acquired genes from fungal and prokaryotic donors encode cellulolytic enzymes in the bdelloid rotife

Gene566(2015)125–137

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MultiplehorizontallyacquiredgenesfromfungalandprokaryoticdonorsencodecellulolyticenzymesinthebdelloidrotiferAdinetaricciae

L.Szydlowski,C.Boschetti,A.Crisp,E.G.G.Barbosa,A.Tunnacliffe

DepartmentofChemicalEngineeringandBiotechnology,UniversityofCambridge,CambridgeCB23RA,UnitedKingdom

articleinfoabstract

Thebdelloidrotifer,Adinetaricciae,ananhydrobioticmicroinvertebrate,exhibitsahighrateofhorizontalgenetransfer(HGT),withasmuchas10%ofitstranscriptomebeingofforeignorigin.Approximately80%oftheseforeigntranscriptsareinvolvedinmetabolicprocesses,andthereforebdelloidsrepresentausefulmodelforassessingthecontributionofHGTtobiochemicaldiversity.Tovalidatethisconcept,wefocusedoncellulosedigestion,anunusualactivityinanimals,whichisrepresentedbyatleast16genesencodingcellulolyticenzymesinA.ricciae.Thesegeneshavebeenacquiredfromavarietyofdifferentdonororganismsamongthebacteriaandfungi,demonstratingthatbdelloidsusediversegeneticresourcestoconstructanovelbiochemicalpathway.Avariablecomplementofthecellulolyticgenesetwasfoundin veotherbdelloidspecies,indicatingadynamicprocessofgeneacquisition,duplicationandlossduringbdelloidevolution.Forexample,inA.ricciae,genedupli-cationshaveledtotheformationofthreecopiesofageneencodingaGH45familyglycosidehydrolase,atleastoneofwhichencodesafunctionalenzyme;allthreeofthesegenecopiesarepresentinacloserelative,Adinetavaga,butonlyonecopywasfoundineachoffourRotariaspecies.Furthermore,analysisofexpressionlevelsofthecellulolyticgenessuggeststhatabacterial-origincellobiaseisupregulatedupondesiccation.Insummary,bdelloidrotifershaveapparentlydevelopedcellulolyticfunctionsbytheacquisitionanddomesticationofmulti-pleforeigngenes.

©2015ElsevierB.V.Allrightsreserved.

Articlehistory:

Received9December2014

Receivedinrevisedform1April2015Accepted3April2015

Availableonline8April2015Keywords:

HorizontalgenetransferLateralgenetransferCellulase

EndoglucanaseCellobiohydrolasesCellobiase

1.Introduction

Bdelloidrotifers,microscopicmetazoansinhabitingmainlyfresh-waterandsemi-aquaticecosystems,areabletoundergoanhydrobiosisandsurviveanalmostcompletelossofwaterforlongperiodsoftime(Ricci,1987).Althoughover400specieshavebeenidenti ed(Segers,2007),thereisnoevidenceofsexormeiosisinbdelloidrotifers,sug-gestingtheyrelysolelyonasexualreproduction(MarkWelchandMeselson,2000;BirkyandBarraclough2009).Ahighrateofhorizontalgenetransfer(HGT,i.e.acquisitionofgenesfromanotherspecies)hasbeenreportedinthegenomeofAdinetavaga(Gladyshevetal.,2008;Flotetal.,2013),andasmuchas10%oftheexpressedgenesinAdineta

Abbreviations:aLRT,approximatelikelihoodratiotest;BLAST,BasicLocalAlignmentSearchTool;CAZy,CarbohydrateActiveEnzymes(database);CBA,cellobiase;CBH,cellobiohydrolase;CBM,cellulose-bindingmodule;CMC,carboxymethylcellulose;DEPC,diethylpyrocarbonate;EC,EnzymeCommission;EDTA,ethylenediaminetetraaceticacid;ENG,endoglucanase;GH,glycosidehydrolase;HGT,horizontalgenetransfer;NADES,natu-raldeepeutecticsolvents;Ni-NTA,nickel-nitrilotriaceticacid;ORF,openreadingframe;(q)PCR,(quantitative)polymerasechainreaction;RACE,rapidampli cationofcDNAends;RT,reversetranscriptase;SL,splicedleader;UTR,untranslatedregion.

Correspondingauthorat:DepartmentofChemicalEngineeringandBiotechnology;UniversityofCambridge,NewMuseumsSite,PembrokeStreet,CambridgeCB23RA,UnitedKingdom.

E-mailaddress:at10004@cam.ac.uk(A.

Tunnacliffe).

ricciaeareofforeignorigin(Boschettietal.,2011,2012).Theseforeigngenesarelargelyinvolvedinmetabolicpathways,someofwhichareunusualinanimals(Boschettietal.,2012);onesuchpathwayiscellu-losedigestion.

Cellulose,apolysaccharideofβ-D-glucose,isthemostabundantpolymeronEarth.Aswellasbeingamajorcomponentofplantandalgalcellwalls,celluloseisalsosynthesizedbysomebacteria,fungi(Delmer,1999)andevenurochordates(Matthysseetal.,2004).Duetoitsphysicalproperties,cellulosemicro brilsareinsolubleinwaterandthusarerecalcitranttothemajorityofpolysaccharide-digestingenzymes(DaviesandHenrissat,1995).Cellulose-digestingenzymes(cellulases)includeseveralglycosidehydrolases(GHs)thatactsyner-gisticallyonthevariouspartsofthepolymer(Fig.1)andaresometimesorganisedinmultiproteincomplexescalledcellulosomes(BéguinandLemaire,1996;Bayeretal.,1998).AccordingtotheCarbohydrateActiveEnzymes(CAZy)onlinedatabase,133differentfamiliesofGHshavebeenidenti edtodate,ofwhichatleast18aredirectlyinvolvedincel-lulosedigestion(Lombardetal.,2014).GHscontainacatalyticdomainandalsousuallycarbohydrate-bindingmodules(CBMs)thatfacilitateenzymeattachmenttothepolymer,recognisespeci cpolysaccharidesandevenenhancetheirdegradationbydisruptingthecomplexpolysac-charidestructures(Borastonetal.,2004).Sixty-eightdifferentCBMfamiliesarelistedintheCAZyonlinedatabase,ofwhichatleast20areassociatedwithcellulosedigestion(Lombardetal.,2014).

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