micb-08-02501 Decembe 16, 2017 Time: 17:4 # 1
ORIGINAL RESEARCH
published: 19 Decembe 2017
doi: 10.3389/ micb.2017.02501
Edi ed by:
Jesus L. Romalde,
Uni e sidade de San iago
de Compos ela, Spain
Re iewed by:
Hai ham Sghaie ,
Cen e Na ional des Sciences e
Technologies Nucléai es, Tunisia
Tomoo Sawabe,
Hokkaido Uni e si y, Japan
*Co espondence:
Ma ia del Ca men Mon e o-Calasanz
[email p o ec ed]
Wen-Jun Li
[email p o ec ed]
Ma kus Göke
[email p o ec ed]
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: 08 Augus 2017
Accep ed: 01 Decembe 2017
Published: 19 Decembe 2017
Ci a ion:
Mon e o-Calasanz MC,
Meie -Kol ho JP, Zhang D-F,
Ya amis A, Rohde M, Woyke T,
Ky pides NC, Schumann P, Li W-J
and Göke M (2017) Genome-Scale
Da a Call o a Taxonomic
Rea angemen
o Geode ma ophilaceae.
F on . Mic obiol. 8:2501.
doi: 10.3389/ micb.2017.02501
Genome-Scale Da a Call o a
Taxonomic Rea angemen o
Geode ma ophilaceae
Ma ia del Ca men Mon e o-Calasanz1,2*, Jan P. Meie -Kol ho 2, Dao-Feng Zhang3,
Adnan Ya amis1,4, Man ed Rohde5, Tanja Woyke6, Nikos C. Ky pides6,
Pe e Schumann2, Wen-Jun Li3*and Ma kus Göke 2*
1School o Biology, Newcas le Uni e si y, Newcas le upon Tyne, Uni ed Kingdom, 2Leibniz Ins i u e, Ge man Collec ion o
Mic oo ganisms and Cell Cul u es, B aunschweig, Ge many, 3S a e Key Labo a o y o Biocon ol and Guangdong P o incial
Key Labo a o y o Plan Resou ces, School o Li e Sciences, Sun Ya -sen Uni e si y, Guangzhou, China, 4Depa men o
Bio echnology, Middle Eas Technical Uni e si y, Anka a, Tu key, 5Cen al Facili y o Mic oscopy, Helmhol z Cen e o
In ec ion Resea ch, B aunschweig, Ge many, 6Depa men o Ene gy, Join Genome Ins i u e, Walnu C eek, CA,
Uni ed S a es
Geode ma ophilaceae (o de Geode ma ophilales, class Ac inobac e ia) o m a
compa a i ely isola ed amily wi hin he phylum Ac inobac e ia and ha bo many s ains
adap ed o ex eme ecological niches and ole an agains eac i e oxygen species.
Cla i ying he e olu iona y his o y o Geode ma ophilaceae was so a mainly hampe ed
by he insu icien esolu ion o he main phylogene ic ma ke in use, he 16S RNA
gene. In conjunc ion wi h he axonomic cha ac e isa ion o a mo ile and ae obic
s ain, designa ed YIM M13156Tand phylogene ically loca ed wi hin he amily, we
he e ca ied ou a phylogene ic analysis o he genome sequences now a ailable o
he ype s ains o Geode ma ophilaceae and e-analyzed he p e iously assembled
pheno ypic da a. The esul s indica ed ha he la ges genus, Geode ma ophilus, is
no monophyle ic, hence he a angemen o he gene a o Geode ma ophilaceae mus
be econside ed. Taxonomic ma ke s such as pola lipids and a y-acids p o ile, cellula
ea u es and empe a u e anges a e indeed he e ogeneous wi hin Geode ma ophilus.
In con as o p e ious s udies, we also add ess which o hese ea u es can be
in e p e ed as apomo phies o which axon, acco ding o he p inciples o phylogene ic
sys ema ics. We hus p opose a no el genus, Klenkia, wi h he ype species Klenkia
ma ina sp. no . and ha bo ing ou species o me ly assigned o Geode ma ophilus, G.
b asiliensis, G. soli, G. aihuensis, and G. e ae. Emended desc ip ions o all species
o Geode ma ophilaceae a e p o ided o which ype-s ain genome sequences a e
publicly a ailable. Ou s udy again demons a es ha he p inciples o phylogene ic
sys ema ics can and should guide he in e p e a ion o bo h genomic and pheno ypic
da a.
Keywo ds: Klenkia, Geode ma ophilus,Modes obac e , Blas ococcus, GBDP, GGDC, phylogene ic sys ema ics,
polyphasic axonomy
INTRODUCTION
The o de Geode ma ophilales (Sen e al., 2014) comp ises he sole amily Geode ma ophilaceae,
which was ini ially p oposed by No mand e al. (1996), al hough no ype genus was designa ed
a ha ime, con i med la e by S ackeb and e al. (1997), o mally desc ibed by No mand
(2006) and la e emended by Zhi e al. (2009). The amily accommoda es he gene a Blas ococcus
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Mon e o-Calasanz e al. Taxonomic Rea angemen o Geode ma ophilaceae
(Ah ens and Moll, 1970;Ske man e al., 1980;Hezb i e al.,
2016b), Modes obac e (Me s e al., 2000) and he ype genus
Geode ma ophilus (Luedemann, 1968;Ske man e al., 1980).
Geode ma ophilus was his o ically poo ly s udied due o
di icul ies in cul u ing no el isola es (U zì e al., 2004).
O e coming hose echnical di icul ies, he numbe o alidly
named species wi hin he genus d ama ically inc eased om a
single species, G. obscu us, in 2011 o wen y-one species a he
ime o w i ing (Pa e, 2014). The numbe o Geode ma ophilus
isola es is expec ed o con inue o aise in coming yea s, as
indica ed by me agenomics s udies ca ied ou in a id and hype -
a id habi a s (Neilson e al., 2012;Giongo e al., 2013). Species
belonging o he genus a e indeed mainly isola ed om a id
soils and cha ac e ized by ole ance agains oxida i e s ess (G a i
e al., 2012;Mon e o-Calasanz e al., 2013a, 2014b, 2015;Hezb i
e al., 2015a, 2016a) al hough some isola es om hizosphe ic
soils and lake sedimen s ha e also been classi ied wi hin he
genus.
In he pos -genomic e a, he in eg a ion o genomic
in o ma ion in mic obial sys ema ics (Klenk and Göke ,
2010) in addi ion o physiological and chemo axonomic
pa ame e s as axonomic c i e ia is s ongly sugges ed o
classi ying p oka yo es (Ramasamy e al., 2014). This pa icula ly
holds in g oups such as Geode ma ophilaceae, which a e
only incomple ely esol ed in phylogenies in e ed om he
mos commonly applied ma ke gene, he 16S RNA gene.
Ne e heless, excep o he genome sequences gene a ed wi hin
ou p ojec only he genome (I ano a e al., 2010) and p o eome
sequence o G. obscu us (Sghaie e al., 2016) we e publicly
a ailable.
Based on phylogenies in e ed om genome-scale da a and on
a e-in e p e a ion o he a ailable pheno ypic e idence acco ding
o he p inciples o phylogene ic sys ema ics (Hennig, 1965;
Wiley and Liebe man, 2011), his s udy in oduces he new genus
Klenkia in o Geode ma ophilaceae, whose ype species is Klenkia
ma ina sp. no . Acco dingly, we also p opose he eclassi ica ion
o G. b asiliensis as Klenkia b asiliensis comb. no ., G. soli as
K. soli comb. no ., G. aihuensis as K. aihuensis comb. no . and
G. e ae as K. e ae comb. no ., as well as emended desc ip ions
wi hin Geode ma ophilus.
MATERIALS AND METHODS
Isola ion
S ain YIM M13156Twas isola ed om a sample collec ed om
he Sou h China Sea (119◦31.949E, 18◦2.114 N), and was
ob ained using he se ial dilu ion echnique. Sedimen sample
(1 g) was added o 9 ml s e ile dis illed wa e and mixed by
o exing. A 10- old dilu ion o his soil suspension was p epa ed
in s e ilized dis illed wa e , and 0.1 ml was sp ead on Fucose-
p oline aga medium [ ucose 5 g; p oline 1 g; (NH4)2SO41 g;
NaCl 1 g;CaCl22 g; K2HPO41 g; B-Vi amin ace (0.5 mg
each o hiamine-HCl (B1), ibo la in, Niacin, py idoxin, Ca-
pan o hena e, inosi ol, p-aminobenzoic acid, and 0.25 mg o
bio in); sea sal 30 g; aga 20 g; pH 7.2; dis illed wa e 1 li e ].
The pla e was hen incuba ed a 28◦C o 30 days.
Pheno ypic Analysis
Mo phological and Physiological Tes s
Mo phological cha ac e is ics o s ain YIM M13156Twe e
de e mined on GYM S ep omyces medium a 28◦C. Colony
ea u es we e obse ed a 4 and 15 days unde a s e eo
mic oscope acco ding o Pelcza (1957). Exponen ially g owing
bac e ial cul u es we e obse ed wi h an op ical mic oscope
(Zeiss AxioScope A1) wi h a 1000- old magni ica ion and
phase-con as illumina ion. G am eac ion was pe o med
using he KOH es desc ibed by G ege sen (1978). Oxidase
ac i i y was analyzed using il e -pape disks (Sa o ius g ade
388) imp egna ed wi h 1% solu ion o N,N,N0,N0- e ame hyl-p-
phenylenediamine (Sigma–Ald ich); a posi i e es was de ined
by he de elopmen o a blue-pu ple colo a e applying biomass
o he il e pape . Ca alase ac i i y was de e mined based
on o ma ion o bubbles ollowing he addi ion o 1 d op o
3% H2O2. G ow h a es we e de e mined on pla es o GYM
S ep omyces medium o empe a u es om 10◦C o 50◦C a 5◦C
inc emen s and o pH alues om 4.0 o 12.5 (in inc emen s
o 0.5 pH uni s) on modi ied ISP2 medium by adding NaOH
o HCl, espec i ely, since he use o a bu e sys em inhibi ed
g ow h o he s ain. The oxida ion o ca bon compounds was
es ed a 28◦C using GEN III Mic opla es in an Omnilog de ice
(BIOLOG Inc., Haywa d, CA, Uni ed S a es) in compa ison wi h
he e e ence s ains G. b asiliensis DSM 44526T,G. soli DSM
45843T,G. aihuensis DSM 45962T,G. e ae DSM 45844Tin
pa allel assays. The GEN III Mic opla es we e inocula ed wi h
cells suspended in a iscous inocula ing luid (IF C) p o ided
by he manu ac u e a a cell densi y o 94% T o G. b asiliensis
DSM 44526T, 83% T o G. soli DSM 45843Tand G. e ae DSM
45844Tand 90% T o G. aihuensis DSM 45962Tand o he
s ain YIM M13156T. Respi a ion a es we e measu ed yielding
a o al unning ime o 5 days in Pheno ype Mic oa ay mode.
Each s ain was s udied in wo independen echnical eplica es.
Da a we e expo ed and analyzed using he opm .1.0.6 package
(Vaas e al., 2012, 2013) o he Rs a is ical en i onmen (R Co e
Team, 2017). Reac ions wi h a dis inc beha io be ween he wo
eplica es we e ega ded as ambiguous.
Chemo axonomic Tes s
Whole-cell amino acids and suga s we e p epa ed acco ding
o Leche alie and Leche alie (1970), ollowed by hin-laye
ch oma og aphy (TLC) analysis (S aneck and Robe s, 1974).
Pola lipids we e ex ac ed, sepa a ed by wo-dimensional TLC
and iden i ied acco ding o p ocedu es ou lined by Minni
e al. (1984) wi h modi ica ions p oposed by K oppens ed and
Good ellow (2006). Fo iden i ica ion he p esumed OH-PE
spo s we e manually sc aped o om uns ained TLC pla es,
ex ac ed wi h me hanol and e apo a ed o d yness. The ex ac s
we e hen dissol ed in Reagen 3 ( a y acid ex ac ion) and
analyzed by MIDI Sys em. Menaquinones (MK) we e ex ac ed
om eeze-d ied cell ma e ial using me hanol as desc ibed
by Collins (1985) and analyzed by high-pe o mance liquid
ch oma og aphy (HPLC) (K oppens ed , 1982). The ex ac ion
and analysis o cellula a y acids was ca ied ou in wo
independen epe i ions om biomass g own on GYM aga pla es
held a 28◦C o 4 days and ha es ed always om he same
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Mon e o-Calasanz e al. Taxonomic Rea angemen o Geode ma ophilaceae
sec o ( he las quad an s eak). Analysis was conduc ed using
he Mic obial Iden i ica ion Sys em (MIDI) She lock Ve sion
4.5 (me hod TSBA40, ACTIN6 da abase) as desc ibed by Sasse
(1990). The anno a ion o he a y acids in he ACTIN6 peak
naming able is consis en wi h IUPAC nomencla u e. Fa y-acid
pa e ns we e isualized as a hea map using he lipid ex ension
o he opm package (Vaas e al., 2013) and clus e ed using he
p clus .1.2.2 package (Suzuki and Shimodai a, 2006) o he R
s a is ical en i onmen . Quan i a i e analysis o he a y acids
used logi - ans o med pe cen ages h oughou (a e se ing ze o
alues o he lowes non-ze o numbe ) because p opo ion da a
a e expec ed o a y s onge a ound 50% han close o 0 o 100%
(C awley, 2007). All chemo axonomic analyses we e conduc ed
unde s anda dized condi ions o s ain YIM M13156Tand he
ype s ains lis ed abo e.
Excep o he sepa a ely s o ed MIDI measu emen s, all
pheno ypic cha ac e s we e collec ed in a s anda dized abula
o ma ha cons i u es Supplemen a y Table S1. Cus om sc ip s
de eloped a DSMZ allow o ex ac ing such da a in ways
sui able o subsequen phylogene ic o o he analysis. Fo
de e mining ea u es speci ic o p ede e mined g oups o
in e es , such as new axa sugges ed by phylogene ic analysis, he
andomFo es unc ion om he eponymous Rpackage . 4.6-12
(B eiman, 2001) was applied in classi ica ion mode unde de aul
se ings excep o inc eased alues o n ee (50,000) and m y
(hal he numbe o analyzed ea u es).
Sequence Analysis
Fo 16S RNA gene sequencing, genomic DNA ex ac ion, PCR-
media ed ampli ica ion o he 16S RNA gene and pu i ica ion
o he PCR p oduc was ca ied ou as desc ibed by Rainey
e al. (1996). Fo genome sequencing, he s ain YIM M13156T
and six een species wi h alidly published Geode ma ophilus
names we e cul i a ed in GYM S ep omyces b o h a 28◦C. The
p ojec in o ma ion is a ailable h ough he Genomes Online
Da abase (Mukhe jee e al., 2017). The d a genomes we e
gene a ed a he DOE Join Genome Ins i u e (JGI) as pa o
Genomic Encyclopedia o A chaeal and Bac e ial Type S ains,
Phase II (KMG-II): om indi idual species o whole gene a
(Ky pides e al., 2014) ollowing he same p o ocol as in Nouioui
e al. (2017). All genomes we e anno a ed using he DOE-JGI
anno a ion pipeline (Hun emann e al., 2015;Chen e al., 2016)
and eleased h ough he In eg a ed Mic obial Genomes sys em
(Chen e al., 2017).
Phylogene ic analysis o he 16S RNA gene sequences om
he ype s ains o all species wi h e ec i ely published names
in Geode ma ophilaceae, as well as he genome-sequenced
s ains C yp ospo angium a um DSM 44712Tand Spo ich hya
polymo pha DSM 43042T o use as ou g oup, was conduc ed as
p e iously desc ibed (Göke e al., 2011;Mon e o-Calasanz e al.,
2014b). Pai wise 16S RNA gene simila i ies we e calcula ed as
ecommended by Meie -Kol ho e al. (2013b) o de e mine
s ains wi h ≥99.0% simila i y, be ween which (digi al)
DNA:DNA hyb idiza ion expe imen s should be conduc ed.
Genome-scale phylogenies we e in e ed om he a ailable
Geode ma ophilaceae (and ou g oup) whole p o eome sequences
using he high- h oughpu e sion (Meie -Kol ho e al., 2014a)
o he genome BLAST Dis ance Phylogeny (GBDP) app oach
(Auch e al., 2010) in conjunc ion wi h Fas ME (Le o e al.,
2015) as desc ibed ea lie (Hahnke e al., 2016). An addi ional
Fas ME ee was in e ed wi hou he wo ou g oup genomes
o de ec po en ial long-b anch a ac ion o he ou g oup, a
p ocess called long-b anch ex ac ion (Siddall and Whi ing,
1999). Addi ionally, he alidi y o he oo ing was es ed by
e-es ima ing he oo (Simon e al., 2017) using leas -squa es
da ing (To e al., 2015). The GBDP ee es ic ed o he well-
suppo ed b anches (≥95% pseudo-boo s ap suppo ) was used
as a backbone cons ain in a u he 16S RNA gene analysis o
in eg a e in o ma ion om genome-scale da a (Hahnke e al.,
2016). Digi al DNA:DNA hyb idisa ions we e conduc ed using
he ecommended se ings o he Genome-To-Genome Dis ance
Calcula o (GGDC) e sion 2.1 (Meie -Kol ho e al., 2013a).
The G+C con en was calcula ed om he genome sequences as
desc ibed by Meie -Kol ho e al. (2014b).
RESULTS
Sequence Analysis
The phylogene ic ee based on he whole p o eomes o he
sequenced ype s ains placed, wi h maximum suppo , he
g oup o med by he s ain YIM M13156T,G. b asiliensis
DSM 44526T,G. soli DSM 45843Tand G. aihuensis DSM
45962Tas a sis e -g oup o all o he Geode ma ophilaceae
gene a (Figu e 1). Hence Geode ma ophilus is ob iously non-
monophyle ic acco ding o his ee, as Modes obac e as well
as Blas ococcus appea ed as mo e closely ela ed o co e
Geode ma ophilus (including he ype species G. obscu us) han
he ou de ia ing Geode ma ophilus s ains. When C. a um
and S. polymo pha we e emo ed, he esul ing un oo ed
opologically was iden ical o he (un oo ed) ing oup opology
shown in Figu e 1. Tha is, i was impossible o oo he
educed ee in a way ha made Geode ma ophilus monophyle ic,
indica ing ha he non-monophyly o Geode ma ophilus was no
caused by a long-b anch a ac ion a i ac . Leas -squa es da ing
con i med he oo ing in bo h he ull and he educed ee.
As expec ed, he uncons ained phylogene ic ee based on 16S
RNA gene sequences was no well esol ed a i s backbone
bu when applying he cons ain de i ed om he GBDP
ee, Modes obac e , Blas occocus and he wo dis inc g oups
o Geode ma ophilus appea ed as monophyle ic (Figu e 2);
suppo o hei monophyly was s ong excep in he case
o Blas ococcus. Mode a e o s ong suppo was ob ained o
Modes obac e and Blas occocus being mo e closely ela ed o
co e Geode ma ophilus han he g oup o med by he s ain YIM
M13156Tas well as he species G. b asiliensis, G. soli, G. aihuensis
and G. e ae.
Wi hin his clade, he 16S RNA gene sequence o s ain
YIM M13156Tshowed simila i ies ≥99.0% wi h he ype s ains
o G. soli (99.1%) and G. b asiliensis (99.0%) only; hose wi h
G. e ae (98.9%) and G. aihuensis (98.0%) we e lowe . Digi al
DNA:DNA hyb idisa ions be ween s ain YIM M13156Tand
G. soli DSM 45843Tand G. b asiliensis DSM 44526T esul ed
in 29.8 and 29.6% simila i y, espec i ely, clea ly below he 70%
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FIGURE 1 | Phylogenomic ee in e ed wi h GBDP. The ee was in e ed wi h Fas ME om GBDP dis ances calcula ed om whole p o eomes. The numbe s abo e
b anches a e GBDP pseudo-boo s ap suppo alues om 100 eplica ions. Tip colo s indica e chemo axonomic cha ac e s ha p o ide apomo phies o g oups o
in e es , genome sizes and he exac G+C con en as calcula ed om he genome sequences (see he embedded legend o de ails). B., Blas ococcus;C.,
C yp ospo angium; G., Geode ma ophilus; M., Modes obac e ; S., Spo ich hya; NA, no applicable.
FIGURE 2 | Maximum likelihood phylogene ic ee in e ed om 16S RNA gene sequences, showing he phylogene ic posi ion o he s ain YIM M13156T ela i e o
he ype s ains wi hin Geode ma ophilaceae. The b anches a e scaled in e ms o he expec ed numbe o subs i u ions pe si e (see size ba ). Suppo alues om
maximum-likelihood cons ained ( i s ), maximum-pa simony cons ained (second), maximum-likelihood uncons ained ( hi d) and maximum-pa simony
uncons ained ( ou h) boo s apping a e shown abo e he b anches i equal o o la ge han 60%.
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h eshold ecommended by Wayne e al. (1987) o con i m he
species s a us o no el s ains. Addi ional digi al DNA:DNA
hyb idisa ions we e no conduc ed, based on he obse a ion o
Meie -Kol ho e al. (2013b) ha an Ac inobac e ia-speci ic 16S
RNA h eshold o 99.0% yielded a maximum p obabili y o e o
o only 1% o ob ain DNA:DNA hyb idiza ion alues ≥70%.
The genome size ange ound in he sequenced ype s ains
a ied be ween 4.2 Mbp o he s ain YIM M13156Tand 5.9
Mbp o G. ama gosae DSM 46136T(Figu e 1). The genomic
G+C con en o s ain YIM M13156Twas 74.4%. Fo he o he
genomes i anged be ween 74.0% o G. obscu us, G. ube
and G. sabuli and 75.9% o G. nig escens (Figu e 1). Because
G+C con en alues do no di e mo e han 1% wi hin bac e ial
species (Meie -Kol ho e al., 2014b), s onge de ia ions a e due
o a i ac s in con en ionally de e mined G+C con en alues.
Hence, we acco dingly p opose o emend he species desc ip ions
o hose species o which we obse ed a de ia ion om published
G+C con en alues >1%.
Pheno ypic Analysis
Mo phology and Physiology
S ain YIM M13156Tshowed mo ile, od-shaped and G am-
posi i e cells. These obse a ions a e in line wi h hose desc ibed
by Jin e al. (2013),Qu e al. (2013), and Be azzo e al. (2014)
o G. soli and G. e ae,G. aihuensis, and G. b asiliensis,
espec i ely. In con as o hese ou species, nei he high
agg ega es o ming mul ilocula spo angia no zoospo es we e
iden i ied in YIM M13156Tcul u es (Supplemen a y Table S1).
Colonies we e pink-colo ed, con ex, ci cula and opaque wi h
a smoo h su ace and an en i e ma gin, an appea ance simila
o o he Geode ma ophilus species when cul i a ed unde he
same g ow h condi ions. Cell g ow h anged om 20 o
35◦C (op imal g ow h empe a u e 25–30◦C) and om pH
6.0 o 8.5 (op imal pH 6.5–8.0). Resul s om pheno ype
mic oa ay analysis a e shown as a hea map in he Supplemen a y
Ma e ial (Supplemen a y Figu e S1) in compa ison o he ype
s ains o he ou mos closely ela ed species. Di e ences
be ween species we e much mo e p onounced han be ween
eplica es. A summa y o selec ed pheno ypic cha ac e is ics is
p esen ed in Table 1 ( o an o e iew o pheno ypic p o iles in
Geode ma ophilus see Supplemen a y Table S1).
The andomFo es analysis indica ed ha among he
p e iously men ioned pheno ypic ea u es he cell shape well
disc imina es be ween he g oup o med by s ain YIM M13156T,
G. b asiliensis, G. soli, G. aihuensis and G. e ae, which p oduce
ods, on he one hand and co e Geode ma ophilus, which is
cha ac e ized by pleomo phic cells (and seldom cocci), on he
o he hand (Supplemen a y Figu e S2).
Chemo axonomy
Analysis o whole-cell componen s e ealed he p esence o meso-
diaminopimelic acid (Cell-wall ype III), which is consis en wi h
he o he ep esen a i es o Geode ma ophilaceae (No mand and
Benson, 2012).
S ain YIM M13156Tdisplayed p ima ily menaquinone
MK-9(H4) (52.7%), in ag eemen wi h alues epo ed o
Geode ma ophilaceae (No mand, 2006), and MK-9(H0) (39.2%).
The p esence o a signi ican amoun o MK-9(H0) was al eady
men ioned in he o iginal desc ip ions o G. b asiliensis,G. soli,
G. aihuensis, and G. e ae, bu also in o he Geode ma ophilus
species (Mon e o-Calasanz e al., 2015). In con as o he al eady
desc ibed p o iles o isop enologs o G. soli, G. aihuensis and
G. e ae, aces o MK-9(H2) (6.6, 1.5, and 2.8%, espec i ely)
we e now addi ionally iden i ied in hose ype s ains.
MK-8(H4) (5.7%) and MK-10(H4) (3.4%) we e also de ec ed in
he pa e ns o G. aihuensis DSM 45962Tand G. e ae DSM
45844T, espec i ely.
The pola lipid pa e n o s ain YIM M13156Tconsis ed
o diphospha idylglyce ol (DPG), phospha idyle hanolamine
(PE), phospha idylinosi ol (PI), glycophospha idylinosi ol
(GPI), an uniden i ied aminolipid (AL) and aces o
hyd oxyphospha idyle hanolamine (OH-PE) (Figu e 3a). I is in
acco dance wi h pa e ns ob ained o he closely ela ed species
in es iga ed in his s udy (Figu es 3b–d) and he phospholipid
pa e n e ealed by Be azzo e al. (2014) o G. b asiliensis. The
andomFo es analysis de ec ed he lack o phospha idylcholine
and he p esence o glycophospha idylinosi ol as excellen ly
p edic i e o he p ospec i e new genus, whe eas he
absence o phospha idylglyce ol was also obse ed in o he
Geode ma ophilus species (Be azzo e al., 2014;Mon e o-
Calasanz e al., 2014b, 2015;Hezb i e al., 2015a,b,c, 2016a). The
unambiguous p esence o hyd oxyphospha idyle hanolamine is
a sligh ly less ele an axonomic ma ke o he p ospec i e new
genus; among co e Geode ma ophilus i was only de ec ed in he
pola lipid p o ile o G. pul e is DSM 45839Tby Hezb i e al.
(2016a).
E en hough bo h absence o phospha idylcholine and
p esence o glycophospha idylinosi ol we e also obse ed in he
pola -lipid p o iles o some Modes obac e species (Mon e o-
Calasanz e al., in p epa a ion), he addi ional p esence o
hyd oxyphospha idyle hanolamine o ms a unique pa e n o
s ain YIM M13156Tand i s ou mos closely ela ed species. In
addi ion, based on ou esul s and he ch oma og aphic mobili y
o he pola lipid labeled as phospha idylme hyle hanolamine
in he o iginal desc ip ions o G. soli and G. e ae by
Jin e al. (2013), i is s ongly sugges ed ha i was no
co ec ly iden i ied in he o iginal wo k, since a e binding
a me hyl-g oup o phospha idyle hanolamine he esul an
componen would show a highe apola i y and he e o e a
highe mobili y on he pla e han phospha idyle hanolamine
i sel . Hyd oxyphospha idyle hanolamine is no known om he
ou g oup species C yp ospo angium a um (Tamu a e al., 1998).
Majo a y acids we e he sa u a ed b anched-chain iso-C16:0
(36.8 ±1.1%), he monounsa u a ed C17:1ω8c (13.4 ±0.4%)
and he sa u a ed b anched-chain iso-C15:0(11.5 ±0.5%)
complemen ed by iso-C16:1H (5.0 ±0.4%), C17:0(4.5 ±0.3%)
and C18:19ωc (5.0 ±0.2%) in ag eemen wi h he closes
ela ed species (Table 1; o an o e iew o a y-acid p o iles
in Geode ma ophilus see Supplemen a y Table S2). In addi ion,
he occu ence o 2-hyd oxy a y acids (mainly iso-C17:02OH)
is also wo h men ioning as i suppo s he p esence o OH-PE
obse ed in he pola lipids p o iles o s ain YIM M13156Tand
i s ou mos closely ela ed species ( o an o e iew o a y-acids
p o iles in Geode ma ophilus see Supplemen a y Table S2). The
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FIGURE 3 | Pola lipids p o ile o s ain YIM M13156T(a),G. soli DSM 45843T(b),G. aihuensis DSM 45962T(c) and G. e ae DSM 45844T(d) a e sepa a ion by
wo-dimensional TLC using he sol en s chlo o o m:me hanol:wa e (65:25:4; : : ) in he i s dimension and chlo o o m:me hanol:ace ic acid:wa e (80:12:15:4;
: : : ) in he second one. Pla es we e sp ayed wi h molybda ophospho ic acid (3.5%; Me ckTM) o de ec ion o he o al pola lipids. DPG, diphospha idylglyce ol;
PG, phospha idylglyce ol; PE, phospha idyle hanolamine; OH-PE, hyd oxyphospha idyle hanolamine; PI, phospha idylinosi ol; GPI, glycophospha idylinosi ol; GPL,
uniden i ied glycophospholipid; AL, aminolipid; L1-7, uniden i ied lipids. All da a a e om his s udy.
2-hyd oxy a y acids a e a p e- equisi e o he syn hesis o he
hyd oxyla ed pola lipid (Kämp e e al., 2010).
The clus e ing analysis o he logi - ans o med a y-acid
p o iles e ealed ha hose o YIM M13156T,G. b asiliensis,
G. soli, G. aihuensis, and G. e ae sepa a ed i s . Hence
he p o iles o he o he Geode ma ophilus species we e mo e
simila o he ones o Blas ococcus and Modes obac e (Figu e 4).
Acco dingly, he andomFo es analysis iden i ied h ee mino
componen s (iso-C17:010-me hyl, C17:03OH and iso-C16:0
10-me hyl) highly p edic i e o he g oup o med by s ain YIM
M13156T,G. b asiliensis, G. soli, G. aihuensis, and G. e ae.
The a y-acid p o iles hus e en independen ly suppo ed he
assignmen o hese i e species o a new genus (Supplemen a y
Figu es S3, S4).
Whole-cell suga analysis e ealed hamnose, ibose,
mannose, glucose and an uniden i ied suga showing a simila
ch oma og aphic mobili y han he uniden i ied suga ound in
G. no mandii DSM 45417Tby Mon e o-Calasanz e al. (2013b).
On he o he hand, G. soli DSM 45843T,G. aihuensis DSM
45962Tand G. e ae DSM 45844Tshowed he same suga p o ile
as G. b asiliensis DSM 45426T(Be azzo e al., 2014), consis ing
o ibose, mannose, glucose and galac ose (Leche alie and
Leche alie , 1970). The absence o galac ose in he p o ile o
s ain YIM M13156Tmigh di e en ia e his species om o he s
wi hin he g oup.
In o de o s anda dize he pheno ypic da a a ailable o
he genus Geode ma ophilus, analyses o pola lipids, whole-cell
suga s and menaquinones we e also ca ied ou o he species
G. obscu us DSM 43160T,G. ube DSM 45317Tand G. nig escens
DSM 45408T. The pola lipid and menaquinone p o iles o hose
species we e al eady speci ied in he Table 1 o he o iginal
desc ip ion o G. a ena ius DSM 45418Tby Mon e o-Calasanz
e al. (2012), ne e heless hey we e ne e p ope ly desc ibed
no he species emended. The h ee species showed he ypical
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TABLE 1 | Pheno ypic cha ac e is ics o s ain YIM M13156Tin compa ison o hose o he ype s ains o he mos closely ela ed Geode ma ophilus species.
Cha ac e is ics 1 2 3 4 5
Mo ili y +−+++
Oxida ion o :
S achyose − − + +/− −
α-D-Lac ose − + + +/− +
D-Melibiose − +/− + + −
N-Ace yl-D-Galac osamine −+−−−
D-Mannose +++−+
L-Rhamnose −-++−+
L-Aspa ic acid −−−−+
Quinic acid −++++
L-Lac ic acid −−+−+
Ci ic acid +−+−−
α-Ke o-Glu a ic acid +−−−+
D-Malic acid +−+−+
L-Malic acid +−+−+
Pola lipids DPG, PE, OH-PE,
PI, GPI
DPG, PE, OH-PE,
PI, GPI
DPG, PG, PE,
OH-PE, PI, GPI,
GPL
DPG, PG, PE,
OH-PE, PI, GPI
DPG, PG, PE,
OH-PE, PI, GPI
Suga s Rham, Rib, Man,
Gluc, US
Rib, Man, Gluc, Gal Rib, Man, Gluc, Gal Rib, Man, Gluc, Gal Rib, Man, Gluc, Gal
Menaquinonesa# MK-9(H4),
MK-9(H0)
MK-9(H4),
MK-9(H2),
MK-9(H0),
MK-10(H4)
MK-9(H4),
MK-9(H2),
MK-9(H0)
MK-9(H4),
MK-9(H2),
MK-9(H0),
MK-8(H4)
MK-9(H4),
MK-9(H2),
MK-9(H0),
MK-10(H4)
Fa y acidsb# iso-C16:0,
C17:1ω8c, iso-C15:0
iso-C15:0,iso-C16:0iso-C15:0,iso-C16:0,
iso-C17:0
iso-C15:0,iso-C16:0iso-C16:0,iso-C15:0,
C18:1ω9c
S ains: 1, s ain YIM M13156T; 2, G. b asiliensis DSM 44526T; 3, G. soli DSM 45843T; 4, G. aihuensis DSM 45962T; 5, G. e ae DSM 45844T. All da a a e om his s udy.
+, posi i e eac ion; −, nega i e eac ion; +/−, ambiguous; DPG, diphospha idylglyce ol; PE, phospha idyle hanolamine; PE-OH, hyd oxy-phospha idyle hanolamine; PG,
phospha idylglyce ol; PI, phospha idylinosi ol; GPL, uniden i ied glycophospholipid; APL, uniden i ied amino-phospholipid; Rham, hamnose; Rib, ibose; Man, mannose;
Gluc, glucose; US, uniden i ied suga ; MK, menaquinones; iso-, iso-b anched. aonly componen s making up ≥1% peak a ea a io a e shown; bonly componen s making
up ≥10% peak a ea a io a e shown; #, he componen s a e lis ed in dec easing o de o quan i y.
pola -lipid p o ile obse ed in Geode ma ophilus consis ing o
diphospha idylglyce ol (DPG), phospha idyle hanolamine (PE),
phospha idylcholine (PC), phospha idylinosi ol (PI) and mino
amoun s o phospha idylglyce ol (PG) (see Supplemen a y
Figu e S5). Simila o he g oup o med by G. b asiliensis
DSM 44526T,G. soli DSM 45843T, G. aihuensis DSM 45962T
and G. e ae DSM 45844T,G. ube DSM 45317Tdisplayed
glycophospha idylinosi ol (GPI) in addi ion o an uniden i ied
phospholipid (PL). The pola -lipid p o iles o G. obscu us
DSM 43160Tand G. nig escens DSM 45408Tcon e sely
e ealed he ypical uniden i ied glycolipid al eady desc ibed
o mos Geode ma ophilus species (See supplemen a y Table
S1). Rega ding he MK pa e n o G. obscu us DSM 43160T,
apa om MK-9(H4) (64.3%) al eady indica ed by Zhang e al.
(2011), MK-9(H2) (8.7%), MK-9(H0) (4.9%) and MK-8(H4)
(4.3%) we e also e ealed. Ne e heless, in con as o Zhang
e al. (2011) and in addi ion o MK-9(H4), ou s udies did no
iden i y MK-9(H0) in he p o ile o G. ube DSM 45317T. The
whole-cell suga pa e ns o G. obscu us DSM 43160T e ealed
he p esence o ibose, xylose, mannose, glucose and galac ose.
G. ube DSM 45317Tdisplayed a p o ile consis ing o ibose
and glucose. Di e en ly om Nie e al. (2012) who iden i ied
galac ose, a abinose and glucosamine as he whole-cell suga
pa e s o G. nig escens DSM 45408T, ou esul s showed a p o ile
comp ising mannose, glucose, galac ose and aces o hamnose
and ibose. These p o iles a e consis en wi h hose p e iously
desc ibed in he genus, al hough i is wo h men ioning he
absence o galac ose in he p o iles o G. ube DSM 45317T,
a ea u e sha ed, as men ioned p e iously, wi h he s ain YIM
M13156T. The p esence o xylose was al eady desc ibed o
G. saha ensis DSM 45423T(Mon e o-Calasanz e al., 2013c).
DISCUSSION
Phylogene ic analysis based on whole genome and 16S RNA
gene sequences e ealed wi h s ong suppo ha s ain YIM
M13156Tand he species G. b asiliensis DSM 44526T,G. soli DSM
45843T,G. aihuensis DSM 45962Tand G. e ae DSM 45844T
o med a sepa a e lineage wi hin Geode ma ophilaceae, hence
Geode ma ophilus is no monophyle ic. Since he main goal o
phylogene ic sys ema ics is o ob ain monophyle ic axa (Hennig,
1965;Wiley and Liebe man, 2011) axonomic consequences
a e necessa y. A lumping app oach would equi e me ging all
Geode ma ophilaceae gene a in o Geode ma ophilus, which has
p io i y, and hus he gene a ion o 13 new names (i.e., new
combina ions o Blas ococcus and Modes obac e ). In con as ,
placing he abe an Geode ma ophilus species in o a sepa a e
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FIGURE 4 | Fa y-acids dend og am es ima ed om he logi - ans o med pe cen alues as measu ed wi h he MIDI sys em using he Wa d algo i hm o
agglome a i e hie a chical clus e ing and euclidean dis ances using he p clus package o he Rs a is ical en i onmen . App oxima ely unbiased (AU, le ) and
boo s apping (BP, igh ) suppo alues a e shown abo e he b anches.
genus equi e he gene a ion o only ou new names, one o he
new genus and ou new combina ions. Taxonomic conse a ism,
mos easily be measu ed as in e sely p opo ional o he numbe
o new names o be c ea ed (B eide e al., 2014), hus clea ly
a o s he spli ing solu ion. These wo a gumen s alone jus i y
he need o in oduce a new genus o Geode ma ophilaceae.
Ne e heless, he pheno ype also p o ided ich in o ma ion
on he in e ela ionships o he en isaged new genus. S ain YIM
M13156Tand i s ou neighbo ing species we e dis inguished
om o he gene a in he amily Geode ma ophilaceae
by cell mo phology, he lack o spo es, he absence o
phospha idylcholine and he ypical uniden i ied glycolipid ound
ins Geode ma ophilus, he p esence o glycophospha idylinosi ol
and hyd oxyphospha idyle hanolamine in hei pola lipids
p o iles ( o an o e iew o he cha ac e is ics ha di e en ia e
he s ain YIM M13156T,G. b asiliensis, G. soli, G. aihuensis
and G. e ae om closely ela ed Geode ma ophilaceae gene a
see Supplemen a y Table S1 and andomFo es p edic ions
Supplemen a y Figu e S2) and he occu ence o iso-C17:02OH
and o he mino compounds (Supplemen a y Figu e S4 and
Supplemen a y Table S2) in hei a y-acids pa e ns.
Howe e , in phylogene ic sys ema ics diagnos ic ea u es o a
g oup a e insu icien o es ablish i as a axon because when hese
ea u es we e plesiomo phic (ances al) ins ead o apomo phic
(de i ed) hen hey could well diagnose a pa aphyle ic g oup
(Hennig, 1965;Wiley and Liebe man, 2011); ep iles a e a
classical example. Fo his eason, we s udied he dis ibu ion
o he abo e lis ed ea u es among Geode ma ophilaceae
and he ou g oup species C yp ospo angium a um (Tamu a
e al., 1998) and Spo ich hya polymo pha (Supplemen a y
Figu e S4 and Supplemen a y Table S2) o de e mining wi h
maximum-pa simony econs uc ions which cha ac e s a e
was apomo phic o which g oup (Figu e 1). Acco dingly,
p esence o hyd oxyphospha idyle hanolamine, iso-C17:0
10-me hyl, C17:03OH and iso-C16:010-me hyl appea ed as
synapomo phies o YIM M13156Tand i s ou neighbo ing
species; p esence o glycophospha idylinosi ol appea ed as
au apomo phy o Geode ma ophilaceae; seconda y absence o
glycophospha idylinosi ol and p esence o phospha idylcholine
as synapomo phy o co e Geode ma ophilus and Blas ococcus;
and p esence o he uniden i ied glycolipid as au opomo phies
o co e Geode ma ophilus. Hyd oxyphospha idyle hanolamine
is p esen in G. pul e is, oo, bu was gained independen ly;
he uniden i ied glycolipid is missing in G. ube bu was
seconda ily los (Figu e 1). The s a us o cell mo phology and
spo e o ma ion was unclea due o missing da a (Supplemen a y
Table S1) bu he al eady assembled e idence clea ly suppo s he
en isaged eclassi ica ion.
The cu en ly s ill domina ing p ac ice o polyphasic
axonomy (Vandamme e al., 1996) in mic obial sys ema ics
has inc easingly been called in o ques ion in ecen yea s
(Su cli e e al., 2012;Vandamme and Pee e s, 2014;Su cli e,
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Mon e o-Calasanz e al. Taxonomic Rea angemen o Geode ma ophilaceae
2015;Thompson e al., 2015). C i ics mainly emphasize ha
mo e genomic in o ma ion should be inco po a ed and ha
some o he nowadays ou inely conduc ed pheno ypic es s
migh ac ually be unnecessa y. I was also ob ious in he p esen
s udy ha genome-scale da a yielded high esolu ion (Figu e 1),
which ia a backbone cons ain (Hahnke e al., 2016) could
also in o m a mo e comp ehensi ely sampled 16S RNA gene
analysis (Figu e 2).
Whe eas phylogenomics is expec ed o yield mo e s ongly
esol ed ees, hese migh in heo y also yield mo e con lic
be ween dis inc analyses (Je oy e al., 2006;Klenk and Göke ,
2010). Ho izon al gene ans e is a known cause o opological
con lic be ween analyses o single genes ha has e en been be
used o a gue agains hie a chical classi ica ion (Bap es e and
Bouche , 2009;Klenk and Göke , 2010). Howe e , he inc ease
o suppo in phylogenomic analyses a e adding genes up o
i ually all a ailable genes indica es a s ong hie a chical signal
(B eide e al., 2014), whe eas he selec ion o p e-de ined se
o ew genes does no yield genome-scale da a and elies on
a p io i assump ions abou he ela i e sui abili y o genes o
analysis (Lienau and DeSalle, 2009;Klenk and Göke , 2010).
Me hods such as GBDP, which in e ees a he di ec ly om
comple e genomes, a e mo e p omising o ob aining a uly
genome-based classi ica ion, bu con lic be ween single genes
aises he ques ion o how o no o e es ima e phylogene ic
con idence (Taylo and Piel, 2004). This issue can ha dly be
o e es ima ed because in phylogene ic sys ema ics axa mus
be as well suppo ed as monophyle ic as possible (Vences
e al., 2013), which ules ou all phylogenomic me hods ha
do no e en yield s a is ical suppo alues. Ins ead o s anda d
boo s apping, he pa i ion boo s ap, which esamples en i e
genes, is supposed o educe con lic and p o ide mo e eliable
suppo alues (Siddall, 2010;Simon e al., 2017). Wi hin
he GBDP pseudo-boo s apping amewo k, he g eedy-wi h-
imming algo i hm (Meie -Kol ho e al., 2014a) as applied in
he p esen s udy is he equi alen o he pa i ion boo s ap
(Hahnke e al., 2016).
P ope ly analyzed genome-scale da a hus add ess he cu en
sho coming o polyphasic axonomy ha i s s a ing poin is an
o en poo ly esol ed 16S RNA gene ee. A e choosing axon
bounda ies om such a ee, he polyphasic app oach would hen
p oceed wi h de e mining diagnos ic ea u es o he new axa.
Tools such as andomFo es as used he e can assis in he ask
o selec ea u es p edic i e o a ce ain g oup o in e es om
la ge numbe s o cha ac e s.
Howe e , a mo e se ious p oblem wi h he cu en ly
domina ing polyphasic app oach is ha such diagnos ic
ea u es canno p o ide independen e idence o axon
bounda ies when hese bounda ies we e al eady used o
choose he ea u es. Independen e idence can ins ead be
ob ained by de ec ing he same g oups independen ly when
analyzing he addi ional ea u es (Figu e 3). Mo eo e , in
phylogene ic sys ema ics diagnos ic ea u es a e insu icien
o jus i ying a axon because axa mus be monophyle ic,
whe eas diagnos ic cha ac e s a es can be plesiomo phic
and hus diagnose a pa aphyle ic g oup (Hennig, 1965;
Wiley and Liebe man, 2011). To he bes o ou knowledge,
publica ions applying polyphasic axonomy ha dly e e add ess
hese wo issues, e en hough phylogene ic sys ema ics is he
app op ia e pa adigm o mic obial axonomy, oo (Klenk
and Göke , 2010). Ou g oup and ing oup compa isons migh
some imes be di icul because o incomple e cha ac e sampling
bu in he p esen s udy succeeded in de e mining ha
some cha ac e s a es we e apomo phies o he en isaged
new axa. We hus belie e ha mic obial axonomy would
no only bene i om inco po a ing genomic in o ma ion
bu also om adhe ing o he p inciples o phylogene ic
sys ema ics.
Taxonomic Consequences
Based on pheno ypic and geno ypic da a p esen ed, we p opose
ha s ain YIM M13156T ep esen s a no el species o a new
genus o Geode ma ophilaceae, o which he name Klenkia
ma ina gen. no ., sp. no . is p oposed. In addi ion we p opose
he eclassi ica ion o Geode ma ophilus b asiliensis as Klenkia
b asiliensis comb. no ., Geode ma ophilus soli as Klenkia soli
comb. no ., Geode ma ophilus aihuensis as Klenkia aihuensis
comb. no . and Geode ma ophilus e ae as Klenkia e ae comb.
no . The emenda ion o he genus Geode ma ophilus and
he species G. a icanus, G. ama gosae, G. aquaeduc us, G.
dic yospo us, G. nig escens, G. no mandii, G. obscu us, G.
poikilo ophus, G. pul e is, G. ube , G. sabuli, G. saha ensis, G.
sicca us, and G. ellu is a e also p oposed in his s udy.
Desc ip ion o Klenkia gen. no .
Klen’ki.a (N. L. em. n. Klenkia, named in hono o Hans-Pe e
Klenk, P o esso a Newcas le Uni e si y (Uni ed Kingdom) in
ecogni ion o his con ibu ions o bac e ial sys ema ics including
he p omo ion o s udies in Geode ma ophilaceae).
Cells a e mo ile, od-shaped and G am- eac ion-posi i e. The
pep idoglycan in he cell-wall con ains meso-diaminopimelic
acid. The p edominan menaquinones a e MK-9(H4) and
MK-9(H0)bu MK-9(H2), MK-8(H4) and MK-10(H4) may
also be p esen in mino amoun s. The basic pola -lipids p o ile
includes diphospha idylglyce ol, phospha idyle hanolamine,
hyd oxyphospha idyle hanolamine, phospha idylinosi ol
and glycophosphoinosi ol. In some species an uniden i ied
glycophospholipid may be p esen . Phospha idylcholine is
absen . Majo cellula a y acids a e iso-C16:0and iso-C15:0. The
basic whole-cell suga pa e n includes ibose, mannose and
glucose. The p esence o galac ose is equen . Rhamnose may
occu in some species. The genomic G+C con en is 74.0–75.0%.
The ype species o Klenkia is Klenkia ma ina, sp. no .
Desc ip ion o Klenkia ma ina sp. no .
K. ma. i’na. (L. em. adj. ma ina, o he sea, ma ine).
Colonies a e pink-colo ed, con ex, ci cula and opaque wi h
a smoo h su ace and an en i e ma gin. Cells a e mo ile, od-
shaped and G am- eac ion-posi i e. Acco ding o he BIOLOG
Sys em: dex in, D-mal ose, D- ehalose, D-cellobiose, suc ose,
u anose, β-me hyl-D-glucoside, N-ace yl-D-glucosamine,
D-glucose, D-mannose, D- uc ose, D-galac ose, inosine,
D-manni ol, glyce ol, L-alanine, L-glu amic acid, L-py oglu amic
acid, pec in, me hyl py u a e, ci ic acid, α-ke o-glu a ic acid,
F on ie s in Mic obiology | www. on ie sin.o g 9Decembe 2017 | Volume 8 | A icle 2501