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
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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
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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).
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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
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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
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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
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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.
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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
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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
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