Comparative genomic analysis of nine Sphingobium strains ....

Comparative genomic analysis of nine Sphingobium strains: insights into their evolution and hexachlorocyclohexane (HCH) degradation pathways.

Vermaetal.BMCGenomics2014,15:1014

http://wendang.chazidian.com/1471-2164/15/1014

Comparativegenomicanalysisofnine

Sphingobiumstrains:insightsintotheirevolutionandhexachlorocyclohexane(HCH)degradationpathways

HelianthousVerma1?,RoshanKumar1?,PhoebeOldach1,NaseerSangwan1,JitendraPKhurana2,

JackAGilbert3,4andRupLal1*

*Correspondence:ruplal@http://wendang.chazidian.com?Equalcontributors1RoomNo.115,MolecularBiologyLaboratory,DepartmentofZoology,

UniversityofDelhi,Delhi110007,India

Fulllistofauthorinformationisavailableattheendofthe

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Comparative genomic analysis of nine Sphingobium strains: insights into their evolution and hexachlorocyclohexane (HCH) degradation pathways.

Vermaetal.BMCGenomics2014,15:1014

http://wendang.chazidian.com/1471-2164/15/1014

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Background

ThefamilySphingomonadaceaehasbeensubdividedintofivegenera:Sphingomonas,Sphingobium,Novosphingobium,SphingopyxisandSphingosinicella[1,2].Todate,thege-nomesofnearly40sphingomonadshavebeensequenced,whichhasrevealedthegeneticbasisforthedegradationofabroadrangeofpolycyclicaromatichydrocarbons(PAH)andpolysaccharides[3].However,Sphingobiumspp.areofparticularinterestduetotheirabilitytodegradehexa-chlorocylcohexane(HCH).ThemajorityofHCHisomers(i.e.α,β,δ,ε)areformedduringtheproductionoftheinsecticidelindane(γ-HCH),andhavebeenactivepol-lutantssincethe1950s[4].Amongalltheseisomers,onlyγ-HCHhasinsecticidalproperties.Purificationofγ-HCH(10-12%)fromthemixtureleadstotheforma-tionofHCHmuck(88-90%ofthetotalHCHmixture)havingmainlyα(60-70%),β(5-12%),δ(6-10%),andε(3-4%)isomers[5].ThishasbeengenerallydiscardedintheopenbythesideofindustrialunitscreatingalargenumberofHCHdumpsitesbetweenthe1960stothe1980saroundtheworld[6].Sphingobiumspp.areoftenenrichedinHCHdumpsitesandhavebeenshowntoacquireandmaintaingenesassociatedwithHCHdegradation[7-10].

ThedegradationpotentialforHCHisomershasbeenattributedtothelinpathway(Figure1),whichhasbeenstudiedindetailinbothSphingobiumjaponicumUT26S[11,12]andSphingobiumindicumB90A[6].ThelinpathwayissubdividedintoanupperdegradationpathwayconsistingofHCHdehydrochlorinase(LinA),haloalkanedehalogenase(LinB)anddehydrogenase(LinC/LinX),andalowerdegradationpathwayconsistingofreductivedechlorinase(LinD),ringcleavageoxygenase(LinE),maleylacetatereductase(LinF),anacyl-CoAtransferase(LinG,H),athiolase(LinJ)andtranscriptionfactors(LinIandLinR).TheLinK,LinL,LinMandLinNi.e.,a

permease,

Comparative genomic analysis of nine Sphingobium strains: insights into their evolution and hexachlorocyclohexane (HCH) degradation pathways.

Vermaetal.BMCGenomics2014,15:1014

http://wendang.chazidian.com/1471-2164/15/1014

ATPase,periplasmicproteinandalipoproteinrespectively,togetherconstituteaputativeABC-typetransporter[6].Thereisevidencethatindicateshighlevelsofpoly-morphismsintheaminoacidsequencesofthelinAandlinBgenes.Furtherstudieshaverevealedthatthesedif-ferencescontributetotheefficacyofHCHdegradationandsubstratespecificity[13].WhilethereareseveralstrainsofsphingomonadsisolatedfromHCHdumpsiteswithdemonstrateddifferencesinHCHdegradationabil-ity[8,14],genome-widecomparativeanalysestobetterunderstandthelinpathway,localizationoflingenesinthegenomeandmethodsofrecruitmenthavenotyetbeenundertaken.

InordertounderstandtheevolutionoftheHCH-degradationpathway,thedraftgenomesofsixSphingobiumspp.isolatedfromHCHdumpsitesandthecompletege-nomesofthreepreviously-sequenced,well-studiedstrainswereanalysed.Here,wecharacterizethegeneticdivergencebetweenthesestrainsinreferencetothelincatabolicsystemandauxiliarycharacteristicsassociatedwithbioremediationpotential.WealsopresentevidenceforpossibleplasmidandIS6100basedhorizontalgenetransfer(HGT)asthemethodforspreadofthelinsystemgenesamongsphingomonads.Additionally,variationinthelingenesequencesisamatteroffurtherinvestigationforimproveddegradationabilityofthesestrains.

Resultsanddiscussion

GenomicfeaturesofSphingobiumstrains

ThegenomesizesforthesixnewlysequencedSphingobiumspp.averaged4.83Mbpandrangedfrom4.08to5.89Mbp,withS.chinhatenseIP26maintainingthelargestgenome(Table1).ThesesizesareconsistentwithexistingSphingo-biumspp.[15].Thevariationingenomesizecanbepar-tiallycorrelatedtothepresenceofgenomicislands;IP26maintainedthelargestgenomeandthehighestgenomicislandcontent,whileLL03hadtheleast(Table1).ThispotentiallyreflectsdifferentialdegreesofHGTandmobilegeneticelementacquisitionamongthesestrains.UT26S,B90A,IP26andHDIPO4allsharedhighsequenceidentity(>97%),whereasLL03,P25,RL3andDS20haveaccumu-latedmoresequencevariationdespitebeingundersimilarselectionpressures(90-70%)(Figure2).

CRISPRelementswereonlyfoundassociatedwithS.baderiLL03(22spacers)http://wendang.chazidian.comctosutensDS20(5spacers).Thesespacersequencesareknownbac-terialdefensemechanismsagainstviralandplasmidchallengesacquiredfromforeigninvadingDNA,withthenumberofnewphage-derivedspacersbeingcor-relatedwithphageresistance[16].However,theirspacersequenceshadnosimilaritytoknownviralphagese-quences.Furthermore,LL03maintainedatypeIICRISPRelementwiththecas9geneinvolvedintargetinterference,whereasDS20hadtypeICRISPRelementswiththecas3

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gene[17].StrainsLL03andDS20wereisolatedfromHCHdumpsitesintheCzechRepublicandIndia,respect-ively,andthesestrainshadtwodifferentCRISPR/CASsystems,thatmaycorrespondtotheirdifferentgeograph-icallocations.ThesedataalsoreflectedthatLL03shouldhavethegreatestphageresistance.

Comparativephylogeneticanalysis

Fourdifferentphylogeneticmethods(16SrRNAgenese-quence,singlecopygenesequences,tetranucleotidefre-quencybasedcorrelation,andaveragenucleotideidentity(ANI))wereusedtoanalyzetherelationshipsoftheninestrainsunderstudy(Figure3).Theconsensustreetop-ologyobtainedbythesemethodsclusteredS.indicumB90A,S.japonicumUT26S,S.chinhatenseIP26,andSphingobiumsp.HDIPO4,withtheexceptionofthesinglecopygeneapproach.Notably,thesefourstrainsweretheonlyoneswithanentirelycompletelinpathway,thussuggestingconvergentevolutionthroughHCHselec-tionpressure.Furthermore,ANItopologysupportedthegroupingofSphingobiumsp.HDIPO4andS.chinhatenseIP26assubspecies(≥99.34%)(ANIvalueswithinthesub-species>98%)[18].Theotherfivestrainsi.e.,LL03,DS20,RL3,P25andSYK6didnotproduceaconsensusphyl-ogeny,withrelationshipsdifferingbetweentheseap-proaches;inshort,strainswiththecompletelinpathwayformedaclosedgroupwhereas,theothershavediverged.Inaddition,16SrRNAandsinglecopygeneapproachesmaybeproblematicfordifferentiationamonghighlyre-latedstrains(asthesemethodologiesdonotconsidertheinfluenceofHGT).However,ANIbasedpairwisecompari-sonhasclusteredLL03andRL3(partiallingenedeficientbutHCHdegraders)inamonophyleticcladewithP25(partiallingenedeficientandslowHCHdegrader)formingacloserelationship.Moreover,DS20andSYK6(non-HCHdegraders)wereclusteredtogether.ThissuggeststhatANIbasedphylogenyismoreappropriateandmirrorstheirre-lationshipwithrespecttoHCHdegradation.

CommongenecontentandfunctionalprofilingofSphingobiumspp.

Coregenomeanalysisidentified322orthologsconservedbetweentheninegenomes.Themajorityofthesegeneswereinvolvedinhousekeepingfunctionssuchasthesynthesisofribosomalproteins,DNAreplication,tran-scription&translationmachinery,aminoacidmetabol-ismandmembranetransporters.CoregenomeanalysisfortheeightstrainsthatwereeitherisolatedfromanHCHdumpsiteorshowedHCHdegradationpotential(i.e.allexceptSYK6)predicted880orthologs(Figure2),whichsuggestsasignificantincreaseingenomicconserva-tion(~2.7times)resultingfromtheselectivepressureofHCHexposure.Thisconservationisalsoseeninthedeg-radationpotentialforotheraromaticcompoundssuchas

Comparative genomic analysis of nine Sphingobium strains: insights into their evolution and hexachlorocyclohexane (HCH) degradation pathways.

Comparative genomic analysis of nine Sphingobium strains: insights into their evolution and hexachlorocyclohexane (HCH) degradation pathways.

Vermaetal.BMCGenomics2014,15:1014

http://wendang.chazidian.com/1471-2164/15/1014

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benzoate,1,4-dichlorobenzene,1,2-methylnapthalene,capro-lactam,tolueneandxylene,trinitrotoluene,biphenylandstyrenedegradation(Figure4).Genesinvolvedinthedegrad-ationofp-hydroxybenzoate,benzoate,quinate,gentisare,andcatecholwerealsoidentifiedinthenineSphingobiumgenomes(Additionalfile1:TableS1).Thepresenceofdegradationpathwaysforphenol/toluene,chlorophenol,anthranilate,andhomogentisateareidentifiedinUT26S[19].Thesepathwayswereobservedinatleasttwoofthenewlysequencedstrains(Additionalfile1:TableS1).ThissuggeststhattheseSphingobiumspp.possessbroadaro-maticcompounddegradationpotential,althoughwedidnotobservethepresenceofthesecompoundsattheHCHdumpsite[20].Thelinkbetweenthesearomaticdegrad-ationpathwaysandtheHCHdegradationpathwayre-quiresfurtherinvestigation.Functionalprofilingwasusedtoanalyzepathwaysthatweredifferentiallyenrichedinthesestrains.Forthis,aden-dogramwasconstructedbaseduponthetop50subsystemsat0.8%minimumabundanceusingpearsoncorrelationdis-tance.Theanalysisrevealedthatthetwo-componentsystemforgeneexpressionwashighlyabundantinalloftheSphingobiumgenomes(Figure4).Thissystemisknowntofacilitateadaptationtoextremeenvironmentalcondi-tionsandlikelycontributestotheabilitytosurviveincon-ditionsofhighHCHpressure,salinity,andaciditythatexistattheHCHdumpsite[9].Additionally,theninestrainscollectivelyshowedanabundanceofABCtrans-porterswithintheirgenomes.Theabundanceofthesetransportersimpliesthatthesestrainsarehighlyengagedintransportofawidevarietyofsubstratesacrossextra-andintracellularmembranes[21],whichisconsistent

with