Downloaded from www.microbiologyresearch.org by IP: 138.232.79.190 On: Thu, 08 Sep 2016 12:50:59 atryangtzensis bacter anaeL989-N997 htfrte1S2SISsqec sL989.Teacsinnmesfrtegnm seque genome the for numbers accession The LN998998. is sequence ITS 16S-23S the for that LN998990-LN998997; are mann n upeetr al saalbewt h nieSplmnayMaterial. Supplementary online the with available is table supplementary One 001073 the for numbers accession GenBank/EMBL/DDBJ The Abbreviations: Microbiology Evolutionary and Systematic of Journal International hsi nOe cesatcedsrbtdudrtetrso h raieCmosAtiuinLcne(http://creativecommons.org/licenses/by/4.0/). License Attribution Commons Creative the of terms the under distributed article Access Open an is This [email protected] Hahn W. Martin Correspondence ã 06IUMS 2016 N,aeaencetd dniy T,itrei rncie spacer. transcribed intergenic ITS, identity; nucleotide average ANI, MWH-JaK3 rne nGetBritain Great in Printed T and oyuloatrsinensis noyboti h memn utr bv rblwtetrsodfrspecies for threshold (R the correlation below significant or A above demarcation. culture Ammermann the in endosymbiont iiaiyadAIvle fgnm-eune tan nbe h rdcinta ti highly species is the it to that belong prediction strains the four enabled these strains that genome-sequenced unlikely of values ANI and similarity eoecmaiosbsdo vrg uloieiett AI auso orsriscurrently strains four of values (ANI) identity as nucleotide classified average on based comparisons Genome =S 21495 (=DSM 3 2 1 Lang Elke and Hahn, W. Martin of description sinensis Polynucleobacter yangtzensis Polynucleobacter duraquae Polynucleobacter asymbioticus Polynucleobacter necessarius four of Reclassification elsiiaino tan QLW-P1DMWA-1 strains of reclassification utlcssqec oprsnapoc a sdt siaei h orsriscurrently strains four the cultures. if two estimate the to in used contained as was endosymbionts classified approach the comparison of sequence identity multilocus the A proved and 30859 ATCC with pce s oee,hmee ytefc,ta hsseisi ae nyo ecito and another culture description found a We the on available. Furthermore, only culture. based pure is as species aediculatus available this strain that type fact, a the lacks by hampered however, is, species 84% ugsigta aho hs tan ersnsasprt pce.Tespecies The species. separate a represents strains those of P each that suggesting %, 78.4 noybot fclae fiitdwt h genus the with affiliated of endosymbionts oyuloatrduraquae Polynucleobacter oyuloatrsinensis Polynucleobacter HuW1 ebi ntttDM-etceSmln o irognse n elutrnGmbH, Zellkulturen Braunschweig, D-38124 und 7B, Mikroorganismen von Inhoffenstraße Sammlung DSMZ-Deutsche Institut P Leibniz Helsinki, of University Station, Biological Mondsee, Lammi A-5310 9, Mondseestrasse Innsbruck, of University Limnology, for Institute Research . necessarius T =S 21492 (=DSM TC389 nwihtedsrpino h pce a ae,i olonger no is based, was species the of description the which on 30859, ATCC oyuloatrnecessarius Polynucleobacter oyuloatrnecessarius Polynucleobacter rpoB T a rpsdb ekanadShiti 97t comdt obligate accommodate to 1987 in Schmidt and Heckmann by proposed was CP110977 =CIP , W-u1 r OI0000adLJ0000,respectively. LOJJ00000000, and LOJI00000000 are MWH-HuW1T 3 trpE 1 , oan Schmidt, Johanna T icdA (2016), tan srpeettvsof representatives as strains CP110978 =CIP p o. respectively. nov., sp. , oyuloatrnecessarius Polynucleobacter p nov., sp. glnA ultsaediculatus T ,MWH-JaK3 ), 66, , mdh 2883 , oyuloatryangtzensis Polynucleobacter fbp T as ) 2 – , 2892 msbA 0.98, subsp. subsp. T oyuloatrasymbioticus Polynucleobacter T Polynucleobacter =S 18221 (=DSM 1 =S 21493 (=DSM and P lxnr Pitt, Alexandra 000)btenmliou sequence multilocus between <0.0001) a € utr Amran hrn h aeorigin same the sharing (Ammermann) culture Euplotes aj € asymbioticus asymbioticus gyrA p o. n emended and nov., sp. rete30 60 am,Finland Lammi, 16900 320, arventie € P p nov., sp. . ee of genes necessarius p o.and nov. sp. h eurdrvso fthis of revision required The . T ob nov., comb. T CP109841 =CIP CP110976 =CIP oyuloatrnecessarius Polynucleobacter eutdi N auso 75.7 of values ANI in resulted hr N auswt the with values ANI share 1 p o.and nov. sp. epropose We . DOI aiJ Taipale J. Sami 10.1099/ijsem.0.001073 Euplotes ob nov., comb. T T ,MWH-MoK4 ), n MWH- and ) csof nces Polynucleo- 2 Ammer- 2883 T – Downloaded from www.microbiologyresearch.org by IP: 138.232.79.190 On: Thu, 08 Sep 2016 12:50:59 n rmteedsminspoonl nterlifestyle their in profoundly endosymbionts (Hahn differ- the thus ponds, from and lakes ing freshwater of column water the necessarius RAgn eunesimilarities sequence gene rRNA a ifcl (Hahn 2003) (Hahn, difficult habitats freshwater was iso- from cultures previously pure strains as of lated classification limita- taxonomic these the to tions, Owing analyses. are morphological comparisons to phenotypical restricted and traits, chemotaxonomic eoi N fteendosymbiotic the of DNA genomic al. et 6 RAgn re n lotietclGCvle ftheir of (Hahn values DNA G+C identical genomic almost and trees gene rRNA 16S essarius ai esn,fu relvn tan eepreliminarily were strains free-living as classified four prag- for reasons, However, species. matic same endosym- the the represent and strains free-living biotic the if test rigorously to possible epciey a rpsd(Hahn proposed was respectively, free- and living endosymbiotic of separation lifestyle, in differences eesqec iiaiy( similarity sequence gene hmtxnmclyadcasfe as classified and chemotaxonomically th representing genus strains free-living four study, taxonomic ous ius soito xeiet (DNA experiments association utr iisdtriaino e aab DNA by taxa new pure of a determination of limits lack pur- culture The many research. for taxonomic suitable comparative not of reasons poses several for is culture This asymbioticus n atraadedsminso h genus the of free- endosymbionts various and ciliate, the ing for food as serving algae of ciliate, consists the culture mixed This 1987). Schmidt, & (Heckmann 07.Tu,tegenus the Thus, 2007). etdb ecito fteedsminscnandi the aediculatus in Euplotes contained endosymbionts the of description a by sented yatp tan h yeo h species the of type The strain. type a by necessarius mosbet sals uecluerpeetn h yeof necessarius type it the representing P. made culture pure hosts a their establish to and impossible bacteria tight these This between endosymbionts. relationship obligate represent thus and 2007) hi otcls(ekan&Shit 97 Vannini 1987; Schmidt, on & rely (Heckmann cells obligately host endosymbionts their these genus that demonstrated the with iated affil- ciliates freshwater of endosymbionts bacterial for 1987 in necessarius 2884 necessarius P. as classified of previously position strains taxonomic the of Evaluation genus The others and Hahn W. M. tan rvosydsrbdas free-living described two of previously traits ecological strains and genomic Recently, subsp. 07 u&Hh,20) wn otelc fpure of lack the to Owing 2006). Hahn, & Wu 2007; , .necessarius P. Polynucleobacter ih hlgntccutrn ihedsminsin endosymbionts with clustering phylogenetic tight , tal. et r w ftefwpoaytctx o represented not taxa prokaryotic few the of two are eepooe yK ekanadH .Schmidt J. H. and Heckmann K. by proposed were necessarius noybot u wl sfe-iigsrisin strains free-living as dwell but endosymbionts Polynucleobacter .necessarius P. ihsri QLW-P1DMA-1 strain with 09 02;Jezberova 2012b; 2009, , Hcmn cmd,18;Vannini 1987; Schmidt, & (Heckmann tan notosbpce,i.e. subspecies, two into strains ‘ tc 15 stock tal. et subsp. and tal. et Polynucleobacter eecaatrzdpeoyial and phenotypically characterized were Euplotes  tan u otehg 6 rRNA 16S high the to due strains 09.Teesrissae16S share strains These 2009). , .necessarius P. 9% ihendosymbiotic with %) 99 09.Bcueo h profound the of Because 2009). , n h species the and ’ utr =2=TC30859) (=E24=ATCC culture needn investigations Independent . asymbioticus – N yrdzto)o by or hybridization) DNA tal. et .necessarius P. .necessarius P. 09.I hsprevi- this In 2009). , .necessarius P. tal. et n t yespecies type its and T .necessarius P. subsp.  stetp strain. type the as 9%with % 99 Polynucleobacter 00 Vannini 2010; , Polynucleobacter asymbioticus twsnot was it , .necessar- P. – N re- DNA srepre- is .nec- P. subsp. subsp. tal. et tal. et liv- P. P. e , , . , bioticus eemndtegnm euneo h eann two remaining the of sequence (MWH-HuW1 strains genome the determined h orsrispeiul lsiidas asymbioticus classified previously strains four The 1). (Table EMBL/GenBank Sequenc- 1). (Table 42 Mbp about 2 was about coverage is ing size genome the cases, MWH-MoK4 strain of characterization phenotypic the elsinn rudislnt xs speiul reported, previously As MWH-MoK4 axis. microscopical strain length single its a recent of around observation spinning in cell a resulted for strain this required Interestingly, of genes investigation of synthesis. set days, whole 17 the within flagella encoded mm 50 strain of this diameter and a to up swarms building annotation IMG/ER the (Markowitz using pipeline annotated were sequences 17 about MWH-JaK3 oiino l orfe-iigsriso h genus the taxonomic of the strains evaluated free-living cleobacter four we all here, of presented position study the In iy(N)vleo 56%frcmlt eoesqecsof sequences genome complete QLW-P1DMWA-1 for strains % 75.6 of value (ANI) identity nucleotide average an revealed investigation This 2016). ee o ytei fa nxgncpooytei system photosynthesis anoxygenic an of synthesis for genes asymbioticus ide 05;Konstantinidis 2005a; Tiedje, & etdfrpoaytcseis(Kim species prokaryotic for gested rwho raa oentoe orewsfudprevi- found was source (Hahn nitrogen strains two sole conflict in as ously partial strains. urea the in of on are features Growth data phenotypic content reported gene previously with cases, proteins. nitrogen flagella three as encoding all urea genes In of and genes utilization catalase for source, coding previ- genes to presence/ of included related absence This were traits. features phenotypic determined content 2). ously gene (Table DNA these content gene their of in of Some differed content they However, G+C 1). and (Table only size differed genome and in similarity sequence marginally gene rRNA 16S % >99 MoK4 genus the of cleobacter strain (Hahn another medium for previously NSY bio- described liquid from in extracted was grown sequencing mass genome for used DNA 95 of threshold demarcation species the below ardedrasrsle o tan MWH-HuW1 strains for resulted reads Germany). paired-end instrument MiSeq Genomics, Illumina (Eurofins an with sequenced paired-end ltsa en o-oie u nacoe ok strain look, closer a agar on soft But using two non-motile. by in MWH-MoK4 tested being been detected genes as had only had but plates strains were strains four positive, All four trait catalase strains. All this be 2). encoding to (Table putatively previously strains four tested the detected been only of were one transporters urea in and urease a encoding nentoa ora fSseai n vltoayMicrobiology Evolutionary and Systematic of Journal International T MWH-HuW1 , (Hahn  rvosycasfe as classified previously (Meincke T ..srisQLW-P1DMWA-1 strains i.e. , (Hahn o tanMWH-JaK3 strain for T n1 n 2cnis epciey nboth In respectively. contigs, 42 and 19 in ooismysol pedo uhplates, such on spread slowly may colonies tal et tal. et T ,20)b eoecmaio.We comparison. genome by 2009) ., tal. et tal. et T loecdsacmlt lse of cluster complete a encodes also 09 eecmae (Hahn compared were 2009) , n MWH-JaK3 and T T 02 n eoie nDDBJ/ in deposited and 2012) ,  tal. et 02.Sognlbaiswere libraries Shotgun 2012). , n MWH-MoK4 and n MWH-JaK3 and o tanMWH-HuW1 strain for tal et 09,btgnsputatively genes but 2009), , .necessarius P. tal et ,2006). ., enovo De T h rf genome draft The . ,21;Konstantinidis 2014; ., .necessarius P. T n completed and ) – tal. et 6%AIsug- ANI % 96 T hc sfar is which , subsp. sebyof assembly T T MWH- , shared , 04 as 2004) , Polynu- Polynu- subsp. T asym- T tal. et T . and and 66 - , Downloaded from www.microbiologyresearch.org by IP: 138.232.79.190 On: Thu, 08 Sep 2016 12:50:59 otesm pce.Dtriaino arieAIvalues ANI pairwise of belong Determination to species. considered same be to the have to strains, strains four which the tested of first position we taxonomic the of or evaluation errors For annotation from the tests. result phenotypic of cases, insufficient could other expression In discrepancies used. of conditions the lack cultivation from the under result phe- genes and simply content gene may in differences notype these of some that ered h al ersnsa nopeels fdfeecsbtengenomes. between differences of list incomplete an represents table The http://ijs.microbiologyresearch.org 2. Table pigmentation any showed (Hahn never trait strain this the revealing of cultures but 1. Table lsiidas classified h pe orsrisaecretycasfe as classified currently are strains four upper The tal. et xdtv stress Oxidative Motility nxgncphotosynthesis Anoxygenic Other xdtv hshrlto/nrymetabolism phosphorylation/energy Oxidative MWH-JaK3 MWH-MoK4 ee uaieyencoding: putatively Genes TR ( STIR1 ’ nrai nutrients Inorganic QLW-P1DMWA-1 tanLfsyeGenome Lifestyle Strain MWH-HuW1 eaproteobacterium beta 03 u lsesi 6 RAgn (Wang gene rRNA 16S in clusters but 2013) , ultsaediculatus Euplotes ifrne ngn otn ewe h orsriso h genus the of strains four the between content gene in Differences genus the of taxa six the of characteristics genome Major .necessarius P. T Catalase lglagenes Flagella htsnhssgn cluster gene Photosynthesis ells ytaeoeo rti + + [UDP-forming] subunit catalytic synthase Cellulose C protein operon synthase Cellulose ctt permease Acetate feoAB abnmnxd dehydrogenase monoxide Carbon yohoeb- emnloiae(yA)+ (CydAB) oxidase terminal bd-I Cytochrome raeadACtp raetasotr+ transporter urease ABC-type and Urease uaaereductase Fumarate B-yeFe ABC-type irt euts asmltr)++ + + + + + NH (releases lyase Cyanate (assimilatory) reductase Nitrite (assimilatory) reductase Nitrate transporter nitrate/nitrite/cyanate ABC-type T =S 21493 (=DSM T =S 21495 (=DSM =S 21492 (=DSM ee utk fFe of (uptake genes T =S 18221 (=DSM ’ BFe-iig20 61C044 55558Hao 2565956558 CP004348 46.1 2.05 Free-living CB 3+ subsp. noybot15 56C011 53804Boscaro 2503982034 CP001010 45.6 1.56 Endosymbiont ) rnpr system transport T tal. et relvn .54. OI000020627 hsstudy This 2608642177 LOJI00000000 45.4 2.05 Free-living ) T actP T relvn .34. P05120330 Hahn 2505313000 CP007501 45.2 2.03 Free-living ) relvn .24. OJ000023993 hsstudy This 2630969031 LOJJ00000000 45.5 2.32 Free-living ) necessarius 06.I a ob consid- be to has It 2016). , T relvn .64. P06560219Meincke 640427129 CP000655 44.8 2.16 Free-living ) 2+ 3 + + + ) n CO and (Boscaro .necessarius P. 2 tal. et rmcaae + + cyanate) from tal. et 09 n te hlgntctes(is1ad2 with 2) and 1 (Figs trees phylogenetic other and 2009) , 03,adstrain and 2013), , (Mbp) subsp. size QLW-P1DMWA-1 asymbioticus N G+C DNA Polynucleobacter (mol%) content ee gene 1 genes 2 ewe 57ad7. Tbe3.Tu,alvle r far are values 95 all range of Thus, value the 3). threshold in (Table the values below % 78.4 in Rosselló-Móra, and combinations 75.7 & all between (Richter for JSpecies resulted software 2009) the using by eacto fpoaytcseis(Kim species prokaryotic of demarcation yteedsminso h genus the of endosymbionts free-living species the four the those by of to one belong least may at if strains test to aimed we Next, Konstantinidis 2005; Tiedje, & stantinidis mixed À À À À À À À À À + ‘ eaproteobacterium beta tan (Hahn strains Polynucleobacter ultsaediculatus Euplotes ceso number accession T DDBJ/EMBL/ GenBank MWH-JaK3 oprdi hsstudy this in compared tal. et À À À À À À À À À À À À À + + + + + 09,teolgt noybotSI1is STIR1 endosymbiont obligate the 2009), , ’ Bi akn on lsiiain(Hao classification sound a lacking is CB eiinof Revision netgtdtaxonomically investigated T MWH-HuW1 Genome .necessarius P. IMG ‘ ID – tc 15 stock 6%AIsgetdfrthe for suggested ANI % 96 oyuloatrnecessarius Polynucleobacter .necessarius P. À À À À À À À À À À À À + Polynucleobacter T ’ strains. tal. et tal. et utr (ATCC culture tal. et Reference MWH-MoK4 tal. et 2006). , 04 Kon- 2014; , tal. et tal. et represented (2013) (2016) À À + + + + (2013) (2012) nthe in 2885 T Downloaded from www.microbiologyresearch.org by IP: 138.232.79.190 On: Thu, 08 Sep 2016 12:50:59 utr rmMH n etdwehrteendosym- the as whether here tested designated and bionts, MNHN from culture an osse h ae16S same the possessed mann, culture the genus from the should of both descend endosymbionts bacter thus, same 1969; the both in contain Ammermann cul- D. 30859 the by and established culture ATCC This mate- signed. a ture if is institution agreement this transfer from rial obtained be can and , al 1(vial nteoln upeetr Material). Supplementary 16S online the in The in listed (available are culture. sequencing S1 and ATCC Table amplification previous for used the Primers in contained endosymbionts salse yD memn ssilmitie tthe at maintained still is Ammermann of Mus D. culture by the established former the to Importantly, as similar highly species. corrected is which later 1971), (Ammermann, but 1970) mann, an h iit ern h noyboi atrawas bacteria endosymbiotic as the identified bearing initially Heck- Klaus ciliate to The provided mann. later was and 1970) Ammermann, cmd 18)hdbe sltdb itrAmmermann Dieter by isolated ‘ been had (1987) Schmidt & nmr,adcluerqet oti nvriyi June in exist The university not this does to culture, & unsuccessful. requests were the 2015 Heckmann culture of by and source a anymore, mentioned as was (1987) Schmidt which Mun- unsuccessful. of Germany, University were the ster, 1987 at collection in culture ATCC Heckmann The at Schmidt Heckmann o te ore fteculture the searches of Recent sources time. of other the periods for maintain longer to over able not culture were ciliate we laboratories, two in efforts (Vannini eeunigo h 6 RAgn n yetbihetof establishment by and gene 16S rRNA 16S the of resequencing 2886 staff ATCC enquiry, On ago. years culture stated, few ciliate a catalogue this the removed from ATCC Unfortunately, 30859). 3. Table others and Hahn W. M. utr ial eevdealdu obte characterize The better 2003. to in us us enabled of of type one the of by received order previously finally an for placed culture months 10 about culture of this period delivery a for ble owr ihadalrcn tepshv eutdi com- in resulted have difficult attempts very recent failure was all plete item and the with avail- work beginning be to ever the not From likely again. will and able lot distribution new a make rmpnsna asil,France Marseille, near ponds from MWH-JaK3 TR ( STIR1 QLW-P1DMWA-1 MWH-MoK4 –  u ainld National eum ultsaediculatus Euplotes 3 negnctasrbdsae IS sequences (ITS) spacer transcribed intergenic 23S eotie the obtained We . ‘ tm389hspoe o ifcl ob beto able be to difficult too proved has 30859 item ultsaediculatus Euplotes oprsno eoi iiaiyo aab ariecluaino N values ANI of calculation pairwise by taxa of similarity genomic of Comparison – 3 T euneof sequence ITS 23S tal. et .necessarius P. T ’ T Arl21) hs ifcliswr responsi- were difficulties These 2014). (April =S 21493 (=DSM =S 21495 (=DSM 07.Dsieitnieindependent intensive Despite 2007). , T =S 18221 (=DSM ’ ultseurystomus Euplotes itieNtrle(NN,Paris, (MNHN), Naturelle Histoire 807. 4176.3 84.1 76.1 78.0 ) utr netgtdb Heckmann by investigated culture otie nti iit utr by culture ciliate this in contained ultsaediculatus Euplotes T ) T ) .necessarius P. T – ) 3 T eune sthe as sequences ITS 23S ‘ tc 15 stock .necessarius P. ’ ultsaediculatus Euplotes ultsaediculatus Euplotes n16 Ro& (Rao 1969 in Ro&Ammer- & (Rao QLW-P1DMWA-1 tanAmmer- strain ’ Ammermann eoie by deposited Polynucleo- strain T h genus the al. et engnm eunigo h noybot contained endosymbionts the the of in have the sequencing would of genome endosymbionts the been comparison with for strains free-living strategy four straightforward most The 30859. utr of culture ersnsasnnmu usiuin(/)a third a ( at gene (T/C) the of substitution position synonymous codon polymorphism a nucleotide single represents base This single a similarity). in % only (99.98 differed STIR1 and Ammermann of strain sequences the while differences), nucleotide 77 ueotie rmteMH r nedietclwith identical of indeed material are type MNHN the the from obtained ture TR eoeaddsge pcfcpiesfrpartial of loci for eight sequencing the primers enabled of primers specific These S1). designed (Table amplification the and around genome scattered sequenced STIR1 genes completely housekeeping representing the loci with necessarius similarity P. genome high un eunecmaiosadpyoeei analyses. we phylogenetic sequencing, and subse- that with Assuming comparisons genome approach sequence sequencing quite whole quent multilocus be a of would employed coverage Instead acceptable an laborious. endosymbi- with the yielding genome of for sequencing ont cultivation for sufficient mass DNA endosym- amounts and total DNA culture, the the of the because fraction in small task contained very a non-routine only comprise a bionts be would this memn.Teeaeteedsminscnandin contained endosymbionts and 30859 the ATCC are These Ammermann. ftetoclae r oae bu 0 maati France in apart km 400 about isolation located of are sites ciliates the since two surprising the quite of revealed is loci similarity eight sequence the high at very The dehydrogenase. bandrvae htaogte24ISsqecsof sequences ITS genus 224 the the representing among strains that revealed obtained rmtegnmso h orfe-iigsrispossessed 81.8 strains of range free-living the four in the similarities sequence of extracted genomes sequences concatenated the the The and from bp. endosymbiont 6087 the of of length sequence sequence total a in ing hr nietclsqec with sequence identical organisms two an only share DDBJ/EMBL/GenBank, in deposited the to 30859 1) ATCC culture (Fig. from identical previously (Vannini be obtained to sequence found was Ammermann nentoa ora fSseai n vltoayMicrobiology Evolutionary and Systematic of Journal International – MWH-MoK4 7% hssget htteedsminsi h cul- the in endosymbionts the that suggests This %. 97 07.AlohrISsqecso tan representing strains of sequences ITS other All 2007). , ultsaediculatus Euplotes 577. 75.7 78.3 75.7 tal. et Polynucleobacter ultsaediculatus Euplotes TR (Boscaro STIR1 2007). , T .necessarius P. .necessarius P. .necessarius P. BLAST .necessarius P. MWH-JaK3 memn utr.However, culture. Ammermann hr iiaiisi h ag of range the in similarities share erhswt h T sequence ITS the with searches 6478.4 76.4 tal. et icdA tanAmransae a shares Ammermann strain (Petroni TR otie nanother in contained STIR1 Polynucleobacter tanAmranresult- Ammermann strain noigteisocitrate the encoding ) 03,w eetdeight selected we 2013), , T .necessarius P. otie nATCC in contained tal. et – 84%(1108 % 88.4 02 Vannini 2002; , MWH-HuW1 .necessarius P. 76.7 currently strain – 704 T 66 Downloaded from www.microbiologyresearch.org by IP: 138.232.79.190 On: Thu, 08 Sep 2016 12:50:59 http://ijs.microbiologyresearch.org of isolation the culture before years Ammermann in aediculatus 30 the about and of place Parma), took near establishment Stirone the (River addition, Italy and (Marseille) octntdsqecshmlgu oteegtlc and loci values. eight the ANI to whole-genome homologous with sequences Concatenated correlate multilo- of sequences similarities cus sequence whether analysed we Next, of DNA considered genomic unavailable be sarius the could for similarities. STIR1 surrogate a sequence of as sequence genome-wide genome high the strains Thus, very two the share that also however, suggests, similarity sequence i.1. Fig. ee fbcei ftegenus the of bacteria of genes 2. Fig. ehd;teebosrpvle r eitdi h re JM/P a,00 usiuin e uloieposition. nucleotide per substitutions 0.05 Bar, NJ/ML/MP. order the in depicted are values bootstrap these methods; insrpeetn h genus the representing bionts rmaaye ytemxmmlklho M)admxmmprioy(P ehd r loidctd otta values Bootstrap indicated. also are of methods threshold (MP) the maximum-parsimony above and replicates) (ML) of maximum-likelihood (percentage the by analyses from eune rvosy(Hao previously sequenced anwr bandb sn pcfcpies hra l te eune eeetatdfo hl eoesequences. genome whole from extracted were sequences other all whereas primers, specific Strain using by obtained were mann ‘ tc 15 stock TR (Petroni STIR1 ‘ eaproteobacterium beta egbu-onn N)te ae n16S on based tree (NJ) Neighbour-joining egbu-onn recluae ihcnaeae utlcssqecso ih oirpeetn housekeeping representing loci eight of sequences multilocus concatenated with calculated tree Neighbour-joining ’ AC 30859). (ATCC 100/100/99 0.05 0.05 63/67/72 tal. et 71/ -/55 tal. et ’ Brpeet tanaflae ihtegenus the with affiliated strain a represents CB Polynucleobacter Polynucleobacter 02.Ti eyhigh very This 2002). , 80/ -/59 2013). , 100/100/100 100/96/97 97/96/- 53/67/64 100/99/99 100/100/100 88/ -/59

100/98/94 74/ -/35 100/100/99 100/99/99 100/100/100 otie nteclueof culture the in contained P.difficilis – TbeS) eune fteendosymbiont the of Sequences S1). (Table .neces- P.  Euplotes 3 T eune eosrcigtepyoeei oiino endosym- of position phylogenetic the reconstructing sequences ITS 23S 0%aesonfrtoendsspotdi tlatoeo h three the of one least at in supported nodes those for shown are % 60 Cupriavidusmetallidurans MWH-HuW1 ‘beta proteobacterium’ CB‘beta proteobacterium’ (CP004348) MWH-JaK3 MWH-MoK4 P. acidiphobus QLW-P1DMWA-1 AM-8B5 P. necessarius P. necessarius P. necessarius 100/100/100 P. cosmopolitanus metallidurans Cupriavidus metallidurans Cupriavidus taiwanensis arieAIvle o h eoe ftefu free-living four and the of investigated genomes strains the for values ANI pairwise h rdce ausfl nterneo 77 of range the in fell values predicted The hudb lsiida ebro h species the of member a as strains free-living classified four necessarius the be of none should that confirmed which eunesmlrt fteegtcnaeae oiadANI and of loci correlation (R concatenated tight eight values a the revealed of This similarity sequence 3). (Fig. analysed were tanAmranadSI1wr lotietcl tcan it identical, almost were STIR1 and Ammermann strain orsrisrpeetn h genus the representing strains four eoesqecso i tan ersnigtegenus the representing ynucleobacter strains six of sequences genome T (AJ550657) T T T (AJ550654) (FM208181) (AJ550666) MWH-PoolGreenA3 Ralstoniasolanacearum P.rarus ‘beta proteobacterium’ CB(CP004348) Ammermann (LN998998) STIR1 (AM398078) ATCC 30859 (AM397067) MWH-MoK4 T MWH-JaK3 MWH-HuW1 ultsaediculatus Euplotes (AJ879783) QLW-P1DMWA-1 P. necessarius P. necessarius 2 ic h utlcssqecsof sequences multilocus the Since . MT-CBb6A5 0.98, Polynucleobacter CH34 MWH-MoIso2 (including ATCC 43291 P T T (NC 007973) 000) hc nbe rdcin of predictions enabled which >0.0001), T (LOJI01000000) (CP007501) T LMG 19424 CH34 H1130 (AXBU00000000) (LOJJ01000000) T (FM208180) Ammermann STIR1 (CP000655) T eiinof Revision (FM208182) .necessarius P. T GMI1000 (AL646053) .necessarius P. T (CP000655) T (AJ550672) ‘ (CP000352) eaproteobacterium beta T tanAmran Results Ammermann. strain hs eoehsbeen has genome whose (CP002877) T (NC_010528)

oyuloatrnecessarius Polynucleobacter Cupriavidus tanAmmer- strain tanAmmermann. strain .necessarius P. – 5%ANI, % 85 ’ B and CB) Fg 2) (Fig. 2887 Pol- P. 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Polynucleobacter Cupriavidus h phylogenetic The . tanAmmermann strain respectively. , Polynucleobacter Polynucleobacter T n MWH- and Polynucleo- l four All . p nov., sp. resulted and P. o.dfesfo h tesb h eeto ftehydroxyl- C the acid of detection fatty the ated by others the from differs nov. rmtetreohrtp tan ytedtcino h satu- the of C detection acid the fatty by rated strains type other three the from oe h yesri of strain type the more, int u o both not but pionate yangtzensis P. oyuloatrnecessarius Polynucleobacter Hahn emend. necessarius of description Emended con- acids. acids fatty fatty total the both to since % reliable 1 than very less tributed be not may trait this polys ecie rvosy(Hahn previously described HuW1 nt.Tetp tanof strain propi- type of assimilation The no onate. showed which strain only the is nov. spsil yuigteciei rsne nTbe5 h type The 5. Table of in presented strain criteria other the each using from by possible species is novel four the of discrimination The far. assimilate te pce ftegenus the of species other pce ope nc ihntegenus the Jezbera 2003; within (Hahn, PnecC complex species (Hahn taxa scribed necessarius P. eeto ftetoftyaisC acids fatty two the of detection sinensis bet siiaebt xlaeaeand oxaloacetate both assimilate to able ifrfo tan representing strains four strains All from criteria. differ chemotaxonomic using by possible akn o tanMWH-MoK4 strain for lacking ily bus lxi hrceie ytepeec fadiagnostic a of presence the by (5 characterized sequence is plex oyuloatrcosmopolitanus genus Polynucleobacter the of species novel Polynucleobacter four the of Differentiation 2009). 01,b 02)btcno edsrmntdb hsfeature of this by strains discriminated be from cannot but 2012a) b, 2011a, hrceitc o ifrnito fsrisaflae with affiliated strains genus of the differentiation for Characteristics based be sequences. cannot ribosomal complex on species solely cryptic this within PnecC-specific species the (Hahn using 445 by detected be fluoresence can sequence tic fteftyai C presence the acid by fatty characterized the are of species latter the of Strains acidiphobus coli nentoa ora fSseai n vltoayMicrobiology Evolutionary and Systematic of Journal International , oiin 445 positions oyuloatrdifficilis Polynucleobacter ueos .ms.n. masc. L. numerous; T p o.dfesfo h te he yesrisb the by strains type three other the from differs nov. sp. epciey hs rpsdtp tan were strains type proposed These respectively. , .asymbioticus P. L- Polynucleobacter satt,wietetp tanof strain type the while aspartate, tal et nsitu in nteGCcneto N (Hahn DNA of content G+C the in ¢ p o.i h nysri bet siiaepro- assimilate to able strain only the is nov. sp. -GAGCCGGTGTTTCTTCCC-3 h ornvlseisadsvrlunde- several and species novel four the , ,20) oee,asgmn oacertain a to assignment However, 2005). ., 6:0 : 16 0:0 : 20 – .cosmopolitanus P. ekan&Shit1987 Schmidt & Heckmann 2:0 : 12 6)i h 6 RAgn.Ti diagnos- This gene. rRNA 16S the in 463) tal et rmtepeiul ecie species described previously the from yrdzto FS)poePnecC-16S- probe (FISH) hybridization tal et eepbihdpeiul (Hahn previously published were hl h yesri of strain type the while , -H(al ) h yesri of strain type The 4). (Table 2-OH tal et -H hc a o eetdi any in detected not was which 3-OH, L- ltmt n -satt.Further- L-aspartate. and glutamate ,21)tgte omteso-called the form together 2016) ., ob o.i h nysri beto able strain only the is nov. comb. 2009 . .sinensis P. .asymbioticus P. ,21) hscytcseiscom- species cryptic This 2011). ., Polynucleobacter rmohrmmeso h fam- the of members other from tal. et nucleous (Po.ly.nuc T and , r rsne nTbe4. Table in presented are 7:0 : 17 09,addt previously data and 2009), , .rarus P. Polynucleobacter oyuloatracidipho- Polynucleobacter oyuloatrrarus Polynucleobacter p o.i h oestrain sole the is nov. sp. n C and u,kre;NL masc. N.L. kernel; nut, (Hahn ¢ eobctr r adj. Gr. le.o.bac.ter. , ob o.differs nov. comb. L- .difficilis P. 5:1 : 15 ltmt,while glutamate, hrceie so characterized Polynucleobacter .duraquae P. .duraquae P. ¢ tal. et ! , 6 c Escherichia however, ; 2010). , and tal. et tal. et sp. sp. 66 P. P. is , , Downloaded from www.microbiologyresearch.org by IP: 138.232.79.190 On: Thu, 08 Sep 2016 12:50:59 h ehdue o eemnto fasmlto aaiiiswsdsrbdpeiul (Hahn previously described was capabilities assimilation of determination for used method The .masc. L. http://ijs.microbiologyresearch.org 5. Table n. 4. Table enpeae n esrdudrtesm odtosa o tanMWH-MoK4 strain for as conditions same the under measured and prepared been sar hsseisblnst h family the to belongs species This aafrsrisQLW-P1DMWA-1 strains for Data ue npr utr (Vannini culture pure in tured th of genus ciliates in living strains endosymbiotic obligatory bours 07,adte1SrN eesqec otissome of strains contains free-living sequence in far gene so rRNA found not 16S mutations unusual the and 2007), otpoal ecne rmfe-iigsriso h genus Polynucleobacter the of strains free-living from descended probably Most 1987). Schmidt, & (Heckmann penicillin-sensitive nucleoid-likestructures; multiple with morphology elongated have Cells + otn s44 is content G+C nini ubro suoee (Boscaro pseudogenes of number C C C C C C C C C C C 11-Methyl C C C hrceitcQLW-P1DMWA-1 Characteristic C siiainof: Assimilation umdfaue7(nldn C (including 7 feature Summed C (including 2 feature Summed C (including 1 feature Summed C acid Fatty bacter ¢ 6:0 : 16 0 : 12 1 : 18 1 : 18 1 : 16 1 : 16 1 : 15 1 : 14 0 : 20 0 : 18 0 : 17 0 : 16 0 : 15 0 : 14 0 : 12 rpoi cd+++ + + acid Oxaloacetic acid Propionic L L .s .adj. L. i.us. Guaae+ -Glutamate Aprae+ -Aspartate tal. et Euplotes. ! ! ! ! ! ! 2-OH 2-OH 7 9 7 5 6 5 c c c c c c hrceitc o ifrnito ftefu rpsdnvlseiso h genus the of species novel proposed four the of differentiation for Characteristics genus the of species novel four the representing strains of contents acid fatty Major 07.Gnm iei bu . b n t DNA its and Mbp 1.6 about is size Genome 2007). , h qiaeto h r et n. neut. Gr. the of equivalent the Polynucleobacter 8:1 : 18 necessarius ofr noyboi tan ol o ecul- be not could strains endosymbiotic far, So ! (Boscaro 7 c – 6ml.Tegnm nldsalarge a includes genome The mol%. 46 tal. et nipnal,necessary). indispensable, o ihmn ulod;nec.es. nucleoids; many with rod 9:1 : 19 0 : 14 0 : 12 tal et T 03 Hahn 2013; , MWH-JaK3 , ! -H . . . 11.9 9.9 1.0 9.2 0.5 9.6 0.4 3-OH) ALDE?) 6 tal. et ,20;Hahn 2007; ., c Burkholderiaceae . . 0.3 2.0 0.4 ) À 03 Vannini 2013; , T bactron tal. et n MWH-HuW1 and T QLW-P1DMWA-1 09 Van- 2009; , tal., et o;N. rod; a n har- and 2009). 221. 9615.9 29.6 15.5 22.2 292. . 19.8 38.6 1.1 45.0 20.4 35.6 12.9 41.3 . . . 0.5 0.3 3.8 2.4 0.3 5.5 0.5 1.2 3.7 1.1 1.2 0.3 0.9 3.4 . . . 4.2 1.1 8.1 0.4 3.1 0.9 . . . 1.3 1.3 2.2 2.5 tal et – – – – – – – – – – ., e T MWH-JaK3 eetknfo Hahn from taken were T À À À À + + + ne h ceso ubr M907 AM398078, AM397067, numbers accession CP001010. LN998990-LN998998 and the available are under endosymbionts these characterizing sequences h genus the andi the in tained 05.Edsminso h genus ATCC the culture of unavailable Endosymbionts currently 30859. the in endosymbionts species the TC389 noybot ftegenus the the in of Endosymbionts culture in 30859. endosymbionts ATCC the with identical be to considered (Mus eamme ftespecies the of the to member considered also a in is be sequence, genome endosymbionts complete a by the terized charac- endosymbiont, this to Therefore, culture. genetically Ammermann similar highly T T  u ainld National eum MWH-JaK3 . ultsaediculatus Euplotes Polynucleobacter . 0.2 0.6 – – – – .necessarius P. T tal. et tal. et ultsaediculatus Euplotes MWH-HuW1 2009). , Polynucleobacter 20) oee,ftyai ehletr had esters methyl acid fatty however, (2009); ’ itieNtrle ai,Fac)are France) Paris, Naturelle, Histoire eiinof Revision Polynucleobacter srpeetdb ecito fthe of description a by represented is MWH-HuW1 + (Vannini TR utr eefudt be to found were culture STIR1 .necessarius P. 0.5 0.3 . 0.3 0.4 0.6 – T oyuloatrnecessarius Polynucleobacter tal. et T Polynucleobacter memn culture Ammermann eeadgenome and Gene . 07.Tetp of type The 2007). , Polynucleobacter MWH-MoK4 MWH-MoK4 0.4 1.8 À À – – – – – – 2889 con- T T Downloaded from www.microbiologyresearch.org by IP: 138.232.79.190 On: Thu, 08 Sep 2016 12:50:59 i aapeetdi al .Cnan relvn strains free-living Contains 1. genus Table the of in presented Hahn data of mic data Hahn phenotypical of on Hahn based and (2003) is description The of concentrations higher limestone). with dissolved water i.e. water, hard from/of fakln rcru-eta rswtrsses Never (Hahn systems. waters freshwater acidic circum-neutral in found or alkaline of ad .fm n. fem. L. hard; oyuloatrduraquae Polynucleobacter nov. sp. of Description CP000655 numbers accession AJ879783. the and type under the available characterizing are sequences strain genome and Gene 2011). eie yasz f22MpadaDAGCcnetof 5 content sequence G+C diagnostic DNA character- the a are species by and this Mbp ized with 2.2 affiliated Strains of mol%. size 44.8 a by terized 2890 109841 QLW-P1DMWA-1 is strain type The C is compound 2-hydroxylated sole The C asymbioticus Polynucleobacter asymbioticus of Description others and Hahn W. M. odlctdi h utinAp ta liueo 30m 1300 of altitude an at Alps Austrian (Hahn the in located pond TGATTGTTTA-3 o;NL ac adj. masc. N.L. not; Basonym: adj. masc. rctae ao ellrftyaisaeC are acids fatty cellular Major citrate. or rswtrhbtt.Clsaesotrd,0.7 rods, short circum-neutral or are acidic Cells of habitats. column genus water freshwater the the in of dwelling strains free-living Contains al. et Hahn of Hahn of Hahn data and phenotypical (2009) on based is description The Hahn eghad0.4 and length eoi n auttvl neoi.Cnb utvtdon cultivated Luria be Can R2A, anaerobic. NSY, facultatively and aerobic osntasmlt lclt,oaae oxaloacetate, oxalate, glycolate, assimilate not Does 34 to up at 0 occurs with Growth con- occurs surface. and Growth circular smooth non-pigmented, with are vex agar NSY on grown ihrcnetain.Asmltsaeae propionate, or acetate, fumarate, NaCl succinate, (w/v) Assimilates malate, % 0.6 pyruvate, with concentrations. not but (w/v) higher % 0.5 with occurs L- bevdfrsm oesbtae (Hahn substrates more some for observed 8:1 : 18 cysteine, ! 21) swl sdt rsne nTbe n 2. and 1 Tables in presented data as well as (2012), tal et 7 tal. et T c ,wihwsioae rmasalaii freshwater acidic small a from isolated was which ), tal. et tal. et n umdfaue2(nldn C (including 2 feature summed and 2009. . asymbioticus L- oyuloatrnecessarius Polynucleobacter 05.Tegnm ftetp tani charac- is strain type the of genome The 2005). , ltmt and glutamate Polynucleobacter 20) aapeetdi al n geno- and 4 Table in presented data (2009), – 20) n ngnmcdt fMeincke of data genomic on and (2009), . mi it.Chemo-organotrophic, width. in µm 0.5 ¢ aqua tal et – tal. et ob nov. comb. etn n etn ei.Colonies media. peptone and Bertani nte16S the in oyuloatrduraquae Polynucleobacter Polynucleobacter yboiu a-um -a symbioticus 21a,o hmtxnmcldata chemotaxonomical on (2012a), . o symbiotic). not – ae;NL e.fm n. fem. gen. N.L. water; 20) nceoaooia data chemotaxonomical on (2009), . wv al ekgrowth Weak NaCl. (w/v) % 0.4 (dur.a L- satt.Wa siiainis assimilation Weak aspartate. wligi h ae column water the in dwelling (a.sym.bi.o – tal et 3 RA(Jezbera rRNA 23S ¢ ue .adj. L. quae. ¢ -ACTAAGCGATCTAA ,21;Jezbera 2016; ., T =S 18221 (=DSM iigtgte;N.L. together; living D- 2:0 : 12 subsp. ¢ aatrncacid, galacturonic ics r pref Gr. ti.cus. Polynucleobacter 2-OH. 6:1 : 16 tal. et durus asymbioticus – 4:0 : 14 ! . min µm 1.2 7 duraquae c 2009). , C , L- 3-OH). a-um -a T tal. et serine tal. et =CIP tal. et 6:0 : 16  C. a , , , cuswt 0 with occurs ae xlaeae ucnt,fmrt and fumarate succinate, oxaloacetate, vate, int,mlnt,oaae levulinate, oxalate, malonate, (Hahn substrates pionate, more some al. for et observed is assimilation D- 01.Clsaecre os 0.9 rods, curved are Cells 2011). L- at cd r C are acids fatty r C are L- loyae xlt,levulinate, oxalate, glyoxylate, and fumarate (Hahn substrates succinate, malate, L- oxaloacetate, pyruvate, e ytm.Clsaesotrd,0.5 rods, short genus are the Cells systems. of ter strains free-living Contains Polynucleobacter 2). Hahn (Table of study data Hahn phenotypical of on Hahn based and (2003) is description The isolated). was strain type the where from yangtzensis ufc.Got cusa pt 35 to up at 0 NSY smooth with occurs on with convex grown Growth and Colonies surface. circular media, media. non-pigmented, soy are R2A agar tryptic and extract, agar yeast Standard nutrient soytone, NSY, peptone, on cultivated broth, be Can anaerobic. facultatively o bevd a ecliae nNY etn,yeast peptone, NSY, on Luria cultivated and R2A far. be extract, usually so Can is observed motility observed. but been flagella not of not synthesis has for genes a system expression Encodes encodes photosynthesis but strain a photosynthesis type of anoxygenic The for observed. cluster not gene was anaer- aerobic; growth and obic Chemo-organotrophic width. in µm 0.5 0.3 mohsrae rwhocr tu o30 to up with at convex occurs and Growth circular surface. non-pigmented, smooth are agar NSY D- ue2(nldn C (including 2 ture ltdcmon sC is compound ylated 110977 oyuloatryangtzensis Polynucleobacter yangtzensis of Description numbers accession of the content under G+C DNA available AJ550654. a and are CP007501 and the strain characterizing Mbp sequences 2.0 genome type and character- of Gene is mol%. size strain 45.2 type a the by of ized genome The 2003). (Hahn, h yesri sMWH-MoK4 is strain type The icuigC (including nentoa ora fSseai n vltoayMicrobiology Evolutionary and Systematic of Journal International alanine, serine, ytie ekasmlto sosre o oemore some for observed is assimilation Weak cysteine. galactose, galactose, – . mi it.Ceoogntohc eoi and aerobic Chemo-organotrophic, width. in µm 0.5 09.De o siiaegyoae loyae pro- glyoxylate, glycolate, assimilate not Does 2009). , 6:1 : 16 – T . wv al siiae ctt,propionate, acetate, Assimilates NaCl. (w/v) % 0.3 ,wihwsioae rmakln aeMondsee Lake alkaline from isolated was which ), tal. et L- ! L- saaieo irt.Mjrclua at acids fatty cellular Major citrate. or asparagine 7 fo eogn oteYnteRvr h river the River, Yangtze the to belonging or of serine, 4:0 : 14 c D- L- – C , 20)ado eoi aapeetdi this in presented data genomic on and (2009) fucose, sorbitol, . wv al siiae ctt,pyru- acetate, Assimilates NaCl. (w/v) % 0.3 6:1 : 16 -Hadiso-C and 3-OH p nov. sp. 8:1 : 18 tal. et wligi h ae ouno freshwa- of column water the in dwelling tal. et 4:0 : 14 L- Polynucleobacter ! ! saaieo irt.Mjrcellular Major citrate. or asparagine c C 7c, 2:0 : 12 09.De o siiaeglycolate, assimilate not Does 2009). , 7 D- – 3-OH). 20) nceoaooia data chemotaxonomical on (2009), c L- etn ei.Clne rw on grown Colonies media. Bertani C , sorbitol, glutamate, 2-OH. 8:1 : 18 6:0 : 16 (yang.tzen ! – c C 7c, 6:0 : 16 . mi eghad0.4 and length in µm 2.9 n umdfaue2 feature summed and T L- D- glutamate, – =S 21495 (=DSM ) h oe2-hydrox- sole The I). mannose, L- 6:0 : 16 . mi eghand length in µm 1.5 ¢ aspartate,  i.NL ac adj. masc. N.L. sis. .Got occurs Growth C. n umdfea- summed and L- ytie Weak cysteine.  D- .Growth C. L- D- mannose, aspartate, L- glucose, T alanine, CIP = 66 – Downloaded from www.microbiologyresearch.org by IP: 138.232.79.190 On: Thu, 08 Sep 2016 12:50:59 C C od(31 pond ainld National at cdmaueet.Ti td a upre yteAustrian the by the out supported carrying was for study Pötter This Gabriele measurements. thank acid We fatty culture. this of nance etakAn uusnFer o epn st idthe Mus find the from to obtained culture providing the us for of (MNHN) history Dellinger helping the Marc on to for grateful information also are Fleury We mann. Aubusson aediculatus Anne Euplotes thank We Acknowledgements and LOJJ01000000) (version accession the AJ550666. under LOJJ00000000 available are numbers strain type char- sequences G the genome DNA and acterizing a Gene and mol%. 45.5 Mbp 2.3 of the content of of +C size genome a by The characterized area. is geographic strain type certain a to taxon restricted the of distribution is the that indicate not spe- does The epithet 2003). cies (Hahn, PR Suzhou, in located (Huqiu) http://ijs.microbiologyresearch.org MWH-JaK3 is strain type The h yesri sMWH-HuW1 is strain 110978 type The OH. 110976 oyuloatrsinensis Polynucleobacter nov. sp. of Description LOJI00000000 numbers mol%. AJ550657. accession 45.4 and LOJI01000000) strain the of (version type content under a the G+C available characterizing by DNA are sequences characterized a genome is and and strain Mbp Gene type 2.0 the of of size geographic certain genome a The to restricted the area. is that taxon indicate the not of does distribution epithet species The 2003). (Hahn, L- L- h ae ouno rswtrsses el r uvdrods, curved 0.6 are Cells systems. freshwater of column water the osntasmlt lclt,gyxlt,oaae levulinate oxalate, glyoxylate, D- glycolate, assimilate not Does ngnmcdt rsne nti td Tbe2.Contains genus 2). the (Table of study strains this free-living in presented data genomic on 20) nceoaooia aao Hahn Hahn of of data chemotaxonomical data on phenotypical (2009), on based is description The was bacterium the where country isolated). the China, to pertaining and fumarate succinate, oxaloacetate, pyruvate, malate, malonate, ih0 with 35 convex with to and up at circular occurs non-pigmented, Growth surface. smooth are agar grown Colonies NSY media. R2A on and NSY on growth good observed.Shows not is growth anaerobic aerobic; and organotrophic 6:0 : 16 0 : 16 saaieo irt.Mjrclua at cd r C are acids fatty cellular Major citrate. or asparagine fucose, mannose, – . mi eghad0.4 and length in µm 1.4 L- ioI.Tesl -yrxltdcmon sC is compound 2-hydroxylated sole The I). -iso ltmt.Wa siiaini bevdfor observed is assimilation Weak glutamate. n umdfaue2(nldn C (including 2 feature summed and – T T . wv al siiae ctt,propionate, acetate, Assimilates NaCl. (w/v) % 0.5 ,wihwsioae rmasihl laie artificial alkaline, slightly a from isolated was which ), ,wihwsioae rmteYnteRiver Yangtze the from isolated was which ), ’  itieNtrle swl sfravc eadn mainte- regarding advice for as well as Naturelle, Histoire 20 D- D sorbitol, ¢ -glucose, 14.81 utr rgnlyetbihdb itrAmmer- Dieter by established originally culture oyuloatrsinensis Polynucleobacter † 120 N L- (sin.en D- aspartate, galactose,  – Polynucleobacter 34 ¢ . mi it.Chemo- width. in µm 0.5 i.NL e.adj. fem. N.L. sis. ¢ T T 34.20 =S 21493 (=DSM =S 21492 (=DSM D- L- lyxose, alanine, †  .Got occurs Growth C. )a ie Hill Tiger at E) tal. et 4:0 : 14 wligin dwelling 20)and (2009) -Hand 3-OH D- L- fructose, cysteine. L- 6:1 : 16 sinensis 2:0 : 12 T T serine, =CIP =CIP tal. et !  eum 7 2- c , , ocr,V,Flet,M,Vnii . cemn .S,Can .S., bacteria. symbiotic (2013). P. and authors ing other Chain, & G. S., T. M. Doak, necessarius M., Polynucleobacter Ackerman, Shin, M., C., L. Vergez, Vannini, S., Malfatti, M., Felletti, V., Boscaro, 33 lio h ciliates the of clei (1971). D. Ammermann, References in diversification FREDI. ect (Ecological I482-B09 project Polynucleobacter (FWF) Fund Science an .W (2003). W. M. Hahn, clmtzto ehdfrioaino nipratfato ftenot the of fraction (2004). important M. an Pöckl, bacteria. & of cultivable readily L. isolation Q. for Wu, method P., acclimatization Stadler, W., M. Hahn, ebr,J,Jezberov J., Jezbera, diversification. bacter agnee,S an .W (2010). W. M. Hahn, & S. Langenheder, o. p o. nolgtl noyboi atru iigi h cyto- the in Living bacterium endosymbiotic of obligately plasm an nov., sp. nov., Jezberov lntno rswtrpond. freshwater a of (2005). L. Q. Wu, polynucleobacter & M. Pöckl, W., M. Hahn, lmtczones. climatic necessarius ynucleobacter a,Z,L,L,Lu . e,Y,Wn,L,Brlm . gi .&Wn,Y. CB. Wang, & strain T. Egli, Betaproteobacterium M., Bartlam, rium, L., Wang, Y., Ren, J., (2013). 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Polynucleobacter 60 plEvrnMicrobiol Environ Appl nio Microbiol Environ 166 , ucutrpplto ueial oiaigbacterio- dominating numerically population subcluster ,adteErpa cec onain(S)proj- (ESF) Foundation Science European the and ), tlnci mytilus Stylonychia n ytEo Microbiol Evol Syst J Int p o. lntncfehae atru affiliated bacterium freshwater planktonic a nov., sp. slto fsrisblnigt h cosmopolitan the to belonging strains of Isolation n rpslo w subspecies, two of proposal and – ,J,Bad,U an .W (2011). W. M. Hahn, & U. Brandt, J., a,  173. 59 ,J,Kl,U,Sursi-ek .&Shit J. Schmidt, & T. 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