionhistidinekinase SpermidinePutrescinetransportATP-binding LSUribosomalproteinL4p(L1e) SSUribosomalproteinS14p(S29e) Malatedehydrogenase(EC ) Cellsurfaceprotein Flagellarhook-lengthcontrolproteinfliK ZincABCtransporter,periplasmic-binding Oxidoreductase,aldo>ketoreductasefamily Cellsurfaceprotein transcriptionalregulator,gntrfamily 2-hydroxymuconicsemialdehydehydrolase(EC 4-oxalocrotonatedecarboxylase(EC ) Acetaldehydedehydrogenase(acetylating) 4-hydroxy-2-oxovaleratealdolase(EC ) 2-polyprenylphenolhydroxylaseandrelated Catechol2,3-dioxygenase(EC ) Probableextracellularnuclease Predictedmembraneprotein Majorheadprotein(LateproteinGp8) Putativeanti-terminatorregulatoryprotein membraneprotein HDfamilyhydrolase,diverged Adenylatecyclase(EC ) gnl TPRrepeat UncharacterizedproteinTM_0929 putativeabctransporter,periplasmic MultiplesugarABCtransporter, ProbableABCtransporterpermeaseprotein Transcriptionalrepressorofthearabinose Alpha-mannosidase(EC ) DNA-bindingresponseregulatorDegU Flavodoxin HeavymetaltranslocatingP-typeATPase 2-dehydropantoate2-reductase(EC ) Responseregulator Cytochromec-typebiogenesisproteinCcs1>ResB Multiantimicrobialextrusionprotein Metallo-beta-lactamasesuperfamilyhydrolase HTHtranscriptionalregulatorTetRfamily Diaminopimelateepimerasehomolog Transporter Histidineammonia-lyase(EC ) GlycinebetainetransporterOpuD Histidineammonia-lyase(EC ) MethylaspartatemutaseSchain(EC )

176 3 75 METHYLASPARTATEMUTASE(EC ) PutativeglutamatemutasesubumitE Methylaspartateammonia-lyase(EC ) DNAfor3-methylaspartateammonia-lyase, Transcriptionalregulator,GntRfamily> TAP1protein MutatormutTprotein sensoryboxhistidinekinase>response HDdomainprotein Mll1436protein Single-strandedDNA-bindingprotein Dihydrolipoamideacetyltransferasecomponent RiboseABCtransportsystem,permeaseprotein D-xylosetransportATP-bindingproteinxylG RiboseABCtransportsystem,periplasmic Galactose>methylgalactosideABCtransport SerinephosphataseRsbU,regulatorofsigma SerinephosphataseRsbU,regulatorofsigma Flagellarhook-lengthcontrolproteinfliK Adenylatecyclase(EC ) ATPsynthasedeltachain(EC ) containspfamdomainpf04685:proteinof Transcriptionalregulator,MerRfamily Mll3043protein Alanineracemase(EC ) Pirin Putativecytoplasmicprotein Threoninesynthase(EC ) D-ornithineaminomutaseScomponent Methylaspartatemutase(EC ) alpha-arabinosidesabctransportsystem, alpha-arabinosidesabctransportsystem, alpha-arabinosidesabctransportsystem, Sucrosephosphorylase(EC ) sensoryboxhistidinekinase>response Nucleotidyltransferase(EC ) domainprotein Branched-chainaminoacidtransportATP-binding Branched-chainaminoacidtransportATP-binding Branched-chainaminoacidtransportsystem High-affinitybranched-chainaminoacid Branched-chainaminoacidABCtransporter, 4-hydroxybenzoyl-CoAthioesterasefamilyactive GDP-L-fucosesynthetase(EC ) putativebacteriophageprotein ADAregulatoryprotein ATPase Sensoryboxhistidinekinase>response DNArecombinationandrepairproteinRecF

177 3 76 Ribokinase(EC ) Sodium-dependentphosphatetransporter ATPase Probablepoly(beta-D-mannuronate)O-acetylase RiboseABCtransportsystem,ATP-binding TaurinetransportATP-bindingproteintauB ABCtransporterpermeaseprotein RNApolymerasesigmafactorRpoE Transcriptionalregulator MethionineABCtransporterATP-bindingprotein MultiplesugarABCtransporter, MultiplesugarABCtransporter, Cytotoxictranslationalrepressorof DNA-bindingprotein GGDEF Histoneprotein Ureacarboxylase Putativedeoxyribose-specificABCtransporter, bmpfamilyprotein Isochorismatasefamilyprotein Isochorismatasefamilyprotein Methioninegamma-lyase(EC ) Phosphatebutyryltransferase(EC ) Glutathioneperoxidase(EC ) membraneprotein ChaperoneproteinDnaK ChaperoneproteinDnaJ transcriptionalregulator,marrfamily Nitroreductase FlagellarbiosynthesisproteinflhA HuntingtininteractingproteinE-likeprotein Two-componentsensorhistidinekinase FlaAhomolog-1 DNA-bindingresponseregulator Flagellarhook-lengthcontrolproteinfliK Adenosylhomocysteinase(EC ) Mo>Fe-nitrogenase-specifictranscriptional Nitrogenaseironprotein(EC ) 2-isopropylmalatesynthase(EC ) Xaa-Prodipeptidase(EC ) ABCtransporterATP-bindingprotein- AminoacidABCtransporter,permeaseprotein ABCtransporter,substrate-bindingprotein Cysteinesynthase(EC ) PROBABLEACETYLTRANSFERASE(EC ) putativetransferase PutativeABC-typeamino-acidtransporter Aminoacid(Glutamine)ABCtransporter Amino-acidABCtransporterATP-bindingprotein

178 3 77 CRISPR-associatedproteinCas2 ProbableAAAfamilyATPase Threonyl-tRNAsynthetase(EC ) putativebacteriophageprotein Single-strandedDNA-bindingprotein probablemembraneproteinb2001 putativetransposase Lactate-responsiveregulatorLldRin Methioninebiosynthesisandtransportregulator Transposase,mutatorfamily Cellsurfaceprotein serine>threoninekinase serine>threoninekinase Leaprotein-soybean ReplicativeDNAhelicase(EC ) ProbableAAAfamilyATPase Putativekinaseprotein Putativetelluriumresistanceprotein Probableextracellularnuclease Lysophospholipase(EC );Monoglyceride Aspartyl-tRNAsynthetase(EC ); PTSsystem,mannitol-specificIICcomponent(EC Mannitoloperonactivator,BglGfamily PTSsystem,mannitol-specificIIAcomponent(EC Putativeoxidoreductase putativemembraneprotein LactosetransportsystempermeaseproteinlacF alpha-arabinosidesabctransportsystem, putativetwo-componentsystemsensorkinase Methyl-acceptingchemotaxisprotein Sugar>maltosefermentationstimulationprotein Probableextracellularnuclease Sensoryboxhistidinekinase>response Putativepheromoneprecursorlipoprotein PutativesugarABCtransportsystem, MSM(multiplesugarmetabolism)operon InositoltransportATP-bindingprotein RiboseABCtransportsystem,permeaseprotein RiboseABCtransportsystem,periplasmic Pyruvateformate-lyase(EC ) 2-keto-3-deoxy-D-arabino-heptulosonate-7- Two-componentsystemsensorhistidine BatB Lipase Sensoryboxhistidinekinase>response RiboseABCtransporter,periplasmic Sensoryboxhistidinekinase>response FOG:CheY-likereceiver bmpfamilyprotein

179 3 78 FOG:CheY-likereceiver Sensoryboxhistidinekinase>response Transcriptionregulatorcontains Lactoylglutathionelyase(EC ) RNA-bindingprotein Putativeinnermembraneprotein Dihydroxyacetonekinase,ATP-dependent(EC Heavy-metal-associateddomain(N-terminus)and XyloserepressorXylR(ROKfamily) RiboseABCtransportsystem,ATP-binding RiboseABCtransportsystem,permeaseprotein RiboseABCtransportsystem,periplasmic Largerepetitiveprotein RNDmultidrugeffluxtransporter;Acriflavin ProbableCo>Zn>Cdeffluxsystemmembranefusion RecAprotein RNApolymerasesigmafactorRpoD Molybdate-bindingdomainofModE Possiblemembranetransportprotein Transcriptionalregulator,GntRfamily PutativegntRfamilyregulatoryprotein PeroxidestressregulatorPerR,FURfamily PutativedeoxyribonucleaseYcfH DipeptidetransportATP-bindingproteindppF DipeptidetransportATP-bindingproteindppD Dipeptidetransportsystempermeaseprotein Dipeptidetransportsystempermeaseprotein Methyltransferase(EC ) Ubiquinone>menaquinonebiosynthesis ZincABCtransporter,innermembranepermease ZincABCtransporter,ATP-bindingproteinZnuC CationABCtransporter,periplasmicbinding Putativehemolysin TonB-dependentreceptor T. primitia str. ZAS-2 O-acyltransferase,putative probablepoly(beta-d-mannuronate)o-acetylase(alginatebiosynthesisproteinalgi) lipopolysaccharidebiosynthesis,putative NAD-dependentepimerase-dehydratase dtdp-rhamnosyltransferaserfbg glycosyltransferase,group capsularpolysaccharidebiosynthesisprotein membraneprotein,putative membraneprotein,putative transporter glycosyltransferase glycosyltransferasesugar-bindingregioncontainingdxdmotif O-unitflippase,putative putativepyruvate-formatelyase

180 3 79 tetratricopeptidetpr_ tetratricopeptiderepeatdomainprotein lipoprotein,putative probableextracellularnuclease methyl-acceptingchemotaxisprotein anti-anti-sigmafactor phageshockproteina,pspa PotDprotein spermidine-putrescineimportatp-bindingproteinpota PotBprotein binding-protein-dependenttransportsystemsinnermembranecomponent putativephosphodiesterase lipoprotein,putative lipoprotein,putative lipoprotein,putative serine-threonine-proteinkinasepkn transcriptionalregulator,deorfamily sorbosereductasesou D-xylulosereductase(Xylitoldehydrogenase)(XDH) dihydropteridinereductase phosphopantothenoylcysteinesynthetase-decarboxylase peptidasefamilyt domainprotein EF-handcalcium-bindingdomain-containingprotein signaltransductionhistidinekinase,nitrogenspecific,ntrb,putative radicalsamdomainprotein,putative ABCtransportercomponentA taurinetransportsystempermeaseproteintauc nitratetransportatp-bindingproteinnrtc multi-sensorhybridhistidinekinase sulfatasedomainprotein methyltransferasedomainfamily ClpX,ATPaseregulatorysubunit,putative lipopolysaccharidebiosynthesisprotein,putative glycosyltransferase,group WfbF methyltransferasefkbm methyltransferasetype methyltransferasetype membraneprotein,putative methyltransferasefkbmfamily glyoxalasefamilyprotein riboseimportatp-bindingproteinrbsa nitrogenaseironprotein adenosine transcriptionalregulator,lacifamily,putative ribosetransportsystempermeaseproteinrbsc riboseimportatp-bindingproteinrbsa multi-sensorhybridhistidinekinase

181 3 80 putativesignalingprotein aldo-ketoreductase VanZfamilyprotein virulenceassociatedproteinc bifunctionalacetyltransferase-isomerase(wxcm) methyltransferasetype methyltransferasetype putativeacyltransferase,putative -deoxy-d-xylulose- pyrophosphataseppax lipopolysaccharidebiosynthesisprotein putativeglycosyltransferase membraneprotein,putative capsularpolysaccharidebiosynthesisprotein lipopolysaccharidebiosynthesis,putative VIpolysaccharidebiosynthesisproteinVipA-tviB alpha-d-quinacalpha- UDP-glucose undecaprenyl-phosphategalactosephosphotransferase polysaccharidebiosynthesisproteincapd ATPase polysaccharidebiosynthesis-exportprotein DHHsuperfamilyprotein,subfamily histidinetriad glyceraldehyde- phosphoglyceratekinase(pgk) Myo-inositolcatabolismproteinIolE probablemalonicsemialdehydeoxidativedecarboxylase myo-inositolcatabolismprotein xyloseisomerasedomainproteintimbarrel D-ribose-bindingperiplasmicprotein catabolitecontrolprotein N-carbamyl-L-cysteineamidohydrolase binding-protein-dependenttransportsystemsinnermembranecomponent binding-protein-dependenttransportsystemsinnermembranecomponent taurineimportatp-bindingproteintaub extracellularligand-bindingreceptor inner-membranetranslocator high-affinitybranchedchainaminoacidabctransporter,permeaseprotein highaffinitybranched-chainaminoacidabctransporter,atp-bindingprotein high-affinitybranched-chainaminoacidtransportatp-bindingproteinlivf(liv-iproteinf) nitroreductase branched-chainaminoacidtransportatp-bindingproteinlivg highaffinitybranched-chainaminoacidabctransporter,atp-bindingprotein inner-membranetranslocator,putative inner-membranetranslocator,putative receptorfamilyligandbindingregion fattyacyl-coahydrolase,mediumchain(thioesteraseb) ggdefdomainprotein

182 3 81 D-serineammonia-lyase(dsdA) phosphoenolpyruvate-proteinphosphotransferase(ptsp) phosphocarrierproteinhpr(histidine-containingprotein) lichenan-specificphosphotransferaseenzymeiiacomponent(ptssystemlichenan-specificeiiacomponent)(eiia-lic)(eiii-lic) lichenan-specificphosphotransferaseenzymeiibcomponent(ptssystemlichenan-specificeiibcomponent)(eiib-lic) PTSsystem,IIccomponent beta-glucosidase(gentiobiase)(cellobiase)(beta-d-glucosideglucohydrolase)(amygdalase) anaerobicdimethylsulfoxidereductasechaina(dmsoreductase) putativexaa-proaminopeptidase acyl-coenzymea: uroporphyrinogendecarboxylase,putative oligopeptidetransportatp-bindingproteinappf dipeptidetransportatp-bindingproteindppd oligopeptidetransportsystempermeaseproteinappc binding-protein-dependenttransportsystemsinnermembranecomponent extracellularsolute-bindingprotein,family methyl-acceptingchemotaxisprotein methyl-acceptingchemotaxisprotein ribonucleasepproteincomponent(rnpa) NAD-dependentdeacetylase(RegulatoryproteinSIR sirohydrochlorincobaltochelatase TonB-dependentreceptor,putative cobalaminbiosynthesisproteincbid(cbid) CbiG precorrin- threonine-phosphatedecarboxylase(cobd) cobalt-precorrin- HmuUprotein ferricenterobactintransportatp-bindingproteinfepc cobyricacidsynthasecobq(cobq) nitrogenfixationproteinnifh-nife oxidoreductase-nitrogenase,component bifunctionaladenosylcobalaminbiosynthesisproteincobp AcrBfamilymembranetransportprotein,putative TonB-dependentreceptorplugdomainprotein effluxtransporter,rndfamily,mfpsubunit outermembraneeffluxprotein lipolyticenzyme,gdsldomain listeria-bacteroidesrepeatdomain(list_bact_rpt)family ficfamilyprotein theglugmotifdomainprotein heavymetaltransport-detoxificationprotein trapdicarboxylatetransporter,dctmsubunit tripartiteatp-independentperiplasmictransporter,dctqcomponent,putative transcriptionalregulator,gntrfamily oxidoreductase virulence-associatedprotein,putative oligoendopeptidase,pepf-m MutT-nudixfamilyprotein,putative

183 3 82 ferrousirontransportproteinb cystathioninegamma-lyase(gamma-cystathionase)(probasin-relatedantigen)(prb-ra) RRF glycosyltransferasefamily OmpAfamilyprotein transcriptionalregulator,lacifamily sugarkinase,fggyfamily ironabctransportersubstrate-bindingprotein spermidine-putrescineabctransporteratp-bindingsubunit inositol- aspartyl-glutamyl-trna(asn-gln)amidotransferasesubunitb(asp-glu-adtsubunitb),putative ABCtransporterpeptide-bindingprotein,putative glutathionetransportsystempermeaseproteingsid oligopeptidetransportatp-bindingproteinoppd oligopeptidetransportatp-bindingproteinappf radicalsamdomainprotein radicalsamdomainprotein radicalsamdomainprotein anti-anti-sigmafactor PAS innermembraneabctransporterpermeaseproteinyddr oligopeptidetransportatp-bindingproteinoppd oligopeptidetransportatp-bindingproteinappf dipeptide-bindingprotein creatininase aldo-ketoreductase -methylthioadenosine-s-adenosylhomocysteinedeaminase(mta-sahdeaminase),putative aminoacidabctransporter,periplasmicaminoacid-bindingprotein,putative glutaminetransportatp-bindingproteinglnq galactosidetransportsystempermeaseproteinmglc mateeffluxfamilyprotein RRF nitroreductase outermembraneautotransporterbarreldomain,putative uroporphyrinogendecarboxylase RNA-bindingprotein multiphosphoryltransferprotein ABCtransportercomponentA cellsurfaceprotein glyoxalasedomain-containingprotein ATPaseassociatedwithvariouscellularactivities,AAA_ smallgtp-bindingprotein typeisite-specificdeoxyribonuclease typeirestriction-modificationsystemssubunit typeirestrictionmodificationsystemmsubunit thermostablecarboxypeptidase D-methioninetransportsystempermeaseproteinMetI methionineimportatp-bindingproteinmetn D-methionine-bindinglipoproteinMetQ

184 3 83 cystathioninegamma-synthase(cgs)(o-succinylhomoserine(thiol)-lyase) lipoprotein,putative pyridoxamine zinctransporter,zipfamily sensorytransductionhistidinekinase GH trapdicarboxylatetransporter,dctmsubunit filamentoushaemagglutininfamilyoutermembraneprotein,putative surfaceantigenbspa,putative transposase acetyltransferase,gnatfamily RNApolymerasebeta-subunit sugar-phospahtenucleotidyltransferase DNArepairproteinRadC AAAATPase transcriptionalregulator,putative proteinofunknownfunction DNApolymeraseIII,alphasubunit plasmidpartitionprotein,putative multi-sensorhybridhistidinekinase oligopeptidetransportatp-bindingproteinappf oligopeptidetransportatp-bindingproteinappd probablepeptideabctransporterpermeaseproteiny glutathionetransportsystempermeaseproteingsid bacterialextracellularsolute-bindingprotein,family pyrrolidone-carboxylatepeptidase( probablel-aspartatedehydrogenase peptidasem leucinerichrepeatdomainprotein glutaminetransportatp-bindingproteinglnq glutamineabctransporter,permease-substrate-bindingprotein polaraminoacidabctransporter,innermembranesubunit phospho- amidohydrolasefamilyprotein domainofunknownfunction,putative ggdef-eal-pas-pac-domaincontainingprotein lipoprotein,putative transposase,is transposase,mutatorfamily transcriptionalregulator,badm-rrf putativenadhdehydrogenase-nad(p)hnitroreductase lipoprotein,putative ThlR,HTHtranscriptionalregulatorTetR-AcrRfamily thiolperoxidase majormembraneimmunogen sensoryboxhistidinekinase-responseregulator membraneprotein,gpr CrcBprotein,putative outermembraneprotein

185 3 84 hypotheticalcytosolicprotein crispr-associatedrampprotein,csm crispr-associatedprotein,family,putative plasmidstabilityprotein,putative TonBfamilyC-domainprotein proteinofunknownfunction Igdomainprotein,group lipoprotein,putative lipoprotein,putative methyltransferasetype cobalaminsynthesisprotein,p ABCtransporter,ATP-bindingprotein transcriptionalregulator,marrfamily addictionmoduleantidoteprotein,higafamily,putative methioninegamma-lyase(l-methioninase) transcriptionalregulator lipoprotein,putative aminoglycosideadenyltransferase putativenadhdehydrogenase-nad(p)hnitroreductase mateeffluxfamilyprotein membraneprotein,bmpfamily sensorproteingacs hybridsensorykinase adenylatecyclase sensorproteingacs sensoryboxhistidinekinase-responseregulator adenylate-guanylatecyclasecatalyticdomainprotein acetyl-coacarboxylase,carboxyltransferase,alphasubunit(acca) acetyl-coacarboxylase,carboxyltransferase,betasubunit(accd) acetyl-coacarboxylase,biotincarboxylcarrierprotein membraneprotein,putative membraneprotein,putative aminobenzoyl-glutamateutilizationproteinb citratelyase,alphasubunit(citf) citrate(pro- meioticexpressionup-regulatedprotein vonwillebrandfactor,typea,putative lipoprotein,putative aminobenzoyl-glutamateutilizationproteinb methylaspartatemutase,ssubunit(mama) MutLprotein methylaspartatemutase,esubunit methylaspartateammonia-lyase putativemethyltransferasecmuc tripartiteatp-independentperiplasmictransporter,dctqcomponent,putative APendonuclease,family SalB catechol2,3-dioxygenase binding-protein-dependenttransportsystemsinnermembranecomponent

186 3 85 ABCtransporter,ATP-bindingprotein para-nitrobenzylesterase(pnbcarboxy-esterase)(intracellularesteraseb)(pnbce) para-nitrobenzylesterase(pnbcarboxy-esterase)(intracellularesteraseb)(pnbce) NAD+synthetase probableextracellularnuclease transcriptionalregulator ABC-typeaminoacidtransport-signaltransductionsystem,periplasmiccomponent-domain HAD-superfamilyhydrolase,subfamilyIIB proteinusha MutLprotein D-ornithineaminomutaseScomponent pyridoxal- dihydrodipicolinatereductase alanineracemase,n-domainprotein ribosomallargesubunitpseudouridinesynthased(rlud) transglutaminasedomainprotein ABC-typemultidrug-protein-lipidtransportsystem,ATPasecomponent prepilin-typen-cleavage-methylationdomainprotein peptidase,a putativeigdomainfamily Coffamilyprotein probableextracellularnuclease,putative addictionmoduleantidoteprotein,higafamily(higa) PilTproteindomainprotein,putative tetratricopeptiderepeatdomainprotein tetratricopeptiderepeatdomainprotein HAD-superfamilyhydrolase macrolideexportatp-binding-permeaseproteinmacb effluxtransporter,rndfamily,mfpsubunit helix-turn-helixdomainprotein transposase,mutatorfamily ISPsy lipoprotein,putative bacterialextracellularsolute-bindingprotein,putative sugarabctransporterpermease high-affinityironpermease kdamembraneantigen(pathogen-specificmembraneantigen) membraneprotein,putative ABCtransporterpermeaseprotein permeasedomainprotein macrolideexportatp-binding-permeaseproteinmacb kdalipoprotein parallelbeta-helixrepeat outermembraneautotransporterbarreldomain spermidine-putrescineabctransporteratp-bindingsubunit addictionmoduletoxin,rele-stbefamily,putative ribosomalproteins phosphoesterasephpdomainprotein,putative ABCtransporterpermeaseprotein

187 3 86 transcriptionalregulator,putative transcriptionalregulator,aracfamilyprotein riboseimportatp-bindingproteinrbsa ribosetransportsystempermeaseproteinrbsc probablesugarabctransporter,substrate-bindingprotein,putative hypotheticalabctransporterperiplasmicsolute-bindingprotein ABCtransporter,permeaseprotein tobedomainfamily proteinadeh_ membraneprotein,putative vanillate:corrinoidproteinmethyltransferase transporter,majorfacilitatorfamily veratrol:corrinoidproteinmetyltransferase,putative transcriptionalregulator,lysrfamily,putative cobalaminsynthesisprotein,p putativeaminoacidtransporter ferredoxin,putative mateeffluxfamilyprotein NADPH-dependentfmnreductase,putative carboxylesterasefamily para-nitrobenzylesterase(pnbcarboxy-esterase)(intracellularesteraseb)(pnbce) dihydroxy-aciddehydratase(dad) oxidoreductaseydhf sugarabctransporterpermease beta-galactosidase GntRdomainprotein glycosyltransferasefamily stageiisporulationproteine(spoiie) stageiisporulationproteine(spoiie) signaltransductionhistidinekinase methyl-acceptingchemotaxisprotein macrolideexportatp-binding-permeaseproteinmacb ABCtransporter,permeaseprotein heminimportatp-bindingproteinhmuv HmuUprotein trapdicarboxylatetransporter-dctpsubunit,putative plasmidstabilizationsystemantitoxinprotein ABCtransporter,ATP-bindingprotein transcriptionalregulator,crp-fnrfamily sulfitereductase,subunita(asra) sulfitereductase,subunitc(asrc) responseregulatorreceivermodulatedmetaldependentphosphohydrolase methyl-acceptingchemotaxisprotein bacterialextracellularsolute-bindingprotein,putative transcriptionalregulator,luxrfamilyprotein hybridsensorykinase proteintyrosine-serinephosphatase lipoprotein,putative tetratricopeptiderepeatdomainprotein

188 3 87 bacterialextracellularsolute-bindingproteins,family putatived-aminoacylase X-Prodipeptidase oligopeptidetransportatp-bindingproteinappf oligopeptidetransportatp-bindingproteinappd ATPase putativesucrosephosphorylase(sucroseglucosyltransferase) hypotheticalcytosolicprotein proteinofunknownfunction UvrD-REPhelicase,putative AAAATPase,putative RNApolymerasesigmafactorRpoD(Sigma-A)(Sigma- XylR signaltransductionhistidinekinase,putative surfaceantigenbspa PAS flagellarfilament NADPH-dependentfmnreductase,putative ABCtransporterpeptide-bindingprotein glutathioneabctransporter,permeaseprotein(gsic) glutathionetransportsystempermeaseproteingsid oligopeptidetransportatp-bindingproteinoppd oligopeptidetransportatp-bindingproteinappf cobalaminsynthesisprotein-p veratrol:corrinoidproteinmetyltransferase transcriptionalregulator,merrfamily putativeribokinase hypotheticalabctransporterperiplasmicsolute-bindingprotein tobedomainfamily binding-protein-dependenttransportsystemsinnermembranecomponent HAD-superfamilyhydrolase,subfamilyIA,variant hydrolase PHPdomain,putative sensoryboxsensorhistidinekinase-responseregulator sensorproteingacs HADsuperfamilyhydrolase glycosyltransferasegroup membraneprotein,putative sugarabctransporter,putative transcriptionalregulator,luxrfamilyprotein caudovirusproheadprotease,putative endonucleaseandmethylasellagi peptidase,s DNA-directedDNApolymerase UvrD-REPhelicase,putative binding-protein-dependenttransportsystemsinnermembranecomponent,putative transposase,mutatorfamily mutt-nudixfamilyprotein multi-sensorhybridhistidinekinase

189 3 88 multi-sensorhybridhistidinekinase,putative DNApolymeraseIV(dinB) MutT-nudixfamilyprotein(mutT) lipoprotein,putative phagecapsidfamily,putative phageportalprotein phageterminase,largesubunit,pbsxfamily transcriptionalregulator,alpafamily fibroin OmpA theglugmotifdomainprotein nucleotidyltransferasesubstratebindingproteinlike oligopeptidetransportatp-bindingproteinappf dipeptidetransportatp-bindingproteindppd oligopeptidetransportsystempermeaseproteinappb lipoprotein,putative radicalsamdomainprotein radicalsamdomainprotein lipoprotein,putative radicalsamdomainprotein lipoprotein,putative transposase,mutatorfamily ISSod transposase,mutatorfamily transposase,mutatorfamily transcriptionalregulator,tetrfamily iron-sulfurcluster-bindingprotein SMI probablenadph:quinoneoxidoreductase caenorhabditisproteinofunknownfunction phagetranscriptionalregulator,alpa phage glycoproteing transposase,mutatorfamily putativetranscriptionalregulator proteinofunknownfunction halomucin multi-sensorhybridhistidinekinase transcriptionalregulator,luxrfamilyprotein bacterialextracellularsolute-bindingprotein,putative methylphosphotriester-dnaalkyltransferase membraneprotein nlpalipoprotein D-methioninetransportsystempermeaseproteinMetI lipoprotein,putative MutT-nudixfamilyprotein(mutT) dtdp- paratosesynthase glycosyltransferase,group

190 3 89 proteincontainingnucleotide-diphospho-sugartransferasedomain UDP-glucuronicaciddecarboxylase transposase,mutatorfamily glycosyltransferasegroup innermembraneprotein innermembraneprotein two-componentsystemhybridsensorhistidinekinase-responseregulatorprotein endoglucanasem(egm)(endo- oligopeptidetransportatp-bindingproteinappf dipeptidetransportatp-bindingproteindppd glutathionetransportsystempermeaseproteingsid binding-protein-dependenttransportsystemsinnermembranecomponent putativedipeptide-bindingabctransporterprotein putativeabctransportercomponent adenylate-guanylatecyclasecatalyticdomainprotein probableextracellularnuclease,putative lipoprotein,putative domainprotein lipoprotein,putative prolipoproteindiacylglyceryltransferase membraneprotein,putative lipoprotein,putative YheOdomainprotein glycosylhydrolase,family TPRrepeat glycosyltransferase,family O-antigenexportsystempermeaseproteinRfbA teichoicacidsexportatp-bindingproteintagh(teichoicacid-transportingatpase) citratelyaseligasec-domainprotein citratelyaseligasec-domainprotein citratelyaseligasec-domainprotein citratelyaseligasec-domainprotein coiled-coildomaincontaining transcriptionalregulator,xrefamily exopolygalacturonatelyase,putative MDR-typeABCtransporter ABC-typemultidrug-protein-lipidtransportsystem,ATPasecomponent ATPasefamilyassociatedwithvariouscellularactivities(AAA)protein transcriptionalregulator,marrfamily RRF effluxtransporter,rndfamily,mfpsubunit acriflavinresistanceprotein,putative outermembraneeffluxprotein lipoprotein,putative excisionase-xis,dna-binding transposase,putative putativetypeirestrictionenzymehindviipmprotein(m.hindviip) divergentaaaatpase restrictionmodificationsystemdnaspecificitydomain

191 3 90 putativetypeirestrictionenzymehindviiprprotein HDfamilyhydrolase,diverged NADPH-dependentfmnreductase tetratricopeptiderepeatdomainprotein tetratricopeptiderepeatdomainprotein fibronectintypeiiidomainprotein probableextracellularnuclease,putative fibronectintypeiiidomainprotein regulatoryprotein,arsr positiveregulatorofsigmae,rsec-mucc,putative mateeffluxfamilyprotein lipoprotein,putative transcriptionalregulator hypotheticalcytosolicprotein lipoprotein,putative chondroitinsulfate-heparinutilizationregulationprotein carboxymethylenebutenolidase -isopropylmalatesynthase(alpha-isopropylmalatesynthase)(alpha-ipmsynthetase) glutamineamidotransferaseclass-i transcriptionalregulator,nifasubfamily,fisfamily cobalaminsynthesisproteincobw transporter,majorfacilitatorfamily methioninesynthase MmoS methioninesynthase domainprotein PadR aldo-ketoreductase aldo-ketoreductase lipoprotein,putative antibioticbiosynthesismonooxygenasedomainprotein phospholipase-carboxylesterasefamily methyl-acceptingchemotaxisprotein phosphoenolpyruvate-proteinphosphotransferase(ptsp) '-Nucleotidasedomainprotein,putative phosphoglyceratemutasefamilyprotein YcsE transcriptionalantiterminator,bglg cyclicnucleotide-bindingprotein ptssystemmannitol-specificeiicbacomponent(eiicba-mtl)(eii-mtl) HTHdomainfamily phosphocarrierproteinhpr(histidine-containingprotein) ptssystemmannitol-specificeiicbacomponent(eiicba-mtl)(eii-mtl) Mcp- binding-protein-dependenttransportsystemsinnermembranecomponent binding-protein-dependenttransportsystemsinnermembranecomponent PfkBdomainprotein uroporphyrinogendecarboxylase(uro-d)superfamily methyltransferasetype

192 3 91 Xaa-Hisdipeptidase oligopeptideabctransportersubstrate-bindingprotein oligopeptideabctransportersubstrate-bindingprotein oligopeptidetransportsystempermeaseproteinappb dipeptidetransportatp-bindingproteindppd oligopeptidetransportatp-bindingproteinappf extracellularsolute-bindingprotein,family -carboxyhexanoate--coaligase(biow) Hptsensorhybridhistidinekinase L-cystinetransportsystempermeaseproteinTcyB probableamino-acidabctransporter,substrate-bindingprotein L-cystineimportATP-bindingproteinTcyN activatorof(r)- molybdopterin-guaninedinucleotidebiosynthesisproteina ironhydrogenase proteinaega phenylacetylcoa formatedehydrogenase,alphasubunit(fdha) proteint RecF-RecN-SMCNdomain,putative ferredoxin cobalttransportpermease,cbiqfamily,putative cobaltimportatp-bindingproteincbio flagellarmotorswitchproteinflim(flim) lipoprotein,putative PINdomaincontainingprotein PAS-PACsensorhybridhistidinekinase twocomponenttranscriptionalregulator,luxrfamily tetratricopeptidetpr_ lipoprotein,putative lipoprotein,putative lipoprotein,putative probableextracellularnuclease,putative lipoprotein,putative multi-sensorhybridhistidinekinase cyclasefamilyprotein bacterialpre-peptidasec-domainfamily YbaK-ebsCprotein(ybaK) mureinhydrolaseexporter LrgBfamilyprotein rubredoxin,putative withinp.aerophilum nickelimportatp-bindingproteinnike oligopeptide-dipeptideabctransporter,atp-bindingprotein binding-protein-dependenttransportsystemsinnermembranecomponent binding-protein-dependenttransportsystemsinnermembranecomponent peptide-opine-nickelabctransporter ferrichrometransportatp-bindingproteinfhuc transportsystempermeaseprotein

193 3 92 ABC-typeFe FwdEfamilyprotein iron(iii)dicitratetransportatp-bindingproteinfece transportsystempermeaseprotein periplasmicbindingprotein ubiquinone-menaquinonebiosynthesismethyltransferaseubie methyltransferasetype transcriptionalregulator,putative DNApolymeraseIIIsubunitalpha glutathioneimportatp-bindingproteingsia hydrogenase NAD-dependentformatedehydrogenasebetasubunit peroxiredoxinhyr permease RelA-SpoTdomainprotein chaperoneproteindnak(heatshockprotein molecularchaperone,dnajfamily Zn-fingercontainingprotein transcriptionalregulator,lysrfamily aconitasefamily(aconitatehydratase) citratetransportersuperfamily bacterialcapsulesynthesisproteinpga_cap L-cystineimportATP-bindingproteinTcyN ABC-typeaminoacidtransportsystem,permeasecomponent bacterialextracellularsolute-bindingprotein,family antibioticbiosynthesismonooxygenase ABC-typeaminoacidtransportsystem,permeasecomponent ABC-typeaminoacidtransportsystem,permeasecomponent ExpA two-componentsystemsensorkinase peptidase,m OmpAfamilyprotein lipoprotein,putative cobalaminb transcriptionalregulator,aracfamilyprotein transcriptionalregulator,putative sperm-activatingpeptidesfamily virulencegenerepressorrsal PINdomainprotein prevent-host-deathfamilyprotein,putative pirindomainprotein DNApolymerase,betadomainproteinregion hydroxylaminereductase(hcp) transcriptionalregulator lipoprotein,putative lipoprotein,putative hybridsensorykinase Nif-specificregulatoryprotein(nifA) HicB

194 3 93 deoxyribonuclease,tatdfamily heavymetaltranslocatingp-typeatpase heavymetaltranslocatingp-typeatpase nitrogenaseironprotein(nifh) probablepeptideabctransporterpermeaseproteiny glutathioneimportatp-bindingproteingsia high-affinitynickel-transporter carbonmonoxidedehydrogenaseaccessoryproteincooc extracellularsolute-bindingprotein,family putativehelix-turn-helixprotein AsnCfamilytranscriptionalregulator transcriptionalregulator,lysrfamily,putative '-Nucleotidasedomainprotein,putative transporter,majorfacilitatorfamily vanillate:corrinoidproteinmethyltransferase nitroreductase lipoprotein,putative probableextracellularnuclease,putative two-componentresponseregulator ferricuptakeregulator,furfamily nitroreductase polymorphicoutermembraneprotein,putative RNApolymerasesigmafactorRpoD(Sigma- HmuUprotein heminimportatp-bindingproteinhmuv periplasmicbindingprotein TfoXN-domainsuperfamily carboxymuconolactonedecarboxylase transposase,mutatorfamily aspartyl-trnasynthetase(aspartate--trnaligase)(asprs) transportergatedomainprotein transcriptionalregulator lipoprotein,putative oligopeptidetransportatp-bindingproteinoppd oligopeptidetransportatp-bindingproteinappf isochorismatasefamilyprotein transcriptionalregulator,merrfamily carboxymuconolactonedecarboxylase serine-threonine-proteinkinase PINdomainprotein DnaJdomainprotein transposase,mutatorfamily transposasetpna peptidase,s dnamethylase-typeirestriction-modificationsystem ATPaseoftheAAA+class ATPaseoftheAAA+class NADH-ubiquinoneoxidoreductasechain transcriptionalregulator,tetrfamily

195 3 94 Xaa-Proaminopeptidase thiaminesprotein cellulose-binding,familyii polysaccharidebiosynthesis-exportdomainprotein,putative lipopolysaccharidebiosynthesisprotein,putative thiogalactosideacetyltransferase proteincontainingnucleotide-diphospho-sugartransferasedomain putativesugartransferase UDP-glucose epimerase-dehydratase,putative putativelpsbiosynthesisrelatedglycosyltransferase membraneprotein,putative filamentationinducedbycampproteinfic protein,withaweakd-galactaratedehydratase-altronatehydrolasedomain YwbO,putative indigoidinesynthaseafamilyprotein kinase,pfkbfamily domainprotein peptidase,m hemerythrinfamilyprotein mateeffluxfamilyprotein,putative phageinfectionprotein metallo-beta-lactamasesuperfamilyhydrolase transcriptionalregulator,tetrfamily flavodoxin lipoprotein,putative PINdomainprotein lipoprotein,putative amidohydrolase membraneprotein,putative membraneprotein,putative TPRrepeat TPRrepeat TPRrepeat TPRrepeat flagellarbiosynthesisproteinflha exonuclease,putative Cl-channel,voltage-gatedfamilyprotein radicalsamdomainprotein O-methyltransferase acyltransferase peptidases T. azotonutricium str. ZAS-9 responseregulator biotincarboxylase ribosetransportsystempermeaseproteinrbsc riboseimportatp-bindingproteinrbsa L-rhamnose ferricuptakeregulator,furfamily

196 3 95 activatorof PilTproteindomainprotein prevent-host-deathfamilyprotein isochorismatasehydrolase Smfprotein domainprotein transcriptionalregulator,luxrfamilyprotein domainprotein transcriptionalregulator,luxrfamilyprotein sugardehydrogenase,putative APsuperfamily legionellavirregionprotein transposaseis outermembraneautotransporterbarrel,putative probableextracellularnuclease,putative majormembraneimmunogen Zn-dependentalcoholdehydrogenase endo-arabinase helix-turn-helix-domaincontainingprotein,aractype extracellularsolute-bindingprotein,family IstBdomainproteinATP-bindingprotein IstBdomainproteinATP-bindingprotein transcriptionalregulator,lacifamily L-fucoseisomerasedomainprotein probablefructose-bisphosphatealdolase inositol probablemalonicsemialdehydeoxidativedecarboxylase inososedehydratase( adenylate-guanylatecyclase transcriptionalregulator probableinsertionsequencetransposaseprotein,is transposaseis transposase,mutatorfamily tetratricopeptiderepeatdomainprotein glucose- dsbaoxidoreductase,putative tetratricopeptiderepeatdomainprotein pirindomainprotein integrasedomainprotein proteinofunknownfunctionduf DNA-directedDNApolymerase,putative DNApolymeraseIII,alphasubunit mateeffluxfamilyprotein APendonuclease,family sporulationinitiationinhibitorproteinsoj transposaseis TetR-familyregulator adenylate-guanylatecyclasecatalyticdomainprotein transcriptionalregulator,arsrfamily

197 3 96 arabinoseoperonproteinaram cyclicnucleotide-bindingprotein TrksystempotassiumuptakeproteinTrkH transposase,is PINdomainprotein transposase,mutatorfamily,putative TonB-dependentreceptor cationabctransporter,periplasmicbindingprotein,putative zincimportatp-bindingproteinznuc methyl-acceptingchemotaxisprotein cationicoutermembraneprotein transposase,is integrasedomainprotein glutamine-bindingperiplasmicprotein-glutaminetransportsystempermeaseprotein sensorproteingacs ATP-dependenthelicaseHrpB(hrpB) integrasedomainprotein phagecapsidfamily domainprotein CTPpyrophosphohydrolase PemKfamilyprotein transposaseinsiforinsertionsequenceelementis glycosyltransferase glycosyltransferase,group CDP-Glycerol:Poly(glycerophosphate)glycerophosphotransferasefamily innermembraneprotein glycosyltransferase,group CDP-alcoholphosphatidyltransferasefamily nitroreductase NADPH-dependentfmnreductase NAD-dependentepimerase-dehydratase adenylatekinase transposase,is tetratricopeptiderepeatdomainprotein transposaseis ATPaseassociatedwithvariouscellularactivities,AAA_ swimzincfingerdomainprotein ankyrin tetratricopeptiderepeatdomainprotein transcriptionalregulator,tetrfamily,putative PAS ribosetransportsystempermeaseproteinrbsc riboseimportatp-bindingproteinrbsa mateeffluxfamilyprotein radicalsamdomainprotein sensorproteingacs responseregulatorreceiver:metal-dependentphosphohydrolase,hdsubdomain transcriptionalregulator,luxrfamilyprotein

198 3 97 sensorproteingacs outermembraneprotein PAS lipoate-proteinligasea methyl-acceptingchemotaxisprotein(mcp)signalingdomain responseregulatorreceiver:metal-dependentphosphohydrolase,hdsubdomain bacterialextracellularsolute-bindingprotein,putative sugartransportsystem sugarabctransporter,permeaseprotein sugarphosphateisomerases-epimerases xyloseisomerasedomainproteintimbarrel,putative dehydrogenase binding-protein-dependenttransportsystemsinnermembranecomponent L-arabinoseABCtransportpermeaseprotein hexulose- transcriptionalrepressoroftheriboseoperon glyoxalase-bleomycinresistanceprotein-dioxygenase alpha-galactosidase(melibiase) glucose--fructoseoxidoreductase(gfor),putative ribosetransportsystempermeaseproteinrbsc riboseimportatp-bindingproteinrbsa haloaciddehalogenasedomainproteinhydrolase PfkBdomainprotein prolylaminopeptidase L-fucoseisomeraserelatedprotein chitinase proteinofunknownfunction typeiiirestrictionenzyme,ressubunit phageportalprotein,hk site-specificrecombinase,phageintegrasefamilyprotein aldo-ketoreductase oxidoreductase,aldo-ketoreductasefamily integrasedomainprotein majorfacilitatorfamilytransporter,putative transcriptionalregulator,tetrfamilyprotein fkbp-type aryl-alcoholdehydrogenase mateeffluxfamilyprotein aspartyl-trnasynthetase(asps) metallophosphoesterase glycosyltransferases,putative glycosyltransferase,group fibronectintypeiiidomainprotein fibronectintypeiiidomainprotein TPRrepeat,putative lipolyticproteing-d-s-lfamily mucin-desulfatingsulfatase,putative membraneprotein,putative glucanendo-

199 3 98 AlgI glycosyltransferase,involvedincellwallbiogenesis CotHprotein fibronectintypeiiidomainprotein sensorytransductionhistidinekinase heminimportatp-bindingproteinhmuv transportsystempermeaseprotein HmuUprotein periplasmicbindingprotein LcoC -hydroxythreonine- extracellularsolute-bindingprotein,family zincfingerprotein acetyl-coacarboxylase,biotincarboxylcarrierprotein(accb) helix-turn-helixdomainprotein cobalttransportprotein SfsA tetratricopeptidetpr_ cystathioninegamma-synthase(cgs)(o-succinylhomoserine(thiol)-lyase) transcriptionalregulator,badm-rrf extracellularligand-bindingreceptor HmgE high-affinitybranchedchainaminoacidabctransporter,permeaseprotein highaffinitybranched-chainaminoacidabctransporter,atp-bindingprotein abctransporter,hydrophobicaminoaciduptaketransporter(haat)family,atp-bindingprotein polaraminoacidabctransporter,innermembranesubunit polaraminoacidabctransporter,innermembranesubunit glutaminetransportatp-bindingproteinglnq helicase,snf aspartyl-glutamyl-trna(asn-gln)amidotransferasesubunitb(asp-glu-adtsubunitb),putative zinctransporter,zipfamily PAS ROKdomaincontainingprotein ClcA voltage-gatedchloridechannel radicalsamdomainprotein oxidoreductase ferritin OmpAfamilyprotein,putative HmuUprotein TonBfamilyprotein proteinofunknownfunction methyltransferase cobalaminsynthesisprotein,p ABCtransporter,ATP-bindingprotein methylcobamide:commethyltransferasemtba(methylcobamide:commethyltransferaseiiisozymea)(mt nitrogenaseironprotein(nifh) transposase,mutatorfamily acetyltransferase,gnatfamily

200 3 99 RNApolymerasesigma- surfaceantigenbspa novelprotein oxaloacetatedecarboxylase,gammasubunit Rrf cytoplasmicfilamentproteina ferricuptakeregulatoryprotein transcriptionalregulator,aracfamilyprotein ADP-ribosylglycohydrolasesuperfamily bacterialextracellularsolute-bindingprotein binding-protein-dependenttransportsystemsinnermembranecomponent binding-protein-dependenttransportsystemsinnermembranecomponent regulatoryproteinlaci glutamatesynthasenadph]smallchain(nadph-gogat) methioninegamma-lyase(l-methioninase) mureinhydrolaseexportregulator LrgAfamilyprotein,putative responseregulatorreceiver:metal-dependentphosphohydrolase,hdsubdomain Hptsensorhybridhistidinekinase APendonuclease,family bacterialextracellularsolute-bindingprotein,putative transcriptionalregulator membraneprotein,putative twocomponenttranscriptionalregulator,luxrfamily nitroreductase transcriptionalregulator,marrfamily,putative peroxiredoxinhyr TPRDomaincontainingprotein translocase beta-lactamase transcriptionalregulator,cro-cifamily,putative transcriptionalregulator NifK oxidoreductase-nitrogenase,component nitrogenaseironprotein aliphaticsulfonatesimportatp-bindingproteinssub radicalsamdomainprotein phosphoadenosinephosphosulfatereductase GH TPRrepeatprotein trapdicarboxylatetransporter,dctmsubunit cellwallsurfaceanchorfamilyprotein,putative oxidoreductasedomainprotein glucose- transcriptionalregulator L-ribulokinase(araB) periplasmicbindingprotein-lacitranscriptionalregulator,putative phosphateacetyltransferase(phosphotransacetylase) RNApolymerasesigmafactorRpoD(rpoD)

201 3 100 isochorismatasehydrolase ferrousirontransportproteina signaltransductionhistidine-proteinkinasebara extracellularsolute-bindingprotein,family L-arabinosetransportsystempermeaseproteinAraP antibioticbiosynthesismonooxygenasedomainprotein PAS two-componentsystemsensory-regulatoryprotein mateeffluxfamilyprotein,putative transcriptionalregulator,tetrfamily uroporphyrinogendecarboxylase(uro-d)superfamily transcriptionalregulator transcriptionalregulator,arsrfamily DNA-bindingprotein phagemajorcapsidprotein,hk transcriptionalregulator transposaseandinactivatedderivatives membraneprotein,putative integrasedomainprotein macrolideexportatp-binding-permeaseproteinmacb ABCtransporter,permeaseprotein -hexulose- dihydropteridinereductase fggyfamilyofcarbohydratekinases,domainprotein proteinofunknownfunction deathoncuringprotein,putative HDdomainprotein metaldependentphosphohydrolase D-mannonateoxidoreductase(Fructuronatereductase) extracellularsolute-bindingprotein,family binding-protein-dependenttransportsystemsinnermembranecomponent oligopeptidetransportatp-bindingproteinoppd riboseimportatp-bindingproteinrbsa periplasmicsugar-bindingproteins integrasedomainprotein periplasmicbindingproteinsandsugarbindingdomainofthelacifamily,putative extracellularsolute-bindingproteinfamily sugarabctransporterpermease sugarabctransporterpermease methyl-acceptingchemotaxisprotein multi-sensorhybridhistidinekinase sensorytransductionhistidinekinase -oxoacyl-acyl-carrier-protein]reductase( integrasedomainprotein ribose- sugarabctransporter,permeaseprotein extracellularsolute-bindingprotein,family alpha-l-rhamnosidase regulatoryproteinlaci

202 3 101 ROKfamilyprotein extracellularsolute-bindingproteinfamily binding-protein-dependenttransportsystemsinnermembranecomponent binding-protein-dependenttransportsystemsinnermembranecomponent OmpAfamilyprotein polymorphicoutermembraneprotein bacterialextracellularsolute-bindingprotein,putative ABCtransporter,carbohydrateuptaketransporter- binding-protein-dependenttransportsystemsinnermembranecomponent innermembraneabctransporterpermeaseproteinycjp sensoryboxhistidinekinase-responseregulator ribonucleasepproteincomponent(rnpa) PAS ATPase bacterialpre-peptidasec-domainfamily ATPase,AAAfamily,putative ABCsugartransporter,periplasmicligandbindingprotein PAS-PACsensorhybridhistidinekinase peptidase,m aquaporin diguanylatecyclase sensorproteingacs membraneproteincontaining putativesignalingprotein beta-lactamase integralmembraneprotein HipAprotein,putative xyloseisomerasedomainproteintimbarrel rhomboidfamilyprotein transcriptionalregulator,gntrfamily tripartiteatp-independentperiplasmictransporter,dctqcomponent,putative trapdicarboxylatetransporter,dctmsubunit methyl-acceptingchemotaxisprotein polymorphicoutermembraneprotein nitrogenfixationproteinnifh-nife oxidoreductase-nitrogenase,component bifunctionaladenosylcobalaminbiosynthesisproteincobp methyltransferasemtaa-cmua methyltransferasemtaa-cmua transposase,mutatorfamily putativecelldivisionproteaseftshhomolog myo-inositolcatabolismprotein galactosidetransportsystempermeaseproteinmglc two-componenthybridprotein HAD-superfamilyhydrolase,subfamilyIA,variant multiplesugar-bindingtransportsystemmultiplesugar-bindingprotein,putative ribosetransportsystempermeaseproteinrbsc xyloseimportatp-bindingproteinxylg uroporphyrinogendecarboxylase(uro-d)superfamily

203 3 102 ATPase helix-turn-helixdomainprotein lactosetransportsystem leucinerichrepeatdomainprotein binding-protein-dependenttransportsystemsinnermembranecomponent multi-sensorhybridhistidinekinase PINdomainprotein ATPase integrasedomainprotein domainprotein phosphoglycolatephosphatase(pgpase)(pgp),putative transcriptionalregulator,gntrfamily Zn-dependentalcoholdehydrogenase transposase,mutatorfamily transposase,mutatorfamily transposase,mutatorfamily transposaseforinsertionsequenceelementisrm transposaseis transposaseis peptidasem transposase,is PINdomainprotein multi-sensorhybridhistidinekinase putativetranscriptionalregulator multi-sensorhybridhistidinekinase spfh-band transposaseforinsertionsequenceelementisrm transposase,mutatorfamily domainprotein transcriptionalregulator,xrefamily domainprotein phageterminase,smallsubunit,putative glutamineabctransportersubstrate-bindingprotein proteaseprsw(proteaseresponsibleforactivatingsigma-w) extracellularsolute-bindingproteinfamily binding-protein-dependenttransportsystemsinnermembranecomponent responseregulatorreceiverprotein oxidoreductasedomainprotein glucose-inhibiteddivisionproteina radicalsamdomainprotein periplasmicbindingprotein-lacitranscriptionalregulator,putative insertionsequenceputativeatp-bindingprotein(orf binding-protein-dependenttransportsystemsinnermembranecomponent responseregulatorreceiverprotein xyloseisomerasedomainproteintimbarrel uroporphyrinogendecarboxylase PINdomain,putative two-componentresponseregulatoryesn bacterialextracellularsolute-bindingprotein

204 3 103 methyl-acceptingchemotaxisproteindmcb transcriptionalregulator,marrfamily transposase,mutatorfamily putativednajhomologsubfamilybmember metallo-beta-lactamasesuperfamilyprotein glycosidehydrolase,family YbaC transcriptionalregulator,tetrfamilyprotein innermembraneprotein innermembraneprotein oxidoreductasedomainprotein,putative sorbitoldehydrogenase(l-iditol isochorismatasehydrolase proteinofunknownfunction extracellularsolute-bindingproteinfamily binding-protein-dependenttransportsystemsinnermembranecomponent binding-protein-dependenttransportsystemsinnermembranecomponent ribosomalproteins transposase,mutatorfamily flavodoxin proteincontaininganddnajdomain,putative phageshockproteina,pspa anti-anti-sigmafactor chainn,architectureofmammalianfattyacidsynthase Igdomainprotein,group repressor,putative proteinrelatedtomifh-dopdproteinfamily,functioninbacteriaisunknown,putative alpha-d-mannosidase transcriptionalregulator,putative sigma- signaltransductionhistidinekinase,nitrogenspecific,ntrb YkrA radicalsamdomainprotein,putative transcriptionalregulator,deorfamily,putative crispr-associatedproteincas crispr-associatedrampprotein,cmr hydrolaseofthehdsuperfamily crispr-associatedrampprotein,cmr crispr-associatedprotein,cmr crispr-associatedrampprotein,cmr crispr-associatedprotein,family,putative transposaseis kdalipoprotein permeasedomainprotein membraneprotein,putative kdamembraneantigen(pathogen-specificmembraneantigen) high-affinityironpermease thymidylatesynthase(thya) ABCtransporter,ATP-bindingprotein,MsbAfamily

205 3 104 amidohydrolase transcriptionalregulator,aracfamilyprotein ribulose-phosphate sorbitoldehydrogenase(l-iditol putatived-erythrulose- ROKdomaincontainingprotein putativeoxidoreductase YurM sugarabctransporterpermease bacterialextracellularsolute-bindingprotein,putative L-threonine sensoryboxhistidinekinase-responseregulator integrasedomainprotein fumaratereductase-succinatedehydrogenaseflavoproteindomainprotein FMN-bindingdomainprotein binding-protein-dependenttransportsystemsinnermembranecomponent nitratetransportatp-bindingproteinnrtd dgqhrdomain,putative proteinofunknownfunction transcriptionalregulator,nifasubfamily,fisfamily cobaltimportatp-bindingproteincbio transcriptionalregulator,trmb sugartransportersugarbindingprotein ABC-typesugartransportsystems,permeasecomponents sugarabctransporterpermease glutaminase multi-sensorhybridhistidinekinase proteinr methyl-acceptingchemotaxisprotein spermidine-putrescineimportatp-bindingproteinpota SoxRprotein phospho- deoxyribose-phosphatealdolase(deoc) VanZlikeprotein domainofunknownfunction dtdp- CDP-abequosesynthase WbyH putativeparatosetransferase glycosyltransferase,putative EpsT glycosyltransferase,group membraneprotein,putative putativerhamnosyltransferase EpsD GDP-mannose responseregulator transcriptionalregulator,trmb alpha-n-arabinofuranosidase(arabinosidase)

206 3 105 periplasmicbindingprotein binding-protein-dependenttransportsystemsinnermembranecomponent binding-protein-dependenttransportsysteminnermembranecomponent oligopeptidetransportatp-bindingproteinappf anti-anti-sigmafactor hemolysin- peptidasem oxidoreductasedomainprotein transcriptionalregulator,tetrfamilyprotein long-chain-fatty-acid--coaligase,putative HipAN-domain,putative domainprotein NAD-dependentepimerase-dehydratase cellsurfaceprotein O-acetylhomoserine(thiol)-lyase(O-acetylhomoserinesulfhydrylase)(OAHsulfhydrylase)(Homocysteinesynthase) cystathioninegamma-synthase(cgs)(o-succinylhomoserine(thiol)-lyase) D-methionine-bindinglipoproteinMetQ methionineimportatp-bindingproteinmetn D-methioninetransportsystempermeaseproteinMetI hydrogenase- -isopropylmalatesynthase(alpha-isopropylmalatesynthase)(alpha-ipmsynthetase) domainprotein periplasmicmolybdate-bindingprotein-domain oxidoreductasedomainprotein,putative transcriptionalregulator,aracfamily,putative probableoxidoreductase,putative sulfatase,putative APendonuclease,family Hptsensorhybridhistidinekinase responseregulator,narl-family membraneprotein,putative bacterialextracellularsolute-bindingprotein,putative transcriptionalregulator,luxrfamilyprotein transcriptionalregulator,luxrfamily listeria-bacteroidesrepeatdomain(list_bact_rpt)family Igdomainproteingroup outermembraneautotransporterbarreldomain,putative outermembraneautotransporterbarrel,putative cellsurfaceprotein,putative cellsurfaceprotein,putative elongationfactortu ribosomalproteinl lactoylglutathionelyase sensorproteingacs transposaseinsiforinsertionsequenceelementis ABCtransporter,permeaseprotein ABCtransporter,ATP-bindingprotein L-serinedehydratase,iron-sulfur-dependent,betasubunit(sdaAB) PAS

207 3 106 membraneprotein,putative mateeffluxfamilyprotein phosphonatesimportatp-bindingproteinphnc ROK,putative cobalaminb putativetranscriptionalregulator methylphosphotriester-dnaalkyltransferase methylated-dna--protein-cysteinemethyltransferase sorbosereductasesou mannosyltransferase polymorphicoutermembraneprotein,putative transcriptionalregulator,aracfamily,putative oligopeptidetransportatp-bindingproteinappd oligopeptidetransportsystempermeaseproteinappc oligopeptidetransportatp-bindingproteinappf alpha-galactosidase(melibiase) trimethylaminecorrinoidprotein oxidoreductase proteinofunknownfunction two-componentsensorhistidinekinasewithresponseregulatorreceiverdomain D-methionineABCtransporter,periplasmicD-methionine-bindingprotein(metQ) purinenucleosidephosphorylase(deod) peptidaseu sensorproteingacs responseregulatorreceiversensorhybridhistidinekinase PASfoldfamily multi-sensorhybridhistidinekinase YbaK-ebsCprotein(ybaK) transporter,majorfacilitatorfamily nachtnucleosidetriphosphatase tetratricopeptiderepeatdomainprotein glutathioneperoxidase bacterialextracellularsolute-bindingprotein,putative binding-protein-dependenttransportsystemsinnermembranecomponent,putative sugarabctransporterpermease penicillin-bindingprotein insertionsequenceputativeatp-bindingprotein(orf ribose dihydroxyacetonekinase,lsubunit PTS-dependentdihydroxyacetonekinase,dihydroxyacetone-bindingsubunitDhaK glucose Zn-dependentalcoholdehydrogenase invasionproteinibea invasionproteinibea bacterialextracellularsolute-bindingprotein,putative ABCtransporter,permeaseprotein thiaminepyrophosphateenzyme,centraldomainfamily iron-containingalcoholdehydrogenase,putative methyl-acceptingchemotaxisprotein

208 3 107 tetratricopeptiderepeatdomainprotein domainprotein PilTproteindomainprotein,putative

209 3 108

210 3 109 Fig.3A 1:PFAMdomainsandconservedfunctionalresiduesandsequencemotifsin Treponemaprimitiastr.ZAS 1andZAS 2meta cleavagepathwayproteins.the primarystructurespanofmeta cleavagepathwayproteinsisrepresentedbyblack lines,andpfamdomainswithinthemeta cleavagepathwayproteinsare representedbygreyrectangles.symbolsrepresentingconservedfunctional residuesarecenteredoverthelocationofthefunctionalresidues.activesite residuesarerepresentedbyblackinvertedtriangles,metal bindingresiduesare representedbywhitediamonds,residuescontributingtoproteinstructureare representedbylightgreysquares,andsubstrate bindingresiduesarerepresented bygreycircles.conservedsequencemotifsarerepresentedbywhitebars.

211 3 110 Fig.3A 2:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 ferredoxin likepeptide(pf00111,step1 ).Bayesianproteinphylogeneticanalysis (183treesfrom73,000generations;PSRF=1.001;averagestandarddeviationof splitfrequencies= )isbasedon85unambiguouslyalignedaminoacid positionsofa98aminoacid longprotein.bayesvalues(whengreaterthan50)are reportedabovethenodes.phylipprotparsmaximumparsimonysupportafter analysisof1000bootstraps(whengreaterthan50%)isreportedbelownodes. Shadedcircles( )indicatenodessupportedbybothmaximumparsimonyand Fitchdistancematrixmethods.Opencircles( )indicatenodessupportedbyone ofthosemethods.scalebarindicatesdistancedepictedas0.1aminoacidchanges peralignmentposition.

212 3 111 Fig.3A 3:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 24 hydroxy 2 oxopentanoatealdolase(pf00682andpf07836,step4).bayesian proteinphylogeneticanalysis(375treesfrom150,000generations;psrf=1.000; averagestandarddeviationofsplitfrequencies= )isbasedon331 unambiguouslyalignedaminoacidpositionsofa340aminoacid longprotein. Bayesvalues(whengreaterthan50)arereportedabovethenodes.Phylip PROTPARSmaximumparsimonysupportafteranalysisof1000bootstraps(when greaterthan50%)isreportedbelownodes.shadedcircles( )indicatenodes supportedbybothmaximumparsimonyandfitchdistancematrixmethods.open circles( )indicatenodessupportedbyoneofthosemethods.scalebarindicates distancedepictedas0.1aminoacidchangesperalignmentposition.

213 3 112 Fig.3A 4:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 acetaldehydedehydrogenase(pf01118andpf09290,step5).bayesianprotein phylogeneticanalysis(279treesfrom111,500generations;psrf=1.004;average standarddeviationofsplitfrequencies= )isbasedon259unambiguously alignedaminoacidpositionsofa291aminoacid longprotein.bayesvalues(when greaterthan50)arereportedabovethenodes.phylipprotparsmaximum

214 3 113 parsimonysupportafteranalysisof1000bootstraps(whengreaterthan50%)is reportedbelownodes.shadedcircles( )indicatenodessupportedbyboth maximumparsimonyandfitchdistancematrixmethods.opencircles( ) indicatenodessupportedbyoneofthosemethods.scalebarindicatesdistance depictedas0.1aminoacidchangesperalignmentposition.

215 3 114 Fig.3A 5:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 catechol2,3 dioxygenasen terminaldomain(pf00903).bayesianprotein phylogeneticanalysis(220treesfrom88,000generations;psrf=0.999;average standarddeviationofsplitfrequencies= )isbasedon56unambiguously alignedaminoacidpositionsofa64aminoacid longdomain.bayesvalues(when greaterthan50)arereportedabovethenodes.phylipprotparsmaximum parsimonysupportafteranalysisof1000bootstraps(whengreaterthan50%)is reportedbelownodes.shadedcircles( )indicatenodessupportedbyboth maximumparsimonyandfitchdistancematrixmethods.opencircles( ) indicatenodessupportedbyoneofthosemethods.scalebarindicatesdistance depictedas0.1aminoacidchangesperalignmentposition.

216 3 115 Fig.3A 6:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 catechol2,3 dioxygenasen terminaldomain(pf00903)withextra domainregion. Bayesianproteinphylogeneticanalysis(600treesfrom240,000generations;PSRF =1.000;averagestandarddeviationofsplitfrequencies= )isbasedon67 unambiguouslyalignedaminoacidpositionsofa68aminoacid longregion.bayes values(whengreaterthan50)arereportedabovethenodes.phylipprotpars maximumparsimonysupportafteranalysisof1000bootstraps(whengreaterthan 50%)isreportedbelownodes.Shadedcircles( )indicatenodessupportedby bothmaximumparsimonyandfitchdistancematrixmethods.opencircles( ) indicatenodessupportedbyoneofthosemethods.scalebarindicatesdistance depictedas0.1aminoacidchangesperalignmentposition.

217 3 116 Fig.3A 7:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 catechol2,3 doxygenasen terminaldomain(pf00903)withintra domainregion. Bayesianproteinphylogeneticanalysis(375treesfrom150,000generations;PSRF =1.000;averagestandarddeviationofsplitfrequencies= )isbasedon88 unambiguouslyalignedaminoacidpositionsofa139aminoacid longregion.bayes values(whengreaterthan50)arereportedabovethenodes.phylipprotpars maximumparsimonysupportafteranalysisof1000bootstraps(whengreaterthan 50%)isreportedbelownodes.Shadedcircles( )indicatenodessupportedby bothmaximumparsimonyandfitchdistancematrixmethods.opencircles( )

218 3 117 indicatenodessupportedbyoneofthosemethods.scalebarindicatesdistance depictedas0.1aminoacidchangesperalignmentposition.

219 3 118 Fig.3A 8:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 catechol2,3 dioxygenasec terminaldomain(pf00903).bayesianprotein phylogeneticanalysis(350treesfrom140,000generations;psrf=1.000;average standarddeviationofsplitfrequencies= )isbasedon115unambiguously alignedaminoacidpositionsofa116aminoacid longdomain.bayesvalues(when greaterthan50)arereportedabovethenodes.phylipprotparsmaximum parsimonysupportafteranalysisof1000bootstraps(whengreaterthan50%)is reportedbelownodes.shadedcircles( )indicatenodessupportedbyboth maximumparsimonyandfitchdistancematrixmethods.opencircles( ) indicatenodessupportedbyoneofthosemethods.scalebarindicatesdistance depictedas0.1aminoacidchangesperalignmentposition.

220 3 119 Fig.3A 9:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 catechol2,3 dioxygenasec terminaldomain(pf00903)withextra domainregion. Bayesianproteinphylogeneticanalysis(450treesfrom180,000generations;PSRF =1.000;averagestandarddeviationofsplitfrequencies= )isbasedon155 unambiguouslyalignedaminoacidpositionsofa158aminoacid longregion.bayes values(whengreaterthan50)arereportedabovethenodes.phylipprotpars maximumparsimonysupportafteranalysisof1000bootstraps(whengreaterthan 50%)isreportedbelownodes.Shadedcircles( )indicatenodessupportedby bothmaximumparsimonyandfitchdistancematrixmethods.opencircles( ) indicatenodessupportedbyoneofthosemethods.scalebarindicatesdistance depictedas0.1aminoacidchangesperalignmentposition.

221 3 120 Fig.3A 10:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 catechol2,3 dioxygenasec terminaldomain(pf00903)withintra domainregion. Bayesianproteinphylogeneticanalysis(500treesfrom200,000generations;PSRF =1.000;averagestandarddeviationofsplitfrequencies= )isbasedon140 unambiguouslyalignedaminoacidpositionsofa141aminoacid longregion.bayes values(whengreaterthan50)arereportedabovethenodes.phylipprotpars maximumparsimonysupportafteranalysisof1000bootstraps(whengreaterthan 50%)isreportedbelownodes.Shadedcircles( )indicatenodessupportedby bothmaximumparsimonyandfitchdistancematrixmethods.opencircles( ) indicatenodessupportedbyoneofthosemethods.scalebarindicatesdistance depictedas0.1aminoacidchangesperalignmentposition.

222 3 121 Fig.3A 11:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 24 hydroxy 2 oxopentanoatealdolasehmgl likedomain(pf00682).bayesianprotein phylogeneticanalysis(313treesfrom125,000generations;psrf=1.000;average standarddeviationofsplitfrequencies= )isbasedon243unambiguously alignedaminoacidpositionsofa232aminoacid longdomain.bayesvalues(when greaterthan50)arereportedabovethenodes.phylipprotparsmaximum parsimonysupportafteranalysisof1000bootstraps(whengreaterthan50%)is reportedbelownodes.shadedcircles( )indicatenodessupportedbyboth maximumparsimonyandfitchdistancematrixmethods.opencircles( ) indicatenodessupportedbyoneofthosemethods.scalebarindicatesdistance depictedas0.1aminoacidchangesperalignmentposition.

223 3 122 Fig.3A 12:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 24 hydroxy 2 oxopentanoatealdolasedmpg likecommunicationdomain(pf07836). Bayesianproteinphylogeneticanalysis(480treesfrom192,000generations;PSRF =1.000;averagestandarddeviationofsplitfrequencies= )isbasedon65 unambiguouslyalignedaminoacidpositionsofa66aminoacid longdomain.bayes values(whengreaterthan50)arereportedabovethenodes.phylipprotpars maximumparsimonysupportafteranalysisof1000bootstraps(whengreaterthan 50%)isreportedbelownodes.Shadedcircles( )indicatenodessupportedby bothmaximumparsimonyandfitchdistancematrixmethods.opencircles( ) indicatenodessupportedbyoneofthosemethods.scalebarindicatesdistance depictedas0.1aminoacidchangesperalignmentposition.

224 3 123 Fig.3A 13:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 acetaldehydedehydrogenase,nadbindingdomain(pf01118).bayesianprotein phylogeneticanalysis(123treesfrom205,000generations;psrf=1.000;average standarddeviationofsplitfrequencies= )isbasedon110unambiguously alignedaminoacidpositionsofa110aminoacid longdomain.bayesvalues(when greaterthan50)arereportedabovethenodes.phylipprotparsmaximum parsimonysupportafteranalysisof1000bootstraps(whengreaterthan50%)is reportedbelownodes.shadedcircles( )indicatenodessupportedbyboth maximumparsimonyandfitchdistancematrixmethods.opencircles( ) indicatenodessupportedbyoneofthosemethods.scalebarindicatesdistance depictedas0.1aminoacidchangesperalignmentposition.

225 3 124 Fig.3A 14:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2 acetaldehydedehydrogenasedimerisationdomain(pf09290).bayesianprotein phylogeneticanalysis(145treesfrom58,000generations;psrf=0.999;average standarddeviationofsplitfrequencies= )isbasedon135unambiguously alignedaminoacidpositionsofa137aminoacid longdomain.bayesvalues(when greaterthan50)arereportedabovethenodes.phylipprotparsmaximum parsimonysupportafteranalysisof1000bootstraps(whengreaterthan50%)is reportedbelownodes.shadedcircles( )indicatenodessupportedbyboth maximumparsimonyandfitchdistancematrixmethods.opencircles( ) indicatenodessupportedbyoneofthosemethods.scalebarindicatesdistance depictedas0.1aminoacidchangesperalignmentposition.

226 3 125 T. primitia str. ZAS GntR * * * * * * TAAAATCAATTTTTCAGAATAGTTCATAGACAAAAGGAGAAACTTATTATATG T. primitia str. ZAS * * * * * ATAGTA AAATTCTTCATGTTCGTTTTGTATGTAATGAGGGGTGAATG GntR * * * * * * * * * * * * TAAAGATTGAAATCTAAGGTAGGGCCTCGATTACAGAGAAAGATTTATAGGATAAATATG GCTTCTGATTGAAATATACAAGGTGTATTGCAATAAAGGGGTGATTGGTAATG 1000 bp Fig.3A 15:TranscriptionalregulatoryelementswithintheTreponemaprimitiastr. ZAS 1andZAS 2meta cleavagepathwaygeneneighborhoods.promoterregions areblackrectanglesandarrowsindicatedirectionoftranscription.nucleotide basesofthepromoterregionareshownwithconservedpromoterelementsinblue andtranscriptionalstartsitesinred.stemloopstructureisrepresentedbya hairpin likesymbol.ferredoxin likepeptide(step1,red),catechol2,3 dioxygenase (step1,orange),2 hydroxymuconicsemialdehydehydrolase(step2,lightblue),2 oxopent 4 enoatehydratase(step3,darkgreen),4 hydroxy 2 oxopentanoate aldolase(step4,darkblue),acetaldehydedehydrogenase(step5,purple),putative regulatorygenes(darkgrey),andhypotheticalproteins(lightgrey)aredepicted. Regulatoryfamiliesarenoted.

227 3 126

228 3 127 Fig3A 16:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2metapathwayassociatedGntRfamilytranscriptionalregulator(PF00392).Bayesian proteinphylogeneticanalysis(488treesfrom195,000generations;psrf=1.001; averagestandarddeviationofsplitfrequencies= )isbasedon228 unambiguouslyalignedaminoacidpositionsofa249aminoacid longprotein. Bayesvalues(whengreaterthan50)arereportedabovethenodes.Phylip PROTPARSmaximumparsimonysupportafteranalysisof1000bootstraps(when greaterthan50%)isreportedbelownodes.shadedcircles( )indicatenodes supportedbybothmaximumparsimonyandfitchdistancematrixmethods.open circles( )indicatenodessupportedbyoneofthosemethods.scalebarindicates distancedepictedas0.1aminoacidchangesperalignmentposition.

229 3 128

230 3 129 Fig.3A 17:PhylogeneticpositionofTreponemaprimitiastr.ZAS 1andZAS 2metapathwayassociatedGntRfamilytranscriptionalregulator,Bacterialregulatory proteinsgntrfamilydomain(pf00392).bayesianproteinphylogeneticanalysis (950treesfrom380,000generations;PSRF=1.000;averagestandarddeviationof splitfrequencies= )isbasedon61unambiguouslyalignedaminoacid positionsofa63aminoacid longdomain.bayesvalues(whengreaterthan50)are reportedabovethenodes.phylipprotparsmaximumparsimonysupportafter analysisof1000bootstraps(whengreaterthan50%)isreportedbelownodes. Shadedcircles( )indicatenodessupportedbybothmaximumparsimonyand Fitchdistancematrixmethods.Opencircles( )indicatenodessupportedbyone ofthosemethods.scalebarindicatesdistancedepictedas0.1aminoacidchanges peralignmentposition.

231 3 130 Fig.3A 18:YellowTreponemaprimitiastr.ZAS 1cultures.T.primitiastr.ZAS 1 growingin2yacomediumunderan80%n2/20%co2headspace(leadbetteretal. 1999;Graber&Breznak2004;Graberetal.2004)reformulatedtocontainno dithiothreitol(dtt)orotherreducingagent,noresazurin(aredoxindicator). Maintainingsterileandanoxiccultures,10mMmaltosewasaddedtotheright culture.maintainingsterileandanoxiccultures,10mmmaltose,0.5mmcatechol, androomairtoachieveafinalconcentrationof0.5%o2vol/volwereaddedtothe leftculture.thesecultureswereincubatedat25 Cinahorizontalpositionwithout agitation.after5 7daysofgrowthfluidsintheleftcultureturnedadistinctlemonyellowcolor.Analysisofculturefluidsrevealedthatthecatechol dependentyellow materialhadanabsorbancemaximumat375nmconsistentwiththepresenceofthe expecteddioxygenase mediatedringcleavageproduct,2 hydroxymuconic semialdehyde.uv/visabsorbancespectrawereobtainedusingacarywinuv Spectrophotometer.

232 3-131 Absorbance "2YACo Media" "2YACo Media + ZAS- 1" "2YACo Media + ZAS- 1 + O2" "2YACo Media + ZAS- 1 + Catechol" "2YACo Media + ZAS- 1 + Catechol + O2" "2YACo Media + ZAS- 1 + Catechol + O2 INITIAL" Wavelength (nm) Fig. 3A- 19: Absorbance spectra of Treponema primitia str. ZAS- 1 cultures. T. primitia str. ZAS- 1 was grown in 5mL of anaerobic, 2YACo, liquid media with an 80% N2/20% CO2 headspace in 25mL butyl rubber- stoppered Balch tubes. Where noted in the accompanying legend, cultures were supplemented with 0.5mM catechol and/or sterile air added to achieve 0.5% vol/vol O2 in the headspace. For comparison, an initial spectra of T. primitia str. ZAS- 1 with 0.5mM catechol and 0.5% vol/vol O2 (upon inoculation and 4 days before culture fluid turned yellow) is shown. Media lacked both DTT and resazurin, and no other reducing agents were added. Cultures were incubated at 25 C in a horizontal position and still. Data taken 4 days after inoculation. UV/Vis absorbance spectra data was obtaind with a Cary WinUV Spectrophotometer.

233 3 132 a

234 3 133 b Fig.3A 20:Treponemaprimitiastr.ZAS 1andZAS 2O2/catecholgradientcultures andcontrols.oxygengradienttubesunderaheadspaceofn2/co2/o2(70:10:4)and depictedafter7daysofincubation.gradienttubespreparedasdescribedin MaterialsandMethodsabovebutinsteadofaddingT.primitiastr.ZAS 1andZAS 2 toculturesalongtheverticallengthoftheagarose,t.primitiastr.zas 1andZAS 2 wasaddedhomogenously(a)fromlefttoright:uninoculatedcatechol(10mmplug atbottomoftube)ando2control,andtriplicatesofo2/catecholgradienttubes inoculatedwithoft.primitiastr.zas 2.(b)Fromlefttoright:triplicatesof O2/catecholgradienttubesinoculatedwithofT.primitiastr.ZAS 2asseenin(a) comparedtotriplicatesofo2/catecholgradienttubesinoculatedwithoft.primitia str.zas 1.(formorethoroughanalysesofgrowthofT.primitiastr.ZAS 1in O2/aromaticgradienttubesseeTable3 4.Fig.3 7andAppendixFig.3A 21aandb).

235 3 134 a

236 3 135 b Fig.3A 21:Treponemaprimitiastr.ZAS 1(a)catecholgradientculturesand controlsand(b)watergradientculturesandcontrols.gradienttubesundera headspaceof80%n2/20%co2(unlessnotedotherwise)depictedafter7daysof incubation.gradienttubespreparedasdescribedinmaterialsandmethodsabove. Atthebottomofeachtubethereisaplugof(a)10mMcatecholor(b)water.(a) Fromlefttoright:uninoculated4%vol/volO2andcatecholcontrol;uninoculated catechol onlycontrol;triplicatesoft.primitiastr.zas 1inoculatedingradient tubesinwhicha10mmcatecholplugislocatedatthebottombuttowhichnoo2 wasadded.orange=catechol,white=nocells,red=o2.(b)fromlefttoright: uninoculated4%vol/volo2andwatercontrol;uninoculatedwater onlycontrol; triplicatesoft.primitiastr.zas 1inoculatedingradienttubesinwhicha10mM plugofwaterislocatedatthebottombuttowhichnoo2wasadded.blue=water, white=nocells,red=o2.

237 3 136 a

238 3 137 b

239 3 138 c

240 3 139 d Fig.3A 22:TransmissionelectronmicroscopyimagesofsectionsofZootermopsis nevadensishindgut(a,b,c,andd).thehindgutofamedium sizedzootermopsis nevadensisworkerspecimenwasdissectedoutandimmediatelyfixedin2.5% glutaraldehydein100mmphosphatebufferatph7.0for24hrs.postfixation,the hindgutwaswashedin200mmphosphatebufferandtreatedin1%osmium tetroxidein100mmphosphatebufferat4 Cfor2hrs.Next,thehindgutwaswashed indistilledwaterandstainedwith2%aquaeousuranylacetateat4 Cfor2hrsinthe dark.then,thehindgutsampleunderwentaseriesofstepsfordehydrationthat consistedof30%acetonefor15min,50%acetonefor15min,70%acetonefor 15min,90%acetonefor15min,andthreechangesof100%acetonefor30mineach. Priortoresinembedding,thehindgutsamplewasexposedtopropyleneoxidetwice for15minseach.thenthehindgutsampleunderwentaresininfiltrationregime thatconsistedof2:1mixofpropyleneoxide:resinfor1hr,1:1mixofpropylene oxide:resinfor1hr,1:2mixofpropyleneoxide:resinfor1hr,100%resinovernight, freshresin1hr,andpolymeriseat60 Cfor24hrs.Unlessnotedotherwise,sample