Three Novel Species with Peptidoglycan Cell Walls form the New Genus Lacunisphaera gen. nov. in the Family Opitutaceae of the Verrucomicrobial Subdivision 4.

micb-08-00202 Feb ua y 10, 2017 Time: 15:47 # 1 ORIGINAL RESEARCH published: 13 Feb ua y 2017 doi: 10.3389/ micb.2017.00202 Edi ed by: Damien Paul De os, Pablo de Ola ide Uni e si y, Spain Re iewed by: Seong Woon Roh, Ko ea Basic Science Ins i u e, Sou h Ko ea Geo ge Liech i, Uni o med Se ices Uni e si y o he Heal h Sciences, USA *Co espondence: Ch is ian Jogle ch is ian@jogle .de Ma eike Jogle ma eike@jogle .de Special y sec ion: This a icle was submi ed o E olu iona y and Genomic Mic obiology, a sec ion o he jou nal F on ie s in Mic obiology Recei ed: 28 Sep embe 2016 Accep ed: 27 Janua y 2017 Published: 13 Feb ua y 2017 Ci a ion: Ras P, Glöckne I, Boedeke C, Jeske O, Wiegand S, Reinha d R, Schumann P, Rohde M, Sp ing S, Glöckne FO, Jogle C and Jogle M (2017) Th ee No el Species wi h Pep idoglycan Cell Walls o m he New Genus Lacunisphae a gen. no . in he Family Opi u aceae o he Ve ucomic obial Subdi ision 4. F on . Mic obiol. 8:202. doi: 10.3389/ micb.2017.00202 Th ee No el Species wi h Pep idoglycan Cell Walls o m he New Genus Lacunisphae a gen. no . in he Family Opi u aceae o he Ve ucomic obial Subdi ision 4 Pa ick Ras 1, Ines Glöckne 2, Ch is ian Boedeke 1, Olga Jeske1, Sand a Wiegand1, Richa d Reinha d 3, Pe e Schumann4, Man ed Rohde5, S e an Sp ing6, F ank O. Glöckne 7, Ch is ian Jogle 1,8*and Ma eike Jogle 1* 1Mic obial Cell Biology and Gene ics, Leibniz-Ins i u DSMZ-Deu sche Sammlung on Mik oo ganismen und Zellkul u en GmbH, B aunschweig, Ge many, 2Ins i u e o Pha macology, Toxicology and Clinical Pha macy, Uni e si y o Technology, B aunschweig, Ge many, 3Max Planck Genome Cen e , Max Planck Ins i u e o Plan B eeding Resea ch, Köln, Ge many, 4Depa men o Cen al Se ices, Leibniz-Ins i u DSMZ-Deu sche Sammlung on Mik oo ganismen und Zellkul u en GmbH, B aunschweig, Ge many, 5Cen al Facili y o Mic oscopy, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many, 6Depa men Mic oo ganisms, Leibniz-Ins i u DSMZ-Deu sche Sammlung on Mik oo ganismen und Zellkul u en GmbH, B aunschweig, Ge many, 7Depa men o Molecula Ecology, Max Planck Ins i u e o Ma ine Mic obiology, B emen, Ge many, 8Depa men o Mic obiology, Ins i u e o Wa e and We land Resea ch, Facul y o Science, Radboud Uni e si y, Nijmegen, Ne he lands The cell wall o ee-li ing bac e ia consis s o pep idoglycan (PG) and is c i ical o main enance o shape as dissol ed solu es cause osmo ic p essu e and challenge cell in eg i y. Su p isingly, he subdi ision 4 o he phylum Ve ucomic obia appea s o be excep ional in his espec . O ganisms o his subdi ision a e desc ibed o be de oid o mu amic o diaminopimelic acid (DAP), usually ound as componen s o PG in bac e ial cell walls. He e we desc ibe h ee no el bac e ial s ains om a eshwa e lake, IG15T, IG16bT, and IG31T, belonging o a new genus in he subdi ision 4 o Ve ucomic obia which we ound o possess PG as pa o hei cell walls. Biochemical analysis e ealed he p esence o DAP no only in hese no el s ains, bu also in Opi u us e ae PB90- 1T, he closes desc ibed ela i e o s ains IG15T, IG16bT, and IG31T. Fu he mo e, we ound ha nea ly all genes necessa y o pep idoglycan syn hesis a e p esen in genomes o subdi ision 4 membe s, as well as in he comple e genome sequence o s ain IG16bT. In addi ion, we isola ed and isualized PG-sacculi o s ain IG16bT. Thus, ou esul s challenge he concep o pep idoglycan-less ee-li ing bac e ia. Ou polyphasic axonomy app oach places he no el s ains in a new genus wi hin he amily Opi u aceae, o which he name Lacunisphae a gen. no . is p oposed. S ain designa ions o IG15T, IG16bTand IG31Ta e Lacunisphae a pa ula sp. no . (=DSM 26814 =LMG 29468), L. limnophila sp. no . (=DSM 26815 =LMG 29469) and L. ana is sp. no . (=DSM 103142 =LMG 29578) espec i ely, wi h L. limnophila IG16bTbeing he ype species o he genus. Keywo ds: pep idoglycan, subdi ision 4, Ve ucomic obia, Lacunisphae a, o ni hine F on ie s in Mic obiology | www. on ie sin.o g 1Feb ua y 2017 | Volume 8 | A icle 202 micb-08-00202 Feb ua y 10, 2017 Time: 15:47 # 2 Ras e al. No el Subdi ision 4 Ve ucomic obia Possess Pep idoglycan INTRODUCTION In aqua ic en i onmen s, abio ic ac o s such as salini y and empe a u e, bu also in insic me abolism- ela ed mechanisms challenge he cellula in eg i y o mic oo ganisms and hei abili y o p oli e a e. P o ec i e elemen s may be o a s uc u al na u e, such as S-laye s, o he a oidance o osmo ic s ess by li ing in dependency o hos o ganisms which p o ide s able condi ions o su i al (Miles, 1992;Engelha d , 2007). Membe s o he class Mollicu es o example lack a pep idoglycan cell wall (Razin, 2006), a e osmo ically agile and exhibi pleomo phism (Miles, 1992). Thus, hey depend on an euka yo ic hos o p o ide an osmo ically s able en i onmen o li ing. On he o he hand, ee-li ing bac e ia usually possess cell wall s uc u es including h ee dimensionally c oss-linked polyme ic glycan s ands, in e connec ed by sho pep ide elemen s, a s uc u e commonly known as pep idoglycan (PG) o p o ec cellula in eg i y. Among bac e ia only ew excep ions a e desc ibed while all con o e sy discussed species belong o he Planc omyce es-Ve ucomic obia-Chlamydiae (PVC) supe phylum (Wagne and Ho n, 2006). In many espec s, his PVC-supe phylum seems o challenge ou concep o he p oka yo ic cell (Lee e al., 2009;Fue s and Sagulenko, 2011; Jacquie e al., 2015;Ri as-Ma ín e al., 2016). In pa icula , he sugges ed absence o PG in Planc omyce es (König e al., 1984), Chlamydia (Fox e al., 1990) and subdi ision 4 Ve ucomic obia (Yoon, 2011) is ema kable. While he assumed lack o PG seems o be associa ed wi h he lack o he o he wise uni e sal bac e ial cell di ision p o ein F sZ in Planc omyce es (Pilho e e al., 2008;Jogle e al., 2012) and Chlamydia (S ephens e al., 1998), subdi ision 4 Ve ucomic obia encode he ubulin homolog F sZ (Pilho e e al., 2008). Howe e , Planc omyce es we e ecen ly ound o possess a PG cell wall (Jeske e al., 2015; an Teeseling e al., 2015). Fo Chlamydia, he exis ence o PG was demons a ed bu a canonical PG sacculus was no isola ed (Liech i e al., 2014;Packiam e al., 2015). Howe e , o some o he membe s o he phylum Chlamydiae a PG sacculus was iden i ied (Pilho e e al., 2013). Chlamydia a e obliga e in acellula pa hogens (Jacquie e al., 2015) and hus dwell in an en i onmen iso onic o hei cy oplasm, hey do no necessa ily equi e a pep idoglycan sacculus o main ain cell shape. Acco dingly, ecen e idence sugges s ha PG o ms an M eB egula ed ing a mid-cell o allow cell di ision in pa hogenic Chlamydia (Liech i e al., 2016). In con as a ypical bac e ial sacculus was epo ed o he ee-li ing Planc omyce es ha ha e o wi hs and a ious osmo ic challenges in hei na u al habi a s (Jeske e al., 2015; an Teeseling e al., 2015), while ee-li ing bac e ia o he e ucomic obial subdi ision 4 a e s ill conside ed o lack a PG sacculus. This bac e ial g oup belongs o he phylum Ve ucomic obia which is di ided in o six so-called subdi isions. Thus a , cul u ed ep esen a i es a e a ailable o subdi isions 1–4. Recen ly o subdi ision 5 he new Phylum Ki i ima iellaeo a was p oposed, wi h one cha ac e ized isola e (Sp ing e al., 2016). Playing a c ucial ole in en i onmen al nu ien cycles, membe s o he Ve ucomic obia ha e no only been ound o deg ade a a ie y o complex polyme ic compounds in, e.g., soil communi ies (Wang e al., 2014, 2015), some we e also iden i ied as me hano ophs (Sha p e al., 2013; an Teeseling e al., 2014). Inc easing e o s o ex end he knowledge abou his en i onmen ally impo an phylum ha e led o he success ul isola ion and desc ip ion o se e al new species in ecen yea s (Lee e al., 2014;Kim e al., 2015). Howe e , he majo i y o new s ains b ough in o pu e cul u e is a ilia ed wi h subdi ision 1. The e o e, he sca ce da a exis ing o da e lea es inconclusi e esul s abou he suspec ed pep idoglycan anomaly o subdi ision 4 Ve ucomic obia. Fu he mo e, hus a only wo genomes om alidly desc ibed species (Opi u us e ae and Co alioma ga i a akajimensis) a e a ailable. Bo h genomes we e no ye analyzed o PG ela ed genes wi h s a e-o - he-a bioin o ma ic me hods (Jeske e al., 2015). Some membe s o his subdi ision ha e been ound o be esis an o a ious β-lac am an ibio ics, indica ing ei he absence o PG o an esis ance mechanism such as β-lac amases. Fo o he s ains he p esence o ypical cellula PG building blocks was no in es iga ed a he ime o hei desc ip ion (Shieh and Jean, 1998;Choo e al., 2007), lea ing open he ques ion whe he pep idoglycan exis s in e ucomic obial subdi ision 4. Membe s o his subdi ision ha e been isola ed om soil communi ies and lea s, while mos s ains o igina e om aqua ic habi a s, including eshwa e lakes, ma ine wa e s and ex eme habi a s such as ho sp ings (Shieh and Jean, 1998;Chin e al., 2001;Choo e al., 2007; Yoon e al., 2007c, 2010). He e we desc ibe he a ge ed isola ion o subdi ision 4 Ve ucomic obia, using an ibio ic agen s as selec i e ma ke s o β-lac am esis an bac e ia. Ou s a egy led o he success ul cul i a ion o h ee no el s ains om su ace esh wa e samples. By biochemical, mic oscopic and compu a ional analysis we ound ha he no el and p e iously epo ed membe s o he e ucomic obial subdi ision 4 possess PG as pa o hei cell walls. Ou indings challenge he p oposed absence o pep idoglycan among subdi ision 4 Ve ucomic obia, while a he same ime ex ending he sca ce pool o cul i a ed species in his en i onmen ally impo an phylum. MATERIALS AND METHODS Sample Collec ion and P epa a ion Su ace eshwa e samples we e collec ed in iplica es om a local pond (52◦903800 N, 10◦3204000 E, Wol enbü el, Ge many) on Augus 30 h, 2012 a e he obse a ion o a massi e cyanobac e ial blooming e en . Wa e was collec ed in s e ile polyp opylene bo les, immedia ely ans e ed o he labo a o y, homogenized and p ocessed wi hin 2 h. Cul u e Media and Bac e ial Isola ion Cul i a ion medium M1H was p epa ed wi h double dis illed wa e con aining 0.25 g/l pep one (Bac oTM), 0.25 g/l yeas ex ac (Bac oTM), 2.38 g/l HEPES (Se a), 20 ml/l mine al sal solu ion and a pH adjus ed o 8.0 wi h 5 M KOH. A e s e iliza ion, he medium was complemen ed wi h 10 ml/l o a 2.5% glucose solu ion, 5 ml/l double concen a ed i amin solu ion, 1 ml/l o 100 mg/ml ca benicillin and 20 mg/ml cycloheximide s ock solu ions, espec i ely. Solid medium was F on ie s in Mic obiology | www. on ie sin.o g 2Feb ua y 2017 | Volume 8 | A icle 202 micb-08-00202 Feb ua y 10, 2017 Time: 15:47 # 3 Ras e al. No el Subdi ision 4 Ve ucomic obia Possess Pep idoglycan p epa ed wi h h ee imes washed 12 g/l aga (Bac oTM) and cooled o 55◦C p io o he addi ion o hea sensi i e solu ions. Bo h, mine al sal solu ion and double concen a ed i amin solu ion we e p epa ed acco ding o DSMZ medium 621, while me al sal s solu ion consis ed o 250 mg/l Na–EDTA, 1095 mg/l ZnSO4.7H2O, 500 mg/l FeSO4.7H2O, 154 mg/l MnSO4.H2O, 39.5 mg/l CuSO4.7H2O, 20.3 mg/l CoCl2.6H2O, and 17.7 mg/l Na2B4O7.10H2O o which 50 ml we e added pe li e o mine al sal solu ion. Fo ini ial bac e ial isola ion, solid M1H medium was supplemen ed wi h 100 µl o ca benicillin s ock solu ion (100 mg/ml), d ied o 30 min and inocula ed wi h 100 µl homogenized sample ma e ial pe pla e in a 10–10−2dilu ion se ies and incuba ed a 20◦C in he da k un il colony o ma ion became isible. Single colonies we e inocula ed on esh solid medium wi h espec i e an ibio ics. Pu e cul u es we e c yop ese ed in M1H medium supplemen ed wi h 50% glyce ol o 5% DMSO and s o ed a −80◦C. S ains isola ed and la e iden i ied as membe s o he e ucomic obial subdi ision 4 we e designa ed IG15T, IG16bTand IG31T. Unless o he wise indica ed, e ucomic obial s ains we e cul i a ed a 28◦C o ensu e ep oducibili y o cul i a ion dependen expe imen s. Cul i a ion medium o hin laye ch oma og aphy (TLC) e e ence s ains, Bacillus sub ilis DSM 10 and Esche ichia coli DSM 498, was s anda d LB medium con ained 10 g/l yp one, 10g/l sodium chlo ide and 5 g/l yeas ex ac a pH 7.0 (Be ani, 1951). Fo O. e ae PB90-1Tcul i a ion was pe o med ollowing he ecommenda ions o he Leibniz Ins i u e DSMZ (DSMZ medium no. 295). Molecula Iden i ica ion and Phylogene ic Analysis No el isola es we e iden i ied by di ec sequencing o he 16S RNA gene a e ampli ica ion wi h he op imized uni e sal p ime s 8 (50–AGA GTT TGA TCM TGG CTC AG–30) and 1492 (50–GGY TAC CTT GTT ACG ACT T–30) modi ied om (Lane, 1991). PCR eac ions we e pe o med di ec ly on single colonies o iden i ica ion o liquid cul u es o check o pu i y, using he Taq DNA Polyme ase Ki (Qiagen) wi h one eac ion o 25 µl con aining 11 µl PCR–g ade H2O, 2.5 µl 10x Co alLoad bu e , 2.5 µl Q-Solu ion, 0.5 µl dNTPs (10 mM each), 1 µl s e ile bo ine se um albumin solu ion (20 mg/ml), 0.5 µl MgCl2 solu ion (25 mM), 0.125 µlTaq–Polyme ase (1 U/µl) and 1 µl o each p ime (10 pmol). The employed p o ocol consis ed o wo s eps, he i s s ep wi h an ini ial dena u a ion a 94◦C, 5 min, 10 cycles o dena u a ion a 94◦C, 30 s, annealing a 59◦C, 30 s, elonga ion a 72◦C, 1 min, ollowed by he second s ep wi h 20 cycles dena u a ion a 94◦C, 30 s, annealing a 54◦C, 30 s, elonga ion a 72◦C, 1 min and a inal elonga ion s ep a 72◦C, 7 min. All PCRs we e ca ied ou in an Applied Biosys emsR Ve i iR  he mal cycle (The mo Fishe Scien i ic) and PCR p oduc s we e s o ed a 4◦C un il Sange sequencing. To gene a e nea ull leng h 16S sequences, addi ional p ime s (compa e Supplemen a y Table S1) we e used o sequencing and assembly o he esul ing sequences was pe o med wi h he Con igExp ess applica ion o he Vec o NTIR Ad ance 10 so wa e (The mo Fishe Scien i ic). Alignmen o nea ull leng h 16S RNA sequences was pe o med using he SINA web aligne (P uesse e al., 2012), co ec ed manually and used o phylogene ic ee econs uc ion. T ee econs uc ion was pe o med wi h he ARB so wa e package (Ludwig e al., 2004) using he Maximum Likelihood RAxML module and a e dis ibu ion model GTR GAMMA unning he apid boo s ap analysis algo i hm, he Neighbo Joining ool wi h Felsens ein co ec ion o DNA and Maximum Pa simony me hod employing he Phylip DNAPARS module. Boo s ap alues o all h ee me hods we e compu ed wi h 1,000 esamplings including he E. coli 16S RNA gene posi ions 101–1,371. The analysis in ol ed 68 nucleo ide sequences o desc ibed ype s ains and uncul u ed clones, ela ed o he no el s ains (compa e Supplemen a y Table S2). 16S RNA gene iden i y alues o no el isola es and ela ed ype s ains we e calcula ed using neighbo joining clus e ing o he ARB package. Cha ac e iza ion o No el Isola es Mo phological, Physiological, and Biochemical Analysis Bac e ial cells we e immobilized on a 1% aga ose–pad in Ma Tek 35 mm glass-bo om dishes and imaged unde phase–con as illumina ion using a Nikon Eclipse Ti in e s mic oscope a 100× magni ica ion and he Nikon DS–Ri2 came a. To de e mine he cell size o he no el s ains, 100 indi idual cells o each s ain we e measu ed using he NIS-Elemen s so wa e V4.3 (Nikon Ins umen s). Fo ield emission scanning elec on mic oscopy (FESEM) bac e ia we e ixed in 1% o maldehyde in HEPES bu e (3 mM HEPES, 0.3 mM CaCl2, 0.3 mM MgCl2, 2.7 mM suc ose, pH 6.9) o 1 h on ice and washed one ime wi h HEPES bu e . Co e slips wi h a diame e o 12 mm we e coa ed wi h a poly- L-lysine solu ion (Sigma–Ald ich) o 10 min, washed in dis illed wa e and ai -d ied. 50 µl o he ixed bac e ia solu ion was placed on a co e slip and allowed o se le o 10 min. Co e slips we e hen ixed in 1% glu a aldehyde in TE bu e (20 mM TRIS, 1 mM EDTA, pH 6,9) o 5 min a oom empe a u e and subsequen ly washed wice wi h TE–bu e be o e dehyd a ing in a g aded se ies o ace one (10, 30, 50, 70, 90, and 100%) on ice o 10 min a each concen a ion. Samples om he 100% ace one s ep we e b ough o oom empe a u e be o e placing hem in esh 100% ace one. Samples we e hen subjec ed o c i ical-poin d ying wi h liquid CO2(CPD 300, Leica). D ied samples we e co e ed wi h a gold/palladium (80/20) ilm by spu e coa ing (SCD 500, Bal–Tec) be o e examina ion in a ield emission scanning elec on mic oscope (Zeiss Me lin) using he E e ha Tho nley HESE2–de ec o and he inlens SE–de ec o in a 25:75 a io a an accele a ion ol age o 5 kV. Tempe a u e op ima o he no el isola es we e de e mined by op ical densi y measu emen s o g owing cul u es a 600 nm (OD600nm). S ains we e inocula ed 1:10 om ea ly s a iona y phase cul u es in glass ubes wi h M1H medium and incuba ed unde cons an agi a ion in empe a u e con olled shake s ( o exac empe a u es es ed, compa e Supplemen a y Figu e S1). F on ie s in Mic obiology | www. on ie sin.o g 3Feb ua y 2017 | Volume 8 | A icle 202 micb-08-00202 Feb ua y 10, 2017 Time: 15:47 # 4 Ras e al. No el Subdi ision 4 Ve ucomic obia Possess Pep idoglycan Measu emen s we e pe o med in iplica es and each ube se ed as i s own blank p io o inocula ion. Resul ing g ow h cu es we e analyzed by plo ing change o OD600nm du ing exponen ial g ow h phases (slope alues), o each indi idual empe a u e agains empe a u e alues in ◦C. To de e mine he pH op imum, M1H medium was bu e ed o pH alues o 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 10.0 using 10 mM MES, HEPES, HEPPS and CHES bu e s, co esponding o hei indi idual bu e ange. OD600nm was de e mined in glass ubes, incuba ed a 28◦C, wi h h ee eplica es as measu e o g ow h. Ca alase ac i i y was de e mined by bubble o ma ion wi h esh 3% H2O2solu ion. Cy och om oxidase ac i i y was de e mined using Bac iden R Oxidase es s ipes (Me ck Millipo e) ollowing he manu ac u e ’s ins uc ions. G am p ope ies we e de e mined by eac ion o esh biomass wi h esh 3% KOH solu ion (Suslow e al., 1982). Subs a e u iliza ion o he isola ed s ains was in es iga ed using he Biolog GN2 Mic oLogTM es panel o G am-nega i e bac e ia. S e ile glass ubes we e p epa ed in duplica es wi h a basic medium mix u e con aining 15.7 ml IF-0a inocula ion luid (Biolog), 160 µl o 1 M HEPES bu e (pH 8.0) and 80 µl double concen a ed i amin solu ion. Tubes we e inocula ed wi h bac e ial colony ma e ial om exponen ially g owing cul u es o a u bidi y o 56–68%. Two indi idual pla es pe s ain we e e alua ed. To enable he compa ison o he de i ed da a, he da a o each single expe imen we e no malized o 100. Only alues co esponding o >25% u iliza ion we e conside ed as posi i e. The hea map g aphic was ob ained in he R en i onmen (R Co e Team, 2015) by using he hea map.2() unc ion o he gplo s package. Analysis o Cellula Fa y Acids Biomass o he isola ed s ains was ob ained om liquid cul u es g own in M1H medium a 28◦C un il s a iona y phase. The ob ained biomasses we e s o ed a −20◦C. Fo a y acid analysis, 30 mg o lyophilized biomass was p ocessed acco ding o he s anda ds o he Iden i ica ion Se ice o he Ge man Collec ion o Mic oo ganisms and Cell Cul u es (DSMZ) (Mille , 1982; Kuykendall e al., 1988). De e mina ion o Mola G +C Con en S ains we e g own in liquid cul u e o s a iona y phase and biomass was ob ained by cen i uga ion. Fo s ains IG15T and IG31T, he mola G +C con en was de e mined by he se ice acili ies o he DSMZ. In b ie , genomic DNA is isola ed (Cashion e al., 1977), hyd olyzed, dephospho ylized (Mesbah e al., 1989) and analyzed by HPLC (Tamaoka and Komaga a, 1984) in compa ison o DNA s anda ds om o ganisms wi h published genome sequences and a G +C con en ange om 43 o 72 mol%. G +C con en o s ain IG16bTwas de e mined du ing genome sequencing wi h he Paci ic Bioscience sequence . An ibio ic Suscep ibili y Tole ance o IG15Tand IG16bT owa d β-lac am an ibio ic agen s was in es iga ed in a ea men assay using ca benicillin. S ains we e inocula ed as iplica es 1:10 in glass ubes wi h M1H medium and inal concen a ions o 0, 500, 1000, o 2000 mg/l ca benicillin we e added. Tubes we e incuba ed a 28◦C and g ow h was measu ed as change in op ical densi y a 600 nm. A e 120 h o incuba ion, cell iabili y was in es iga ed by FESEM and cell numbe s pe ml we e calcula ed by coun ing wi h a Neubaue chambe . Genome Sequencing o S ain IG16bT DNA Ex ac ion and Pu i y Con ol To ob ain high molecula weigh DNA o s ain IG16bT, nucleic acid was ex ac ed om whole-cells using a weaked Genomic DNA ki p o ocol wi h Genomic ips 100/G (Qiagen). The p o ocol was pe o med as ecommended by he manu ac u e wi h one excep ion: incuba ion ime wi h diges i e enzymes was p olonged o an o e nigh s ep o ensu e comple e lysis o bac e ial cells. An aliquo o he ex ac ed DNA was used o p epa e 16S RNA clone lib a ies (Ze o Blun R PCR Cloning ki ; In i ogen) and esul ing clones we e sequenced o ensu e pu i y o he ex ac ed DNA. Sequencing and Gene Con en Analysis De no o genome sequencing o s ain IG16bTwas pe o med using a PacBio RS sequence . Single molecule eal- ime (SMRT) bellTM lib a ies (Paci ic Bioscience) we e p epa ed using ∼10 µg genomic DNA. Sequencing da a was p ocessed and assembled using he SMRT analysis so wa e. The closed and comple e ch omosome o s ain IG16bTwas anno a ed using he P okka anno a ion ool (Seemann, 2014) and subjec ed o analysis o pu a i e genomic islands and phage egions using IslandViewe 3 (Dhillon e al., 2015) and PHAST (Zhou e al., 2011), espec i ely. The e ucomic obial genomes o he gene con en analysis we e de i ed om NCBI and IMG (Ma kowi z e al., 2012) in Ap il 2016 and had o ma ch he ollowing c i e ia upon CheckM analysis (Pa ks e al., 2015): comple eness >90, con amina ion <5 and s ain he e ogenei y <20. O hologs we e de ec ed by P o eino ho (Lechne e al., 2011), a ool ha iden i ies he ecip ocal bes hi s om he gi en p o ein sequences. The genome plo was hen gene a ed wi h BRIG (Alikhan e al., 2011). Pep idoglycan Analysis Iden i ica ion o Pep idopglycan Syn hesis Genes and β-lac amase P o ein Homologs The p esence o pep idoglycan syn hesis genes was analyzed using blas p (Al schul e al., 1997), while p o ein sequences o Phycisphae a miku ensis FYK2301M01To Gimesia ma is 534- 30Tse ed as que y and we e compa ed wi h p o ein sequences encoded in he genomes o O. e ae PB90-1T,C. akajimensis 04OKA010-24Tand s ain IG16bT.β-lac amase encoding genes we e de ec ed in IG16, O. e ae and C. akajimensis as p e iosly desc ibed (Bush, 2013;Jeske e al., 2015). Fo bo h analysis, homologous p o eins equi ed an iden i y >30%, an e- alue lowe han 1e−6and a conse ed domain a chi ec u e. Lysozyme Assay Suscep ibili y o lysozyme was in es iga ed by incuba ion o he no el s ains in M1H medium. Since s ain IG31Tshowed no lysis a e 24 h in M1H medium, osmo ic s ess was F on ie s in Mic obiology | www. on ie sin.o g 4Feb ua y 2017 | Volume 8 | A icle 202 micb-08-00202 Feb ua y 10, 2017 Time: 15:47 # 5 Ras e al. No el Subdi ision 4 Ve ucomic obia Possess Pep idoglycan inc eased by incuba ion o cells in ddH2O (nega i e con ols as well as lysozyme ea ed cells). Lysozyme was added o a inal concen a ion o 10 mg/ml and cells we e incuba ed o up o 24 h a 37◦C unde cons an agi a ion a 300 pm. Bac e ial cells we e immobilized on a 1% aga ose–pad in Ma Tek 35 mm glass–bo om dishes and imaged unde phase–con as illumina ion using a Nikon Eclipse Ti in e s mic oscope a 100×magni ica ion and he Nikon DS–Ri2 came a (Nikon Ins umen s). Cell iabili y was checked by mic oscopy a e 1, 3, 6, and 24 h o incuba ion in M1H medium o ddH2O un il cell lysis was obse ed. Biochemical Analysis o Pep idoglycan Building Blocks The p esence o diaminopimelic acid (DAP) was in es iga ed employing hin-laye ch oma og aphy and gas ch oma og aphy/mass spec ome y (GC/MS). Thin-laye ch oma og aphy o whole-cell hyd olysa es o s ains IG15T, IG16bT, IG31Tas well as e e ence s ains B. sub ilis DSM 10 and E. coli DSM 498 was pe o med as p e iously desc ibed (S aneck and Robe s, 1974). No el isola es we e g own in M1H medium a 28◦C o s a iona y phase and cells we e ha es ed by cen i uga ion. B. sub ilis and E. coli se ed as o ganismic con ols, g own in 50 ml LB medium a 37◦C o e nigh and ha es ed by cen i uga ion, while a mix u e o pu i ied DAP isome s (Sigma) was used as de ec ion s anda d. Whole-cell hyd olysa es o s ains IG15T, IG16bT, IG31T as well as o O. e ae PB90-1T(DSM 11246) we e analyzed using a gas ch oma og aphy/mass spec ome y (GS/MS)-based me hod (Schumann, 2011), p e iously employed o quan i y he pep idoglycan ma ke DAP and in addi ion o ni hine in a new p oposed Ve ucomic obia ela ed phylum (Sp ing e al., 2016). In b ie , cell pelle s we e ob ained om liquid cul u es (g own as desc ibed abo e) and biomass was lyophilized. Samples we e s anda dized o he quan i ica ion o diagnos ic diamino acids by supplemen ing lyophilized biomass wi h 2 µmol o no leucine as in e nal s anda d. The hyd olysa es (200 µl 4N HCl, 100◦C, 16 h) o he samples we e d ied in a acuum desicca o . Amino acids de i a ized o N-hep a luo obu y yl isobu yles e s and we e esol ed in e hyl ace a e and analyzed by GC/MS (Singlequad 320, Va ian; elec on impac ioniza ion, scan ange 60 o 800 m/z). The DAP de i a i e was de ec ed in ex ac ed ion ch oma og ams using he cha ac e is ic agmen ion se 380, 324, 306, and 278 m/z a a e en ion ime o 22.17 min. A agmen ion o 266 m/z wi h a e en ion ime o 15.13 min was indica i e o he p esence o o ni hine. P epa a ion o IG16bTSacculi Cells o IG16bTwe e ha es ed om 2 l o s a iona y phase cul u es g own in M1H medium a 28◦C, by cen i uga ion a oom empe a u e ollowing a p o ocol es ablished by an Teeseling e al. (2015). In b ie , cells we e boiled a 100◦C o 1 h wi h 4% SDS, while being gen ly mixed by in e ing he eac ion ube se e al imes in 15 min in e als. Lysa es we e ans e ed o Floa -a-Lyze R dialysis ubes (Spec umLabs, DG B eda, Ne he lands) and dialyzed agains deionized wa e in a i e-li e beake o e he cou se o 3 days (wa e was exchanged wo imes). Dialyzed samples we e s o ed a RT un il analysis by ansmission elec on mic oscopy (TEM). Nega i e S aining o IG16bTSacculi Thin ca bon suppo ilms we e p epa ed by sublima ion o a ca bon h ead on o a eshly clea ed mica su ace. Lysa e con aining he sacculi was adso bed on o a ca bon ilm o 1 min and nega i ely s ained wi h 1% (w/ ) aqueous u anyl ace a e, pH 5.0 (Valen ine e al., 1968). A e ai -d ying, samples we e examined in a TEM 910 ansmission elec on mic oscope (Ca l Zeiss, Obe kochen, Ge many) a an accele a ion ol age o 80 kV and calib a ed magni ica ions using a line eplica. Images we e eco ded digi ally wi h a Slow-Scan CCD-Came a (P oScan, 1024x1024, Scheu ing, Ge many) wi h ITEM-So wa e (Olympus So Imaging Solu ions, Müns e , Ge many). Nucleo ide Sequence Accession Numbe s Nea ull-leng h sequences o he 16S ibosomal RNA genes as well as he comple e genome sequence o s ain IG16bT we e deposi ed wi h he Na ional Cen e o Bio echnology In o ma ion (NCBI) and a e a ailable unde KX058881 (IG15T), KX058882 (IG16bT), KX058883 (IG31T) and CP016094 (IG16bT whole genome). RESULTS No el Species o he Ve ucomic obial Subdi ision 4 Isola ion and Iden i ica ion Su ace wa e samples om a local duck pond we e used o he a ge ed isola ion o no el subdi ision 4 Ve ucomic obia. Gi en ha membe s o subdi ision 4 we e hough o lack pep idoglycan, β-lac am an ibio ics we e used as selec ion p essu e o en ich a ge bac e ia. Ob ained colonies o β-lac am esis an bac e ia we e sc eened by 16S RNA gene sequencing analysis and h ee isola es we e iden i ied as membe s o he e ucomic obial subdi ision 4. Phylogene ic ee econs uc ion based on nea ull-leng h 16S RNA gene sequences (Figu e 1) e ealed ha s ains IG15T, IG16bT, and IG31Tbelong o he amily o Opi u aceae, sha ing 92.21, 92.39, and 92.90% sequence iden i y wi h he closes ela ed species O. e ae PB90-1T, espec i ely (Table 1). Based on ecen h eshold alues o 16S RNA gene sequence compa ison (Rosselló-Mó a and Amann, 2015), he no el s ains ep esen h ee dis inc species ha o m a no el genus wi hin he amily Opi u aceae, wi h IG15T, IG16bT, and IG31Tbeing he ype s ains. Mo phological, Physiological, and Biochemical Cha ac e iza ion o No el S ains Cells o s ains IG15T, IG16bT, and IG31Twe e in es iga ed using ligh mic oscopic and elec on mic oscopic echniques, e ealing a coccoid cell shape wi h cells p esen as mono- o diplococci (Figu es 2 and 3). No chain o ose e o ma ion was obse ed. IG15Tcells we e he smalles o he h ee s ains in a e age, measu ing 0.6 ±0.1 µm (diame e o single cocci F on ie s in Mic obiology | www. on ie sin.o g 5Feb ua y 2017 | Volume 8 | A icle 202 micb-08-00202 Feb ua y 10, 2017 Time: 15:47 # 6 Ras e al. No el Subdi ision 4 Ve ucomic obia Possess Pep idoglycan FIGURE 1 | Maximum likelihood 16S RNA gene-based phylogene ic ee. All h ee s ains clus e wi hin he subdi ision 4 o Ve ucomic obia wi h Opi u us e ae as he closes ep esen a i e wi h alidly published name. Rela ed sequences o uncul u ed bac e ia a e shown o compa ison and p onounce he dis inc phylogene ic posi ion o s ains IG15T, IG16bT, and IG31T. Boo s ap alues based on h ee di e en ee building me hods (Maximum Likelihood: ML; Neighbo Joining: NJ; Maximum Pa simony: MP). Black do s indica e suppo alues abo e 70% o all h ee me hods while g ay do s show suppo alues o mo e han 50% o all h ee me hods and less han 70%, a leas o one me hod. B anches ha we e no suppo ed by all h ee me hods show no do . Scale ba indica es 10% es ima ed sequence di e gence. wi h s anda d de ia ion; n=100 cells) while cells o IG16bT and IG31Tmeasu ed 0.9 ±0.2 and 0.6 ±0.1 µm in diame e , espec i ely (Figu e 2D). In wide- ield mic oscopy expe imen s, cell size a iabili y o all h ee s ains (compa e Figu e 2) became mo e e iden han in scanning elec on mic oscopy, whe e cells appea ed smalle in size (compa e Figu e 3) due o osmo ic s ess du ing ixa ion. Du ing exponen ial g ow h, cells o s ain IG15T and IG31Twe e highly mo ile, while IG16bTshowed only e y ew mo ile cells. While cul u e agi a ion was no necessa y o g ow h, cells o s ain IG15Tp oduced an ex acellula ma ix when g own unde cons an agi a ion (90 pm) (Figu es 3A,B) wi h cells embedded in loose agg ega es. No ex acellula ma ix o ma ion was obse ed o s ain IG16bTand IG31T (Figu es 3C–F). All s ains g ow ae obically. Tempe a u e and pH op ima measu emen s e ealed a mesophilic g ow h p o ile wi h g ow h empe a u es om 13–38, 13–36, and 20–36◦C o s ains IG15T, IG16bT, and IG31T, espec i ely. Op ical densi y changes du ing exponen ial g ow h poin ed o op imum g ow h empe a u es o 33, 32, and 30◦C, espec i ely (Supplemen a y Figu e S1). IG15Tand IG16bTwe e able o g ow in pH anges om 6.0 o 9.0, wi h an op imum be ween 7.5 and 8.0. The pH op imum o s ain IG31Twas no de e mined, since i s pH g ow h p ope ies a e likely o be simila o s ains IG15Tand IG16bT. Addi ionally, esul s o he oxidase assays we e posi i e and de e mina ion o ca alase ac i i y showed nega i e esul s o all h ee s ains. S ains we e ound o be G am-nega i e by eac ion wi h 3% KOH solu ion (Suslow e al., 1982). Subs a e TABLE 1 | 16S RNA gene sequence iden i y ma ix indica ing simila i y be ween he h ee e ucomic obial isola es and he nex ela i e Opi u us e ae PB90-1. 16S RNA gene sequence simila i y [%] Opi u us e ae PB90-1T IG15TIG16bTIG31T Opi u us e ae PB90-1T 100 92.21 92.39 92.90 IG15T92.21 100 97.07 97.55 IG16bT92.39 97.07 100 97.71 IG31T92.90 97.55 97.71 100 Simila i y alues we e calcula ed using neighbo joining clus e ing. u iliza ion p o iles o s ain IG15Tand IG16bTshowed simila pa e ns in e ms o suga and suga acid u iliza ion, while s ain IG31Twas clea ly dis inc , u ilizing subs a es such as glycyl- L-glu amic acid, L- hamnose and succinic acid mono-me hyl es e (Figu e 4). Cellula a y acid analysis iden i ied iso-C15:0 as majo componen o IG15Tand IG16bTcell walls wi h 33.3 and 48.6%, espec i ely, while IG31Tonly con ained 9.1% o his pa icula a y acid (Supplemen a y Table S3). Fu he mo e, IG31Tpossessed iso-C14:0as majo componen (15.4%). An ibio ic Suscep ibili y o S ains IG15Tand IG16bT An ibio ic suscep ibili y o s ains IG15Tand IG16bT owa d β-lac ams was in es iga ed by ea men wi h ca benicillin. Op ical densi y (OD600nm) measu emen s indica ed g ow h a F on ie s in Mic obiology | www. on ie sin.o g 6Feb ua y 2017 | Volume 8 | A icle 202 micb-08-00202 Feb ua y 10, 2017 Time: 15:47 # 7 Ras e al. No el Subdi ision 4 Ve ucomic obia Possess Pep idoglycan FIGURE 2 | In es iga ion o cell mo phology and size by ligh mic oscopy. The mo phology and a e age cell size o IG15T(A), IG16bT(B), and IG31T(C) was in es iga ed by ligh mic oscopy unde phase-con as illumina ion. Cells o s ain IG15T(A), IG16bT(B), and IG31T(C) a e o coccoid mo phology and g ow as mono- o diplococci. Cell size was de e mined by measu ing 100 indi idual cells pe s ain (D) and a e age cell size wi h s anda d de ia ion di e ed om 0.6 ±0.1 µm (IG15T), 0.9 ±0.2 µm (IG16bT) o 0.6 ±0.1 µm (IG31T). Scale ba indica es 2 µm. all es ed an ibio ic concen a ions o bo h s ains, as alues inc eased o e ime (Supplemen a y Figu es S2A,B). Howe e , size measu emen s based on SEM mic og aphs e ealed ha ea ed cells o bo h s ains we e signi ican ly inc eased in size when compa ed o un ea ed samples (Figu e 5;p=0.0001). Fu he mo e, he numbe o cells pe ml was signi ican ly lowe (abou 10- old) in ea ed samples (Supplemen a y Figu e S2C; p=0.001). Thus, he inc ease o OD600nm was a he caused by swelling o he cells, han by mul iplica ion a e cell di ision. Genome Sequencing and Gene Con en Analysis o IG16bT The genome o s ain IG16bTwas ob ained solely wi h single molecule eal- ime sequencing (PacBio). Sequencing ead leng h was 3823 bp in a e age and yielded 616 mega bp o sequencing da a om 6 SMRT cells wi h a co e age o ∼80×pe base. Ch omosome size was de e mined a 4,199,284 bp in leng h and bea a GC con en o 66.5 mol%. Anno a ion wi h P okka e ealed he p esence o 3575 coding sequences, 3 RNA and 50 RNA en ies (Table 2). In Figu e 6 he esul s o gene con en analysis based on ecip ocal blas a e shown in a ci cula plo . Known genomes o subdi ision 4 Ve ucomic obia a e compa ed o he IG16bTch omosome, he eby e ealing i s unique genomic egions (Figu e 6, g ay boxes). Some o hese egions we e also p edic ed o be genomic islands (Figu e 6, g ay zones, ou e im), o igina ing om ho izon al gene ans e , and mainly hold hypo he ical p o eins o p o eins wi h domains o unknown unc ion. All p edic ed p ophage egions (Figu e 6, yellow zones, ou e im) we e incomple e (Supplemen a y Table S4), hus no in ac p ophage exis s in he ch omosome o s ain IG16bT. Pep idoglycan in he Ve ucomic obial Subdi ision 4 Bioin o ma ic Analysis o Pep idoglycan Syn hesis Genes and β-lac amase Homologs Using compa a i e genomics, we analyzed he genomes o s ain IG16bT,O. e ae PB90-1Tand C. akajimensis 04OKA010-24T (compa e Table 2) wi h espec o genes equi ed o he syn hesis o pep idoglycan (PG). Resul s o ou blas -based app oach led o he conclusion ha all in es iga ed o ganisms ha bo almos all genes essen ial o he syn hesis o PG (Supplemen a y Table S5). In e es ingly, o he penicillin binding p o eins only sI was iden i ied abo e h eshold. Gene p oduc s o mu B and mu C we e encoded polycis onic in IG16bT,O. e ae and C. akajimensis (compa e Supplemen a y Table S5, o ange boxes) leading o he iden i ica ion o he same p o ein when in es iga ed wi h he que y p o ein sequences o Mu B and Mu C. F on ie s in Mic obiology | www. on ie sin.o g 7Feb ua y 2017 | Volume 8 | A icle 202 micb-08-00202 Feb ua y 10, 2017 Time: 15:47 # 8 Ras e al. No el Subdi ision 4 Ve ucomic obia Possess Pep idoglycan FIGURE 3 | Field emission scanning elec on mic oscopy. S ains show coccoid mo phology wi h o ganiza ion as mono- o diplococci. Mic og aphs o IG15Tcells (A, o e iew; B, close up) illus a e he o ma ion o mul icellula agg ega es embedded in an ex acellula ma ix subs ance (whi e a owheads). In con as , cells o IG16bT(C, o e iew; D, close up) o IG31T (E, o e iew; F, close up) did no p oduce an ex acellula ma ix. Scale ba indica es 2 µm. Tole ance o β-lac am-de i ed an ibio ic agen s in bac e ia is o en ela ed o one o se e al modes o esis ance, including e lux o exclusion mechanisms, al e a ions in a ge p o eins o he mos common cause being he p esence o β-lac amases o deg ade he an ibio ic compound (Poole, 2004). G ow h o s ains IG15T, IG16bT, and IG31Ton solid media supplemen ed wi h he β-lac am ca benicillin ga e ise o he assump ions ha hese s ains possess a mode o ole ance agains β-lac ams. Employing compa a i e genomics, we analyzed he p esence o β-lac amase genes in he genomes o s ain IG16bT,O. e ae and C. akajimensis (see Supplemen a y Tables S6 and S7). Fo IG16bTand O. e ae, h ee β-lac amases we e iden i ied, while o C. akajimensis no β-lac amase was ound wi h he es ed c i e ia. Ou indings sugges ha a ole ance mechanism agains ca benicillin exis s in s ain IG16bTand is a leas pa ially due o he p esence o β-lac amases, leading o he su i al o he o ganism un il he an ibio ic agen is decayed om he cul i a ion medium. Lysozyme Suscep ibili y Assay T ea men wi h lysozyme leads o he dis up ion o he cell en elope by hyd oly ic clea age o β-1,4-linkages in he pep idoglycan complex (Johnson e al., 1968). Un ea ed cells o s ains IG15T, IG16bT, and IG31Tmain ained ypical coccoid cell mo phology, while all h ee s ains displayed a loss o mobili y du ing incuba ion a 37◦C (Figu es 7A–C, espec i ely). Cells ha we e ea ed wi h lysozyme o up o 24 h a 37◦C in ei he cul u e medium (IG15Tand IG16bT) o ddH2O (IG31T) showed di e en suscep ibili y le els owa d he lysozyme ea men . Cells o s ain IG15Tshowed no lysis in M1H medium a e 1, 3, o 6 h, bu we e lysed a e 24 h o incuba ion (Figu e 7D; whi e a owheads). Cells o s ain IG16bTwe e des oyed a e 3 h incuba ion in M1H medium (Figu e 7E; whi e a owheads). Since s ain IG31Tshowed no lysis a e 24 h in M1H medium, osmo ic s ess was inc eased by incuba ion o cells in ddH2O and cells we e dis up ed in ddH2O a e 24 h (Figu es 7D,F; whi e a owheads). Biochemical E idence o he P esence o Pep idoglycan Building Blocks Fi s , he p esence o DAP was in es iga ed o s ains IG15T, IG16bT, and IG31Tby TLC and no DL-DAP was de ec ed. In con as , G am-nega i e and G am-posi i e e e ence s ains, E. coli DSM498 and B. sub ilis DSM10, espec i ely, showed signals o DAP (Supplemen a y Figu e S3), wi h E. coli gi ing only a weak signal. Howe e , we analyzed whole-cell hyd olysa es o IG15T, IG16bT, and IG31Tusing a mo e sensi i e GC/MS me hod ha p e iously e ealed DAP in Planc omyce es. Despi e nega i e esul s in TLC, we ound he speci ic ion peaks, cha ac e is ic o DAP (compa e Figu e 8B), indica ing he p esence o pep idoglycan in IG15T, IG16bT, and IG31Tand O. e ae PB90-1T. The same ion peaks we e p e iously de ec ed o E. coli DSM 498 (Sp ing e al., 2016), he iden ical E. coli s ain we he e used in ou TLC expe imen . In addi ion, o ni hine was de ec ed in he whole cell hyd olysa es o all h ee no el s ains and he closes ela ed ype s ain, O. e ae (Figu e 8A). A quan i a i e es ima ion, based on he in e nal s anda d used, e ealed ha DAP and o ni hine occu ed in nea ly equi alen , albei low amoun s in s ains IG15Tand IG16bT while o ni hine was he dominan subs ance de ec ed o O. e ae and s ain IG31T(Table 3). Howe e , quan i ies o DAP o s ains IG15T(7 nmol), IG16bT(6 nmol), IG31T (3 nmol) and O. e ae (4 nmol) we e nea ly 10- old lowe han hose de ec ed o he con ol E. coli s ain (63 nmol), in es iga ed in he s udy o Sp ing e al. (2016), which explains why no signal o DAP was isible in TLC expe imen s o s ains IG15T, IG16bT, and IG31T, bu a weak signal o E. coli (compa e Supplemen a y Figu e S3). Fu he mo e, p o eins essen ial o DAP biosyn hesis ia he amino ans e ase pa hway a e p esen in he genome o s ain IG16bT(LysC :WP_069962807.1, WP_069963418.1; Asd: WP_069963129.1; DapA: WP_069962952.1; DapB: WP_069962953.1; DapL: WP_069960938.1; DapF: WP_069963382.1) as well as a alanine acemase (WP_069962553.1). Thus, we conclude despi e nega i e esul s in TLC, ha all analyzed s ains con ain DAP as diagnos ic diamino acid o pep idoglycan. Addi ionally, o ni hine was de ec ed which is a pa o he pep idoglycan backbone o ce ain g am-nega i e bac e ia (Yanagiha a e al., 1984;Sp ing e al., 2016). Cell Sacculi o IG16bT To gi e he ul ima e p oo ha PG exis s in he no el s ains isola ed in his s udy cell sacculi we e ex ac ed om s ain F on ie s in Mic obiology | www. on ie sin.o g 8Feb ua y 2017 | Volume 8 | A icle 202 micb-08-00202 Feb ua y 10, 2017 Time: 15:47 # 9 Ras e al. No el Subdi ision 4 Ve ucomic obia Possess Pep idoglycan FIGURE 4 | Hea map illus a ion o subs a e u iliza ion. Subs a e u iliza ion was es ed using he GN2 Mic ologTM pla e sys em. Subs a e spec um o IG15T was mo e simila o IG16bT, while some subs a es such as succinic acid and α-cyclodex in we e almos solely deg aded by IG15T. IG16bTin con as was able o u ilize D-cellobiose and α-D-lac ose, dis inguishing i om s ains IG15Tand IG31T. The u iliza ion pa e n o IG31Twas less b oad, encompassing eigh o he 95 es ed subs a es, bu included o example glycyl-L-glu amic acid, which was no u ilized by IG15To IG16bT. F on ie s in Mic obiology | www. on ie sin.o g 9Feb ua y 2017 | Volume 8 | A icle 202 micb-08-00202 Feb ua y 10, 2017 Time: 15:47 # 16 Ras e al. No el Subdi ision 4 Ve ucomic obia Possess Pep idoglycan owa d o ni hin in PG laye s. FG analyzed sequencing da a and was in ol ed in genome assembly o IG16b’s genome sequence. CJ is PI and oge he wi h MJ unc ions as co esponding au ho . MJ and CJ, along wi h PR designed he s udy and helped wi h expe imen al se ups and design. FUNDING This wo k was kindly unded by he Deu sche Fo schungsgemeinscha (JO 893/3-1). We hank Paci ic Bioscience o genome sequencing. ACKNOWLEDGMENTS We hank Anja Heue o e sa ile and skill ul echnical assis ance. Gab iele Pö e we hank o echnical assis ance in a y acid analysis and hin laye ch oma og aphy o he no el s ains. SUPPLEMENTARY MATERIAL The Supplemen a y Ma e ial o his a icle can be ound online a : h p://jou nal. on ie sin.o g/a icle/10.3389/ micb. 2017.00202/ ull#supplemen a y-ma e ial REFERENCES Alikhan, N. 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Appl. Mic obiol. 56, 213–222. doi: 10.2323/jgam.56.213 F on ie s in Mic obiology | www. on ie sin.o g 17 Feb ua y 2017 | Volume 8 | A icle 202 micb-08-00202 Feb ua y 10, 2017 Time: 15:47 # 18 Ras e al. No el Subdi ision 4 Ve ucomic obia Possess Pep idoglycan Zhou, Y., Liang, Y., Lynch, K. H., Dennis, J. J., and Wisha , D. S. (2011). PHAST: a as phage sea ch ool. Nucleic Acids Res. 39, W347–W352. doi: 10.1093/na / gk 485 Con lic o In e es S a emen : The au ho s decla e ha he esea ch was conduc ed in he absence o any comme cial o inancial ela ionships ha could be cons ued as a po en ial con lic o in e es . Copy igh © 2017 Ras , Glöckne , Boedeke , Jeske, Wiegand, Reinha d , Schumann, Rohde, Sp ing, Glöckne , Jogle and Jogle . This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) o licenso a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. F on ie s in Mic obiology | www. on ie sin.o g 18 Feb ua y 2017 | Volume 8 | A icle 202