Natronobiforma cellulositropha gen. nov., sp. nov., a novel haloalkaliphilic member of the family Natrialbaceae (class Halobacteria) from hypersaline alkaline lakes

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Natronobiforma cellulositropha gen. nov., sp. nov., a novel haloalkaliphilic member of the

family Natrialbaceae (class Halobacteria) from hypersaline alkaline lakes

Sorokin, Dimitry Y.; Khijniak, Tatiana V.; Kostrikina, Nadezhda A.; Elcheninov, Alexander G.; Toshchakov,

Stepan V.; Bale, Nicole J.; Damsté, Jaap S.Sinninghe; Kublanov, Ilya V.

DOI

10.1016/j.syapm.2018.04.002

Publication date

2018

Document Version

Final published version

Published in

Systematic and Applied Microbiology

Citation (APA)

Sorokin, D. Y., Khijniak, T. V., Kostrikina, N. A., Elcheninov, A. G., Toshchakov, S. V., Bale, N. J., Damsté,

J. S. S., & Kublanov, I. V. (2018). Natronobiforma cellulositropha gen. nov., sp. nov., a novel haloalkaliphilic

member of the family Natrialbaceae (class Halobacteria) from hypersaline alkaline lakes. Systematic and

Applied Microbiology. https://doi.org/10.1016/j.syapm.2018.04.002

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Contents lists available atScienceDirect

Systematic

and

Applied

Microbiology

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . d e / s y a p m

Natronobiforma

cellulositropha

gen.

nov.,

sp.

nov.,

a

novel

haloalkaliphilic

member

of

the

family

Natrialbaceae

(class

Halobacteria)

from

hypersaline

alkaline

lakes

Dimitry

Y. Sorokin

a,b,∗

_,

_Tatiana

_V.

_Khijniak

a

_,

_Nadezhda

_A.

_Kostrikina

a

_,

Alexander

G. Elcheninov

a

_,

_Stepan

_V.

_Toshchakov

c

_,

_Nicole

_J.

_Bale

d

_,

Jaap

S. Sinninghe

Damsté

d,e

_,

_Ilya

_V.

_Kublanov

a,c

a_Winogradsky_Institute_of_{Microbiology,}_Research_Centre_of_{Biotechnology,}_Russian_Academy_of_Sciences,_Moscow,_Russia b_Department_of_{Biotechnology,}_TU_Delft,_The_Netherlands

c_Immanuel_Kant_Baltic_Federal_University,_Kaliningrad,_Russia

d_Department_of_Marine_Microbiology_and_{Biogeochemistry,}_NIOZ_Netherlands_Institute_for_Sea_Research,_and_Utrecht_University,_The_Netherlands e_Department_of_Earth_Sciences_–_{Geochemistry,}_Faculty_of_Geosciences,_Utrecht_University,_Utrecht,_The_Netherlands

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received11March2018

Receivedinrevisedform13April2018 Accepted18April2018 Keywords: Hypersaline Sodalakes Haloalkaliphilic Natronoarchaea Cellulotrophic

a

b

s

t

r

a

c

t

Sixstrainsofextremelyhalophilicandalkaliphiliceuryarchaeawereenrichedandisolatedinpureculture fromsurfacebrinesandsedimentsofhypersalinealkalinelakesinvariousgeographicallocationswith variousformsofinsolublecelluloseasgrowthsubstrate.Thecellsaremostlyﬂatmotilerodswithathin monolayercellwallwhilegrowingoncellobiose.Incontrast,thecellsgrowingwithcellulosearemostly nonmotilecoccicoveredwithathickexternalEPSlayer.Theisolates,designatedAArcel,areobligate aerobicheterotrophswithanarrowsubstratespectrum.Allstrainscanuseinsolublecelluloses,cellobiose, afewsolubleglucansandxylanastheircarbonandenergysource.Theyareextremehalophiles,growing withintherangefrom2.5to4.8MtotalNa+_(optimum_at₄_M)_and_obligate_{alkaliphiles,}_with_the_pH_range

forgrowthfrom7.5to9.9(optimumat8.5–9).ThecorearchaeallipidsofstrainAArcel5T_were_dominated

byC20–C20dialkylglycerolether(DGE)(i.e.archaeol)andC20–C25DGEinnearlyequalproportion.The16S

rRNAgeneanalysisindicatedthatallsixisolatesbelongtoasinglegenomicspeciesmostlyrelatedtothe generaSaliphagus-Natribaculum-Halovarius.Takingtogetherasubstantialphenotypicdifferenceofthe newisolatesfromtheclosestrelativesandthephylogeneticdistance,itisconcludedthattheAArcelgroup representsanovelgenus-levelbranchwithinthefamilyNatrialbaceaeforwhichthenameNatronobiforma cellulositrophagen.nov.,sp.nov.isproposedwithAArcel5T_as_the_type_strain_(JCM₃₁₉₃₉T₌_UNIQEM

U972T_).

Introduction

Hypersalinehabitats,suchasinland salt and sodalakes and salterns with salt concentrations close to saturation are usu-ally inhabited by a dense population of haloarchaea, which represent the extremely halophilic branch of the phylum Eur-yarchaeota.Accordingtothecurrentknowledge,haloarchaeaare mostlyaerobicheterotrophs,withafewexceptionsoffacultative anaerobescapableofutilizingsimplesolubleorganicmonomers

∗ Correspondingauthorat:WinogradskyInstituteofMicrobiology,Research Cen-treofBiotechnology,RussianAcademyofSciences,Moscow,Russia.

E-mailaddresses:soroc@inmi.ru,d.sorokin@tudelft.nl(D.Y.Sorokin).

[7,8,1,16,17,6].Afewhaloarchaealspeciesarecapableof

hydrolyz-ingpolymeric substances,suchas starch,proteinsand oliveoil

[3,5,14,19,2].However,thepotentialfunctioning ofhaloarchaea

in themineralization ofinsolubleorganicpolymershasnotyet beenconsideredandthisfunctioninhypersalinehabitatsisusually attributedtohalophilicbacteria[1,16].Inparticular,nexttonothing isknownabouttheabilityofhaloarchaeatoutilizenative insolu-blecelluloseasagrowthsubstrate.Glycosyl-hydrolase(GH)genes encoding putativecellulases havebeennotedinseveral haloar-chaealgenomes and thepresence offunctional endoglucanases weredemonstratedintwogeneraofneutrophilichaloarchaea,i.e. HalorhabdusandHaloarcula[10,11,26].However,itremainstobe investigatedwhetherthesearchaeaareactuallycapableofusing nativeformsofcelluloseasgrowthsubstrates.

https://doi.org/10.1016/j.syapm.2018.04.002

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2 D.Y.Sorokinetal./SystematicandAppliedMicrobiologyxxx(2018)xxx–xxx

Table1

Cellulotrophicnatronoarchaeisolatedfromhypersalinealkalinelakes.

Strain Isolatedfrom: Chemicalparametersofbrines Celluloseformusedin

theenrichment

Lake Area Salt(gl−1) pH Alkalinity(M)

AArcel2 Bitter-1 KulundaSteppeAltai,

Russia

330 10.3 4.0 Amorphous

AArcel4 Sodacrystallizer 380 9.6 3.1 Avicel

AArcel5T _Tanatar-1 ₄₀₀ _11.0 _4.9 _Sigma₂₀_␮m

AArcel9 Mixedfrom3lakes 330–400 9.6–11.0 3.1–4.9 Filterpaper

AArcel6 Shar-Burdiin,Hotontyn n-eMongolia 220–360 9.6–9.9 0.9–1.2 Amorphous

AArcel8-1 Owenslake California 180 9.7 1.0 Amorphous

Sofar,asinglestudyfocusedonthefunctionalaspectofcellulose degradationbyhaloarchaeahasbeenpublished[22].Inthatwork, fortheﬁrsttimewewereabletoenrichandisolateinpureculture anumberofhaloarchaealstrainsutilizingcelluloseasthegrowth substrate.Oneofthemostactivegroupsincludedsix natronar-chaealisolatesfromvariousalkalinehypersalinelakeswhich,to ourknowledge,representtheﬁrstexampleofnatronoarchaeawith suchametabolictrait.Thispaperdescribesphenotypicand phylo-geneticpropertiesofthenovelgroupandproposestoassignitinto anovelgenusandspeciesNatronobiformacellulotropha.

Materialandmethods

Samples

Surfacesedimentsandnear-bottombrinesfromvarious hyper-salinealkalineinlandlakesfromCentralAsia,EgyptandUSAwith saltconcentrationof200–400gl−1,pHfrom9.3to11and solu-blecarbonatealkalinityfrom0.1to4Mwereusedtoenrichfor cellulotrophicnatronoarchaea[22].

Enrichment,isolationandcultivationconditions

Thealkaline(pH9.5)basemedium,containing4MtotalNa+₍₂_M

Na+_as_sodium_carbonates₊₂_M_NaCl)_also_included₁_g_l−1_K₂_HPO₄

and5g/lKClandwassupplementedaftersterilizationwith1mll−1 oftracemetalsolutionandvitaminmix[18],1mMMgCl2,2mM

(NH4)2SO4and20mgl−1ofyeastextract.Additionof200mgl−1of

streptomycinservedtoinhibitgrowthofbacteria.Variousforms ofinsolublecellulosewereusedastheonly carbonand energy sourceataﬁnalconcentrationof1gl−1 (Table1).Before inocu-lation,thesedimentswereresuspended1:10inthebasicmedium andafter5–10minprecipitationofthecoursefractions,a1ml por-tionfromthetopfractioncontainingmostlycolloidalsediments andmicrobialcellswasusedtoinoculate20mlculturesin100ml closedserum bottles placed ona rotary shaker at37◦C and at 120rpm.Thedevelopmentof cellswasmonitoredbythevisual extentofcellulosedegradation,appearanceofpinkcolorandby microscopy.Aftervisiblecellulosedegradationandbiomassgrowth becameevident(20–40days),thecultureswereseriallydilutedin thesamemediumbutwithamorphouscelluloseassubstrateand themaximalpositivedilutionswereplatedontoasolidmedium preparedbymixing3partsoftheliquidmedium(withadditional solidNaCladditiontocompensatefordilutionwithagar) and2 partsof5%extensivelywashedagarat55◦C.After2–6weeksof incubationinclosedplasticbagsat37◦Cthecolonieswith clear-ancezonesweretransferredtotheliquidmediawithamorphous celluloseandthepositivecultureswerefurtherpuriﬁedby sev-eralroundsofplating-liquidculturecultivationwithamorphous cellulose.This,eventually,resultedinisolationof6purecultures ofcellulotrophicnatronoarchaeawitha commondesignationas AArcel(Table1).Thepuritywascheckedmicroscopically(Zeiss

AxioplanImaging2microscope,Göttingen,Germany)andbythe 16SrRNAgenesequencing.

Phenotypiccharacterization

Forthetotalcellelectronmicroscopy,thecellswerecentrifuged andresuspendedin3MNaCl,fixedwithparaformaldehyde(final concentration3%,v/v)for2hatroomtemperature,thenwashed againwiththesameNaClsolutions.Thefixedcellswerepositively contrastedwith1%(w/v)uranylacetate.Forthinsectioning,thecell pelletswerefixedin1%(w/v)OsO4containing3.0MNaClfor1week

at4◦C,washedand resuspendedin3MNaCl,stainedovernight with1%(w/v)uranylacetate,dehydratedinethanol series,and embeddedinEponresin.Thinsectionswerepost-stainedwith1% (w/v)leadcitrate.

Thecoremembranelipidswereobtainedbyacidhydrolysis(5% HClinmethanolbyreﬂuxfor3h)ofthefreeze-driedcellsand sub-sequentanalysisbyHPLC-MSforGDGTsandarchaeolderivatives accordingto[24].IntactpolarlipidswereobtainedbyBlighDyer extractionoffreeze-driedcellsandsubsequentHPLC-MSanalysis asdescribedinRef.[20].

Genomesequencing

FragmentgenomiclibrariesofstrainsAarcel5T_and_Aarcel2_were

preparedby NEBNext® UltraTM _kit _(New_England _Biolabs, _USA)

accordingtothemanufacturer’sinstructionsandsequencedon Illu-minaMiseqTM _System_using_2*150_bp_paired-end_read_cartridge.

PreliminaryassemblyandgenepredictionwasperformedbyCLC GenomicsWorkbench10.5(Qiagen,Germany)withrecommended parameters.Draftgenomeassemblieswereusedonlyforobtaining 16SrRNAandrpoBgenesequencesandwillbepublisheduponthe improvementofassembliesbysequencingoflonginsert(jumping) genomiclibraries.

Phylogeneticanalysis

16SrRNAandrpoB genesequenceswereextractedfromthe draft genome assemblies and deposited in the Genbank under theaccessionnumbers:(MG938052-MG938053for16SrRNAand MG940906andMG940907forrpoBgenesofstrainsAArcel5and AArcel2,respectively).

Toperform16SrRNAgenesequence-basedphylogenetic analy-sis,thesequencesofalltypespeciesoftheNatrialbaceaegenera were obtained from the Genbank and aligned together with completesequencesof strainsAArcel5T _and _AArcel2_and_nearly

completesequencesofstrainsAArcel9andAArcel8-1inMuscle, implementedinMega6package[23].Thephylogeneticanalysis wasperformedinMega6usingMaximumLikelihoodalgorithm andtheGeneralTimeReversible(GTR)model(G+I,4categories)

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Fig.1. CellmorphologyofstrainAArcel5growingonamorphouscelluloseatpH9.5,4MtotalNa+_and₃₇◦_C._(a)_colonies;_(b)_phase_contrast_and_(c)_electron_microscopy_of thinsectionsofcellsduringabsorptionphaseoncellulose;(d)electronmicroscopyofthinsectionsand(e)andwholefreesuspendedcellsfromthesecondgrowthphaseon cellulose.

AArcel5sequenceandwerenotanalyzedbutplacedonthetree togetherwiththetypestrain.

ToperformtherpoB’-basedphylogeneticanalysis,full-length nucleotidesequencesoftherpoB’ geneoftypespeciesfromthe NatrialbaceaewereobtainedfromGenBank or IMGand aligned usingtheG-INS-imethodinMAFFTserverv7[9].Phylogenetictree wasconstructedinMega6usingMaximumLikelihoodalgorithm withGTRmodel(G+I,4categories.Forphylogenyreconstruction basedonRpoBproteins,thenucleotidesequencesweretranslated, alignedinMAFFTserverv7usingtheG-INS-ialgorithm,andthetree wasconstructedusingMaximumLikelihoodalgorithmwiththeLG model.ToestimatetherpoBgenedistancesofallvalidlypublished Natrialbaceaerepresentatives,thepairwisedistancesmatrixbased onpercentageofsequenceidentitieswasconstructedusingMega 6.

Resultsanddiscussion

Phenotypicproperties

Onplateswithamorphouscellulose,allstrainsformedpin-point pinkcoloniesafter4–6weeksincubationwitha largeclearance aroundthem,indicativeofcellulosehydrolysis(Fig.1a).Growth inliquidculturewithallformsofcellulosesstartedwithamassive attachmentofcellstothesolidphasecellulosesurface,followedby gradualdissolutionofcelluloseandappearanceofcellsinthe liq-uidphase.Adramaticchangeincellmorphologywasobservedin thosetwophases.Thecellsaggregatedwiththecelluloseparticles werenon-motilecocci(Fig.1b)coveredwithathickelectrondense externallayer(Fig.1c),whilethefreesuspendedcellsinthesecond growthphaseweredominatedbymotilethinﬂatrodswithathin

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4 D.Y.Sorokinetal./SystematicandAppliedMicrobiologyxxx(2018)xxx–xxx 0 10 20 30 40 50 60 70 80 90 100 7 7.5 8 8.5 9 9.5 10 10.5 final pH ) m u mi x a m f o %( et ar ht w or G 0 10 20 30 40 50 60 70 80 90 100 2 2.5 3 3.5 4 4.5 5 total Na+_(M) ) m u mi xa m f o %( et ar ht w or G

a

b

Fig.2.InﬂuenceofpHat4MtotalNa+_(a)_and_Na+_at_pH₉_(b)_on_growth_of_strain_AArcel5_with_cellobiose_at₃₇◦_C._The_results_are_mean_values_from_two_biological_replicate experiments.

cellwall(Fig.1d,e).Thecellsinthecoloniesresembledthecoccoid cellsfromtheﬁrstphaseinliquidcultures,whilethecellsgrownon cellobioseinliquidcultureweresimilartothosefromthesecond growthphaseoncellulose.

Thepolarmembranelipidswereanalyzedin thetype strain AArcel5T _grown _with_cellobiose _at₃₇◦_C. ₄_M_total _Na+ _and _pH

9.3 harvested in the mid-exponential growth phase. The core membrane lipids were represented by two dominant compo-nents:archaeol(C20–C20dialkylglycerolether(DGE),58%ofthe

total) and extended archaeol (C20–C25 DGE, 40% of the total).

Tracesofthemonoglycerolether(MGE)lipids(1-C20MGE,2-C20

MGE,and2-C25MGE)werealsodetected.Theintactpolarlipids

were dominated (in order of abundance) by phosphatidylglyc-erophosphatemethylester(PGP-Me),phosphatidylglycerol(PG),a phosphatidylglycose(GL-PG),adiglycosyl(2GL),and phosphatidyl-glycerophosphate(PGP)(SupplementaryFig.S1).

TheAArcelstrainsareobligatelyaerobicsaccharolyticarchaea withalimitedrangeofsubstratessupportinggrowth.Theyarethe firstnatronoarchaeareportedasbeingspecializedintheutilization ofnativeinsolublecelluloses(buttheycannotuseanartificial sol-ubleanaloguecarboxymethylcelluloseCMC)[22].Allstrainscan growwithxylan(frombirchandbeech)andbarleybeta-glucan. AArcel strains2, 4, and 5 also utilized lichenan, glucomannan and _{␤-1,4-mannan,} but growth was much slower. Amorphous chitinalso seemstobe a substratefor these 3strains, but the growthwasunstable:thetransitionfromcellulosetochitinwas onlyrandomlysuccessful,failingonmanyoccasions.Apparently growthonthispolymerisnotoptimalforthesearchaea. Alpha-glucans,suchasstarchandstarch-likepolymers,werenotutilized. Amongthesugars,onlytwodimerswereused—cellobioseand maltose(lessactively).Sugarsnotutilizedincluded:glucose, fruc-tose,galactose,mannose,rhamnose,arabinose,raffinose,sucrose, trehalose,maltose,glucosamin,N-acetylglucosamine,glucouronic acid,halacturonicacid,lactose,ribose,xylose,melezitoseand meli-biose. Sugar alcohols which tested negative included glycerol, sorbitolandmannitol.NegativeorganicacidswereC2–C8saturated

fattyacids,lactate,pyruvate,succinate,malateandfumarate.The organicnitrogencompoundsnotutilizedwereglutamate, aspar-tatemeatandcaseinpeptonsandyeastextract.Ammoniumwas utilized as the N-source with cellobiose as carbon and energy substrate,while nitrateand ureawerenegative. Anaerobic fer-mentativegrowth witharginine,cellobiose or maltose wasnot observed,norwasanaerobicrespiration(with cellobioseasthe electrondonor)withnitrate,sulfur,fumarateorDMSOaselectron acceptors.

Thesearchaeabelongtothegroupofextremehalophiles, grow-ingoptimallyat4MtotalNa+₍_Fig.₂_a)_and_are_moderate_alkaliphiles

withapHoptimumaround9(Fig.2b).Itisimportanttostressthat, whenworkingwithalkaliphiles,thepHproﬁlingshouldbe per-formedwithanobligatorycheckofthepHchangeduringgrowth

[21].Forthisparticulargroupofnatronoarchaeamineralizing sug-arsandproducingmetabolicacids,weobservedadropofthepH from11to9.5evenwhenusingahighlybufferedsodiumcarbonate system.Inourexperience,ifanatronarchaeonisgrowingatpH6 itwillnotgrowatpH10,whichleadstotheimpressionthatinthe case,forexample,ofNatribaculumbreve,theﬁnalpHcheckatthe highestpHrangewasnotperformed[12].ThetypestrainAArcel5T

grewwithinarelativelywidetemperaturerangefrom20to53◦C withanoptimumat40◦C.However,itshouldbementionedthat thegrowthathightemperature(above43◦C)wasonlypossibleat thelowestgrowthpH(8.5).Themostprobableexplanationisthat acombinationofhightemperatureandhighalkalinityresultsin instabilityofthehaloarchaealS-layer.

Phylogeneticanalysis

Theresultsof16SrRNAgenesequenceanalysisdemonstrated that the AArcelisolates formed a separate, single species-level groupwithinthefamilyNatrialbaceaewitharecentlydescribed neutrophilicSaliphagusinfecundisoliastheclosestrelative(Fig.3a). StrainAArcel2 wasmostremote from thetype strainAArcel5T

(99.1%ofsequenceidentity),butstillwithinthecurrently recog-nizedspeciesborder.CalculatedoftheAverageNucleotideIdentity (ANI)betweenthesetwogenome-sequencedstrainsgaveavalue closetothestatisticallyaveragespeciesborder(95%).Takinginto accountpracticallyidenticalphenotypes,itcanbeconcludedthat allsixAArcelcellulotrophicisolatesbelongtoasinglespecies.

Anothermarker,widelyusedforphylogeneticreconstructions ofHalobacteria,istheRNA-polymerasesubunitB gene[13].The phylogenetictrees basedoncomparativeanalysisof rpoB gene andRpoBproteinsequencesofthetypespeciesfromNatrialbaceae revealedthatAArcel5T _and_AArcel2_formed_a_branch_positioned

separatelyfromthecloselyrelatedgenera(Fig.3b,c).TherpoBgene sequencesofAArcel5andAArcel2strainswere92.2%similartoeach otherandhad82.5–88.8%identitywiththemembersof Natrial-baceae(SupplementaryTableS1andFig.S2).Phylogeneticanalysis oftherpoB’-geneofallvalidlypublishedNatrialbaceaewasdoneto revealallinter-andintra-genusclustering(SupplementaryFig.S2). ItshowedthatonlyHalovivax,Halostagnicola,Natrialba, Natrono-coccus,SaliphagusandSalinarchaeumgeneraformedmonophyletic clusters. These genera are similar to AArcel5T_-AArcel2 _in _the

intrageneric/intergenericdistances,calculatedbasingonthe per-centageofsequenceidentities(SupplementaryTableS1):Halovivax 89.9–97.8%/82.4–89.6%;Halostagnicola94.4%/81.2–89%;Natrialba

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Fig.3.PhylogenyoftheAArcelstrains.(a)MaximumLikelihood16SrRNAgenesequence-basedphylogenetictreeshowingpositionoftheAArcelstrains(inbold)withinthe familyNatrialbaceae.Branchlengths(seescale)correspondtothenumberofsubstitutionspersitewithcorrections,associatedwiththemodel(GTR,G+I,4categories).All positionswithlessthan95%sitecoveragewereeliminated.Totally1359positionswereusedinthealignmentof32sequences(exceptforthepartialAArcel4andAArcel6 sequences,100%identicaltoAArcel5T_)._Numbers_at_nodes_indicate_bootstrap_values_of₁₀₀₀_repetitions._Halomarina_oriensis_strain_JCM₁₆₄₉₅_(AB663390.1)_was_used_as anoutgroup.(b)MaximumLikelihoodrpoB’genesequence-basedtreeshowingpositionoftheAArcel2andAArcel5strains(inbold)withinfamilyNatrialbaceae.Totally 1827positionswereusedinthealignmentof18sequences.HalomarinaoriensisJCM16495(KJ870934.1)wasusedasanoutgroup.(c):MaximumLikelihoodRpoB’protein sequence-basedtreeshowingpositionAArcel2andAArcel5strains(inbold)withinthefamilyNatrialbaceae.Totally608positionswereusedinthealignmentof18sequences. HalomarinaoriensisJCM16495(KJ870934.1)wasusedasanoutgroup.SequenceswithaccessionnumbersinitalicwereobtainedfromIMG,inroman–fromtheGenbank.

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6 D.Y.Sorokinetal./SystematicandAppliedMicrobiologyxxx(2018)xxx–xxx

Table2

ComparativepropertyofcellulotrophicnatronoarchaeawiththenearestphylogeneticrelativesinNatrialbaceae:Saliphagusinfecundisoli[25],Natribaculumbreve[13]and Halovivaxasiaticus[4].PGP-Me–phosphatidylglycerophosphatemethylester;PG–phosphatidylglycerols;GL-PG–phosphatidylglycose;2GL–diglycosyl;PGS– phos-phatidylglycerolsulfate;PGP–phosphatidylglycerophosphate;GL–glycolipid;PL–phospholipid;glycolipids:TGD-1(galactosylmannosylglucosyldiether),S2-DGD (disulfatedmannosylglucosyldiether).Antibiotics:s,streptomycin;k,kanamycin;a,ampicillin;t,tetracyclin;v,vancomycin;g,gentamycin;r,rifampicin;.e,erythromycin, c,chrolarmphenicol.

Property “Natronobiforma

cellulositropha”(6strains)

Saliphagusinfecundisoli Natribaculumbreve Halovivaxasiaticus

Cellmorphology Thinﬂatmotilerodson

cellobiose;cocciwiththickcell

walloncellulose

Cocci Motilepleomorphic

rods

Pleomorphic

nonmotile,fromrods

todiscs

Pigmentation Pink Pink Red Pale-pink

Anaerobicgrowth -(Withcellobioseassubstrate) – Contradictorya _–

Growthsubstrates:

Polymers Insolublecellulose,xylane, chitin(3strains),␤-1,4glucans andmannan

Starch,dextrin Starchb_,_gelatin

hydrolysis

Proteolyticc

Sugars Cellobiose,maltose Glucose,mannose,

rafﬁnose,sucrose, trehalose

Glucose Lactose,rafﬁnose, xylose,trehalose

Aminoacids – Glutamate,aspartate,

ornithine,lysine

Organicacids – Pyruvate,succinate Pyruvate Acetate

Esteraseactivity – Tween-20 – Tween-80

Catalase/oxidase +/+ +/+ +/Weak Antibioticresistance s,k,a,t,v,g,e,p (50–100mgl−1₎_r,_c (<50mgl−1₎ a,v,g,e,c,p(discs) s,k,a,t,v,g,e,c,p,r (discs) s,k,a,t,v,g,e,c,p (discs)

Salinityrange(opt.)MNa+ _2.5–4.8_(4.0) _2–6_(2.5–3.0) _0.9–5.1_(2.6) _1.6–4.8_(2.5)

Mgrequirement Low Low Low Low

pHrange(opt.) 7.5–9.9(8.5–9.0) 6.0–8.5(7.0–7.5) 6.0–10e_(7.0–7.5) _6.5–8.5

Temperature(◦_C) _18–53d_(opt.₄₃₎ _25–50_(opt.₃₇₎ _30–62_(opt.₃₇₎ _25–45_(opt.₃₇₎

Corelipids C20–C20,C20–C25 nd nd C20–C20,C20–C25

Polarlipids PG,PGP-Me,GL-PG,2GL,PGP PG,PGP-Me,PGS,three GL PG,PGP-Me,TGD-1, S2-DGD PG,PGP-Me,twoPL, fourGL G+C,mol% 65.4–65.5 64.4 63.9 60.3

Habitat Hypersalinealkalinelakes (s–wSiberia,n–eMongolia, California)

Salinesoil(China) Salinesoil(China) Hypersalinelake(Inner Mongolia)

nd–nodata.

a_It_is_stated_that_it_grows_{anaerobically}_with_nitrate,_and_next_–_that_it_can_not_reduce_nitrate_to_nitrite_or_nitrite_to_N 2. b _Since_this_organism_did_not_utilize_maltose,_its_capability_to_grow_with_starch_is_{questionable.}

c _Growth_on_polymers_was_not_{investigated.} d _At_pH_8.5.

e_The_ﬁnal_pH_is_not_measured,_the_high_pH_limit_is_not_justiﬁed.

Table3

Natronobiformacellulositropha:protologue.

Parameter Genus:Natronobiformagen.nov. Species:Natronobiformacellulositrophasp.nov.

Datecreated 2018-03-04 2018-03-04

Taxonnumber(TXNR) TA00433 TA00433

Author(AUTE) DimitryY.Sorokin

Speciesname(SPNA) Natronobiformacellulotropha

Genusname(GENA) Natronobiforma

Speciﬁcepithet(SPEP) “Cellulotropha”fromNatronobiformacellulotropha

Speciesstatus(SPST) sp.nov.

Etymology(GETY/SPTY) Natronobiforma(Na.tro.no.bi.for’maGr.

neutraln.natron,arbitrarilyderivedfromthe

Arabicn.natrunornatron,soda;L.adv.num.

bis,twice;L.fem.n.forma,form,shape;N.L.

fem.n.Natronobiforma,thedimorphic

natronoarchaeon

Cellulositropha(cel.lu.lo.si.tro’phaN.L.n.cellulosum,cellulose;

N.L.fem.n.fromGr.n.fem.trophê,nourishment,food;N.L.

fem.adj.cellulositropha,utilizerofcellulose)

Authors(AUT) DimitryY.Sorokin,TatianaV.Khijniak,NadezhdaA.Kostrikina,AlexanderG.Elcheninov,StepanV.

Toshchakov,NicoleJ.Bale,JaapS.SinningheDamstéd,IlyaV.Kublanov

Title(TITL) Natronobiformacellulotrophagen.nov.,sp.nov.,anovelhaloalkaliphilicmemberofthefamilyNatrialbaceae

(classHalobacteria)fromhypersalinealkalinelakes

Journal(JOUR) SystematicandAppliedMicrobiology

Correspondingauthor(COAU). DimitryY.Sorokin

E-mailofcorrespondingauthor(EMAU) d.sorokin@tudelft;soroc@inmi.ru

Designationofthetypestrain(TYPE) AArcel5

Straincollectionnumbers(COLN) JCM31939;UNIQEMU972

16SrRNAgeneaccessionnumber(16SR) KT247980

Alternativehouse-keepinggenes:gene [accessionnumbers](HKGN)

rpoB_[MG940906]

Genomestatus(GSTA) Draft

GCmol%(GGCM) 65.4–65.5(genomesofAArcel5T_and_AArcel2)

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Table3(Continued)

Regionoforigin(REGI) Altairegion

Dateofisolation(DATI) 2013-08-15

Sourceofisolation(SOUR) Surfacesedimentsandbrinesofhypersaline

alkalinelakes

SurfacesedimentsfromhypersalinesodalakeTanatar-1

Samplingdates(DATS) 2013-07-07

Geographiclocation(GEOL) SouthSiberia,N–EMongolia,California S–WSiberia,KulundaSteppe

Latitude(LATI) 51◦₃₉_N

Longtitude(LONG) 79◦₄₈_E

Depth(DEPT) 0.1m

Temperatureofthesample(TEMS) 25◦C

pHofthesample(PHSA) 11.0

Salinityofthesample(SALS) 40%

Numberofstrainsinstudy(NSTR) 6

Sourceofisolationofnon-typestrains(SAMP) HypersalinealkalinelakesinRussia,MongoliaandCalifornia

Growthmedium,incubationconditions(CULT) Alkalinemedimcontaining4MNa+_with_pH

9–9.5andcelluloseassubstrate

4MtotalNa+_,_equal_mix_of_sodium_carbonate_and_NaCl_on_the

basisofNamolarity,pH9.5;incubation–37◦C;amorphous

celluloseorcellobioseasCandenergysource

Conditionsofpreservation(PRES) Deepfreezingin15%glycerol(v/v)

Gramstain(GRAM) Negative

Cellshape(CSHA) Pleomorphic,fromﬂatmotilerodstononmotilecoccoidcells

Cellsize(CSZI) 0.5–0.8␮mindiameter,lengthisvariable

Motility(MOTY) Motile

Motilitytype(MOTK) Flagellar

Typeofﬂagellation(TFLA) Variable,fromsinglesubpolartoseveralperetrichousﬂagella

Sporulation(SPOR) None

Colonymorphology(COLM) Pink,upto2mm

Temperaturerangeforgrowth(TEMR) 20–53◦_C

Lowesttemperatureforgrowth(TEML) 20

Highesttemperatureforgrowth(TEMH) 53

Optimaltemperatureforgrowth(TEMO) 43

LowestpHforgrowth(PHLO) 7.5

HighestpHforgrowth(PHHI) 9.9

OptimumpHforgrowth(PHOP) 9.0

pHcategory(PHCA) Alkaliphile(optimum>8.5)

LowestNaClconcentrationforgrowth(SALL) 2.5

HighestNaClconcentrationforgrowth(SALH) 4.8

Optimumsaltconcentrationforgrowth(SALO) 4.0

Othersaltsimportantforgrowth Sodiumcarbonates

Salinitycategory(SALC) Extremehalophilic(optimum>15%NaCl)

Relationtooxygene(OREL) Aerobe

O2conditionsforstraintesting(OCON) Aerobic

Carbonsourceused(class)(CSUC) Carbohydrates

Speciﬁccompounds(CSUC) Cellulose,xylan,mannan,cellobiose,maltose

Nitrogensource(NSOU) Ammonium

Terminalelectronacceptor(ELAC) O2

Energymetabolism(EMET) Chemoorganotrophic

Phospholipids(PHOS) Coremembranelipidsarearchaeol(C20–C20

DGE)andC20–C25DGEinequalproportion

Phosphatidylglycerophosphatemethylester(PGP-Me),

phosphatidylglycerol(PG),phosphatidylglycerolsulfate(PGS)

andphosphatidylglycerophosphate(PGP)

Glycolipids(GLYC) Phosphatidylglycose(GL-PG),diglycosyl(2GL)

Habitat(HABT) Hypersalinealkalinelakes

Extraordinaryfeautres(EXTR) Growthwithnativeinsolublecellulose Fastgrowthwithinsolublenativecelluloses;morethan30GH

glucosyl-hydrolasesgenesinthegenome

88.6–98.6%/82–89%;Natronococcus91.4–93%/82.4–90.8%; Salipha-gusnd/81.5–87.1%andSalinarchaeum92.4%/80.2–85.1%.

Furthermore,phenotypiccomparisonshowsaclear physiolog-icaldifferentiationof theAArcelisolatesfromthe threeclosest relativesinNatrialbaceae(Table2).

Inconclusion,thesixAArcelstrainsisolatedfromhypersaline alkalinelakesrepresentaﬁrstexampleofnatronoarchaea special-izedinutilizationofnativeinsolublecellulosesasgrowthsubstrate. Takingintoaccounttheiruniquephenotypicpropertiesandthe phylogeneticdistances(basedontwoconservativephylogenetic markers)fromthenearestgenerainNatrialbaceae,weproposeto classifythegroupasanovelgenusandspeciesNatronobiforma cel-lulotrophawithstrainAArcel5asthetypestrain.Thenovelgenus andspeciesprotologue(diagnosis)isprovidedinTable3.

Funding

ThisworkwassupportedbytheRussianScienceFoundation (grant 16-14-00121) and by the Russian Academy of Sciences

and Federal Agency of Scientiﬁc Organizations (project 0104-2018-0033).ThisprojectalsoreceivedfundingfromtheEuropean ResearchCouncil(ERC)undertheEuropeanUnion’sHorizon2020 researchandinnovationprogram(grantagreementNo.694569– MICROLIPIDS).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttps://doi.org/10.1016/j.syapm.2018.04.

002.

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