A comparative genomics perspective on the genetic content of the alkaliphilic haloarchaeon Natrialba magadii ATCC 43099

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Title:
A comparative genomics perspective on the genetic content of the alkaliphilic haloarchaeon Natrialba magadii ATCC 43099
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English
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Siddaramappa, Shivakumara
Challacombe, Jean F.
DeCastro, Rosana E.
Pfeiffer, Friedhelm
Sastre, Diego E.
Gimenez, Maria I.
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Background: Natrialba magadii is an aerobic chemoorganotrophic member of the Euryarchaeota and is a dual extremophile requiring alkaline conditions and hypersalinity for optimal growth. The genome sequence of Nab. magadii type strain ATCC 43099 was deciphered to obtain a comprehensive insight into the genetic content of this haloarchaeon and to understand the basis of some of the cellular functions necessary for its survival. Results: The genome of Nab. magadii consists of four replicons with a total sequence of 4,443,643 bp and encodes 4,212 putative proteins, some of which contain peptide repeats of various lengths. Comparative genome analyses facilitated the identification of genes encoding putative proteins involved in adaptation to hypersalinity, stress response, glycosylation, and polysaccharide biosynthesis. A proton-driven ATP synthase and a variety of putative cytochromes and other proteins supporting aerobic respiration and electron transfer were encoded by one or more of Nab. magadii replicons. The genome encodes a number of putative proteases/peptidases as well as protein secretion functions. Genes encoding putative transcriptional regulators, basal transcription factors, signal perception/transduction proteins, and chemotaxis/phototaxis proteins were abundant in the genome. Pathways for the biosynthesis of thiamine, riboflavin, heme, cobalamin, coenzyme F420 and other essential co-factors were deduced by in depth sequence analyses. However, approximately 36% of Nab. magadii protein coding genes could not be assigned a function based on BLAST analysis and have been annotated as encoding hypothetical or conserved hypothetical proteins. Furthermore, despite extensive comparative genomic analyses, genes necessary for survival in alkaline conditions could not be identified in Nab. magadii. Conclusions: Based on genomic analyses, Nab. magadii is predicted to be metabolically versatile and it could use different carbon and energy sources to sustain growth. Nab. magadii has the genetic potential to adapt to its milieu by intracellular accumulation of inorganic cations and/or neutral organic compounds. The identification of Nab. magadii genes involved in coenzyme biosynthesis is a necessary step toward further reconstruction of the metabolic pathways in halophilic archaea and other extremophiles. The knowledge gained from the genome sequence of this haloalkaliphilic archaeon is highly valuable in advancing the applications of extremophiles and their enzymes.
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Siddaramappa et al. BMC Genomics 2012, 13:165 http://www.biomedcentral.com/1471-2164/13/165; Pages 3-21
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doi:10.1186/1471-2164-13-165 Cite this article as: Siddaramappa et al.: A comparative genomics perspective on the genetic content of the alkaliphilic haloarchaeon Natrialba magadii ATCC 43099T. BMC Genomics 2012 13:165.

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RESEARCHARTICLEOpenAccessAcomparativegenomicsperspectiveonthe geneticcontentofthealkaliphilichaloarchaeon Natrialbamagadii ATCC43099TShivakumaraSiddaramappa1,JeanFChallacombe1,RosanaEDeCastro2,FriedhelmPfeiffer3,DiegoESastre2, MaraIGimnez2,RobertoAPaggi2,JohnCDetter1,KarenWDavenport1,LynneAGoodwin1,NikosCKyrpides4, RoxanneTapia1,SamuelPitluck4,SusanLucas4,TanjaWoyke4andJulieAMaupin-Furlow5*AbstractBackground: Natrialbamagadii isanaerobicchemoorganotrophicmemberofthe Euryarchaeota andisadual extremophilerequiringalkalineconditionsandhypersalinityforoptimalgrowth.Thegenomesequenceof Nab. magadii typestrainATCC43099wasdecipheredtoobtainacomprehensiveinsightintothegeneticcontentofthis haloarchaeonandtounderstandthebasisofsomeofthecellularfunctionsnecessaryforitssurvival. Results: Thegenomeof Nab.magadii consistsoffourrepliconswithatotalsequenceof4,443,643bpandencodes 4,212putativeproteins,someofwhichcontainpeptiderepeatsofvariouslengths.Comparativegenomeanalyses facilitatedtheidentificationofgenesencodingputativeproteinsinvolvedinadaptationtohypersalinity,stress response,glycosylation,andpolysaccharidebiosynthesis.Aproton-drivenATPsynthaseandavarietyofputative cytochromesandotherproteinssupportingaerobicrespirationandelectrontransferwereencodedbyoneormoreof Nab.magadii replicons.Thegenomeencodesanumberofputativeproteases/peptidasesaswellasproteinsecretion functions.Genesencodingputativetranscriptionalregulators,basaltranscriptionfactors,signalperception/transduction proteins,andchemotaxis/phototaxisproteinswereabundantinthegenome.Pathwaysforthebiosynthesisof thiamine,riboflavin,heme,cobalamin,coenzymeF420andotheressentialco-factorswerededucedbyindepth sequenceanalyses.However,approximately36%of Nab.magadii proteincodinggenescouldnotbeassigneda functionbasedonBLASTanalysisandhavebeenannotatedasencodinghypotheticalorconservedhypothetical proteins.Furthermore,despiteextensivecomparativegenomicanalyses,genesnecessaryforsurvivalinalkaline conditionscouldnotbeidentifiedin Nab.magadii Conclusions: Basedongenomicanalyses, Nab.magadii ispredictedtobemetabolicallyversatileanditcoulduse differentcarbonandenergysourcestosustaingrowth. Nab.magadii hasthegeneticpotentialtoadapttoitsmilieu byintracellularaccumulationofinorganiccationsand/orneutralorganiccompounds.Theidentificationof Nab. magadii genesinvolvedincoenzymebiosynthesisisanecessarysteptowardfurtherreconstructionofthemetabolic pathwaysinhalophilicarchaeaandotherextremophiles.Theknowledgegainedfromthegenomesequenceofthis haloalkaliphilicarchaeonishighlyvaluableinadvancingtheapplicationsofextremophilesandtheirenzymes. *Correspondence: jmaupin@ufl.edu5DepartmentofMicrobiologyandCellScience,UniversityofFlorida,Bldg. 981,MuseumRd.,P.O.Box110700,Gainesville,FL32611-0700,USA Fulllistofauthorinformationisavailableattheendofthearticle 2012Siddaramappaetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Siddaramappa etal.BMCGenomics 2012, 13 :165 http://www.biomedcentral.com/1471-2164/13/165

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BackgroundArchaeaaretheleastwell-characterizedmembersamong theextantthreedomainsoflife,andrecentgenomesequencingeffortshavefacilitatedourunderstandingof theseunusualmicrobes[1].Thephylum Euryarchaeota containsadiversearrayofarchaeacurrentlyclassified undereightnamedclasses( Archaeoglobi,Halobacteria, Methanobacteria,Methanococci,Methanomicrobia, Methanopyri,Thermococci, and Thermoplasmata )andten orders[2].Membersofthe Euryarchaeota ,particularly thoseof Halobacteria ,havereceivedattentionbecauseof theirecologicalandevolutionaryimportance.Halophilic archaeaarephysiologicallyandphylogeneticallydiverse andoccurinawidevarietyofenvironments[3,4].Most halophilicarchaeathriveinhypersalineenvironments ( > 15%NaCl).Tosurviveinsuch extremeconditions,these organismshaveevolvedstrategiestocopewithnotonly osmoticstressanddesiccation,butalsooxygenlimitation andthedamagingeffectsofUVlight[5].Thehaloalkaliphilesconstituteadistinctgr oupofmicroorganismssince theysurviveintwoextremes:highpHandhypersalinity [6,7].Inaddition,haloalkaliphilicarchaeahavean asymmetricC20C25dietherisoprenoidcorelipidthatis uncommonamongneutrophilichalophilicarchaea[8]. Thegenus Natrialba within Halobacteria isaheterogeneousgroupofhalophilesincludingthosethatthrive inneutralaswellasalkalineenvironments[9].Thetype species Natrialbaasiatica ,whichwasisolatedfroma beachinJapan,isnon-alkaliphilicandrequiresapHof 6.6to7.0foroptimumgrowth[10]. Natrialbamagadii (formerly Natronobacteriummagadii ,astrictlyaerobic chemoorganotrophisolatedfromLakeMagadiinKenya) isanobligatelyhaloalkaliphilicarchaeonthatrequires 20%(3.5M)NaCl,pH9.5,and37to40Cforoptimum growth[9,11].Incontrasttothewhite-yellowcolorof Nab.asiatica Nab.magadii isred-orangecoloreddue tothepresenceofcarotenoidpigmentsinthecellmembrane[12].Furthermore, Nab.magadii lacksglycolipids, whereas Nab.asiatica containsbis-sulfatedglycolipid S2-DGD{2,3-diphytanyl-orphytanyl-sesterterpenyl-1[2,6-(HSO3)2-Manp-1 2-Glcp]-sn-glycerol}[9,13]. Previousworkhasdemonstratedthat Nab magadii synthesizesandaccumulates2-sulfotrahaloseasanosmolyteunderhypersalineconditions[14].Thebiochemical featuresoftheflagellarapparatus,anucleosidediphosphatekinase,aleucinedehydrogenase,andanextracellularserineproteaseof Nab.magadi havealsobeen characterizedsinceitsdiscovery[15-18]. Althoughknowledgeonthebiologyofhalophilicarchaeahasgreatlyadvancedduringthelastdecade, attemptstounderstandthephysiologyandgeneticsof thehaloalkaliphilicarchaeaarescarce[19].Thestudyof haloalkaliphilicarchaeaisinterestingfromseveralperspectivesbecausetheseareamongthemostalkaliphilic microorganismsreportedtodate[20].Ofthehalophilic archaearelatedto Nab.magadii ,alowpassgenomic sequenceof Nab.asiatica strainATCC700177hasbeen reported[21].Thegenomeof Haloterrigenaturkmenica strainDSM5511,ahalophilicarchaeonisolatedfrom sulfatesalinesoilinTurkmenistan,iscompleteandcontains5,287proteincodinggenes[22].Furthermore,the completegenomeof Natronomonaspharaonis DSM 2160,ahaloalkaliphilicarchaeonisolatedfromasoda lakeinEgypt,contains2,843proteincodinggenes[23]. Inaddition,adetailedanalysisofthemetabolicpathways ofhalophilicarchaeahasbeendescribed[24].Thegoal ofthecurrentstudywastoexplorethephysiologyof Nab.magadii atthewholegenomelevelandperform comparativegenomicstudieswithotherhalophilicas wellashaloalkaliphilicarchaea.Anexplorationofthe pathwaysofcoenzymebiosynthesisandproteolysis within Nab.magadii wasalsoenvisaged.MethodsNab.magadii wasgrownat37Caerobicallyinliquid mediumcontaining20g/Lyeastextractusingthe methodofTindall etal .[11],andgenomicDNAwas extractedfromthepelletedcellsusingtheprocedure describedbyNg etal .for Halobacteriumhalobium ( salinarum )[25].Genomiclibraryconstruction,sequencing, andfinishingwereperformedattheJointGenomeInstitute(JGI)facilitiesatWalnutCreekandtheGenome SciencefacilitiesatLosAlamosNationalLaboratory. Briefly,thedraftgenomeof Nab.magadii was sequencedusingacombinationofbothSangerand454 technologies.ASangerwholegenomeshotgunlibrary, whichproduced26,484readswithanaverageinsertsize of6.5kb,anda454FLXstandardlibrary,whichgenerated96.3Mbpofdata,wereconstructedforthisgenome.Allgeneralaspectsoflibraryconstructionand sequencingperformedattheJGIcanbefoundathttp:// www.jgi.doe.gov/.ThePhred/Phrap/Consedsoftware package(http://www.phrap.com)wasusedforsequence assemblyandqualityassessment.Aftertheshotgun stage,readswereassembledwithparallelphrap(High PerformanceSoftware,LLC).Possiblemis-assemblies werecorrectedwithDupfinisherortransposonbombing ofbridgingclones(EpicentreBiotechnologies,Madison, WI).Gapsbetweencontigswereclosedbyeditingin Consed,customprimerwalk,orPCRamplification (RocheAppliedScience,Indianapolis,IN).Atotalof594 additionalcustomprimerreactionswerenecessaryto closeallgapsandraisethequalityofthefinishedsequence.Theestimatederrorrateforthecompletedgenomeof Nab.magadii waslessthan1in100,000.The finalassemblywasbasedon19.1MbpofSangerdraft data,whichprovided4.3xcoverageofthegenome,andSiddaramappa etal.BMCGenomics 2012, 13 :165 Page2of21 http://www.biomedcentral.com/1471-2164/13/165

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96.3Mbpof454draftdata,whichprovided21.7xcoverageofthegenome. Preliminaryautomatedannotation,predictionofthe numberofsubsystems,andpairwiseBLASTcomparisons ofproteinsetswithindifferentstrainswereperformed usingtheRapidAnnotationusingSubsystemsTechnology (RAST),whichisafullyautomated,prokaryoticgenome annotationservice[26].Subsequently,adetailedmanual curationwasperformedtoensureconsistencywiththeannotationofotherhalophilicarchaea.Annotationofgenes involvedincoenzymebiosynthesiswasbasedontheinformationavailableinrecentliteratureand/ortheirrelatednesstofunctionallycharacterizedhomologspresentin otherorganisms.Theseannotationdetailsareprovidedat thewebsitehttp://wiki.rzg.mpg.de/HaloferaxWiki.Proteinsdeemedtobespecificto Nab.magadii werecomparedagainsttheNCBInon-redundantproteindatabase todeterminewhethertheywerehypotheticalorconserved hypothetical.Iftherewasnoadequatealignmentwithany protein(lessthan25%identityoralignedregionisless than25%ofthepredictedproteinlength),thetranslated ORFwasnamedahypotheticalprotein. Multiplegenomecomparisonswereperformedusing the ‘ progressivealignment ’ optionavailableintheprogramMAUVEversion2.3.0[27,28].Defaultscoringand parameterswereusedforgeneratingthealignment. Priortothealignment,the Nab.magadii genomesequencewasrearrangedtofacilitatevisualcomparison. ThiswasaccomplishedusingtheArtemisComparison Tooltoidentifyacoordinatewherethesequencewas shiftedrelativetothatof Htg.turkmenica .Thecoordinate waslocatedat1961610bpandthe Nab.magadii sequence wascutstartingatthiscoordinateuntiltheendofthesequenceandplacedatthebeginningofthefastafilesothat thegenomestartwasnearthemajororiginofreplication. Asyntenyplotwasgeneratedusingtheprogram NUCmer,whichusesexactmatching,clustering,and alignmentextensionstrategiestocreateadotplotbased onthenumberofidenticalalignmentsbetweentwogenomes[29].NUCmerwasusedwiththemaxmatchargumentand,tobeconsistentwiththeMAUVEcomparison, therearranged Nab.magadii sequencewasalignedwith thatof Htg.turkmenica .The Nab.magadii genomeprojectisdepositedintheGenomesOnLineDatabase (GOLD)andthecompletegenomesequenceisavailable fromGenBank/EMBL/DDBJwithaccessionnumbers CP001932,CP001933,CP001934,andCP001935.The genomeof Nab magadii isalsoaccessiblethroughHaloLex(www.halolex.mpg.de)andtheUCSCArchaealgenomebrowser(http://archaea.ucsc.edu/).ResultsanddiscussionNab.magadii genomefeaturesandcomparisonwiththe genomesofotherhalophilicarchaeaThecompletegenomesequenceof Nab.magadii consistedoffourreplicons(totalsize4,443,643bp).Three oftheseelementshadaGCcontentof~61%whereas pNMAG02hadaGCcontentof56.82%.Acomparison ofsomeoftherelevantfeaturesofthesefourelementsis showninTable1.ABLASTNanalysisofpNMAG03on theNCBIdatabaserevealed99%identitytohalovirus Ch1(58,498bp;GenBankaccessionnumber NC_004084),abacteriophage-likeelementisolatedfrom Nab.magadii .Sincehalovirus Ch1hasalreadybeen describedelsewhere[30-32],theanalysisofpNMAG03 wasexcludedfromthescopeofthecurrentwork.The largechromosomeof Nab.magadii containedtwogenes encodingputativereplicationfactorC-likeproteins (Nmag_1868and1910).Thelargechromosome, pNMAG01,andpNMAG02werepredictedtoreplicate usingaconservedarchaealmechanism[33],sinceeach oftheserepliconscontainedatleastonegeneencoding anOrc1/Cdc6familyreplicationinitiationprotein.For thelargechromosome,themajorreplicationoriginwas Table1Characteristicsoftherepliconsof Natrialbamagadii ATCC43099RepliconLargechromosomeSmallchromosomeLargeplasmidVirus Ch1 AnnotationNonepNMAG01pNMAG02pNMAG03 TopologyCircularCircularCircularLinear/Circular Size3,751,858bp378,348bp254,950bp58,487bp GCcontent61.42%60.09%56.82%61.90% NumberofRNAgenes537NoneNone Numberofprotein-codinggenes3559(+1790/-1769)340(+183/-157)219(+110/-109)94(+11/-83) Numberofhypotheticalproteins1278966975 Fulllengthorc1/cdc6homologs511None Glycosyltransferasegenes1931None ISelements21213None Overallcodingdensity83%80%76%93% GenBankaccessionnumberCP001932CP001933CP001934CP001935 Siddaramappa etal.BMCGenomics 2012, 13 :165 Page3of21 http://www.biomedcentral.com/1471-2164/13/165

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predictedtobeatca1.9Mb,locatedbetweenOrc1 (Nmag_1930)ontheforwardstrandandathree-gene operononthereversestrand(Nmag_1927-1929).This setoffourhighlyconservedgeneswasfoundadjacentto thereplicationorigininalmostallhalophilicarchaea. Archaealgenomescancontainalargenumberoftransposableelementsandthevarietyofarchaealinsertion sequencesisthoughttoapproximatethatofbacteria[34]. However,mostarchaealgenomeslackprophageelements [35].Manualcurationindicatedthatthegenomeof Nab magadii contained~36full-lengthortruncatedgenesencodingputativetransposases.T heseinsertionsequenceelementswerescatteredthroughoutthechromosomesand about20ofthesebelongtothe IS 605OrfBfamily.The IS 605OrfBtransposase(alsocalled IS 1341-typetransposase)geneswerehighlydiverse,asistypicalofhalophilic archaea[36].Asingle IS 605OrfA(Nmag_4105,alsocalled IS 200-typetransposase)wasidentifiedinthegenome. Othertransposasegenesin Nab.magadii include7ofthe broadcategory IS 4(3 IS 7-typeand4 IS 9-type),asingle IS 240-type,and4relatedto IS Sod10.Thesmallnumberof transposasegenesandtheirhe terogeneitymayindicatethat Nab.magadii isonlyminimallyaffectedbytheseelements. Thegenomealsocontainedseveralgenesrelatedto bacteriophageelements( e.g .,PhiH1repressorprotein, phagetailproteins,andphageproteinD)anda vgr -like generelatedtorecombinationhotspotelements.In addition,therewere13genesencodingintegrase/recombinase-likeproteins(Additionalfile1:TableS1). Archaealgenomesgenerallyhave1 – 4rRNAoperons consistingofthe16S,23S,and5SrRNAgeneswitha tRNAAlagenelocatedintheinternaltranscribedspacer [37].Thelargechromosomeof Nab magadii contained twocopiesof16SrRNA-tRNAAla-23SrRNA-5SrRNA sequences,oneeachontheplusandminusstrands,as wellastwogenesencodingcomponentsoftheRNA guidemachinery(Nmag_0693-0694)withfibrillarin-like RNAmethyltransferaseasthecatalyticcomponent.The smallchromosomepNMAG01containedacopyof16S rRNA-tRNAAla-23SrRNA-5SrRNAsequenceonthe minusstrandandacopyof23SrRNA-5SrRNA sequenceontheplusstrand.Thethree16SrRNAtRNAAla-23SrRNA-5SrRNAsequencesof Nab magadii had99%nucleotideidentitytoeachother.The smallchromosomepNMAG01alsocontainedanorphan 5SrRNAsequencethathad89%nucleotideidentityto theotherfour5SrRNAgenesof Nab.magadii .Since pNMAG02lackedrRNAoperonsandhadalesserGC contentthanthelargeandsmallchromosomes,thisself replicatingelementcouldbeconsideredalargeplasmid. TheheterogeneityoftherRNAoperonswithin Nab magadii isnotauniquefeatureandtheoccurrenceof suchrRNAoperonsamonghalobacterialgenomesis thoughttobeduetorecombinationbetweenrRNA genesofdifferentstrainsorspecies[38].The16SrRNA genesof Nab.magadii werecloselyrelatedtothoseof Nab.asiatica (97%identity), Htg.turkmenica (96%identity),and Nmn.pharaonis (90%identity).Furthermore, thegenomeof Nab.magadii wascomparedto17 completehaloarchaealgenomesavailableinthepublic databases(Additionalfile2:TableS2).Basedonthis analysis, Htg.turkmenica containedthehighestnumber oforthologs(2601symmetricalhits),followedby Halopigerxanaduensis strainSH-6(2533symmetricalhits). Therewerelessernumberoforthologs(1805symmetricalhits)in Nmn pharaonis ,whichhasarelatively smallergenome.However,whenthedataforthepercentageofproteinshavingabidirectionalbestblasthit in Nab.magadii wascomputed(Additionalfile2:Table S2), Nmn pharaonis wasthetop(63%oftheproteins havingabidirectionalbestblastpair),followedby Hpg. xanaduensis (60%)and Htg.turkmenica (51%).Results from Nmn pharaonis and Htg.turkmenica areemphasized inthispapersincetheformerwastheonlyotherhaloalkaliphilicarchaeonwithacompletegenomesequenceandthe lattercontainedthehighestnumberoforthologs. Thecombinedsizeofthecompletegenomeof Nab magadii was1.7Mblargerthanthecompletegenomeof Nmn pharaonis ,whichconsistsofthreereplicons(total size2,749,696bp).However, Nab magadii genomewas 1Mbsmallerthanthecompletegenomeof Htg.turkmenica ,whichconsistsofsevenreplicons(totalsize 5,440,782bp).TheGCcontent(61.42%)ofthelarge chromosomeof Nab magadii wasslightlylesserthan thatofthelargechromosomesof Htg.turkmenica (~66%GC)and Nmn pharaonis(~63%GC).Alignment ofthelargechromosomeof Nmn pharaonis (2,595,221bp)withthatof Nab magadii usingMAUVE showedthepresenceofveryfewshortsyntenicregions (datanotshown),whereasasimilaralignmentusingthe largechromosomeof Htg.turkmenica (3,889,038bp) showedthepresenceofnumerousshortsyntenicregions (Figure1A).Tofurtherdissectthisco-linearity,a BLASTNcomparisonofthelargechromosomesof Nab magadii and Htg.turkmenica wasperformed.This analysisrevealedthepresenceof400homologous regions(226plus/plusand174plus/minus,sequence range > 300bp,but < 2,000bp)withanaverage nucleicacididentityof89%(theidentityrangewas84%98%,E-value=0).Theplusandminusstrandmatches amongthechromosomesof Htg.turkmenica and Nab magadii generatedbyNUCmerareshowninFigure1B. Athree-waycomparisonofallpredictedprotein-coding genesof Nab magadii usingtheTaxPlottoolofNCBI revealedthat Htg.turkmenica contained2387orthologs, whereas Nmn pharaonis containedonly426orthologs (symmetricalhits).Theseanalysesfurtherconfirmedthat Nab magadii wasmorecloselyrelatedto Htg.turkmenicaSiddaramappa etal.BMCGenomics 2012, 13 :165 Page4of21 http://www.biomedcentral.com/1471-2164/13/165

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thanto Nmn.pharaonis .In Nab magadii ,1518genes couldnotbeassignedafunctionbasedonBLASTanalysis andwereannotatedasencodinghypotheticalorconserved hypotheticalproteins.Theisoelectricpoint(pI)ofmostof thepredictedproteinsof Nab magadii wasinthe3 – 5 range,indicatingthatthegeneralproteomeisacidic, whichistypicalofmosthalophilicarchaea.Atwo-way comparisonofthelargechromosomesrevealedthat Nab magadii contained~945putativeprotein-codinggenes thathadnohomologsin Htg.turkmenica .Avastmajority (~75%)ofthese Nab magadii -specificgenesencoded hypotheticalproteins.Othergenome-specificgenesin Nab magadii encodedABC-typetransporters,ATPases, kinases,phosphatases,proteases,andoxidoreductases. Thegenomeof Nab magadii alsocontainedavarietyof simplesequencerepeatsencodingcharacteristicpeptide repeatpatterns.GeneraladaptivefeaturesInadditiontomaintaininganacidicproteomeandacell wallcomposedofacidicglycoproteins,haloalkaliphilic speciesappeartohaveevolvedseveralothermechanisms ofadaptationtotheirniche[39-41].Theseinclude,but arenotlimitedto,intracellularaccumulationofinorganiccationsand/orneutralorganiccompounds [42-44].Halophilicarchaeamaintainthenecessarywater balanceandosmoticpressureevenwhentheextracellular Na+concentrationexceeds5MbypumpingNa+outand K+intothecellusingavarietyofcation/protonantiporters [12,45,46].Thegenomeof Nab magadii containedan Figure1 A.Alignmentofthelargechromosomesof Natrialbamagadii ATCC43099and Haloterrigenaturkmenica DSM5511using MAUVE2. Priortothealignment,the Nab.magadii genomesequencewasrearrangedtofacilitatevisualcomparison.The Nab.magadii sequencewascutstartingat1961610bp(locatedbetweenNmag_1929andNmag_1930,encodingputativeGTP-bindingproteinandORC1 replicationinitiationprotein,respectively)untiltheendofthesequenceandplacedatthebeginningofthefastafilesothatthegenomestart wasnearthemajororiginofreplication.Identicallycoloredboxes,knownaslocallycollinearblocks(LCBs),depicthomologousregionsinthetwo chromosomes.TheedgesofLCBsindicatechromosomerearrangementsduetorecombination,insertions,and/orinversions.Sequencesof Nab. magadii invertedinrelationtothoseof Htg.turkmenica areshownasblocksbelowthehorizontalline.TheverticallinesconnectingtheLCBs pointtoregionsofhomologyamongthetwochromosomes.Numbersabovethemapsindicatenucleotidepositionswithintherespective chromosomes. B .Syntenyplotofthelargechromosomesof Natrialbamagadii ATCC43099and Haloterrigenaturkmenica DSM5511generatedby NUCmer.NUCmerwasusedwiththemaxmatchargumentandthe Nab.magadii genomesequencewasrearrangedasinFigure1Atofacilitate visualcomparison.Regionsofidentitybetweenthetwochromosomeswereplottedbasedonpair-wisealignments.Numbersindicatenucleotide positionswithintherespectivechromosomes.Plusstrandmatchesareslantedfromthebottomlefttotheupperrightcornerandareshownin red.Minusstrandmatchesareslantedfromtheupperlefttothelowerrightcornerandareshowninblue.Thenumberofdots/linesshownin theplotisthesameasthenumberofexactmatchesfoundbyNUCmer. Siddaramappa etal.BMCGenomics 2012, 13 :165 Page5of21 http://www.biomedcentral.com/1471-2164/13/165

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operonofninegenesencodingaputativepHadaptationK+effluxsystem(Nmag_3445-3453).Genesrelatedtothisoperonwerepresentinseveralhalophilicarchaea,indicating thattheymaynotencodeaspecializedsysteminvolvedin stressresponsetoalkalinegrowthconditions.Apartfrom thisoperon,thegenomecontainedthreeothergenes encodingputativecation/protonantiportersandagene encodingaputativeOsmCfamilyprotein(Additionalfile1: TableS1). Lowmolecularweightorganiccompoundssuchas aminoacids,polyols,andsugarsfacilitatecellularadaptationtohigh-osmolarityandarereferredtoasosmoprotectantsorcompatiblesolutes[47].Halophilicspeciesalso accumulateneutralorganiccompoundsasameansof adaptationtotheirniche[14,43,44].Thelargechromosomeof Nab magadii containedalocusencodinga putativetrehalose-phosphatesynthaseandatrehalosephosphatase,whichmaybeinvolvedinthebiosynthesisof theosmoprotectant2-sulfotrehalose.Theorthologsof thesegeneswerefoundinfewotherhalophilicarchaea andtheosmolytehasbeendetectedbynuclearmagneticresonancespectroscopicanalysisin Nab magadii [14]. Nab.magadii alsocontainedgenesencodingthe biosynthesisofspermineaswellastransportersforthe uptakeofcholine/carnitine/betaineandspermidine/ putrescine,whichmayalsoprovideprotectionathighosmolarity(Additionalfile1:TableS1).Therefore,it appearedthat Nab.magadii hadmultiplemechanisms ( e.g. ,intracellularaccumulationofinorganiccationsas wellasneutraland/orchargedorganiccompounds)for osmoticadaptation. Thescarcityofmolecularoxygeninahypersaline milieucouldbeagrowth-limitingfactorforaerobic chemoorganotrophicprokaryotes[48-50].Ithasbeen proposedthatsomearchaealspeciesaccumulateintracellulargasvesiclesthathelpthemfloatonthesurface andperformoxidativerespirationintheirnativesaturatedsaltwaterhabitats[12].Thelargechromosomeof Nab magadii containedaclusterof11genesencoding putativegasvesiclesynthesisfamilyproteins,whichwere relatedtothegasvesiclefamilyproteinsof Hbt.salinarum .However, Nab magadii lackedgenesrelatedto thoseencodingtheminorgasvesicleprotein(GvpC) andtheregulators(GvpDandGvpE).Thegasvesicle geneclustersof Nab magadii and Htg.turkmenica were highlysimilartoeachotherandappearedtocontaina distanthomologofthe Hbt.salinarumgvpI gene (Nmag_0338andHtur_2370,respectively).Nevertheless, thesegenesencodeputativeproteinswithanN-terminal extensionofmorethan200residuesnotfoundinGvpI of Hbt.salinarum .Furthermore,thegasvesicleclusters of Nab magadii and Htg.turkmenica containedanadditionalgene(Nmag_0337andHtur_2371,respectively) thatwasabsentin Hbt.salinarum Nab magadii also containedageneencodingahemAT-typeaerotactic transducerwithaputativeglobin-coupledsensorproteincomprisingofaglobinfolddomainandamethylacceptingchemotaxistransducerdomain(Additional file1:TableS1).Thesetraits,inadditiontotheosmoticadaptationmechanismsdiscussedabove,may playaroleinthesurvivalof Nab magadii initsnaturalenvironment. Otherfeatureslikelytofacilitatetheadaptationof Nab.magadii toitsnicheincludedgenesencodingputativemechanosensitiveionchannels(MscS)thatafford protectionagainsthypoosmoticshock,chaperone proteinsDnaJandDnaK,athermosome,andheatshock proteinsthatmayparticipateinproteinqualitycontrol andcellularresponsetostress. Nab. magadii also contained47genes(26onthelargechromosome,18on pNMAG01,and3onpNMAG02)encodingputative proteinsofvarioussizeswithauniversalstressprotein (UspA)domain.Oneofthesegenes(Nmag_1302) appearedtoformanoperonwithageneencodinga putativeGCN5-relatedN-acetyltransferase(GNAT, Nmag_1303),andasimilargenepairwasfoundin Htg. turkmenica (Htur_3429-3430,80%identity,E-value=2e82to1e-52)and Nmn pharaonis (NP1710A-NP1712A, 49%identity,E-value=2e-41to4e-28).Itispossiblethat theGNATisinvolvedintheacetylationofthelinked universalstressproteininthesespecies.Inaddition,the largechromosomeof Nab magadii containedgenes encodingasuperoxidedismutase( sodA ),twocatalases ( katG ,whichiscommontomosthalophiles,and katE whichiscloselyrelatedto katE ofmethanogens,also presentin Htg.turkmenica and Hpg.xanaduensis ,but notinotherhalophiles),twoalkyl-hydroperoxidase-like proteins,acarbonicanhydrase,andmethioninesulfoxide reductases( msrA and msrB ;Additionalfile1:TableS1). Itispossiblethattheseenzymeshavearoleintheadaptationofthishaloarchaeontovariousoxidativestresses associatedwithenergymetabolism. Furthermore, Nab magadii containedgenesencodingmetaltransportproteinsandaputativecopperresistanceprotein. Nab magadii copperresistance proteinappearstocontainfusedCopC-CopDdomains andadistanthomologofthisproteinoccursin Nmn. pharaonis (NP4610A,39%identity,E-value=2e-105), butnotinotherarchaea.Thesegenesmaybeinvolved inmetalhomeostasisinthehypersalineenvironment that Nab magadii inhabits. Nab magadii alsoencoded DNAmethylases,DNAdamagerepairexcinuclease ABCsubunits,DNAmismatchrepairproteins,and DNArepair/recombinationproteinsRadAandRadB (Additionalfile1:TableS1).Homologsofthesegenes arefoundinseveralotherarchaeaandtheyarepredictedtobeinvolvedinstressresponseandmaintaininggeneticintegrity.Siddaramappa etal.BMCGenomics 2012, 13 :165 Page6of21 http://www.biomedcentral.com/1471-2164/13/165

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Proteases,peptidases,proteaseinhibitors,andprotein translocationAtleast83genesencodingvarioustypesofpeptidases/ proteaseswereidentifiedinthegenomeof Nab magadii bymanualcuration(~1.8%ofallprotein-codinggenes; Additionalfile3:TableS3).Interestingly, Nab.magadii appearstoencodealargersetofproteolyticenzymes comparedtomosthalophilicarchaea,including Nmn. pharaonis Hfx.volcanii and Hbt.salinarum .This suggeststhatthenaturalenvironmentinhabitedby Nab. magadii containsanamplesupplyofproteindebris, whichcouldbeusedasamajorcarbonandnitrogen source.Theclosesthomologsofthevastmajority of Nab.magadii genes(~50%)encodingputative peptidases/proteaseswerefoundin Htg.turkmenica (Additionalfile3:TableS3).Mostofthe Nab.magadii predictedproteasesbelongtothecatalytictypeof metallo-andserineproteases.Otherproteasesinclude variousamino-andcarboxypeptidases,oligopeptidases, signalpeptidases,ATP-dependentproteases,andintramembranecleavingproteases(I-CLiPs). Subtilases(COG1404;subtilisin-likeserineproteases) arealargesuperfamilyoffunctionallydiverseendo-and exo-peptidasesthatoccurinprokaryotesandeukaryotes [51]. Nab magadii containedninegenesencodingputativeS8andS53subtilisinkexinsedolisins(Additional file3:TableS3).Althoughthepredictedsubtilisinsof Nab magadii haddiversesizes(rangingfrom402to 1710aa),theaminoacidmotifscontainingthecatalytic triad(Asp-His-Ser)wereconservedinallofthem.Sixof thepredictedsubtilisins(Nmag_0073,0714,0715,1249, 1874,and3633)of Nab magadii containedputativetargetingsignalsfortranslocationthroughthetwin-argininetransport(Tat)pathway,suggestingthatthese proteasesaremostlikelyexportedoutofthecell.Within thisgroup,Nmag_0715hasbeenbiochemicallycharacterizedanddesignatedasthe Natrialba extracellular protease(Nep)[16].Nepwasdemonstratedtobealkaliresistant,afeaturethatcorrelateswiththeconditions thatpredominateinthenaturalenvironmentof Nab magadii [16].Interestingly,theC-terminaldomainof Nepcontainsanacidicpatchcomposedof12amino acidresiduesthatisabsentinthesubtilasesofneutrophilicorganisms[52].ThisdistinctivefeatureofNep maybeinvolvedinitsstabilityathighsaltand/or highpH.Inaddition,pNMAG01containedageneencodingaputativemicrocystinLRdegradationprotein (Nmag_3774,MlrC-like-prot ein).MlrCpeptidases,initiallyisolatedfromthebacterium Sphingomonas, are aspecializedgroupofmetalloproteasesassignedto M81familyandtheyparticipateinthelaststepof thedegradationpathwayofmicrocystinLR[53]. Theseenzymesrarelyoccurinthearchaealdomain andthehomologsofNmag_3774werenotfoundin Nmn pharaonis and Htg.turkmenica (Additionalfile 3:TableS3). Allarchaealgenomesstudiedtodatearepredicted toencodeself-compartmentalizedproteases(20SproteasomesandLon-typeproteases)likelytofunctionin energy-dependentproteolysisandanubiquitin-type mechanismfortargetingproteinstoproteasomestermed sampylation[54,55].Inarchaea,20Sproteasomesof and -typesubunitsarethoughttofunctionwithAAAATPasessuchastheproteasome-activatingnucleotidase (PAN)indegradingfoldedproteins[54].Inaddition, ubiquitin-likesmallarchaealmodifierproteins(SAMPs) appeartobeconjugatedtoproteintargetsbyanE1-like enzymetermedubiquitin-likeconjugatingenzymeofarchaeaorUbaA(basedonstudyof Hfx.volcanii [56]). Thegenomeof Nab.magadiicontainedanoperonencodingputative20Sproteasome and subunits (Nmag_0515-0514,respectively).Apartfromthisoperon,thegenomecontainedseparategenesencoding 20Sproteasome and subunithomologs(Nmag_3313 andNmag_3351,respectively). Nab.magadii wasalso predictedtoencodehomologsofPAN(Nmag_1362and 2440)andubiquitin-likesmallarchaealmodifierproteins (SAMPs;Nmag_0567,1914,2668,and2971).Thegenomeof Nab.magadi containedtwogenesencodingputativeubiquitin-likeactivatingenzymesofarchaea (UbaA;Nmag_1394and3812).Furthermore,italso encodedadistanthomologofUbaA(Nmag_0356)containingaC-terminalJAB1/MPN/Mov34metalloenzyme (JAMM)domainthatwaspredictedtoremoveSAMPs fromtargetproteins.Incontrast, Hfx.volcanii encodes onlyasingleUbaA-typeproteinthatfunctionsin bothproteinconjugation(sampylation)andsulfur mobilization[56]. Nab.magadii alsoencodedan archaeal-typeLonBprotease(Nmag_2822),whichwas demonstratedinitscellmembranes[57].WhileLonB homologsareconservedandlikelyactaskeyenergydependentproteasesinarchaea,thephysiologicalsignificanceoftheseenzymeshasnotbeenaddressed. Thetetrahedralaminopeptidase(TETprotease)isan energy-independentproteincomplex(withapeptidase domainoftheclanMH,familyM42,accordingtothe MEROPSdatabase)thatwasisolatedfromtheneutrophilichaloarchaeon Har.marismortui [58].Ithasbeen suggestedthatTETdegradesoligopeptidesreleasedby ATP-dependentproteasessuchastheproteasomeand LonB. Nab.magadii encodesahomologofTET (Nmag_1335,peptidaseM42familyprotein),which,in combinationwiththeenergy-dependentproteases,may participateintheintracellularproteinturnoverinthis extremophile.Furthermore,similartothemajorityof haloarchaea, Nab.magadii appearstoencodehomologs ofthethreefamiliesofmembrane-embeddedregulatory proteasesdenotedasI-CLiPs.Theseinclude sppA -typeSiddaramappa etal.BMCGenomics 2012, 13 :165 Page7of21 http://www.biomedcentral.com/1471-2164/13/165

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signalpeptidepeptidases(SPPs,Nmag_2612and2635), site-2proteaseclassofzincmetalloproteasesthatcleave transmembranedomains(S2Ptypepeptidases, Nmag_1508,1514,2136,and3494),andrhomboids (Rho,Nmag_1128,1636,2518,and3579).Furthermore, Nab.magadii containedgenesencodingtypeIsignal peptidases(sec11-type,Nmag_1326,1932,1944,3375, 3743,and4175)andatypeIVprepilinpeptidase (Nmag_1752).ThetypeIsignalpeptidasesandthetype IVprepilinpeptidasearepredictedtobeinvolvedinthe processingofN-terminalsignalpeptidesofexported proteinsandflagellinprecursors,respectively. Cellularproteaseactivityisfrequentlycontrolledby endogenousproteaseinhibitors[59].Genesencoding putativehomologsofproteaseinhibitorsoftheserpin (Nmag_2110)andphosphatidylethanolamine-binding protein(Nmag_0329)typeswerepresentin Nab.magadii .Asubtilisinproteaseinhibitorfromthisarchaeon, denotedNSI,waspreviouslypurifiedandbiochemically characterized[60].Thisproteaseinhibitorremainstobe investigatedatthemolecularlevelandtheavailabilityof itsgenesequencecouldfacilitatecloningandexpression oftherecombinantproteinforfurtheranalysis.Arepresentationofthemajorproteolyticsystems(predicted and/orvalidatedbydetectingmRNAand/orassaying proteinactivity)of Nab.magadii ispresentedinFigure2. Althoughthisdepictionassumesthattheproteolyticsystemsof Nab.magadii areindependentofeachother, theirsynergisticaction invivo cannotberuledout. Proteintranslocationacrossthecellmembranein prokaryotesisfacilitatedbyatleastthreemechanisms includingthegeneralsecretion(Sec)system,thespecializedTatsystem,andthehighlyornate,substrate-specific secretionsystemsfordeliveringeffectorproteinsto targetsites[61-63]. Nab magadii containedgenesthat encodedputativecomponentsoftheSecsystem ( secYEGDF ,Nmag_0233,1140,1564,2707,and2708; srp19 ,Nmag_3604; srp54 ,Nmag_1802; ftsY Figure2 Schematicrepresentationof Natrialbamagadii ATCC43099proteolyticsystems. Themajorproteasesandproteaseinhibitor proteinspredictedfromtheanalysesof Nab.magadii genomearedepicted.NSP( Nab.magadii serineprotease),NMP( Nab.magadii metalloprotease),NSI( Nab.magadii subtilisininhibitor),NEP( Nab.magadii extracellularprotease),andLonBhavebeendescribedintheliterature. SPs,Signalpeptidases;I-CLiPs,Intramembrane-CleavingProteases;SPPs,Signalpeptidepeptidases;S2Ps,Site-2proteases;HtrA,serineprote ase HtrA;HtpX,putativemembrane-boundzincmetalloprotease;TET,Tetrahedralaminopeptidase(PeptidaseM42family);MlrC-likeprotein, MicrocystinLRdegradationprotein;PEBP,Phosphatidylethanolamine-bindingprotein(putativeproteaseinhibitor);TM,Transmembranedomain; Tat,Twinargininetranslocationpathway. Siddaramappa etal.BMCGenomics 2012, 13 :165 Page8of21 http://www.biomedcentral.com/1471-2164/13/165

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Nmag_0182)andoftheSec-independentTatprotein translocasecomplex( tatC ,Nmag_2050-2051; tatA Nmag_3135).WhiletheTatpathwayiscommonlyused forasmallsubsetofexportedproteinsinbacteria,itisa dominantexportrouteinhalophilicarchaea.Manyof theexportedproteinsaresubsequentlyattachedtothe cellmembranebyalipidanchorand Nab.magadii has 119genesencodinglipid-modifiedTattargetproteins,as detectedbyTatLipoanalysis[64].Furthermore, Nab. magadii containedgenesencodingputativecomponents ofatypeIIsecretionsystem(Nmag_3137-3138)andan archaeosortase(Nmag_2750)forwhich17targetswith PGF_CTERMmotifwereidentified[65].N-glycosylation,glycosyltransferases,andpolysaccharide biosynthesisN-glycosylationinarchaeaandeukaryotesusesdolichol phosphateasthelipidbasefortheassemblyofoligosaccharides[66,67].Glycosyltransferases(GTs)arekey componentsofN-glycosylationinallthreedomainsof life,andthegenomeof Nab magadii contained23genes encodingputativeGTs.BasedonBLASTPanalysison theNCBIdatabaseandthepresenceofconserved domains,thesegeneswereassignedintotheGTA (Nmag_0916,0926,1132,1200,2046,2620,2830,3015, 3273,3275,3807,and4148)andGTB(Nmag_ 0132,0135,0432,0925,2541,3017,3285,3431,3512, 3832,and3843)superfamilies.Oneofthesegenes (Nmag_3015)isinanoperonwithNmag_3011 (hexapeptiderepeat-containingtransferase,192aa), Nmag_3012(aminotransferase,481aa),Nmag_3013(oxidoreductase,340aa),andNmag_3014(nucleotidesugar dehydrogenase,544aa). Nab magadii alsocontained genesencodingaputativeoligosaccharyltransferasesubunit(Nmag_0927, aglB homolog)andadolicholkinaselikeprotein(Nmag_1986).Therefore, Nab magadii appearstohavethegeneticpotentialforN-glycosylation. Severalspeciesofhalophilicarchaeaareknowntoproducecopiousamountsofextracellularpolysaccharides [68].Althoughtransmissionelectronmicroscopic(TEM) imagesshowthepresenceofanexopolysaccharide-like materialaround Nab magadii cells(Figure3A),purificationandbiochemicalanalysesofthismaterialareyetto beaccomplished. Nab magadii containedsixgenesencodingputativepolysaccharidebiosynthesisproteins (Nmag_0147,0922,2457,3122,3272,and3437).Other genesinthegenomethatencodedputativeenzymes involvedinpolysaccharidebiosynthesisincludedsix polysaccharidedeacetylases(Nmag_1899,2045, 2647,3024,3271,and3278),twopolyprenylglycosylphosphotransferases(Nmag_0111and1184,65%identity atthepredictedproteinlevel),anO-antigenpolymerase (Nmag_0143),twoUDP-N-acetylglucosamine2-epimerases (Nmag_0149andNmag_06 76),anacylneuraminate cytidylyltransferase(Nmag_0 148),anO-acetyltransferase (Nmag_0150),aN-acylneurami nate-9-phosphatesynthase (Nmag_0151),andtwocapsulesynthesisproteins (Nmag_1511and3999).Itispossiblethatsomeofthese genesareinvolvedinthebiosynthesisof Nab magadii exopolysaccharide-orcapsulelikematerialidentifiedinthe TEMimages.CoenzymebiosynthesisArchaealmetabolicpathwaysareuniqueanddiverse,in comparisontothoseofeubacteria[24].However,the biosynthesisofseveralcoenzymesinvolvedinarchaeal metabolismhasonlybeenpartiallyunderstood.Recent advancesinthisareaincludethediscoveryofanew hemebiosyntheticpathway[69],furtherunderstanding ofthepathwayofcobalaminbiosynthesis,andthe Figure3 Transmissionelectronmicroscopicimagesof Natrialbamagadii ATCC43099stationaryphasecultures.A .Negativestainofa singlecellwith2%phosphotungsticacid. B and C .Ultrathinsectionsstainedwithuranylacetateandleadcitrate,respectively.Thethreadshaped appendagesinallthreeimagesaremostlikelyflagella.ThelightlystainedmaterialaroundthecellinpanelAisprobablyanexopolysaccharide. Siddaramappa etal.BMCGenomics 2012, 13 :165 Page9of21 http://www.biomedcentral.com/1471-2164/13/165

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reconstructionofacompletepathwayforcoenzymeF420biosynthesisinhaloarchaea.Analysesofgenesputatively involvedincoenzymebiosynthesisin Nab.magadii was performedinlightofthesenewdiscoveries.Thissection describesthegenesrelatedtothebiosynthesisof thiamine,riboflavin,NAD,coenzymeF420,folate,heme, andcobalamin. VitaminB1(thiaminepyrophosphate)isinvolvedin severalmicrobialmetabolicfunctions[70].Prokaryotes haveevolvedelaboratemechanismstoeithersynthesize thisimportantco-factor denovo oracquireitfromtheir niche[71].Thiaminebiosyntheticpathwaysamongprokaryotesareverydiverse[70].Thiaminebiosynthesisis accomplishedbyjoiningtwointermediatemoleculesthat aresynthesizedseparately.Oneofthesemoleculesis hydroxymethylpyrimidinepyrophospate(HMP-PP), whichismadefromaminoimidazoleribotide(AIR,an intermediateofpurinebiosynthesis)usingThiCand ThiD.Theothermoleculeishydroxyethylthiazolephosphate(HET-P),whichinbacteriaisgeneratedbyThiGH andTenIandinvolvesthesulfurcarrierThiS.Thesulfur carrierisactivatedforthiolylationviaC-terminaladenylationcatalyzedbytheN-terminalE1-likedomainof ThiI. Nab.magadii containedagene(Nmag_3460)encodingaputativeThiIandseveralubiquitin-like -grasp foldproteins(Nmag_0567,1914,2668,and2971).However, -graspfoldproteinshavemultiplefunctionsin halophilicarchaea,beinginvolvedinsulfurchemistryas wellasubiquitin-likeproteinmodificationbySAMPylation[56].Thespecific -graspfoldproteinlikelyto participateinthiaminebiosynthesisin Nab.magadii remainsunidentified. Furthermore, Nab.magadii lackedhomologsofThiG, ThiH,andTenIinvolvedinHET-Pbiosynthesisinbacteria. Interestingly, Nab.magadii encodedahomologoftheyeast HET-PsynthaseTHI4(Nmag_2419).However,Nmag_2419 iscurrentlyannotatedasribose1,5-bisphosphateisomerase basedonthefunctionalcharacterizationoftheortholog MJ0601from Methanocaldococcusjannaschii .Incontrast, theorthologofNmag_2419in Pyrococcuskodokaraensis (TK0434)doesnothaveribose-1,5-bisphosphateisomerase activity[72].BiochemicalcharacterizationisrequiredtoascertainthepotentialroleofNmag_2419inthiaminebiosynthesis.Thegenomeof Nab.magadii contained purM (Nmag_1281)and thiC (Nmag_2593)homologs,which werepredictedtobeinvolvedinAIRandHMPbiosynthesis,respectively(Table2). Twodistinctproteins,ThiEandThiN,areknownto joinHMP-PPandHET-Ptogeneratethiamine phosphate. Nab.magadii andotherhalophilicarchaea containbothenzymes.WhereasThiE(Nmag_1811)isa monofunctionalprotein,ThiN(Nmag_1282)existsasa C-terminaldomaininaThiDNfusionprotein.Atthe laststep,thiaminephosphateispredictedtobefurther phosphorylatedtothiaminepyrophosphatebyThiL (Nmag_1515).Therefore,theconversionofAIRto HMP-PPin Nab.magadii appearstobesimilartothe bacterialpathwayandmayinvolveThiCandThiD, whereasHET-Pbiosynthesisinthishaloarchaeon appearstobesimilartotheeukaryoticpathwayandmay involveNmag_2419.Inaddition, Nab magadii containedgenesencodingaHETkinase(ThiM, Nmag_1810,predictedtobeinvolvedinthiaminesalvage)andathiaminetransporter(ThiBPQ,Nmag_460462;ThiB2,Nmag_1940). VitaminB2(riboflavin)istheprecursorofcoenzymes flavinmononucleotide(FMN)andflavinadenine dinucleotide(FAD),whicharecofactorsforseveral biochemicalreactions[73].Mostbacteria,fungi,and plantscansynthesizeriboflavin denovo usingonemoleculeofGTPandtwomoleculesofribulose5-phosphate assubstrates[74].Riboflavinbiosynthesishasbeen describedin Mjannaschii [75].Ingeneral,reduction precedesdeaminationinthearchaealriboflavinbiosynthesispathway,whichappearstobesimilartothefungal pathway.In M.jannaschii ,GTPcyclohydrolaseIII,the firstenzymeoftheriboflavinbiosynthesispathway,producesanarchaeal-specificformylatedintermediatethat requiresasubsequentdeformylationstep[75].However, thehaloarchaealhomologofGTPcyclohydrolaseIIIhas notbeenidentifiedthusfar.Furthermore,riboflavin kinasesofhalophilicarchaeaarehomologoustothoseof bacteria,butareunrelatedto M.jannaschii riboflavinkinase.Conversely,riboflavinsynthasesofhalophilicarchaea arerelatedtothoseof M.jannaschii ,butareunrelatedto bacterialriboflavinsynthases.Overall,sixgenesencoding putativeenzymesoftheriboflavinbiosynthesispathway wereidentifiedin Nab magadii andonlytwoofthese (Nmag_0941and0942)wereclusteredtogether(Table2). VitaminB3(nicotinicacid)isthecentralcomponent ofcoenzymesNADandNADP,whichareessential redoxcofactorsinmetabolism.Likemostbacteria,halophilicarchaeasynthesizeNADfromaspartateviaquinolinate. Nab magadii contained7genes(Nmag_29202922formanoperonandencode nadABC ,Nmag_2823 encodes nadM ,andNmag_1544,2475,and2846encode nadE )thatwerepredictedtobeinvolvedinNAD biosynthesis. CoenzymeF420isinvolvedinmethanogenesis[76,77] andothermetabolicpathways( e.g. ,aflatoxinreduction inmycobacteria[78,79])thatrequirehydridetransfer fromthelow-potentialreduceddeazaflavinF420to substrateswithelectron-deficientringsystems.Furthermore,5-amino-6-(D-ribitylamino)uracilisanintermediateofcoenzymeF420andriboflavinbiosynthesis pathways[76,77].AlthoughcoenyzmeF420hasbeen detectedinsomehalophilicarchaea[80],andcoenyzme F420biosynthesisgeneshavebeenidentifiedamongtheSiddaramappa etal.BMCGenomics 2012, 13 :165 Page10of21 http://www.biomedcentral.com/1471-2164/13/165

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Table2 Natrialbamagadii ATCC43099genesinvolvedinthebiosynthesisofvariousvitaminsandsirohemeLocustag,Protein,pI, (Asp+Glu)/(Arg+Lys) AnnotationClosesthomolog (locustag,protein,identity) Thiamine Nmag_0434,309aa,4.31,55/25HTHdomainprotein/thiamine-phosphatesynthase(ThiN)Htur_2542,307aa,81% Nmag_0460,387aa,3.72,92/17ABC-typethiaminetransportersubstrate-bindingprotein(ThiB)Htur_2516,370aa,68% Nmag_0461,663aa,4.87,50/34ABC-typethiaminetransporterpermeaseprotein(ThiP)Htur_2515,634aa,84% Nmag_0462,363aa,4.58,55/29ABC-typethiaminetransporterATPaseprotein(ThiQ)Htur_2514,405aa,68% Nmag_0567,88aa,4.48,18/8MoaDfamilyprotein(MoaD)HacjB3_04780,90aa,55% Nmag_0869,219aa,4.50,42/16Thiaminebiosynthesis/salvageprotein(TenA)Htur_2707,219aa,85% Nmag_1281,345aa,4.16,62/19Aminoimidazoleribotidesynthaserelatedprotein(PurM)Htur_3233,346aa,77% Nmag_1282,461aa,4.33,76/33Phosphomethylpyrimidinekinase/thiamine-phosphatesynthase(ThiDN)Htur_3232,453aa,79% Nmag_1394,278aa,4.25,50/21SAMP-activatingenzymeE1(UbaA)Htur_3268,280aa,86% Nmag_1515,295aa,4.17,54/18Thiamine-monophosphatekinase(ThiL)Htur_3484,307aa,71% Nmag_1810,280aa,4.04,49/14Hydroxyethylthiazolekinase(ThiM)Htur_0247,279aa,75% Nmag_1811,221aa,4.33,35/15Thiamine-phosphatesynthase(ThiE)Htur_0246,222aa,76% Nmag_1914,87aa,4.49,19/10MoaDfamilyprotein(MoaD)Htur_0025,78aa,72% Nmag_1940,383aa,3.94,85/23ABC-typethiaminetransportersubstrate-bindingprotein(ThiB)Halxa_1431,376aa,53% Nmag_2413,299aa,4.28,55/21HTHdomainprotein/thiamine-phosphatesynthase(ThiN)Htur_1469,299aa,85% Nmag_2419,309aa,4.31,54/22THI4-relatedputativethiazolebiosynthesisenzymeHtur_1462,310aa,93% Nmag_2593,503aa,4.37,98/39Hydroxymethylpyrimidinephosphatesynthase(ThiC)Htur_1332,480aa,88% Nmag_2668,111aa,4.09,21/8MoaDfamilyprotein(MoaD)Htur_0699,96aa,78% Nmag_2971,94aa,3.91,24/6MoaDfamilyprotein(MoaD)Htur_0580,93aa,85% Nmag_3460,392aa,4.60,68/38Thiamine/thiouridinebiosynthesisprotein(ThiI)Htur_1671,392aa,78% Nmag_3812,278aa,4.45,45/23SAMP-activatingenzymeE1(UbaA)Hlac_0421,270aa,55% Riboflavin Nmag_0941,235aa,4.45,50/22CTP-dependentriboflavinkinase(RibK)Htur_2982,235aa,87% Nmag_0942,243aa,4.29,45/143,4-dihydroxy-2-butanone4-phosphatesynthase(RibB)Htur_2983,243aa,75% Nmag_1403,208aa,4.25,43/16Riboflavinsynthase(RibE)Htur_3261,201aa,83% Nmag_3078,220aa,4.42,42/222,5-diamino-6-(ribosylamino)-4(3H)-pyrimidinone5'-phosphatereductase(RibG)Htur_5200,220aa,78% Nmag_3262,143aa,4.08,25/96,7-dimethyl-8-ribityllumazinesynthase(RibH)Htur_1120,152aa,83% Nmag_3329,254aa,4.44,44/18GTPcyclohydrolaseIII(Gch3)Htur_1598,253aa,86% NAD Nmag_1544,332aa,4.82,55/39NAD(+)synthase(NadE)Htur_3513,302aa,65% Nmag_2475,284aa,4.26,55/21NAD(+)synthase(NadE)Hbor_04860,277aa,56% Nmag_2823,172aa,4.46,28/13Nicotinamide-nucleotideadenylyltransferase(NadM)Htur_0861,172aa,90% Nmag_2846,264aa,4.53,46/19NAD(+)synthase(NadE)Halxa_1654,264aa,82% Nmag_2920,300aa,4.84,48/30Nicotinate-nucleotidepyrophosphorylase(NadC)Halxa_1866,271aa,78% Nmag_2921,531aa,4.42,101/47L-aspartateoxidase(NadB)Htur_0529,532aa,81% Nmag_2922,377aa,4.21,80/26QuinolinatesynthaseA(NadA)Htur_0530,377aa,89% CoenzymeF420 Nmag_0541,341aa,4.27,67/32ProbableF420-dependentoxidoreductaseHtur_1978,357aa,78% Nmag_0613,330aa,4.00,55/16LPPG:FO2-phospho-L-lactatetransferase(CofD)Halxa_2708,330aa,85% Nmag_1056,336aa,4.28,63/27ProbableF420-dependentoxidoreductaseHtur_2908,333aa,84% Nmag_1142,318aa,4.15,59/13ProbableF420-dependentoxidoreductaseHalxa_4117,318aa,75% Nmag_1562,222aa,4.30,43/16F420H2:NADP+oxidoreductase(NpdG)Htur_3495,222aa,84% Nmag_1581,260aa,4.22,53/23F420-O:gamma-glutamylligase(CofE)Halxa_3409,258aa,85% Siddaramappa etal.BMCGenomics 2012, 13 :165 Page11of21 http://www.biomedcentral.com/1471-2164/13/165

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Table2 Natrialbamagadii ATCC43099genesinvolvedinthebiosynthesisofvariousvitaminsandsiroheme (Continued)Nmag_2083,133aa,4.68,29/16ProbableF420-dependentoxidoreductaseHbor_19440,133aa,79% Nmag_2231,321aa,4.34,64/20ProbableF420-dependentoxidoreductaseSlin_5283,326aa,55% Nmag_2631,248aa,4.56,37/182-phospho-L-lactateguanylyltransferase(CofC)Halxa_1898,226aa,63% Nmag_2633,389aa,4.25,75/30FOsynthasesubunit1(CofG)Halxa_1896,391aa,80% Nmag_2637,459aa,4.61,84/42FOsynthasesubunit2(CofH)Halxa_1693,456aa,86% Nmag_3301,318aa,4.35,57/13ProbableF420-dependentoxidoreductaseHalxa_2620,320aa,83% Folate Nmag_0002,427aa,4.17,83/31Dihydropteroatesynthase(FolP)Htur_1844,397aa,68% Nmag_2300,297aa,4.27,57/24Methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolatecyclohydrolase(FolD) Htur_1699,297aa,92% Nmag_2554,840aa,4.37,157/68Folylpolyglutamatesynthase/7,8-dihydropteroate reductase/dihydropteroatesynthase(FolCP) Htur_3284,847aa,71% Nmag_2792,593aa,4.49,113/61AminodeoxychorismatesynthasecomponentII(PabB)Htur_1366,537aa,70% Nmag_2793,298aa,4.28,58/21AminodeoxychorismatesynthasecomponentI(PabA)Htur_1365,232aa,62% Nmag_2794,310aa,4.42,52/25Aminodeoxychorismatelyase(PabC)Halxa_3973,295aa,75% Nmag_2853,310aa,4.44,68/29GTPcyclohydrolaseI,archaeal-type(MptA)Halxa_1887,311aa,93% Nmag_2988,183aa,4.17,41/11Dihydrofolatereductase(FolA)Htur_1681,181aa,79% Heme/Siroheme/Cobalamin(commonearlypathwaygenes) Nmag_0445,573aa,4.39,131/57Glutamyl-tRNAsynthetase(GltS)Htur_2537,574aa,83% Nmag_1219,457aa,4.17,89/34Glutamyl-tRNAreductase(HemA)Htur_3246,444aa,71% Nmag_1220,238aa,4.49,43/26Precorrin-2oxidase(SirC)[mayalsohaveferrochelataseactivity]Htur_3247,218aa,69% Nmag_2012,242aa,4.00,50/15Uroporphyrinogen-IIIsynthase(HemD)Htur_3677,255aa,74% Nmag_2013,283aa,4.39,50/22Uroporphyrin-IIIC-methyltransferase(SirA)Htur_3676,272aa,87% Nmag_2014,378aa,4.54,77/41Porphobilinogendeaminase(HemC)Htur_3675,380aa,85% Nmag_2017,424aa,4.57,61/29Glutamate-1-semialdehyde2,1-aminomutase(HemL)Halxa_1518,424aa,88% Nmag_2019,333aa,4.69,60/34Porphobilinogensynthase(HemB)Htur_0188,341aa,81% Cobalamin Nmag_0392,177aa,4.34,39/16ATP:cob(I)alaminadenosyltransferase(PudO)Halxa_0779,177aa,88% Nmag_0846,449aa,4.33,90/34CobWdomainproteinHtur_2995,410aa,61% Nmag_0987,340aa,5.33,31/26Adenosylcobinamide-phosphatesynthase(CbiB)Htur_2833,378aa,66% Nmag_0988,253aa,7.90,16/17Cobalamin5'-phosphatesynthase(CobS)Htur_2834,253aa,71% Nmag_0989,243aa,4.50,46/21GTP:adenosylcobinamide-phosphateguanylyltransferase(CobY)Htur_2835,202aa,55% Nmag_0990,388aa,4.08,67/22Nicotinate-nucleotide-dimethylbenzimidazolephosphoribosyltransferase(CobT)Htur_2836,349aa,72% Nmag_0991,383aa,4.46,73/38L-threonine-O-3-phosphatedecarboxylase(CobD)Htur_2837,365aa,66% Nmag_0992,288aa,4.38,39/16Adenosylcobinamideamidohydrolase(CbiZ)Htur_2838,255aa,61% Nmag_1837,391aa,3.76,ABC-typecobalamintransportersubstrate-bindingprotein(BtuF)Hmuk_2791,377aa,42% Nmag_2215,400aa,4.40,83/36CobWdomainproteinHtur_0421,401aa,82% Nmag_2216,448aa,4.43,98/37CobWdomainproteinHtur_0422,442aa,71% Nmag_3068,563aa,4.59,85/47Adenosylcobyricacidsynthase(CbiP)Htur_5212,538aa,71% Nmag_3073,239aa,4.56,45/19cob(I)alaminadenosyltransferase(CobA)Htur_5207,228aa,79% Nmag_3175,453aa,4.37,79/31Cobyrinicacida,c-diamidesynthase(CbiA)Htur_0999,437aa,76% Nmag_3212,301aa,4.27,66/22sirohydrochlorincobaltochelatase(CbiX)[mayinsteadbeaferrochelatase]Htur_1053,299aa,88% Nmag_3605,400aa,3.57,92/13ABC-typecobalamintransportersubstrate-bindingprotein(BtuF)Halxa_3346,406aa,50% Nmag_3606,367aa,6.11,19/15ABC-typecobalamintransporterpermeaseprotein(BtuC)Htur_1831,376aa,78% Nmag_3607,536aa,4.17,95/34ABC-typecobalamintransporterATPaseprotein(BtuD)Halxa_3348,440aa,63% Nmag_4133,424aa,4.01,108/25CobWdomainproteinNP2376A,428aa,81% Siddaramappa etal.BMCGenomics 2012, 13 :165 Page12of21 http://www.biomedcentral.com/1471-2164/13/165

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methanogens[81],itsprecisefunctioninhalophilicarchaeaisunknown. Nab magadii andotherhalophilicarchaeacontainedseveralgenesencodingputative enzymesofthecoenyzmeF420biosynthesispathway. Thesegeneswereidentifiedbasedonthepresenceof theirhomologsamongthemethanogens.Furthermore, usingSIMBALanalysis[82],coenyzmeF420dependent enzymeshavebeenpredictedinhalophilicarchaea( e. g. ,NP1902A,TIGRFam04024,D.Haft,personalcommunication). Nab.magadii predictedproteinswithan assignedF420-relatedTIGRFamareshowninTable2.A possiblefunctionofcoenzymeF420intherespiratory chainof Nab magadii isdiscussedlaterinthispaper. Tetrahydrofolateparticipatesinanumberofbiochemicalreactionsandreducedfolatecofactorsarerequired forthebiosynthesisofavarietyofmoleculesinboth prokaryotesandeukaryotes[83,84].Theproductionof folateinvolvesseveralenzymescatalyzingthepterinand para-aminobenzoicacidbranchesofthepathway[85]. Nab.magadii genesputativelyinvolvedinfolatebiosynthesisweregenerallysimilartothosedescribedin Nmn. pharaonis ,includingthearchaeal-typeGTPcyclohydrolase(Nmag_2853). Nab.magadii containeda pabABC operon(Nmag_2792-2794)andthehomologsofthese geneswerepredictedtobeinvolvedinpara-aminobenzoatebiosynthesisin Nmn,pharaonis [24]. Nab magadii alsocontainedgenesencodingadihydropteroatesynthase,afuseddihydropteroate-dihydrofolate synthase,adihydrofolatereductase,andamethenyltetrahydrofolatecyclohydrolase(Table2).Thelatterenzyme ispredictedtoparticipateintheconversionofthe C1metaboliteattachedtotetrahydrofolate.However, noneoftheothergenesencodingC1-converting enzymesidentifiedin Haloquadratum or Haloarcula werefoundin Nab.magadii and Nmn.pharaonis Furthermore, Nab.magadii containedthreegenesencodingputativeenzymesofthelaterstagesofthefolatebiosynthesispathway(FolP,Nmag_0002;FolC-PrdFolP,Nmag_2554;FolA,Nmag_2988).Nevertheless, genesencodingthebacterialhomologsofFolQBK, theatypicalFolQdescribedin Desulfovibrio ,orthe alternativepathwaybypassingFolQB(describedin Plasmodium andseveralbacteria)wereabsentin Nab. magadii and Nmn.pharaonis .Therefore,precise mechanismsoffolatebiosynthesisinthesespeciesremaintobediscovered. Environmentalbacteriautilizeavarietyofredoxmoleculessuchasporphyrinsandothermodifiedtetrapyrroleslikeheme,siroheme,andadenosylcobalaminfor catalysis,energytransfer,andsignaltransduction[86]. Thesetetrapyrrolesaresynthesized denovo usinga branchedpathwayandaminolevulinicacidastheprecursor[87,88].Inmostprokaryotes,theconversionof glutamatetoaminolevulinicacidiscatalyzedbyglutamyl-tRNAsynthetase,glutamyl-tRNAreductase,and glutamate-1-semialdehydeaminotransferase.Twomoleculesofaminolevulinicacidarecondensedbytheaction porphobilinogensynthasetoformporphobilinogen.Four moleculesofporphobilinogenarepolymerizedbytheactionoftheporphobilinogendeaminasetoformthetetrapyrrolehydroxymethylbilane.UroporphyrinogenIII methyltransferasecyclizeshydroxymethylbilanetoproduceuroporphyrinogenIII.UroporphyrinogenIIIisconvertedtoprecorrin-2inthebiosyntheticpathwayof adenosylcobalaminandsiroheme,whichwasrecently foundtobeanintermediateofhemebiosynthesis(see below). Thecompletepathwayforthebiosynthesisofadenosylcobalaminfromprecorrin-2involvestwomajor branchesandseveralenzymes[89,90],someofwhich arearchaea-specific( e.g. cobY cbiZ )[91,92].Halophilic archaea( e.g. Nmn.pharaonis )usethe “ anaerobic ” branch,whichischaracterizedbyanoxygen-independentringcontractionprocess( cibG )[93].However,ithas beenshownthat Halobacterium synthesizescobalamin denovounderaerobicconditions[91].The “ anaerobic ” branchisalsocharacterizedbyearlycobaltinsertionand Nmn.pharaonis hashomologsoftheATP-independent earlycobaltchelatase(CbiX)from Bacillushalodurans and Archaeoglobusfulgidus [94,95].Inthe “ anaerobic ” branch,sevenarchaealenzymesareknowntobe involvedintheconversionofprecorrin-2intocobyrinic acid( cbiL cbiH cbiF cbiG cbiE cbiT ,and cbiC ),but twopathwaygaps(correspondingto cbiD/cobF and cbiJ/ cobK )stillremain.Asetof11genesisknowntobe involvedinconversionofcobyrinicacidintoadenosylcobalamin( cbiA cobA,pduO,cbiP cbiZ cbiB cobY cobS cobC cobD, and cobT ). Basedongenomeanalyses,itappearedthat Nab magadii wasincapableof denovo cobalaminbiosynthesissinceitlackedthegenesencodingenzymesfor conversionofprecorrin-2intocobyrinicacid.Thisisin Table2 Natrialbamagadii ATCC43099genesinvolvedinthebiosynthesisofvariousvitaminsandsiroheme (Continued)Heme/Siroheme Nmag_0636,283aa,9.00,14/17HemeAsynthase(CtaA)Halxa_2735,289aa,70% Nmag_1221,363aa,4.47,74/34Sirohemedecarboxylase(AhbB/NirGH)Htur_3248,360aa,83% Nmag_2302,495aa,5.92,34/29ProtohemeIXgeranylgeranyltransferase(CtaB)Htur_1181,482aa,79% Nmag_2894,349aa,4.33,65/29Sirohemedecarboxylase(AhbA/NirDL)Htur_0511,346aa,77% Siddaramappa etal.BMCGenomics 2012, 13 :165 Page13of21 http://www.biomedcentral.com/1471-2164/13/165

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contrastto Htg.turkmenica ,whichwaspredictedtobe capableof denovo cobalaminbiosynthesissinceit containedthecorrespondinggenes.However, Nab magadii waspredictedtobecapableofcorrinoidsalvage sinceitcontainedageneencodingaputativecorrinoid transporter. Nab magadii alsocontainedasetofgenes thatwerepredictedtobeinvolvedintheconversionof cobyrinicacidintoadenosylcobalamin,includingagene ( cbiZ )thatisspecifictothearchaealcorrinoidsalvage pathway(Table2). Thehemebiosynthesispathwayinarchaeainvolving uroporphyrinogenIII,precorrin-2,andsirohemeappears tobesimilartothatof Desulfovibrio [69].Conversionof uroporphyrinogenIIIintosirohemerequiresthreefunctions(methylationbySirA,ironchelation,andoxidation bySirC).Theenzymecatalyzingironchelationisunknownsincethehaloarchaealprecorrin-2dehydrogenase mightbemonofunctional(asin Bacillusmegaterium SirC)ormightalsobeaferrochelatase(asintrifunctional Escherichiacoli CysGorthebifunctionalyeast MET8).Fromcomparisonof Nab magadii withother halophilicarchaea,anotherpossibilityemerges:ironchelationmaybeperformedbyoneoftheproteinsannotated asCbiX-typecobaltchelatase. Nmn.pharaonis hasthree cbiX paralogs( cbiX1 ,NP1108A; cbiX2 ,NP1588A; cbiX3 NP0734A),twoofwhichhavecloselyrelatedorthologsin nearlyallotherhalophilicarchaea. Htg.turkmenika ,probablycapableof denovo cobalaminbiosynthesis,hasorthologsofboth cbiX1 and cbiX2 .However, Nab.magadii whichispredictedtobeincapableof denovo cobalamin biosynthesis,andthereforeexpectedtolacktheseproposedearlycobaltchelatases,surprisinglycontaineda cbiX2 ortholog(Nmag_3212).Itispossiblethat cbiX2 functionsasaferrochelataseduringsiroheme/heme biosynthesisratherthanasacobaltochelataseduring denovo cobalaminbiosynthesis. Didecarboxysiroheme,acommonintermediateof hemeandheme d 1biosynthesis,isgeneratedbythedecarboxylationofsirohemeontheC12andC18acetyl groups[69].Sirohemedecarboxylaseactivityisattributedtothe nirDLGH geneset,whichisrepresentedbya pairoftwo-domainproteinsinhalophilicarchaea (AhbA/NirDL,Nmag_2894;AhbB/NirGH,Nmag_1221). Heme d 1isacoenzymeofdissimilatorynitritereductase (alsocalledcytochrome cd1)andisnotrequiredby organismslackingthisenzyme.Thelaststepsofheme biosynthesisincludetheremovalofacetylsidechainsof Fe-coproporphyrinbyAhbC(encodedbyMbar_A1793 in Methanosarcinabarkeri )andtheoxidativedecarboxylationofhemebyAhbD(encodedbyDVU_0855in Desulfovibrio )[69][geneassignments:M.Warren,personalcommunication,includingtheinformationthat SupplementalFigure3showsthesequenceof ahbC and not ahbD ].OrthologsencodingAhbCandAhbDwere presentin Nmn.pharaonis (NP1542AandNP1546A) and Htg.turkmenica (Htur_1726and1728),butnot in Nab.magadii .Thepresenceof ahbC and ahbD in somehalophilicarchaeabutnotinothersisbelieved tobeduetometabolichete rogeneityratherthanincompletehemebiosynthesis.Conversionofheme (alsocalledhemeB)intohemeAin Nab.magadii waspredictedtobecatalyzedbyCtaAandCtaB homologs( ctaA ,Nmag_0636; ctaB ,Nmag_2302). VitaminH,commonlyknownasbiotin,actsasa coenzymeinseveralenzyme-catalyzedcarboxylation anddecarboxylationreactions[96].Mostbacteria cansynthesizebiotin denovo usingpimelicacidasa precursor,andsomeothershaveevolvedmechanismsforimportingthisesse ntialcofactorfromtheir naturalenvironments[97,98].Whereas Nab magadii isabiotinauxotroph, Nmn.pharaonis isabiotin prototrophandthegenomeofthishaloalkaliphilic archaeonhasbeenshowntocontainatleastthree genesputativelyinvolvedinthebiosynthesisofbiotin[24].Theabsenceofgenesforthebiosynthesisof biotinin Nab magadii wasapparentfromtheanalysesofitsgenomesequence.However,thelarge chromosomeof Nab.magadii containedalocusencodingaputativebiotintransporter(BioYMN, Nmag_0886-0888),whichmayfacilitatetheuptakeof biotinfromtheenvironment.Metabolicandco-factorcompetencyNutritionalrequirementsofhalophilicarchaeainthe laboratoryareasdiverseastheirobservedphenotypes, suggestingthatthemetabolicpathwaysintheseorganismsarequiteintricate[24,99].Theanalysisofthegenomesequenceprovidedanunprecedentedopportunity tocomprehendthemetabolicversatilityof Nab magadii .Additionalfile4:TableS4containsacomprehensive listofgenespredictedtobeinvolvedinadiversearrayof functions.Furthermore,gene sencodingputativeenzymes forarchaealmodifiedpathwaysofgluconeogenesisandglycolysisaswellasthoseofribosemetabolismandthetricarboxylicacidcyclewerepresentin Nab magadii .Genes thatencodedputativeenzymesforglycerolutilization,aromaticaminoacidcatabolism,ureagenesis,andureadegradationwerealsoidentifiedin Nab magadii .Otherputative metabolicfeaturesof Nab magadii includedxyloseisomerases,analphaamylase,amethylglyoxalsynthase,sulfatases,achloritedismutase,sarcosineoxidases,and aldehydedehydrogenases(Additionalfile4:TableS4). Molybdenumcofactor(MoCo)isessentialforthe functioningofmolybdoenzymessuchasdimethylsulfoxideandtrimethylamine-N-oxidereductases,formate dehydrogenases,andnitratereductases[24,100].Molybdopterinisthedithiolene-containingtricyclicmoiety foundwithinMoCoofallmolybdoenzymesexceptSiddaramappa etal.BMCGenomics 2012, 13 :165 Page14of21 http://www.biomedcentral.com/1471-2164/13/165

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nitrogenases[101].Inbacteria,genesofthe moa,mob, mod,moe ,and mog locihavebeenimplicatedinthebiosynthesisofMoCousingGTPasthesubstrate[102].The largechromosomeof Nab magadii contained9genes encodingMoCobiosynthesisfunctions(Additionalfile4: TableS4).Itisuncertainifthissubsetofgenesissufficient forMoCobiosynthesisin Nab magadii andbiochemical studiesarerequiredtotestwhetherthishaloarchaeonis molybdenum-dependent. Thehaloarchaeon Haloarculamarismortui converts acetyl-CoAtoglyoxylateviathekeyintermediatemethylaspartate[103].Glyoxylateiscondensedwithasecond moleculeofacetyl-CoAtoformmalate,whichisan intermediateofthetricarboxylicacidcycle.Malatecan subsequentlybeconvertedtooxaloacetate,whichisused byphosphoenolpyruvatecarboxykinaseforgluconeogenesis.In Nab.magadii ,activitiesoftheenzymesofthe methylaspartatecycle,butnotthoseofthekeyenzymes oftheglyoxylatecycle,weredetected[103].Anoperon (Nmag_3333-3338)encodingputativehomologsofthe methylaspartatecycleandageneencodingaputative phosphoenolpyruvatecarboxykinase(Nmag_3507)were presentin Nab magadii .Thesquarearchaeon Haloquadratumwalsbyi containsagene(HQ2709A)encoding aphosphoenolpyruvate-dependentphosphotransferase system(PTS)thatispredictedtobeinvolvedinthe phosphorylationofdihydroxyacetone[104].Homologs ofHQ2709AandgenesencodingadditionalPTScomponentswerepresentin Htg.turkmenica Hfx.volcanii andseveralotherhaloarchaealgenomes.However, Nab. magadii and Nmn pharaonis lackedhomologsofthese genesencodingPTScomponents.RespiratorychainandATPsynthesisRunningaproton-driven,energy-conservingATP synthaseathighextracellularpHisanobviouschallenge.Energycouplingofsodiumionsinsteadofprotons wasproposedtobeanadaptationtoalkaliphilicgrowth conditionsandanATPsynthasedrivenbyNa+isthe hallmarkofsuchanadaptation. Nab.magadii hada clusterofeightgenesthatformthe atpHIKECFAB operonencodingputativeATPsynthasesubunits (Nmag_1370-1377)andanunlinked atpD homolog (Nmag_1366).Similargeneclusterswerefoundin severalhalophilicarchaea.IonspecificityoftheATP synthaseisdeterminedbythec-ring,whichisencoded bythe atpK gene(Nmag_1375)forA-typeATP synthases. Nab.magadii mayhaveaproton-drivenATP synthasesinceitspredictedAtpKlacksthesequencesignatureofNa+-dependentATPsynthases[105].Instead, withintheion-determiningregionofAtpK,thesequence (PETLVIL)isidenticaltothatoftheproton-drivenATP synthasesfrom Hfx.volcanii [106], Hbt.salinarum [107], and Nmn.pharaonis [23]. Reductionofoxygenandtheassociatedprotoncoupledelectrontransfer(respiration)istheprimary sourceofenergyamongaerobicorganisms.Respiratory complexes,whichincludeavarietyofcytochromesand terminaloxidases,areessentialcomponentsofthis process.Biochemicalandcomparativegenomicanalyses oftheelectrontransportchainof Nmn.pharaonis have revealedseveralnovelfeatures,includingageneencodingatypeIINADHdehydrogenase(NP3508A)[23,108]. AhomologofNP3508Ain Acidianusambivalens was proposedtobeinvolvedinNADHreoxidation,feeding intothelipid-solublequinonepool[109].Ahomolog ofNP3508Awasalsopresentin Nab magadii (Nmag_0301)andseveralotherhalophilicarchaea. Nab magadii alsocontainedgenesencodingaputative nuo complex(Nmag_3245-3255),whichwassimilartothe mitochrondrialNADHdehydrogenase(complexI). Although13 nuo clustersubunitswereconservedamong halophilicarchaeaand E.coli ,the nuoEFG subcomplex, whichisinvolvedinacceptingNADH,wasmissingin halophilicarchaea[23].Furthermore,involvementofa typeIcomplexinNADHreoxidationhasbeenruledout in Hbt.salinarum [110].Itisspeculatedthatreduced coenzymeF420,whichissimilartoNADHinitsredox potential,mayinteractwiththe nuo complexinhalophilic archaea.InadditiontotheNADHdehydrogenases, Nab. magadii andotherhalophilicarchaeaarepredictedto encodeasuccinatedehydrogenasethatmayoxidize succinateandreducethequinonepooloftheelectron transportchain. Anumberofcytochromesinvolvedinrespiratory electrontransporthavebeencharacterizedamongthe archaea[12,111].Terminaloxidases,alsoknownasoxygenreductases,canacceptelectronsfromavarietyof donorsandreducedioxygentowater.Thelarge chromosomeof Nab magadii containedlociencoding putativecytochrome c -typeterminaloxidasesubunitsI andII(Nmag_0263-0264)andcytochromeubiquinol oxidasesubunitsIandII(Nmag_1036-1037).Furthermore,pNMAG02containedanoperonencodingputativecytochromeubiquinoloxidasesubunitsIandII (Nmag_4038-4039)thatwererelatedtotheproteins encodedbyNmag_1036-1037(46%identityatthe proteinlevel).ThehomologsofNmag_0263-0264and Nmag_1036-1037werepresentin Htg.turkmenica (Htur_2248-2249andHtur_4570-4571,respectively), butnotin Nmn.pharaonis .Two cbaDBAC operons (plusamonocistronic cbaE )encodingputativecytochrome ba3terminaloxidasecomplexeswereidentified inpNMAG01(Nmag_3754-3758and3802 – 3805), andtheseoperonsappearedtoberelatedtoeachother (38-65%identityatthepredictedproteinlevel).The homologsoftheseORFswerepresentin Htg.turkmenica (Htur_0462-0466)and Nmn.pharaonis (NP2960A-Siddaramappa etal.BMCGenomics 2012, 13 :165 Page15of21 http://www.biomedcentral.com/1471-2164/13/165

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NP2968A),thelatterofwhichhavebeenfunctionally characterized[112].Halocyaninsarepredictedtoactas one-electroncarrierstotheterminaloxidasesinhalophilicarchaea[23,108].Thispredictionissupportedby theobservationthat cbaD isfusedtohalocyaninin Hbt. salinarum and Har.marismortui [113]. Nab magadii containedseveralgenesencodingputativehalocyaninlikeproteins(Nmag_2576,2424,0446,0741,3725,1878, 3525,1774,and3800).Halocyaninsarecoupledtothe reducedquinonepoolbythecytochrome bc1complex. Althoughgenesencodingacytochrome bc1complexare presentin Hbt.salinarum Hfx.volcanii ,andseveral otherhalophilicarchaea,theywereabsentin Htg.turkmenica Nmn.pharaonis ,and Nab.magadii .Theelectrontransferfromthereducedlipid-solublequinone pooltohalocyaninremainsunresolvedinspeciesthat lackacytochrome bc1complex.Basedongenomecomparisons,itappearedthattherespiratorychainof Nab magadii wasmoresimilartothatof Htg.turkmenica thanto Nmn.pharaonis Othergenesencodingputativecytochrome-relatedproteinsinthelargechromosomeof Nab magadii included Nmag_0636(cytochromeoxidaseassemblyprotein), Nmag_1972(cytochromeP450),andNmag_3057(cytochrome c biogenesisprotein).Thesmallchromosome pNMAG01containedageneencodingaputativecytochrome c biogenesisprotein(Nmag_3708)thathad64% identitytotheproteinencodedbyNmag_3057. Nab magadii alsocontainedgenes(Nmag_2430-2432)encodingaputativesulfurutilizationfactor(SUF)system,which wasshowntobeimportantforFe-Sclusterbiogenesis duringstressin E.coli [114].Othergenespredictedtoparticipateinbioenergeticconversionin Nab magadii includethoseencodingelectrontransferflavoprotein subunits(Nmag_0482-0483and1388 – 1389),SCO1/SenC electrontransportproteins(Nmag_0793,3059,and3710), andaredoxindomainprotein(Nmag_3709).Signaltransduction,motility,andtranscriptional regulationTwo-componentsignaltransductionsystemsconsisting ofahistidinekinase(HK)andaresponseregulator(RR) constituteoneofthemostfrequentlyencounteredbacterialandarchaealcommunicationcircuits[115]. Nab magadii containedapairofgenes(Nmag_3535-3536) encodingputativeHK-RRproteins. Nab magadii also containedtwopairsofgenes(Nmag_2042-2043and Nmag_3740-3741)encodingputativeHK-RRproteins withanadditionalRRdomainintheN-terminusofthe predictedHKprotein.Furthermore,anothersetofgenes (Nmag_1129-1130)encodingaHK-likeprotein,which wasdistantlyrelatedtoCheAof Nmn.pharaonis ,anda putativeproteincontainingaRRdomainwasalsopredictedin Nab magadii .Interestingly, Nab.magadii also contained15genesencodingputativeHKproteinswithoutacognateRRgene(Nmag_0168,0323,0435,1296, 1909,2062,3106,3188,3216,3297,3954,4064,4101, 4114,and4119)and11genesencodingputativeRRproteinswithoutacognateHKgene(Nmag_0951,1095, 1797,2147,2692,2693,2877,3151,3379,3389,and3679). Halobacterialperceptionofandresponsetophysical stimulussuchaslight(phototaxis)ismediatedbyphotoreceptors[116].Thegenomeof Nab.magadii contained asinglerhodopsingene(Nmag_2582,thechloridepump halorhodopsin). Nab.magadii alsocontained3genes (Nmag_1701,2879,and2881)encodingdistanthomologsofrhodopsinsthatwererelatedtoeachother(3649%sequenceidentity).Theywerealsorelatedto NP1758Afrom Nmn.pharaonis (33%-42%sequence identity)andpredictedtoencodedistantrhodopsin homologsthatlacktheLysresidueinvolvedincovalent retinalattachment.Whethertheserhodopsinhomologs interactwithretinalnoncovalently,oriftheyinteract withretinalatall,isunknown.Twoofthethreegenes encodingretinalhomologsin Nab.magadii (Nmag_2879 and2881)werelocatedadjacenttogenes(Nmag_2880 and2882)encodingputativemethyl-acceptingchemotactictransducerproteins.In Nmn.pharaonis ,NP1758A andNP3132A,whicharehomologsofNmag_2879and 2881,werealsofoundadjacenttogenes(NP1756Aand NP3134A,respectively)encodingputativemethylacceptingchemotactictransducerproteins[23].Therefore,thisgroupofdistantrhodopsinhomologsmaybe involvedinperceptionofexternalstimuli,althoughit remainstobedeterminediftheyareinvolvedinlight perception.Although Htg.turkmenica lackedgenesencodingbacteriorhodopsinandhalorhodopsin,itcontainedasinglegene(Htur_3663)thatappearedtobea distanthomologofNmag_2879and2881.Furthermore, similarto Nmn.pharaonis Nab.magadii lackedagene encodingaprotonpumpbacteriorhodopsin.However, Nab.magadii containedalocusencodingputativephytoenedesaturase,UbiAprenyltr ansferase,carotenebiosynthesisprotein,andphytoenesynthase(Nmag_1001-1004) aswellasageneencodingapu tativesqualene/phytoene synthase(Nmag_2309,unrelatedtoNmag_1004). Twounrelatedenzymefamiliesareusedforthe cleavageof -caroteneintoretinalinhalophilicarchaea. Distantparalogs,whichbelongtooneofthese -carotenase enzymefamilies,havebeenidentifiedin Hbt.salinarum anddesignatedBrpandBlh[117].Althoughhomologsof brp and blh werepresentin Nmn.pharaonis (NP0650A andNP0206A,respectively),theywereabsentin Nab. magadii .However, Nab.magadiicontainedahomolog (Nmag_4083)of Hqr.walsbyi HQ2020A,whichwas predictedtoencodeadistinct -carotenaseunrelatedto thosementionedabove[104].Interestingly, Htg.turkmenica lackedhomologsof brp and blh aswellasHQ2020A,whichSiddaramappa etal.BMCGenomics 2012, 13 :165 Page16of21 http://www.biomedcentral.com/1471-2164/13/165

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isconsistentwiththeabsence ofallcanonicalrhodopsinsin thisorganism. Microbialresponsetochemicalstimulus(chemotaxis, movementtowardnutrientsorawayfromstressors)is mediatedbychemoreceptors[118].Thelargechromosomeof Nab.magadii containedtwolociencoding putativemotilityandsignaltransductionfunctions. Oneofthemcontainedexclusively “ che genes ” ina cheYBACCDR operon(Nmag_3145-3151),whichisprecededbytwodivergentlytranscribedanddistantly related cheW genes(Nmag_3152-3153).The cheYBACCDR operonencodesaverylongsignaltransductionhistidine kinase(CheA,1576aminoacids),aresponseregulatorreceiverprotein(CheY),aCheR-typeMCPmethyltransferase, andaresponseregulatorreceiver-modulatedmethylesterase(CheB).WhileCheDhasbeenreportedtofunctionas glutaminedeamidaseinsomeorganismsorasmethylesteraseinothers,CheBfunctionsasboth[119].Asimilarlocus wasalsopresentin Htg.turkmenica (Htur_0954-0962)and basedonpredictedproteinhomology,itappearedthatthe twolociwereevolutionarilyverycloselylinked.Highly similargeneclusterswerealsofoundinthegenomesof Halopigerxanaduensis Natrinemapellirubrum ,and Natronobacteriumgregoryi .Thesecondlocus(Nmag_28592889)contained “ che genes ” alongwith “ fla genes ” encodingflagellinbiosynthesisandassemblyfunctions(see below).The “ che genes ” inthislocusencodeputative CheA,CheY,CheR,CheB,CheD,atwo-domainCheC,and twoCheWproteins. Nab.magadii containedtwo cheF geneswithinthislocusandhomologsofthesegeneswere showntobeinvolvedinchemotaxisin Halobacterium [120].Thisgeneclusteralsoencodedthreemethyl-acceptingchemotaxissensorytransducers(Nmag_2880,2882, 2884),twoofwhichwereadjacenttogenesencodingdistantrhodopsinhomologs(Nmag_2879and2881).Other genesencodingputativemethyl-acceptingchemotaxissensorytransducersin Nab.magadii includeNmag_0478, 0937,1253,1386,1542,2639,3325,3638,and3856). Amongthese,two(Nmag_1253,1386)wereadjacentto genesencodingperiplasmicligand-bindingproteins. Archaealflagellaareverydifferentincompositionand assemblyincomparisontobacterialflagella[121].In contrasttothebacterialflagellarmotor,whichisdriven byaniongradient,thearchaealflagellarmotorisdriven byATP,asshownin Hbt.salinarum [122].Within thesecondmotilityandsignaltransductiongenecluster (Nmag_2859-2889)of Nab.magadii isaregion (14,766bp,58.66%GC)with13predictedORFsencodingputativeflagellinbiosynthesisandassemblyproteins (Nmag_2862-2874).ExceptNmag_2871,whichencoded aproteinofunknownfunction,allotherORFswere locatedontheplusstrand.Thisregioncontains4flagellingenes(Nmag_2862-2865),whichencodetheflagella proteinspreviouslyidentified[123]andcharacterized[17]. Furthermore, Nab.magadii containedhomologsof flaF (Nmag_2866), flaG (Nmag_2867), flaH (Nmag_2873), flaI (Nmag_2874),and flaJ (Nmag_2869).Thelattertwogenes (flaI and flaJ )encodeputativeproteinshomologoustothe typeIIsecretionsystemproteinsEandF,respectively.In severalarchaea,FlaIhasbeenshowntobeinvolvedin flagellinassembly[124,125],andwasrecentlyproposedas amotorcomponent[126].Themotilitygeneclustersof halophilicarchaeaaregenerallypolymorphic,probably duetodivergenceofgenomeorganizationanddeletion/ duplicationoftheaccessorygenes[23].Nevertheless, flaH flaI ,and flaJ representacoresetofhighlyconservedgenes presumablycrucialforarchaealmotility. Sincepreviouselectronmicroscopicanalyseshave demonstratedthat Nab.magadii containsdistinctive flagella[127-129],andstruc turesresemblingflagellaare alsovisibleintheTEMimagesinFigure3,itislikelythat the fla locus(Nmag_2862-2874)of Nab.magadii was involvedinflagellinbiosynth esisandmotility.Inaddition, thelargechromosomeof Nab.magadii containedgenes encodingaputativefull-leng thPilTprotein(Nmag_1543) andaprepilinpeptidase(Nmag_1752),whosehomologs werefoundin Nmn pharaonis (NP0198AandNP1276A) and Htg.turkmenica (Htur_3514andHtur_0098). ArchaealbasaltranscriptionmachineryhasmanysimilaritiestotheeukaryoticRNApolymeraseIIapparatus. However,themechanismsoftranscriptionregulation andthetranscriptionalregulatorsamongarchaeaare distinctfromthoseofeukaryotes[130,131]. Nab.magadii contained90genes(~2.2%ofallprotein-coding genes)encodingputativetranscriptionalregulators (TRs).BLASTPanalysesindicatedthatmostofthese predictedproteinswererelatedtobacterialTRs.These TRswerecategorizedintothefollowingfamiliesbased ontheirhelix-turn-helix(HTH)motifs(numbersinparenthesisindicatethenumberofproteinsineachfamily): AsnC(16),IclR(13),PadR(10),ArsR/GntR/Fur(11), XRE(6),TrmB(8),AbrB(3),TetR(3),MarR(3),CopG/ Arc/MetJ/NikR(10),HxlR(2),TenA(1),ModE(1),and unassigned(3).ApartfromtheseTRs, Nab.magadii also contained27genesencodingTRswithanHTH-10domain,whichwasalsofoundinbacterio-opsinactivators. Inaddition, Nab.magadii encodedatwo-domain archaealhistone(Nmag_2205),asingleTATA-binding transcriptioninitiationfactor(Nmag_1863,93%protein sequenceidentitytoTbpEfrom Hbt.salinarum ),asingletranscriptioninitiationfactorTFE(Nmag_3157),and asetofseventranscriptioninitiationfactorsTFB (Nmag_0308,0527,2197,2805,3037,3548,and4179).ConclusionsThisreportdescribesthegenomesequenceof Nab. magadii ,ahaloalkaliphilicarchaeonthatbelongstoa physiologicallydistinctsubgroupofhalophilicarchaea.Siddaramappa etal.BMCGenomics 2012, 13 :165 Page17of21 http://www.biomedcentral.com/1471-2164/13/165

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Although Nab.magadii appearstohavedevelopedstrategiessimilarto Nmn.pharaonis tooptimallythrivein lowwateractivityandhighpHhabitats thegenetic architectureof Nab.magadii ismoresimilartothatof Htg.turkmenica thanto Nmn.pharaonis .Thepresence ofgenesencodingthebiosynthesisoftheosmoprotectant2-sulfotrehaloseisanuncommonfeatureamong halophilicarchaeaandthismayhavecontributedtothe evolutionof Nab.magadii initsnaturalenvironment. Nab.magadii hasgenesencodinganumberofcation/ protonantiportersaswellaspathwaysforthebiosynthesisand/ortransportofvariouscofactorsandvitamins.Theoccurrenceofgenesencodingenzymes involvedinglycolysis,gluconeogenesis,andglycerol utilizationsuggeststhat Nab.magadii ismetabolically versatileandcanusedifferentcarbonandenergy sourcestosustaingrowth.Furthermore,thelargerepertoireofgenesencodingputativeproteases/peptidases andpeptidetransportsystemsisindicativeoftheprotein/peptidecatabolicpotentialof Nab.magadii .Italso appearsthat Nab.magadii canperceiveandprocess physicalandchemicalstimuli,andrespondappropriately bymovingtowardorawayfromthosestimuliusingthe flagellarapparatus.Theinformationobtainedfromthis comparativegenomicanalysiscontributestoouroverall understandingofthebiologyanddiversityofhalophilic archaea.Inparticular,itwillguidecurrentandfutureresearchonthegeneticsandphysiologyof Nab.magadii Suchstudiesareexpectedtofacilitatethemanipulationof thisarchaeonasamodelforhaloalkalphilicmetabolism anditsoptimizationforbiotechnologicalapplications.AdditionalfilesAdditionalfile1: TableS1. Natrialbamagadii ATCC43099genes discussedinthetext.Thistablelists Nab.magadii ATCC43099genes relatedtobacteriophageandrecombinationelements,rRNAgenes,and genesencodingadaptivefeatures. Additionalfile2: TableS2. Bidirectionalbestblastpairsamong proteinsfrom Natrialbamagadii and17otherhaloarchaealgenomes.This tableliststhenumberofbidirectionalbestblastpairsamongproteins from Nab.magadii and17otherhalophilicarchaea.Thefirstcolumnis thenumberoftotalproteinsineachgenome,thesecondcolumnisthe numberofbidirectionalbestblastpairs,andthethirdcolumnisthe percentageofproteinshavingabidirectionalbestblasthitin Nab. magadii Additionalfile3: TableS3. Natrialbamagadii ATCC43099genes encodingputativepeptidases/proteases,proteaseinhibitors,and regulatoryproteins.Thistablelists Nab.magadii ATCC43099genes encodingvarioustypesofproteasesandpeptidasesaswellasprotease inhibitorsandregulatoryproteins. Additionalfile4: TableS4. Natrialbamagadii ATCC43099genes involvedinmetabolism.Thistablelists Nab.magadii ATCC43099genes encodingmolybdenumcofactorbiosynthesisandothermetabolic functions. Competinginterests Theauthor(s)declarethattheyhavenocompetinginterests. Authors ’ contributions SSperformedtheannotationusingRAST,plannedthecomparativeanalysis, anddraftedmostofthemanuscript.JFCcontributedtowholegenome comparisonsandgeneratedFigure1.JCD,KWD,LAG,NK,RT,SP,SL,andTW contributedtoprojectplanning,projectmanagement,genomesequencing, andgenomeannotation.DESandMIGperformedthemanualannotationof proteolyticenzymesandproteaseinhibitorsandgeneratedtheschematic representationof Nab.magadii proteases/inhibitors(Figure2).RAPprepared Nab.magadii samplesusedforTEManalysis.FPparticipatedinthemanual curationof Nab.magadii genome,reconstructedseveralcoenzyme biosyntheticpathways,andhelpeddraftthemanuscript.RDCandJAM conceivedthestudy,participatedingenomeanalyses,andhelpeddraftthe manuscript.Allauthorsreadandapprovedthefinalmanuscript. Acknowledgments ThisworkwasperformedundertheauspicesoftheUSDepartmentofEnergy's OfficeofScience,BiologicalandEnvironmentalResearchProgram,andbythe UniversityofCalifornia,LawrenceBerkeleyNationalLaboratoryundercontractNo. DE-AC02-05CH11231,LawrenceLivermoreNationalLaboratoryunderContract No.DE-AC52-07NA27344,andLosAlamosNationalLaboratoryundercontractNo. DE-AC02-06NA25396.Researchinthela boratoryofJMFwasfundedinpartby grantsfromtheNationalInstitutesofH ealth(R01GM057498)andtheDepartment ofEnergy (DE-FG02-05ER15650).Researchinthela boratoryofRDCwassupportedbygrants fromCONICET(PIP-1783)andUNMDP(EXA542 – 2011),Argentina. Authordetails1DOEJointGenomeInstitute,LosAlamosNationalLaboratory,LosAlamos, NM87545,USA.2InstitutodeInvestigacionesBiolgicas,FacultaddeCiencias ExactasyNaturales,UniversidadNacionaldeMardelPlata,Funes32504to Nivel,MardelPlata,7600,Argentina.3DepartmentofMembrane Biochemistry,MaxPlanckInstituteofBiochemistry,AmKlopferspitz18 D-82152,Martinsried,Germany.4DOEJointGenomeInstitute,WalnutCreek, CA94598,USA.5DepartmentofMicrobiologyandCellScience,Universityof Florida,Bldg.981,MuseumRd.,P.O.Box110700,Gainesville,FL32611-0700, USA. Received:12September2011Accepted:25April2012 Published:4May2012 References1.KooninEV,WolfYI: Genomicsofbacteriaandarchaea:theemerging dynamicviewoftheprokaryoticworld. 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BMCGenomics 2012 13 :165. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Siddaramappa etal.BMCGenomics 2012, 13 :165 Page21of21 http://www.biomedcentral.com/1471-2164/13/165