Natrarchaeobius chitinivorans gen. nov., sp. nov., and Natrarchaeobius halalkaliphilus sp. nov., alkaliphilic, chitin-utilizing haloarchaea from hypersaline alkaline lakes

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Delft University of Technology

Natrarchaeobius chitinivorans gen. nov., sp. nov., and Natrarchaeobius halalkaliphilus sp.

nov., alkaliphilic, chitin-utilizing haloarchaea from hypersaline alkaline lakes

Sorokin, Dimitry Y.; Elcheninov, Alexander G.; Toshchakov, Stepan V.; Bale, Nicole J.; Sinninghe Damsté,

Jaap S.; Khijniak, Tatiana V.; Kublanov, Ilya V.

DOI

10.1016/j.syapm.2019.01.001

Publication date

2019

Document Version

Final published version

Published in

Systematic and Applied Microbiology

Citation (APA)

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

Kublanov, I. V. (2019). Natrarchaeobius chitinivorans gen. nov., sp. nov., and Natrarchaeobius

halalkaliphilus sp. nov., alkaliphilic, chitin-utilizing haloarchaea from hypersaline alkaline lakes. Systematic

and Applied Microbiology, 42(3), 309-318. https://doi.org/10.1016/j.syapm.2019.01.001

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SystematicandAppliedMicrobiology42(2019)309–318

ContentslistsavailableatScienceDirect

Systematic

and

Applied

Microbiology

j o ur na l h o me pa g e :h t t p : / / w w w . e l s e v i e r . c o m / l o c a t e / s y a p m

Natrarchaeobius

chitinivorans

gen.

nov.,

sp.

nov.,

and

Natrarchaeobius

halalkaliphilus

sp.

nov.,

alkaliphilic,

chitin-utilizing

haloarchaea

from

hypersaline

alkaline

lakes

夽

Dimitry

Y. Sorokin

a,b,∗

_,

_Alexander

_G.

_Elcheninov

a

_,

_Stepan

_V.

_Toshchakov

a

_,

_Nicole

_J.

_Bale

c

_,

Jaap

S. Sinninghe

Damsté

c,d

_,

_Tatiana

_V.

_Khijniak

a

_,

_Ilya

_V.

_Kublanov

a

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

c_Department_of_Marine_Microbiology_and_{Biogeochemistry,}_NIOZ_Netherlands_Institute_for_Sea_Research,_The_Netherlands d_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:

Received23October2018 Receivedinrevisedform 30December2018 Accepted2January2019 Keywords: Sodalakes Natronoarchaea Chitin Chitinase Natrialbaceae Natrarchaeobius

a

b

s

t

r

a

c

t

TwogroupsofalkaliphilichaloarchaeafromhypersalinealkalinelakesinCentralAsia,EgyptandNorth Americawereenrichedandisolatedinpurecultureusingchitinasgrowthsubstrate.Thesecultures, termedAArcht,weredividedintotwogroups:group1whichincludeselevenisolatesfromhighly alka-linesodalakesandgroup2whichcontainsasingleisolateobtainedfromthealkalinehypersalineSearles Lake.Thecoloniesofchitin-utilizingnatronoarchaeawerered-pigmentedandsurroundedbylargezones ofchitinhydrolysis.Thefreecellsofbothgroupsweremostlyﬂatnonmotilerods,whilethecellsthat attachedtochitinorformedcoloniesonchitinplatesweremostlycoccoid.Theisolatesareobligate aer-obicsaccharolyticarchaeautilizingchitinandchitosane(lessactively)astheonlysugarpolymersas wellasafewhexosesastheircarbonandenergysource.Bothgroupsareextremelyhalophilic,growing optimallyat3.5–4MtotalNa+_,_but_they_differ_in_their_pH_proﬁles:_the_main_group₁_isolates_are

obli-gatelyalkaliphilic,whilethesinglegroup2strain(AArcht-SlT₎_is_{alkalitolerant.}_The_core_archaeal_lipids_in

bothgroupsaredominatedbyC20–C20andC20–C25dialkylglycerolethers(DGE)inapproximatelyequal

proportion.Phylogeneticanalysisindicatedthattheisolatesformanindependentgenus-levellineage withinthefamilyNatrialbaceaewith3species-levelsubgroups.Theavailablegenomesoftheclosest cul-turedrelativesoftheAArchtstrains,belongingtothegeneraNatrialbaandHalopiger,donotencodeany chitinase-relatedgenes.Onthebasisoftheiruniquephenotypicpropertiesanddistinctphylogeny,we suggestthattheobligatealkaliphilicAArchtisolates(group1)withanidenticalphenotypeareclassiﬁed intoanewgenusandspeciesNatrarchaeobiuschitinivoransgen.nov.,sp.nov.,withstrainAArcht4T_as_the

typestrain(JCM32476T₌_UNIQEM_U966T_),_while_the_{facultatively}_alkaliphilic_strain_AArcht-SlT_(group

2)—asanewspeciesNatrarchaeobiushalalkaliphilussp.nov.(JCM32477T₌_UNIQEM_U969T_).

Introduction

Extremely halophilic euryarchaea form a dominant group withinthe prokaryotic microbial communities in various types

Abbreviations: DGE,dialkylglycerolether;MGE,monoalkylglycerolether; PG,phosphatidylglycerol;PGP-Me,phosphatidylglycerophosphatemethylester;PE, phosphatidylethanolamine;PGP,phosphatidylglycerophosphate.

夽 ThewholegenomeshotgunprojectsofstrainsAArcht4T_,_AArcht7_and_AArcht-SlT

havebeendepositedatDDBJ/ENA/GenBankundertheaccessionsSAMN10160502, SAMN10160503andSAMN10160504,respectively.

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

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

of salt-saturatedterrestrial brines,such asathalassic lakes and seasolarsalterns.Incontrasttootherclasses ofEuryarchaeota, theyaremostlyaerobicheterotrophs,whichutilizesolubleorganic substrates,suchassugars,organicacidsandcomplexrichamino acid-containing substrates such as peptons, and yeast extract

[2,4,12,26–27,29].Untilrecentlythepolymer-degradingpotential of cultivated haloarchaeal species was limited to a few exam-ples,suchasstarch, proteinsandoliveoil[1,3,10,25,34].Asfor the recalcitrant insolublepolysaccharides, such as cellulose or chitin,almost nothinghasbeen describedtodate inthe litera-ture.However,duetotheincreasedavailabilityofmultiplegenome sequencingdataoverthepastfewyears,ithasbecomeapparent thatsomeofthehaloarchaeabelongingtothegeneraHaloarcula, Halobacterium,Halalkalicoccus,Haloferax,Halorhabdus,Halovivax,

https://doi.org/10.1016/j.syapm.2019.01.001

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Halostagnicola, Haloterrigena-Natrinema and Natronococcus may possessthepotentialtohydrolyzesugarpolymers,including cel-luloseandhemicelluloses(GHfamily3,5and9).Thisinference wasrecentlyvalidatedbyphenotypicstudiesofsomeoftheafore mentionedgenera[20–22,36].Indeed,thechitinasegenes(GH fam-ily18)arepresent inthegenomesofthegeneraHalobacterium, Halomicrobium,Natrinema,HaloferaxandSalinarchaeum. Further-more,recentphysiologicalstudiesofpurecultures belongingto thesegenerahaveconﬁrmedtheirabilitytousechitinasgrowth substrate[37,14–15,8,9,24],substantiallychangingprevious con-ceptsoftheecologicalroleoftheseextremophilicarchaea.

Inourrecentworkwewereabletoenrichandisolateinpure culture for the ﬁrst time a number of alkaliphilic haloarchaea (i.e.natronoarchaea)fromhypersalinealkalinelakeswhichutilize chitinastheirgrowthsubstrate[37].Theseincludedtwo phyloge-neticgroups:adominantgroupwithmultipleisolatesfromsoda lakesandasinglestrainfromalessalkalineSearlesLake.Inthis paperwedescribethephenotypicandphylogeneticpropertiesof thesetwogroupsofchitin-utilizingnatronoarchaeaandproposeto classifythemintotwospecieswithinanewgenusNatrarchaeobius.

Materialandmethods

Samples

Toenrichforchitin-utilizingnatronoarchaea,surfacesediments andnear-bottombrinesfromthefollowingalkalinehypersaline inlandlakeswereused:sodalakesinKulundaSteppe(Altairegion, Russia,2011–2012);sodalakesinnorth-easternMongolia(1999) andInnerMongolia(2013);sodaOwensLakeinCalifornia(2008); alkalinelakesinWadialNatrun(Egypt,2000)andalkalineSearles LakeinCalifornia(2005).Thechemicalparametersofthebrines andtheirlocationcoordinatesaregiveninapreviouspublication

[37].Beforeuse,thesedimentslurrieswerehomogenizedby vor-texingandthecoarsesedimentfractionwasremovedbya low speedcentrifugation,whiletheremainingcolloidalfractionwas usedasinoculum(2%v/v).Thebrineswereuseddirectly(10%v/v).

Cultivationandphenotypictests

Thecompositionofthebasicmineralmediumandthedetails ofenrichmentand cultivationofchitin-utilizingnatronoarchaea are described in a previouspublication [37].Briefly, two basic media,onecontaining4MNaClatpH7andanotherone contain-ing4MNaascarbonatesatpH10weremixed1:1,resultingina Cl/carbonatebasicmineralmediumwithapH9.5.Mixingthese twobasesindifferentproportionsalsoallowedforthe investiga-tionofthegrowthpHrangeintherange8–9.5.ForthelowerpH (6–8)theNaClbasemediumbufferingcapacitywasincreasedby adding4gl−1ofHEPES(higherconcentrationsinhibitedgrowth) and,insteadofthealkalinebase,thepHwasadjustedbyadding 1Mfilter-sterilized sodium bicarbonatewith pH8. For thepH above9.5,1partoftheNaClbasewasmixedwiththreepartsof thesodabaseandwasfurthertitratedusing4MNaOHtoreach thepHvalues upto11. Itmustbestressedthatduringgrowth oncarbohydrates,despitethehighbufferingcapacityofthe car-bonatebasemedium,thepHshiftedsignificantlyattheextremes (below8.5andabove10).Wenotethereforethatitisessential, undersuchconditions,tomonitortheactualpHwhiletestingthe influenceofpHongrowth.Forthesaltprofiling,twomedia, con-taininganequalNamolarratioofNaClandsodiumcarbonateatpH 9weremixedindifferentproportionstocreatearangeofsalinities from1to5Mwithstepsof0.5MNa.Thetemperatureprofilewas testedwithintherange 20–60◦C(at5◦C increments).This pro-filewascarriedoutatpH8.5 (toavoidcelllysis atanextreme

alkalinerangeincombinationwithhightemperature),with4M totalNa+ _while_{N-acetylglucosamine} _was_used_as_the_substrate.

Thedetailsofthepreparationofthesolidmediumwithamorphous chitinaredescribedinourpreviouspublication[37].For anaero-bicgrowth,10ml portionsofliquid mediumatpH9.1–9.5(4M totalNa+_and₁₀_mM_{N-acetylglucosamine}_as_carbon_and_N-source)

weredispensedinto23mlsterileserumbottleswhichwereclosed withbutylrubberstoppersandmadeanoxicthroughthreecycles ofevacuation/ﬂushingwithsterileargon.Theutilizationof inor-ganicN-sourceswastestedusingsucroseasasubstrate.Carbon substrateproﬁlingwasdoneforstrainsAArcht4T_,_AArcht7_(at_pH

9.5)andAArcht-SlT _(at_pH₉₎_at₄_M_total _Na+ _using_ammonium

as theN-source.Proteolytic and lipolytic activitieswere tested onsolid mediumwithcasein(clearanceafterﬂoodingwith10% TCA)andemulgatedoliveoil(directclearance),respectively. Cata-laseandoxidaseactivityweredetectedbycolonyassayusing3% H2O2and0.1%N,N,N,N-tetramethyl-p-phenylenediamine(TMPD)

hydrochloride,respectively. Analyses

Thephase contrastandepiﬂuorescencemicroscopyand pho-tography were performed using the Zeiss Axioplan Imaging 2 microscope(Göttingen,Germany).Cellsabsorbedonchitinwere visualized using live-dead staining with SYTO9 (Invitrogen kit L7012).Fortheelectronmicroscopyofthinsections,thecellsof strainsAArcht4T _and _AArcht-SlT _grown _with _amorphous _chitin

wereﬁxedin1%(w/v)OsO4containing3MNaClfor1weekat4◦C,

washedandresuspendedin3MNaCl,stainedovernightwith1% (w/v)uranylacetate,dehydratedinethanolseriesandembedded inEponresin.Afterthinsectioning,thepreparationswere post-stainedwith1%(w/v)leadcitrateandexaminedusingtheJEOL-100 TEM(Japan).

Thecoremembranelipidswereobtainedbyacidhydrolysis(5% HClinmethanolbyreﬂuxfor3h)ofthefreeze-driedcellsand sub-sequentanalysisbyHPLC-MSformembrane-spanninglipidsand archaeolderivativesaccordingtoRef.[40].Intactpolarlipidswere obtainedbyBlighDyerextractionoffreeze-driedcellsand subse-quentHPLC-MSanalysisasdescribedinRef.[35].

Respiratory quinones were recovered from wet biomass by three consecutive extractions with cold acetone for 1h on a magneticstirrer.Thecumulativeextractwasconcentratedby evap-orationandthequinonefractionwasseparatedfromcarotenoids by TLC (Sorboﬁl, Russia) in hexane-diethyl ether (85:15). The obtainedquinone band(Rf=0.52) was recovered by extraction withCCl4–CH3OH(1:1)andsubjectedtomass-spectroscopywith

chemical ionization at atmospheric pressure using quadrupol massspectrometerFinniganLCQAdvantageMAX(Germany)[7].

Phylogeneticanalysis

For strains AArcht4T_,_AArcht7 _and _AArcht-SlT _the _16S _rRNA

andrpoBgenenucleotidesequenceswereobtainedfromthedraft genomeassemblies,whilefortherestoftheAArchtstrainspartial 16SrRNAgenesequenceswerepreviouslyavailable[37].The phy-logeneticanalysiswasperformedinMega7package[18].The16S rRNAgenesequencesofalltypestrainsoftheNatrialbaceae fam-ilyandHalomarinaoriensisJCM16495(asanoutgroup)obtained fromtheGenbankwerealignedtogetherwiththesequencesof AArchtstrainsusing G-INS-imethodinMAFFT serverv.7[17]. ThephylogeneticanalysiswasperformedusingMaximum Like-lihood method and the General Time Reversible (GTR) model (G+I,4categories)[31].FortherpoB-basedphylogenetic analy-sis,thefull-lengthnucleotidesequencesofalltypestrainsfrom theNatrialbaceaeandHalomarinaoriensisJCM16495(asoutgroup) wereobtainedfromtheGenBankandIMGandalignedusingthe

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D.Y.Sorokinetal./SystematicandAppliedMicrobiology42(2019)309–318 311

Table1

Natronarchaealstrainsisolatedfrombrinesandsurfacesedimentsofhypersalinealkalinelakeswithchitinassubstrate.

Strain Isolatedfrom: Phylogeneticgroup Culturecollectionsnumbers

Lake Area

AArcht1 Sodacrystallizer(2012) KulundaSteppe Group1 UNIQEMU967

AArcht5 Tanatar-1 Altai,Russia UNIQEMU968

AArcht6

Sodacrystallizer(2003) AArcht7

AArcht8 Bitter-3 AArcht-St StampLake AArcht3

Mixedbrine-sedimentsfrom6lakes WadiNatrun UNEQEMU966

AArcht4T Egypt JCM32476

AArcht-Mg Shar-Burdiin N–EMongolia

AArcht-Ow OwensLake California,USA

AArcht-Bj BadainJaran InnerMongolia

AArcht-SlT _Searles_Lake _California,_USA _Group₂ _UNIQEM_U969

JCM32477 Boldtextmeansthetypestrainsoftypespecies.

G-INS-imethodinMAFFTserverv.7.Aphylogenetictreewas

con-structedusingMaximumLikelihoodmethodwithGTRmodel(G+I,

4categories).For theconservedproteinsphylogeny,aminoacid

sequencesof33single-copyproteinsderivedfromtherespected

genespresentin49genomesofNatrialbaceaespecies

(Supplemen-taryTableS1), includingAArcht4T_,_AArcht7_and _AArcht-SlT _and

NatronomonaspharaonisDSM2160asanoutgroup,wereobtained

fromIMG[6].The33setsofproteinsequenceswerealignedin MAFFTv.7usingL-INS-ialgorithm,thealignmentswere concate-natedusingFaBoxjoineralignment[39]andthephylogenetictree wasconstructedusingMaximumLikelihoodmethodandtheLG model(G+I,4categories)[20].

PairwiseANIcomparisonwasperformedusingpyanimodule v0.2.7[32]withMUMmer[19]andBLASTn+[5]asalignment meth-ods.TheDDHvaluesbetweenthethreegenome-sequencedAArcht strainsandthetypestrainsofNatrialbaceaewerecalculatedusing theGenome-to-GenomeDistanceCalculator2.1(GGDC)[23]with theBLAST+asalocalalignmenttool.

Resultsanddiscussion

Isolation,morphologyandchemotaxonomy

Overall,elevenstrainsofnatronoarchaeacapableofusingchitin (bothamorphousandcrystalline,originatedeitherfromcrabor shrimpshells)asthegrowthsubstratewerepuriﬁedfrom enrich-mentculturesinoculatedwithbrinesandsurfacesedimentsfrom hypersalinesodalakes(AArchtstrainsgroup1).Inaddition,asingle strainAArcht-SlT_(group₂₎_was_isolated_from_a_less_alkaline

hyper-salineSearlesLake(Table1).AllAArchtisolatesformedred-orange pigmentedcolonies witha largeclearancezonesof amorphous chitinaroundthem,allowingfortheirrecognitionamongst mul-tiplenon-chitinolyticnatronarchaealsatellites(Fig.1a,b).Growth inliquidculturewithchitinhadtwophases:theinitialphasewas characterizedbyanabsorptionofthecellsonchitin,resultinginthe aggregationofamorphouschitinparticlesinlargerconglomerates ortheformationofbioﬁlmsoncrystallinechitinparticles.During thisphase(aswellasinthecoloniesonplateswithamorphous chitin),thecellswereinthecoccoidform(Fig.1e,f).Inthesecond stageofmassivechitinhydrolysis,freecellsstartedtoaccumulate intheculturebrothandweremostlyintheformofnonmotileﬂat rods(Fig.1c,d).Thesametypeof“dimorphism”hasrecentlybeen observedinanothergroupofhydrolyticnatronoarchaeafromsoda lakes,Natronobiformacellulosivorans,whichutilizeinsoluble cellu-losesasasubstrate[36].Thinsectionelectronmicroscopyofstrains AArcht4T _and_AArcht-SlT _revealed_the_presence_of_a _thin

mono-layercellwall, typicalformostofthehaloarchaealspecies,and

alargenucleoid.Manyofthecellsexaminedcontained electron-transparentinclusionbodies,who,althoughtheirnaturewasnot investigated further,are speculatedtobeofeither polyhydrox-yalkanoateorglycogenorigin(Fig.1g,h).Therod-phasecellslyzed afterresuspensionin solutionscontainingless than1.5MNaCl, whilethecellsincoccoidstageweremoreresistanttohypoosmosis andonlystartedtolyzeindistilledwater.

The core membrane lipids in strains AArcht4T _and

AArcht-SlT _were_represented _by_two _dominant _components_commonly

found in haloarchaea: archaeol [C20–C20 dialkyl glycerol ether

(DGE)] and extended archaeol(C20–C25 DGE), approximatelyat

equalproportions.Inaddition,unsaturatedformsofbothC20–C20

DGEandC20–C25DGEweredetectedasminorcomponents.The

C25–C20DGEspecieswasnotdetected.Theintactpolarlipidsin

both strains were dominated by phosphatidylglycerophosphate methylester(PGP-Me)andphosphatidylglycerol(PG),whichare bothcommoninhaloarchaealspecies,includingmembersofthe familyNatrialbaceae.Minoramountsofphosphatidylethanolamine (PE) weredetectedinAArcht4T_,_while _minor_amounts_of

phos-phatidylglycerophosphate(PGP)weredetectedinbothAArcht4T

and AArcht-SlT_. _The _glyco- _and _sulfo-lipids _were _not _detected

(Suppl.Fig.S1).

The respiratory quinone analysis in the two type strains (AArcht4T _and _AArcht-SlT₎ _representing _the _two _groups _of

natronarchaealchitinolyticsrevealedinboththepresenceofa sin-glemenaquinonspecies,identiﬁedasMK-8:0(Suppl.Fig.S2),which iscommonlydetectedinhaloarchaea[11].

Phylogeneticanalysis

BLASTanalysisofthe16SrRNAgenesequenceofthetwelve AArchtstrainsshowedthatallofthemfellintothefamily Natrial-baceaeandthattheyformedtwosubgroupsdividedbya3-species clusterofthegenusNatrialba(Fig.2a).Group1includedten iso-latesfromsodalakeswithahighsequenceidentity(above98%), whilegroup2containedonlystrainsAArcht7andAArcht-SlT_.

How-ever,the16SrRNAgene-basedphylogenyseemedtobeunreliable becausethebootstrapvaluesinthekeynodeswereverylowand thusmostofthebrancheswerenotresolved.Therefore,to fur-therclarifytheAArchtphylogeny,twoadditionalanalyseswere performedforthegenome-sequencedstrainsAArcht4T_,_AArcht7

and AArcht-SlT _together_with _the _{Natrialbaceae} _{representatives}

basedontheRNApolymeraseB-subunit(rpoB)genenucleotide sequences(Fig.2b)andontheconcatenatedalignmentof33 single-copyconservedproteins(Fig.2c).Insharpcontrasttotheresultsof the16SrRNAgene-basedphylogeny,thelatterapproacheswere coherent and reliably showed that the AArcht strainsform an

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Fig.1.MorphologyofstrainsAArcht4T_(a,_c,_e,_g)_and_AArcht-SlT_(b,_d,_f,_h)_growing_at₄_M_total_Na+_,_pH_9.2_and₃₇◦_C_with_chitin._(a–b)_colonies_on_amorphous_chitin

platesforminghydrolysiszones;(c–d)phasecontrastmicrophotographofcellsgrownwithamorphouschitininliquidculture;(e)epifluorescenceimageofcoccoidcellsof AArcht4T_forming_biofilm_on_crystalline_chitin_fiber;_(f)_phase_contrast_{microphotograph}_of_coccoid_cells_of_AArcht-SlT_from_a_colony_on_amorphous_chitin_plate;_(d)_electron

microscopyofthinsectionsofcellsgrownwithamorphouschitin.CW,cellwall;CPM,cytoplasmicmembrane;N,nucleoid,Stg,storagegranule.

independentmonophyleticlineagewithintheNatrialbacea,with AArcht4T _and₇_clustering_together_and_AArcht-SlT _—_as_a

sepa-ratebranch.ItalsodemonstratedthatthegenusNatrialbamost probablyconsistsoftwodifferentgeneraandneedsataxonomic revision.

Further genomic comparison of the 3 genome-sequenced AArchtstrains4,7andSlbetweeneachotherandwiththe mem-bersofNatrialbaceawereperformedusingtwostandardindexes, ANIandDDH.TwovariationsoftheANIcalculationshowedthat thesimilaritylevelbetweentheAArchtstrainsandthemembers of thefamily is higher (but only marginally) than theaverage intragenuslevel(0.86versus0.85)(SupplementaryTablesS2and S3). Likewise,the DDH analysis showedlow values of similar-ity of the 3 AArcht strains (below 24%) with members of the Natrialbacea(Supplementary TableS4)and betweeneach other (below26%).

Growthphysiology

The AArcht isolates are obligately aerobic saccharolytic natronoarchaea(growthbyfermentation,nitrate,DMSOand sul-fur reduction with N-acetylglucosamine as substrate was not observed).Allisolatesgrewwithchitinand(lessactively)with chi-tosaneintheiramorphousorcrystallineforms.Genomeanalysis of3representativestrainsshowedapresenceof3–7 endochiti-nasegenesoftheGH18familyconsistentwiththephysiologyof

theAArchtisolates(Table2).Incontrast,noneofthosegeneswere foundintheavailable genomesfromtherelated generawithin theNatrialbaceaefamilyexcepttheneutrophilicSalinarchaeum,for whichthepotentialtoutilizechitinforgrowthhasalsorecently beendemonstrated[24,37].WetestedtypestrainsofNatrialba asi-aticaandHalopigerxanaduensisfortheirabilitytogrowwithchitin andchitosaneandtheresultswerenegative.BothAArchtgroups alsoutilizedchitinandchitosanemonomers(N-acetylglucosamine and glucosamine, respectively)along witha few other hexoses and glycerol (Table 2). No growth was detected with the fol-lowingpolysaccharides:amorphouscellulose,CMC,various beta-andalpha-glycans, beta-mannan,beta-galactan,betaglyco- and galactomannans, pectin,alginate.The solublesugar compounds whichtestednegativeincludedglucose,galactose,mannose, arabi-nose,rhamnose,glucuronicandgalacturonicacids,xylose,ribose, maltose,lactose,trehalose,melibioze,sorbitolandmannitol.No growthwasdetectedwithorganicacids(C2–C8 fattyacids,

lac-tate,pyruvate,malate,succinate,fumarate)norcomplexorganic aminoacidsubstrates,suchasvariouspeptonsandyeastextract. Lipase and protease were negative. Anaerobic growth with N-acetylglucosaminewasnotobservedeitherbyfermentation,orin thepresence ofelectronacceptors,includingnitrate/nitrite, sul-fur,thiosulfate,DMSO,fumarate,(10mMeach),arsenate,selenate (5mMeach).UtilizationofN-sourceswastestedforthreestrains: AArcht4T_,_AArcht7_and_AArcht-SlT_using_sucrose_as_the_carbon_and

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D.Y.Sorokinetal./SystematicandAppliedMicrobiology42(2019)309–318 313

Fig.2. PhylogenyoftheAArchtstrains.

(a)MaximumLikelihood16SrRNAgene-basedphylogenetictreeofAArchtstrains(inbold)withinthefamilyNatrialbaceaewithHalomarinaoriensisasanoutgroup.Branch lengthscorrespondtothenumberofsubstitutionspersitewithcorrections,associatedwiththemodel(GTR,G+I,4categories).Allpositionswithlessthan95%sitecoverage wereeliminated.Totally1359positionswereusedinthealignmentof93sequences(SupplementaryTableS5a).Numbersatnodesindicatebootstrapvaluesof1000 repetitions.

(b)MaximumLikelihoodphylogenetictreebasedonrpoB_gene_sequences_of_{Natrialbaceae}_{representatives}_together_with_AArcht4T_,_AArcht7_and_AArcht-SlT_strains_(in

bold)withHalomarinaoriensisasanoutgroup.Branchlengthscorrespondtothenumberofsubstitutionspersitewithcorrections,associatedwiththemodel(GTR,G+I,4 categories).Allpositionswithlessthan95%sitecoveragewereeliminated.Totally1830positionswereusedinthealignmentof64sequences(SupplementaryTableS5b). Numbersatnodesindicatebootstrapvaluesof1000repetitions.GeneaccessionnumbersobtainedfromtheIMGdatabaseareunderlined.

(c)MaximumLikelihoodtreebasedonconcatenatedaminoacidsequencesof33single-copyconservedproteinsshowingpositionoftheAArchtlineage(inbold)within theNatrialbaceaefamily.Natronomonaspharaoniswasusedasanoutgroup.Branchlengthscorrespondtothenumberofsubstitutionspersitewithcorrections,associated withthemodel(LG,G+I,4categories).Allpositionswithlessthan95%sitecoveragewereeliminated.Totally6342positionswereusedinthealignmentof49aminoacid sequences.Numbersatnodesindicatebootstrapvaluesof1000repetitions.GeneaccessionnumbersobtainedfromtheIMGdatabaseareunderlined.

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Table2

Comparativepropertyofchitin-utilizingnatronoarchaeawiththerelatedgenerafromthefamilyNatrialbaceaecontainingalkaliphilicspecies.Cumulativecomparativedata aretakenfromRef.[30].Numberofspeciesareindicatedinparenthesis.

Property “Natrarchaeobius” (2) Natrialba(6) Natronolimnobius (2) Natronobacterium (2)

Natronorubrum(6) Natronococcus(4) “Natronobiforma” (1)

Cellmorphology Dimorphic Dimorphic Pleomorphic,ﬂat Rodsorcocci Pleomorphic,ﬂat Cocciinclusters Dimorphic

Motility − V − − V − +

Pigmentation Red-orange Nopigmentor red-orange

Red-orange Red Pink-red Pink,red,orangeor brown Red Celllyzisin distilledwater + + + + + − + Growthwith chitin + −(G) −a _−(G) _-(G) _−(G) ₋ Growthwith insoluble cellulose − −(G) Va _−(G) _-(G) _−(G) ₊ Proteolysis − V V V V V − Starchhydrolysis − V Va ₋ ₋ _V ₋ Anaerobic growth withnitrate − nd − − − − − Minimalsalinity MNa+ 3.0 1.6 Above2.5 1.7 1.7 1.5 2.5 pHtype Facultativeor obligatealkaliphilic Facultative alkaliphilicor alkalitolerant

Obligatealkaiphilic Obligatealkaiphilic Facultativeor obligatealkaliphilic alkalitolerant

Obligatealkaiphilic Obligate alkaiphilic Temperature

max.

55◦_C_(at_pH₈₎ _50-60 ₅₄ ₄₀ _50–55◦_C _50–55 ₅₃_(at_pH_8.5)

Majorcorelipids C20–C20,C20–C25 C20–C20,

C20–C25 C20–C20,C20–C25 C20–C20,C20–C25 C20–C20,C20–C25 C20–C20,C20–C25 C20–C20, C20–C25 Intactmembrane phospholipids PGP-Me,PG (minor):PE,PGP

PGP-Me,PG PGP-Me,PG PGP-Me,PG PGP-Me,PG PGP-Me,PG PGP-Me,PG,

PGP

Glycolipids − S2-DGD(in

alkalitolerant species)

− V(unidentiﬁed) TGA-1(ina

neutrophilic species) − GL-PG,2GL G+C,mol% 61.9–62.3 61.5–64.3 59–64 62.5–65.9 59.9–63.3 62.1–64.0 65.4–65.5 Habitat Hypersaline alkalinelakes

Sodaandsalt lakes

Sodalake Sodalakes Sodaandsaltlakes Sodalakes Sodalakes V,variablepropertyindifferentspecoes;nd,notdetrmined;(G)—genomicdata.

PGP-Me,phosphatidylglycerophosphatemethylester;PG,phosphatidylglycerols;PE,phosphatidylethanolamine;PGP,phosphatidylglycerophosphate;GL-PG, phosphatidyl-glycose;2GL—diglycosyl;S2-DGD—disulfatedmannosylglucosyldiether;TGA-1—triglycosylarchaeol.

Boldtextmeanstheorganismsdescribedinthisarticleincontrasttothereferenceorganismsusedforcomparison.

a_Our_data_{(Natronolimnobius}_{innermongolicus}_and_Nl._baerhaense_can_not_grow_on_chitin,_but_the_latter_can_weakly_grow_with_insoluble_cellulose_and_grow_well_with_xylane

andstarch).

whileurea,nitrateandnitritedidnotsupportgrowthinseveral progressivepassages.

Antibioticresistancewastestedfor AArcht4T _(at_pH_9.5)_and

AArcht-SlT _(at_pH₉₎_in_liquid _culture_with_the_chitin _monomer

assubstrate.Bothwereresistantto100␮gml−1 of penicillinG, ampicilline,kanamycin,streptomycin,gentamicin, erythromycin andvancomicin.Rifampicinandchloramphenicolinhibitedgrowth ofAArcht4T_at₁₀₀_␮g/ml,_while_AArcht-SlT_did_not_grow_already_at

50␮g/mlofthosetwoantibiotics.

ThesaltproﬁleforgrowthintherepresentativestrainsAArcht4T

andAArcht-SlT_was_investigated_using_{N-acetylglucosamine}_as_the

substrateatpH9.Bothstrainsgrewwithina narrowsalt range from3to5MtotalNa+_with_an_optimum_at_3.5–4_M,_which

clas-siﬁesthemasextremehalophiles.InrespecttothepHrangefor growth(tested at4MNa+_), _the_two_groups_were_clearly

differ-ent.FivetestedsodalakeAArchtisolates(1,3,4T_,_5,₇₎_started_to

growonlyatpHaboveneutralwithanoptimumat9.1–9.3and maximum(ﬁnal)pHupto9.9–10,thusbelongingtotheobligate alkaliphilictype.Incontrast,strainAArcht-SlT_showed_growth_at_pH

aslowas6.5,althoughitstillgrewoptimallyatpH8–8.5andup to9.5,characterizingitasafacultativealkaliphile.Thedifference inhighpHresponsebetweenthetwo AArchtgroupscorrelated withtheirdifferentCl−dependence:theAArchtstrainsfromgroup 1demandedatleast1MCl−,while AArcht-SlT _did_not_grow_at

Cl− concentrationsbelow 2.5M.Strainsfrombothgroups grew equallywellatMg2+_{concentrations} _from₁_to₅_mM _(at_higher

concentrationmagnesiumstartedtoprecipitateathighpH).Both

groupsshowedmesophilictemperatureproﬁlestypicalformany haloarchaealspecieswithasomewhatelevatedmaximumupto50 (AArcht4T₎_and₅₅◦_C_(AArcht-SlT₎_which_decreased_with_increasing

pH.Theprobablereasonforthelatterisproteininstabilityatahigh temperature-highpHcombination.

TheNatrialbaceaefamily (theonlyfamily in theorder Natri-albales) currentlyincludestwelverecognizedgenera[30] and a recentlydescribedgenus“Natronobiforma”[36].Mostofthe gen-erainthisfamilyincludealkaliphilicoralkalitolerantspecies[30]. However, as described above, doubt exists asto the reliability of reported maximum pH values as, in most cases, the actual pHchangesduringgrowthwerenottested.Mostprobably,only thosespeciesthatoriginatedfromsodalakesaretruealkaliphiles, including species from the genera Natrialba, Natronobacterium, Natronococcus,Natronolimnobius,Natronorubrumand “Natronob-iforma”.Hence, Table2providesphenotypiccomparison ofthe AArchtisolateswiththosegeneraofthefamilythatcontainmainly speciesoriginatingfromalkaline habitats.Themain phenotypic propertyofthenovelgroupwhichdiscriminatesitfromtheother natronoarchaeaintheNatrialbaceaeistheabilitytoutilizechitin asthegrowthsubstrate.TheonlyothergenusintheNatrialbaceae withsuchcapabilityisthegenusSalinarchaeum[24,37]whichisa neutrophilichalophile.AnotherprominentdifferenceoftheAArcht strainsisthehighvaluesoftheirminimalsaltconcentrationfor growth(3MNa+_)._In_respect_to_cell_morphology,_the_novel_group

sharesatendencyfordimorphism(rodsand cocci)withgenera Natrialbaand“Natronobiforma”.Inrespecttothelipid

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composi-D.Y.Sorokinetal./SystematicandAppliedMicrobiology42(2019)309–318 315

Table3

Comparativepropertyofchitin-utilizingnatronoarchaeawiththerelatedspeciesfromthefamilyNatrialbaceae:1,Natrialbaasiatica[16];2,Natrialbachahannaoensis[41];

3,Halopigerxanaduensis[13].

Property Group1(11strains) AArcht-SlT ₁ ₂ ₃

Cellmorphology Non-motileﬂatrodsin freestate;coccoidsin chitin-attachedstate

Pleomorphic,fromrods tococcoids,non-motile

Rods,motile Rods,non-motile Dimorphic

Pigmentation Red–orange Orange No Red Red

Growthsubstrates: Sugarpolymers

Chitin,chitosane + + −d ₋d

Otherglycans − − − starch

Sugars Glucosamine, N-acetylglucosamine, sucrose,maltose, trehalose,melizitose, cellobiose,glycerol Glucosamine, N-acetylglucosamine, sucrose,maltose, trehalose,melizitose, fructose,glycerol −d −d Glucose,galactose, xylose − − Glucose,fructose, maltose −d −d Glucose,arabinose, xylose Proteins,peptides − − + + + Numberofchitinase GH18genesinthe genome AArcht4(5) 3 0 0 0 AArcht7(7)

Anaerobicgrowth − − − Nitratetonitrite

reduction

+(denitriﬁcation)

Catalase/oxidase +/+ +/+ +/+ +/+ +/+

Salinityrange(opt.)M Na+

3.0–5.0(4.0) 3.0–5.0(3.5) 2.0–5.0(4.0) 1.6–5.2(2.5) 2.5–5.0(4.3)

pHrange(opt.) 7.0–10.0(9.1–9.3) 6.5–9.5(8.0–8.5) 6.0–8.0(6.6–7.0) 8.5–10.5a_(9.0) _6–11b_(7.5–8.0)

Temperature(◦_C)c _20–50_(opt.₄₃₎ _25–55_(opt.₄₅₎ _max.₅₀_(30–40) _20–55₍₅₀₎ _28–45₍₃₇₎

Corelipids C20–C20,C20–C25

(dominant)1-and 2-C20MGEand2-C25

MGE(minor)

C20–C20,C20–C25 C20–C20,C20–C25 C20–C20,C20–C25 C20–C20,C20–C25

Intactmembranepolar lipids

(major):PGP-Me,PG (major):PGP-Me,PG PGP-Me,PG, S2-DGD

PGP-Me,PG PGP-Me,S2-DGD

(minor):PE,PGP (minor):PGP G+C,mol% AArcht4T_:61.9

(genome)

61.1(genome) 62.4(genome) 64.3(Tm) 65.2(genome)

AArcht7:62.3(genome)

Habitat Hypersalinealkaline

lakesinCentralAsia andAfrica

Hypersalinealkaline SearlesLake (California)

Seasaltevaporites Sodalakes,InnerMongolia

Phospholipids:(PG)phosphatidylglycerol,(PGP-Me)phosphatidylglycerophosphatemethylester,(PE)phosphatidylethanolamine,PGP(phosphatidylglycerophosphate);(S2

-DGD)disulfatedmannosylglucosyldiether.

a_Final_pH_is_not_measured_therefore_the_max._growth_pH_is_not_validated.

b_Final_pH_is_not_measured,_therefore_the_max._growth_pH_is_not_validated,_especially_taking_into_account_low_pH_optimum. c _Tested_at_pH_8.5.

d _Tested_in_this_work.

tionallcomparedgeneraaresimilarintheirmajorcoreandintact phospholipids.Thenovelgrouphastwoadditionalminor compo-nents,bothinthecoreandintactlipids,butitispossiblethatthese werenotdetectedinothergenerabecauseoflesssensitiveanalysis (TLCversusHPLC).

Further,moredetailed phenotypiccomparisonof the chitin-utilizing natronoarchaea with related species of the genera NatrialbaandHalopigerisgivenin(Table3).

TheAArchtisolatesrepresentthefirstexampleofalkaliphilic haloarchaeaenrichedandisolatedfromhypersalinealkalinelakes withchitinastheirgrowthsubstrate.Theyarehighlyspecialized intheutilizationofchitin(whichisreflected inthepresenceof multiplechitinasegenesintheirgenomes)andcanonlyutilizea fewsolublesugarsforgrowth.Theirpresenceinhypersaline alka-line(soda)lakesonthreedifferentcontinentsindicatethatchitin mustbeabundantinsuchhabitats—afactnotwellrecognized todate. A mass developmentof chitin-producingbrine shrimp ArtemiamonicainsodalakeMono(California)istheonlyexampleof chitin-producinginvertebratesreportedintheliterature[8]. How-ever,theauthorshavenoticedtheirpresenceenmasseinhighly alkalinesodalakesoftheKulundaSteppe(unpublished,Suppl.Fig. S3a).Anothermassivesourceofchitininsodalakescouldbefrom thesodaflylarvasEphydrahians[38]whichwasalsooftenobserved

bytheauthorsonthelittoralof theKulunda Steppesodalakes (Suppl.Fig.S3b,c).

OneofthepeculiaritiesoftheAArchtstrainsdescribedhere, thatapparentlydrawsattention,istheineffectivenessofthe16S rRNAgene,whichnormallyprovidesreliablephylogenetic recon-structions.Thismarkerhasbeen,sofar,anindisputablebasisfor phylogeneticreconstructions,although,forhaloarchaeain particu-lar,severalproblematicexamplesofmultipledissimilatorycopies of this gene have been documented [28]. This inability to use rrngenesforphylogeneticreconstructionscouldbeovercomeby involvingphylogenomicanalysisofconservedsingle-copyprotein markers[32].InthecaseoftheAArchtstrains,thisapproachgave much moreconsistentresultsindicatinga monophyletic genus-levelgroupwiththreespecies-levelsubgroups:tenisolatesfrom hypersalinesodalakes,AArcht7fromasodacrystallizingpoolin KulundaSteppeandAArcht-SlT_from_the_moderately_alkaline

Sear-lesLake.However,onthephenotypiclevel,strainAArcht7cannot bedistinguishedfromthemainsodalakegroup,incontrasttostrain AArcht-SlT_,_which_clearly_{differentiated}_by_its_lower_pH_optimum

andmaximumandmuchhigherchloridedependence.

Inconclusion,takingintoaccountuniquephenotypicproperties andresultsofphylogenomicanalysis,weproposetoclassifythe majorsodalakegroup1(11isolates)inanewgenusandspecies

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Table4

NatrarchaeobiuschitinivoransandNatrarchaeobiushaloalkaliphilus:protologue.

Parameter Genus:Natrarchaeobiusgen.nov. Species:Natrarchaeobius chitinivoranssp.nov.

Species:Natrarchaeobius halalkaliphilussp.nov.

Datecreated 2018-09-24 2018-09-24 2018-09-24

Taxonnumber(TXNR) GA00091 Author(AUTE) DimitryY.Sorokin

Speciesname(SPNA) Natrarchaeobiuschitinivorans Natrarchaeobiushalalkaliphilus

Genusname(GENA) Natrarchaeobius

Speciﬁcepithet(SPEP) – chitinivorans haloalkaliphilus

Speciesstatus(SPST) – sp.nov. sp.nov.

Etymology(GETY/SPTY) Natr.ar.chae.o’bi.us[N.L.n.natron (arbitrarilyderivedfromArabicn. natrunornatron)soda,sodium carbonate;N.L.pref. natr-pertainingtosoda;Gr.adj.archaios ancient;Gr.masc.n.bioslife;N.L. masc.n.Natrarchaeobius, soda-philicarchaeon]

chitinivorans[chi.ti.ni.vo’ransN.L. neut.n.chitinumchitin;L.pres. part.voransdevouring;N.L.part. adj.chitinivoranschitindevouring]

halalkaliphilus[hal.al.ka.li.phi’lus Gr.n.halshalossalt;N.L.n.alkali sodaash(fromArabical-qalyithe ashesofsaltwort);N.L.adj.philus (fromGr.adj.philos-ê−on)friend, loving;N.L.masc.adj.

halalkaliphilussaltand alkali-loving].

Authors(AUT) DimitryY.Sorokin,AlexanderG.Elcheninov,StepanV.Toshchakov,NicoleJ.Bale,JaapS.SinningheDamsté,TatianaV. Khijniak,IlyaV.Kublanov

Title(TITL) Natrarchaeobiuschitinivoransgen.nov.,sp.nov.,andNatrarchaeobiushalalkaliphilussp.nov.,alkaliphilic, chitin-utilizinghaloarchaeafromhypersalinealkalinelakes

Journal(JOUR) SystematicandAppliedMicrobiology Correspondingauthor(COAU). DimitryY.Sorokin

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

Designationofthetypestrain(TYPE) – AArcht4 AArcht-Sl

Straincollectionnumbers(COLN) – JCM32476;UNIQEMU966 JCM32477;UNIQEMU969 16SrRNAgeneaccessionnumber

(16SR)

– KT247962 KT247971

Alternativehouse-keepinggenes:gene [accessionnumbers](HKGN)

– rpoB

33single-copyconservativeproteingenes Genomestatus(GSTA) – Draft:AArcht4T_(accession

SAMN10160502)

Draft:(accessionSAMN10160504) AArcht7(accession

SAMN10160503)

GCmol%(GGCM) – 61.9–62.3(genomesofAArcht4T

andAArcht7)

61.1(genome) Countryoforigin(COUN) RussianFederation,Mongolia,

China,Egypt,USA

RussianFederation,Mongolia, China,Egypt,USA

USA Regionoforigin(REGI) – Altairegion;N-EMongolia,Inner

Mongolia,WadialNatrun, California

California

Dateofisolation(DATI) – 2011–2013 2012

Sourceofisolation(SOUR) Surfacesedimentsandbrinesof hypersaline

alkalinelakes

Surfacesedimentsandbrinesof hypersalinesodalakes

Surfacesedimentsofhypersaline alkalineSearlesLake

Samplingdates(DATS) 1999–2013 1999–2013 2005

Geographiclocation(GEOL) S–WSiberia,N–EMongolia,Inner Mongolia,NorthernAfrica,North America

S–WSiberia,N–EMongolia,Inner Mongolia,NorthernAfrica,North America

NorthAmerica

Latitude(LATI) – – N35◦₄₄

Longtitude(LONG) – – W117◦₂₀

Depth(DEPT) 0–0.1m 0–0.1m 0–0.1m

Temperatureofthesample(TEMS) 15–25◦C 15–25◦C 20◦C

pHofthesample(PHSA) 9–11.0 9.5–11.0 9.0

Salinityofthesample(SALS) 18–40% 18–40% 35%

Numberofstrainsinstudy(NSTR) 12 11 1

Sourceofisolationofnon-typestrains (SAMP)

– Hypersalinealkalinelakesin Russia,Mongolia,ChinaandUSA

– Growthmedium,incubationconditions

(CULT)

Alkalinemediumcontaining4M Na+_with_pH_9–9.5_and_chitin_as

substrate

4MtotalNa+_,_equal_mix_of_sodium

carbonateandNaClonthebasisof Namolarity,pH9.5;incubation −37◦_C;_amorphous_chitin_as_C,

energyandN-source

4MtotalNa+_,_1:3_mix_of_sodium

carbonateandNaClonthebasisof Namolarity,pH9;incubation −37◦_C;_amorphous_chitin_as_C,

energyandN-source Conditionsofpreservation(PRES) Deepfreezingin15%glycerol(v/v)

Gramstain(GRAM) Negative

Cellshape(CSHA) Pleomorphic,fromﬂatrodstococci

Cellsize(CSZI) – 0.6–1␮mindiameter,lengthis

variablefrom1to4␮m

0.6–1.2␮mindiameter,lengthis variablefrom1to5␮m

Motility(MOTY) – nonmotile

Motilitytype(MOTK) – Typeofﬂagellation(TFLA) – Sporulation(SPOR) none

Colonymorphology(COLM) Pink–orange Pink–orange,upto2mm Paleorange,upto1.5mm Temperaturerangeforgrowth(TEMR) 20–55◦C 20–53◦C 25–55◦C

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D.Y.Sorokinetal./SystematicandAppliedMicrobiology42(2019)309–318 317 Table4(Continued)

Parameter Genus:Natrarchaeobiusgen.nov. Species:Natrarchaeobius chitinivoranssp.nov.

Species:Natrarchaeobius halalkaliphilussp.nov. Highesttemperatureforgrowth(TEMH) 55 50(atpH9) 55(atpH8.5)

Optimaltemperatureforgrowth(TEMO) 43–45◦_C ₄₃◦_C ₄₅◦_C

LowestpHforgrowth(PHLO) 6.5 7.0 6.5

HighestpHforgrowth(PHHI) 10 10 9.5

OptimumpHforgrowth(PHOP) 8.5–9.3 9.1–9.3 8.5

pHcategory(PHCA) Alkaliphile(optimum>8.5) LowestNaClconcentrationforgrowth

(SALL)

3.0MtotalNa+

HighestNaClconcentrationforgrowth (SALH)

5MtotalNa+

Optimumsaltconcentrationforgrowth (SALO)

3.5–4.0MtotalNa+ _4.0_M_total_Na+ _3.5_M_total_Na+

Othersaltsimportantforgrowth Sodiumcarbonates

Salinitycategory(SALC) extremehalophilic(optimum3.5–4MNa+₎

Relationtooxygene(OREL) Aerobe O2conditionsforstraintesting(OCON) Aerobic

Carbonsourceused(class)(CSUC) Carbohydrates

Speciﬁccompounds(CSUC) Chitin,chitosane,hexoses Glucosamine,

N-acetylglucosamine,sucrose, maltose,trehalose,melizitose, cellobiose,glycerol

Glucosamine,

N-acetylglucosamine,sucrose, maltose,trehalose,melizitose, fructose,glycerol

Nitrogensource(NSOU) Ammonium Terminalelectronacceptor(ELAC) O2

Energymetabolism(EMET) Chemoorganotrophic

Phospholipids(PHOS) Coremembranelipidsarearchaeol(C20–C20DGE)andC20–C25DGE

Polarlipidsarephosphatidylglycerophosphatemethylester(PGP-Me),phosphatidylglycerol(PG)

Glycolipids(GLYC) – Phosphatidylglycose(GL-PG),

diglycosyl(2GL)

Respiratoryquinons MK8:0 MK8:0 MK8:0

Habitat(HABT) Hypersalinealkalinelakes

Extraordinaryfeautres(EXTR) Fastgrowthwithchitinandchitosaneinhypersalinealkalinebrines Multiplechitinasegenes(GH18family)inthegenomes

(–),notﬁxedforthetaxon.

Natrarchaeobiuschitinivorans,andtheSearlesLakeisolate

AArcht-SlT_in_a_new_species_{Natrarchaeobius}_{halalkaliphilus.}_The_protologue

summarizingpropertiesofthesethreenoveltaxaispresentedin

Table4.

Fundinginformation

ThisworkwassupportedbytheRussianScienceFoundation (grant16-14-00121)totheRussianauthorsandbytheEuropean ResearchCouncil(ERC)undertheEuropeanUnion’sHorizon2020 researchand innovationprogram(grantagreementno. 694569-MICROLIPIDS)totheDutchauthors.

Acknowledgements

WethankNadezhdaKostrikinaforassistancewiththeelectron microscopy.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttps://doi.org/10.1016/j.syapm.2019.01. 001.

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