Brevitalea aridisoli, B. deliciosa and Arenimicrobium luteum, three novel species of Acidobacteria

12 Pages·2016·1.82 MB·English

InternationalJournalofSystematicandEvolutionaryMicrobiology(2016),66,3355–3366 DOI10.1099/ijsem.0.001199
Brevitalea aridisoli, B. deliciosa and
Arenimicrobium luteum, three novel species of
Acidobacteria subdivision 4 (class Blastocatellia)
isolated from savanna soil and description of the
novel family Pyrinomonadaceae
Pia K. Wüst,1 B€arbel U. Foesel,1 Alicia Geppert,1 Katharina J. Huber,1
Manja Luckner,2 Gerhard Wanner2 and Jörg Overmann1,3
Correspondence 1DepartmentofMicrobialEcologyandDiversityResearch,Leibniz-InstitutDSMZ-Deutsche
PiaK.Wüst SammlungvonMikroorganismenundZellkulturen,Braunschweig,Germany
[email protected] 2DepartmentofBiologyI,Ludwig-Maximilians-Universit€atMünchen,Martinsried,Germany
3TechnischeUniversit€atBraunschweig,Braunschweig,Germany
ThreenovelstrainsofthephylumAcidobacteria(Ac_11_E3T,Ac_12_G8TandAc_16_C4T)were
isolatedfromNamibiansemiaridsavannasoilsbyahigh-throughputcultivationapproachusing
low-nutrientgrowthmedia.16SrRNAgenesequenceanalysisplacedallthreestrainsinthe
orderBlastocatellalesoftheclassBlastocatellia(Acidobacteriasubdivision4).However,16S
rRNAgenesequencesimilaritiestotheirclosestrelativePyrinomonasmethylaliphatogenes
K22Twere(cid:20)90%.CellsofstrainsAc_11_E3T,Ac_12_G8TandAc_16_C4Twere
Gram-staining-negativeandnon-motileanddividedbybinaryfission.Ac_11_E3Tand
Ac_16_C4Tformedwhitecolonies,whilethoseofAc_12_G8Twereorange-yellowish.Allthree
strainswereaerobicchemoorganoheterotrophicmesophileswithabroadpHrangeforgrowth.
Allstrainsusedaverylimitedspectrumofcarbonandenergysourcesforgrowth,witha
preferenceforcomplexproteinaceoussubstrates.ThemajorrespiratoryquinonewasMK-8.The
majorsharedfattyacidwasiso-C .TheDNAG+CcontentsofstrainsAc_11_E3T,
15:0
Ac_12_G8TandAc_16_C4Twere55.9mol%,66.9mol%and54.7mol%,respectively.Based
onthesecharacteristics,thetwonovelgeneraBrevitaleagen.nov.andArenimicrobiumgen.nov.
areproposed,harboringthenovelspeciesBrevitaleaaridisolisp.nov.(Ac_11_E3T=DSM
27934T=LMG28618T),Brevitaleadeliciosasp.nov.(Ac_16_C4T=DSM29892T=LMG28995T)
andArenimicrobiumluteumsp.nov.(Ac_12_G8T=DSM26556T=LMG29166T),respectively.
Sincethesenovelgeneraareonlydistantlyrelatedtoestablishedfamilies,weproposethenovel
familyPyrinomonadaceaefam.nov.thataccommodatestheproposedgeneraandthegenus
Pyrinomonas(Croweetal.,2014).
Members of subdivision 4 of the phylum Acidobacteria (Crowe et al., 2014; Foesel et al., 2013; Huber et al., 2014;
have been only recently cultivated and validly described Pascualetal.,2015;Tank&Bryant,2015).Before,molecu-
lar ecological studies based on 16S rRNA gene analyses
had revealed that subdivision 4 Acidobacteria are abundant
Abbreviations: DDH, DNA–DNA hybridization; DPG, diphosphatidylgly- in almost all types of soils (Barns et al., 1999; Foesel et al.,
cerol;PC,phosphatidylcholine;PE,phosphatidylethanolamine; PI,phos- 2014; Janssen 2006; Jones et al., 2009; Naether et al.,
phatidylinositol.
2012). Recently, the class name Blastocatellia was intro-
TheGenBank/EMBL/DDBJaccessionnumbersforthe16SrRNAgene duced for Acidobacteria subdivision 4 (Pascual et al.,
sequences of Brevitalea aridisoli Ac_11_E3T, B. deliciosa Ac_16_C4T,
2015), currently comprising the sole order Blastocatellales
andArenimicrobiumluteumAc_12_G8TareKF840370,KP638490,and
KM659878,respectively. thataccommodatessixgeneraandninespecieswithvalidly
published names. With the exception of Chloracidobacte-
Three supplementary tables and three supplementary figures are avail-
ablewiththeonlineSupplementaryMaterial. rium thermophilum (Bryant et al., 2007; Tank & Bryant,
001199ã2016IUMS PrintedinGreatBritain 3355
P.K.Wüstandothers
2015) which was isolated from phototrophic microbial (Supplementary Materials and Methods) buffered at pH
mats of a hot spring in Yellowstone National Park, USA, 7.0 (Ac_16_C4T). Media were inoculated with 20 µl soil
all members of the class Blastocatellia originated from ter- suspension per well. Based on in situ pH, soil suspensions
restrial environments. Pyrinomonas methylaliphatogenes were prepared in 10 mM MES pH 6.0 (Ac_11_E3T and
(Crowe et al., 2014; Lee et al., 2015) was obtained from a Ac_12_G8T) and in 10 mM HEPES pH 7.0 (Ac_16_C4T),
geothermally heated volcanic soil in New Zealand. Blasto- respectively. The total cell number in the soil suspensions
catella fastidiosa (Foesel et al., 2013), Aridibacter famidur- was determined after staining with SYBR Green I (Foesel
ans and Aridibacter kavangonensis (Huber et al., 2014), et al., 2014) and a subsample of the soil was diluted
Stenotrophobacter terrae, Stenotrophobacter roseus and accordingly with the respective buffer. After inoculation
Stenotrophobacter namibiensis (Pascual et al., 2015), as well withthesoilsuspension eachwellcontainedapproximately
as Tellurimicrobium multivorans (Pascual et al., 2015) were 10 (Ac_11_E3T and Ac_12_G8T) or 100 (Ac_16_C4T) bac-
isolated from semiarid savanna soils in northern Namibia. terial cells from the original microbial soil community.
C. thermophilum and P. methylaliphatogenes show thermo- After 4 and 7 weeks of incubation at 25(cid:14)C in the dark as
philic traits while all other described representatives of the static cultures, cell extracts were prepared from aliquots of
class Blastocatellia are mesophiles. Members of the class grown wells by freeze–thaw cycles and were used as tem-
Blastocatellia are aerobic chemoorganoheterotrophs, except plates in an Acidobacteria-specific PCR using the primer
for C. thermophilum which is a microaerophilic photohe- pair Acido31f (Barns et al., 1999) and 907r (Lane, 1991).
terotroph (Crowe et al., 2014; Foesel et al., 2013; Huber Acidobacteria-positive enrichments were streaked on the
et al., 2014; Pascual et al., 2015; Tank & Bryant, 2015). same media used before but solidified with 1.5% (w/v)
Results of previous studies suggest that the class Blastoca- purified agar (Oxoid) and strains were isolated by subse-
tellia comprises slow-growing K-strategists that prefer oli- quent streaking. Unless otherwise stated all strains were
gotrophic growth conditions and are able to survive cultivated aerobically at 25(cid:14)C in SSE/HD 1:10 (Supple-
drought and nutrient limitation (Foesel et al., 2013, 2014; mentary Materials and Methods) at pH 7.0 for further
Huber et al., 2014; Pascual et al., 2015). Here we describe physiologicaltestsandbiomassproduction.
three novel isolates that are affiliated with Acidobacteria
After incubation for four weeks on solid media all strains
subdivision 4, but are only distantly related ((cid:20)90% 16S
formed small colonies of (cid:20)1 mm in diameter that were
rRNA gene sequences similarities) to all other so far
smooth, shiny, circular and convex with entire margins.
describedmembers ofthissubdivision. Colonies of Ac_11_E3T and Ac_16_C4T were white while
All strains were isolated from the surface (upper 10 cm) of colonies of Ac_12_G8T exhibited an intense orange-
sandysemiaridsavannasoilssampledinMashare,Kavango yellowish color. Microscopic characterization was per-
region, northern Namibia. Strains Ac_11_E3T and formed as described previously (Foesel et al., 2013; Huber
Ac_12_G8T were enriched from samples obtained from et al., 2014). Strains Ac_11_E3T and Ac_16_C4T formed
two old flood plain soils located in the vicinity of the river short rods with an average size of 1.6(cid:2)0.8 µm and
Okavango. The samples were collected in spring 2011. 1.4(cid:2)0.7 µm, respectively (Table 1 and Fig. S1, available in
Ac_11_E3T was obtained from a slightly acidic bush veld the online Supplementary Material). Cells of Ac_12_G8T
soil (17(cid:14)53¢39.3† S, 20(cid:14)13¢39.5† E; pH 6.5 and 5.7, mea- were ovoid to rod-shaped with an average size of
sured in distilled water and 10 mM CaCl , respectively). 1.6(cid:2)0.8µm.Thethreestrainsoccurredassinglecells,pairs
2
Ac_12_G8T was isolated from a slightly acidic, irrigated or aggregates (Fig. S1), divided by binary fission and were
agricultural soil (17(cid:14)53¢32.4† S, 20(cid:14)11¢15.4† E; pH 6.6 and non-motile. Neither the formation of capsules (as demon-
5.7, measured in distilled water and 10 mM CaCl , respec- strated by India ink staining) nor the formation of spores
2
tively) which was densely covered with Zea mays. (as demonstrated by malachite green staining) (Beveridge
Ac_16_C4T originated from a non-irrigated agricultural et al., 2007) was observed (Table 1). Like all other charac-
sandy Kalahari soil (17(cid:14)54¢9.2† S, 20(cid:14)14¢4.5† E) of near terized acidobacteria, the cells of the three strains stained
neutral pH (pH 7.4 and 6.8, measured in distilled water Gram-negative (Gerhardt et al., 1994) which was also con-
and 10 mM CaCl , respectively) and loosely covered with firmedbytheKOHtest(Buck,1982).
2
Citrullus lanatus and species of the family Fabaceae which
For scanning electron microscopy cells were fixed chemi-
was sampled in spring 2012. Soil samples were transported
callywithglutaraldehydeandfurtherprocessedasdescribed
in air-tight plastic bags in a cool box. After arrival in Ger-
previously (Wanner et al., 2008). To exclude artefacts due
many, they were stored at 4(cid:14)C for 5.5 (2011) and 7 weeks
to shrinking of cells during the chemical fixation
(2012), respectively, until the cultivation experiments
procedureforimagingofultrathinsectionscellswerehigh-
started.
pressurefrozenandcryosubstitutedasdescribedpreviously
All three strains were obtained using a high-throughput (Wanner et al., 2008). Ultrastructural analyses confirmed
cultivation approach set up in sterile 96-well microtiter the Gram-negative cell wall structure of all three strains
plates containing 180 µl of Soil Solution Equivalent (SSE)/ (Fig.1)showinganouterandaninnermembrane(approx-
Cmixmedium(seeSupplementaryMaterialsandMethods) imately 7.5 nm thick) separated by a periplasmic space
buffered at pH 6.0 (Ac_11_E3T and Ac_12_G8T) or con- with a clearly visible peptidoglycan layer (approximately
taining Soil Extract (SE)/HD 1:10 medium 4.0nm) (Fig. 1d–f). In addition, many V-shaped
3356 InternationalJournalofSystematicandEvolutionaryMicrobiology66
Brevitaleaaridisoli,gen.nov.,sp.nov.,B.deliciosasp.nov.andArenimicrobiumluteum,gen.nov.,sp.nov.
Table 1. Characteristics of the novel strains of the class Blastocatellia (Acidobacteria subdivision 4) compared with their closest
relative Pyrinomonas methylaliphatogenes K22T (Crowe et al., 2014). Strains: 1, Ac_11_E3T; 2, Ac_12_G8T; 3, Ac_16_C4T;
4,P.methylaliphatogenesK22T
All strains were Gram-staining-negative, had an aerobic chemoorganoheterotrophic metabolism, divided by binary fission, were non-motile and
formednosporesandcapsules.Allstrainsutilizedcasaminoacids,yeastextractandpeptone.+,positive;(cid:0),negative;(+),weaklypositive;ND,no
dataavailable;P,predictedfromgenomeanalysis(Leeetal.,2015).Onlymajorfattyacids(>5%oftotalfattyacidamountofatleastoneofthe
novelstrains)arelisted.
Characteristics 1 2 3 4
Isolationsource Semiaridsavannasoil Semiaridsavannasoil Semiaridsavannasoil Volcanicsoil
Cellshape Shortrods Ovoidorrods Shortrods Rods
Cellsize(µm) 1.0–2.2(cid:2)0.7–0.9 1.0–2.3(cid:2)0.7–1.0 0.9–1.8(cid:2)0.6–0.8 1.0–4.0(cid:2)0.3–0.6
Pigmentation White Yellow-orange White White-semitransparent
NaCltolerance(%w/v) (cid:20)1.00 (cid:20)0.25 (cid:20)0.50 (cid:20)1.00
Doublingtime(h) 4.4 9.9 7.6 ND
Catalase (cid:0) + (cid:0) +
Cytochrome-coxidase (cid:0) (cid:0) (cid:0) +
Temperaturerange((cid:14)C) 11.0–52.5 8.0–45.0 25.0–37.0 50.0–69.0
Temperatureoptimum((cid:14)C) 35.0–45.0 28.0–45.0 33.0–37.0 65.0
pHrange 4.7–8.1 3.5–9.5 4.7–9.0 4.1–7.8
pHoptimum 5.4–7.0 5.5–7.0 5.4–6.5 6.5
Majorfattyacids
iso-C 43.7 59.8 49.0 40.8
15:0
iso-C 6.3 3.6 4.6 33.8
17:0
anteiso-C 5.8 3.5 8.5 1.1
17:0
iso-C H/C 3-OH 21.9 11.4 16.7 (cid:0)
15:1 13:0
iso-C (cid:0) (cid:0) (cid:0) 12.1
19:0
Substratesforgrowth
Arabinose (cid:0) (cid:0) (cid:0) +
Fructose (cid:0) (+) (cid:0) ND
Glucose (cid:0) (cid:0) (cid:0) +
Lyxose (+) (cid:0) (cid:0) ND
Maltose (+) + (cid:0) P
Mannose (cid:0) (+) (cid:0) +
Sucrose (+) (+) (cid:0) +
Xylose (cid:0) (cid:0) (cid:0) +
Mannitol (+) (cid:0) (cid:0) ND
Ribitol (cid:0) (+) (cid:0) ND
Tyrosine (cid:0) (cid:0) (+) ND
Formate (cid:0) (cid:0) (cid:0) +
Protocatechuate (cid:0) + (+) ND
Succinate (+) (cid:0) (cid:0) (cid:0)
Caseinhydrolysate + + + ND
Hydrolysisofpolymers
Cellulose + (cid:0) (cid:0) P
Starch (cid:0) + + (cid:0)
Xylan + (cid:0) (cid:0) +
DNAG+Ccontent(mol%) 55.9 66.9 54.7 59.4–59.6
Respiratoryquinones MK-8 MK-8,MK-7 MK-8 MK-8
indentations of the outer membrane were apparent which subdivision 4 (Foesel et al., 2013 ; Huber et al., 2014) and
are in contact with the peptidoglycan layer (Fig. 1i). Such were now confirmed the first time, to our knowledge, in
conspicuous structures had been observed previously in high-pressurefrozenandcryosubstitutedcells.Thecellsur-
chemically fixed cells of members of Acidobacteria facesofthethreestrainsweredenselycoveredwithfimbriae
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Fig. 1.Electronmicrographsofultrathinsectionsfromhigh-pressure frozenandcryosubstitutedcellsandscanningelectron
micrographsofconventionallyfixedcellsofstrainsAc_11_E3T(a,d,g,j,m),Ac_12_G8T(b,e,h,k,n)andAc_16_C4T(c,f,i,
3358 InternationalJournalofSystematicandEvolutionaryMicrobiology66
Brevitaleaaridisoli,gen.nov.,sp.nov.,B.deliciosasp.nov.andArenimicrobiumluteum,gen.nov.,sp.nov.
l, o). All strains showed typical Gram-staining-negative cell envelopes with an outer membrane (om), a thin, electron-dense
peptidoglycanlayer(pg)andaninnermembrane(im).Celldivisionwasperformedbyconstriction(b;arrow).Cellsofallstrains
contained polygonal particles in dense packages, most probably representing phage capsides (a, c; circles). Elongate or
sphericalprotrusionsoftheoutermembranewerefrequentlyobserved(f,g,m,n;arrows).Inallstrainsalargeportionofribo-
someswasaffiliatedwiththecytoplasmicmembrane(i;rectangle).TheoutermembraneformednumerousV-shapedindenta-
tions which were in contact with the peptidoglycan layer (i; arrow). Occasionally, vesicles were observed in the cytoplasm
bounded by a unit-membrane (h; arrows). Cells formed little aggregates mediated by a fuzzy layer of fimbriae (j, k, l, m, n).
Aggregatesofsmallgranules(composedofironandphosphorus,asanalyzedbyenergydispersiveX-rayspectroscopy,EDX)
weretypicallylocatedwithincellaggregates(l,o;circles).Bars,100nm(a–i,m–o)or1µm(j–l).
that mediated the formation of cell aggregates (Fig. 1j–n). publishednameswerePyrinomonasmethylaliphatogenesK22T
Within those cell clusters, aggregations of small granules with16SrRNAgenesequencesimilaritiesof(cid:20)90%(88.7%,
composed of iron and phosphorus as analyzed by energy 90.0% and 89.2%, respectively) and Blastocatella fastidiosa
dispersive X-ray spectroscopy (EDX; Kolinko et al., 2013) A2-16T with 16S rRNA gene sequence similarities of <88%
were typically detected (Fig. 1l, o). Moreover, outer mem- (87.7%, 86.4% and 87.9%, respectively). 16S rRNA gene
brane vesicles were discerned resembling those previously sequencesimilaritiestoChloracidobacteriumthermophilumBT
detected in Blastocatella fastidiosa (Foesel et al., 2013). In were(cid:20)84%.
the three novel strains additional protuberances ranging DNA–DNA hybridization (DDH) was performed for
between eighty and several hundred nm in length were
strains Ac_11_E3T and Ac_16_C4T to confirm the separa-
detected (Fig. 1f, g, m, n). Cells exhibited one or two elec-
tion into two distinct species. Cells were disrupted in a
tron-dense DNA regions. In all strains a large portion of
Constant Systems TS 0.75 KW (IUL Instruments) and
ribosomes was affiliated with the cytoplasmic membrane
DNA was purified from the crude lysate by chromatogra-
(Fig.1i).AsrecentlydescribedforafewmembersofAcido-
phy on hydroxyapatite (Cashion et al., 1977). Hybridiza-
bacteria subdivision 1 (Foesel et al., 2016) all three strains
tion was carried out according to established protocols
showed many, very conspicuous granules that most proba-
(De Ley et al., 1970; Huss et al., 1983) using a model Cary
bly represent different stages of phage capsids (Fig. 1a, c).
100 Bio UV/VIS-spectrophotometer equipped with a
Occasionally, vesicles were observed in the cytoplasm
Peltier-thermostat-regulated 6(cid:2)6 multicell changer and a
boundedbyaunit-membrane(Fig.1h).
temperature controller with in situ temperature probe
Thealmost-full-length16SrRNAgenesoftheisolatedstrains (Varian). Hybridization values were 31.5% and 31.0% in
wereamplifiedandsequencedasdescribedpreviously(Foesel a duplicate analysis, which is far below the threshold value
et al., 2013). The resulting sequences comprised 1528 of 70% DNA–DNA similarity recommended for the defi-
(Ac_11_E3TandAc_16_C4T)and1521(Ac_12_G8T)unam- nition of a bacterial species (Wayne et al., 1987). These
biguous nucleotides between E. coli positions 8 and 1510 results confirm that strains Ac_11_E3T and Ac_16_C4T
(Brosius et al., 1978). Sequences were added to the 16S represent separate species.
rRNA-based ‘All-Species Living Tree’ Project (LTP) database
For DNA G+C content determination the DNA was puri-
(Yarza et al., 2008) release 123 (September 2015) using the
fiedasdescribedforDDH(seeabove).Aftertreatmentwith
programpackageARBversion6.0.3(Ludwigetal.,2004).After
P1nucleaseandbovinealkalinephosphatase(Mesbahetal.,
automatedalignmentwiththeFastalignertoolimplemented
1989) the resulting deoxyribonucleosides were analyzed by
inARB,thealignmentwasmanuallyrefinedbasedonsecond-
high performance liquid chromatography (HPLC, Shi-
ary structure information. Phylogenetic trees were recon-
madzu) using conditions adapted from those described by
structed using neighbor-joining, maximum-parsimony and
Tamaoka&Komagata(1984).WhiletheG+C contents for
maximum-likelihood algorithms (40% maximum frequency
strains Ac_11_E3T (55.9 mol%) and Ac_16_C4T (54.7 mol
filtercalculatedfortheconsideredsequencesresultingin1420
%) were within the range reported for other described
valid columns between positions 68 and 1438 of the E. coli
members of the class Blastocatellia (46.5–61.3 mol%; Pasc-
16S rRNA reference gene; 1000 bootstrap resamplings). All
ualetal.,2015),theDNAG+CcontentofAc_12_G8T(66.9
threemethodsrevealedthatthethreenovelisolateswereaffil-
mol%) was considerably higher (Table 1). The DNA G+C
iated with the order Blastocatellales of Acidobacteria subdivi-
contentofthenextmostcloselyrelatedspeciesP.methylali-
sion 4 (class Blastocatellia) (Figs 2, S2). Strains Ac_11_E3T
phatogenes K22T varied between 59.4 and 59.6 mol%,
andAc_16_C4Thada16SrRNAgenesequencesimilarityof
dependingontheanalysismethodused(Croweetal.,2014;
97.9% and formed a well-defined cluster in the neighbor-
Leeetal.,2015).
joining tree (Fig. 2). Strain Ac_12_G8T formed a single,
deeperbranchinglineageandsharedonly92.8%and92.1% Isoprenoid quinones were extracted from dried biomass
of its 16S rRNA gene sequence with Ac_11_E3T and with chloroform/methanol (2:1, v/v) (Collins & Jones,
Ac_16_C4T, respectively. The most closely related species of 1981) and analyzed with HPLC (Tindall, 1990). The major
Ac_11_E3T, Ac_12_G8T and Ac_16_C4T with validly respiratory menaquinone of all three strains was MK-8
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P.K.Wüstandothers
100 SD1/Acidobacteria
30
56
94
3 Stenotrophobacter SD4/Blastocatellia
53
100
2 Aridibacter
10057 Tellurimicrobium multivorans Ac_18_E7T (KP334257)
91 Blastocatella fastidiosa A2-16T (JQ309130)
100 Ac_16_C4T (KP638490)
100 Ac_11_E3T (KF840370)
63 Ac_12_G8T (KM659878)
100 Pyrinomonas methylaliphatogenes K22T (AM749787)
98 Chloracidobacterium thermophilum BT (CP002514)
100
3 SD3
92
773 SD8
73 ‘Thermotomaculum hydrothermale’ AC55 (AB612241)|SD10
Thermoanaerobaculum aquaticum MP-01T (JX420244)|SD23
0.10
Fig. 2.Rootedneighbor-joiningtree(Felsensteincorrection)basedonalmost-full-length16SrRNAgenesequencesillustrat-
ingthephylogeneticpositionofstrainsAc_11_E3T,Ac_12_G8T,Ac_16_C4Tandrelatedtaxa.Thefollowingsequenceswere
used as outgroup: Rubinisphaera brasiliensis DSM 5305T (CP002546), Planctomicrobium piriforme P3T (KP161655) and
PlanctopiruslimnophilaDSM3776T(X62911).Numbersatnodesindicatebootstrapvalues(aspercentages;1000bootstrap
resamplings). Numbers in clusters indicate the number of species constituting the respective cluster. SD, subdivision. Bar,
10%nucleotidedivergence.
(Table 1) which is consistent with the results observed for amount)thanthosereportedpreviouslyformembersofthe
most members of the class Blastocatellia (Pascual et al., familyBlastocatellaceae(Pascualetal.,2015).
2015). Additionally, Ac_12_G8T contained minor amounts
Anadditionaldirectacidhydrolysisofcellmaterialobtained
ofMK-7whichwerealsofoundinrepresentativesofthegen-
from strain Ac_11_E3T revealed the presence of 13,16-
eraAridibacter,Blastocatella,TellurimicrobiumandStenotro-
dimethyl octacosanedioic acid (iso-diabolic acid) and sub-
phobacter (Foesel et al., 2013; Huber et al., 2014; Pascual
stantial amounts of its monoalkyl glycerol ether (MGE)
etal.,2015).
derivative(SinningheDamst(cid:19)eetal.,2014).Iso-diabolicacid
Forfattyacidanalysis,strainswerecultivatedinliquidSSE/ isalsopresentinhydrolyzedcellmaterialofP.methylalipha-
HD 1:10 at pH 5.5 for 7–10 days at 25(cid:14)C (Ac_11_E3T) or togenes K22T, Blastocatella fastidiosa A2-16T, Stenotropho-
28(cid:14)C (Ac_12_G8T and Ac_16_C4T), respectively. About bacter terrae Ac_28_D10T, Aridibacter famidurans
40 mgwetweightoffreshcellswereharvestedandextracted A22_HD_4HT, A. kavangonensis Ac_23_E3T and Chloraci-
(Blight–Dyerextraction)accordingtothestandardprotocol dobacterium thermophilum BT (Sinninghe Damst(cid:19)e et al.,
2014).Thisresultindicatesthatthepresenceofiso-diabolic
(Sasser,1990)oftheMicrobialIdentificationSystem(MIDI
acid is a characteristic feature of members of the class
Inc.; version 6.1). Compounds were identified by compari-
Blastocatellia.
sontotheTSBA40namingtable.Majorfattyacids(i.e.fatty
acids constituting more than 5% of the total fatty acid The polar lipid profiles of strains Ac_11_E3T, Ac_12_G8T
amount)ofstrainAc_11_E3Twereiso-C ,anteiso-C , and Ac_16_C4T were analyzed by two-dimensional thin-
15:0 17:0
iso-C and iso-C H/C 3-OH (summed feature 1) layer chromatography (TLC; modified after Bligh & Dyer,
17:0 15:/1 13:0
(Tables 1, S1). Ac_12_G8T contained iso-C and iso- 1959;Tindalletal.,2007).Theidentityofphosphatidylcho-
15:0
C H/C 3-OH and strain Ac_16_C4T contained iso- line was confirmed using Dragendorff’s reagent. Major
15:1 13:0
C anteiso-C and iso-C H/C 3-OH as major polarlipidsofAc_11_E3TandAc_12_G8Twerediphospha-
15:0, 17:0 15:1 13:0
fattyacids.Themostabundantfattyacidsoftheclosestphy- tidylglycerol(DPG),phosphatidylethanolamine(PE),phos-
logenetic relative P. methylaliphatogenes K22T are iso-C phatidylcholine (PC) and phosphatidylinositol (PI) (Fig.
15:0
iso-C andiso-C (Croweetal.,2014).Thus,thethree S3). Ac_16_C4T contained DPG, PE and an unidentified
17:0 19:0
novelstrainsandP.methylaliphatogenesK22Tshareonlythe glycophospholipid. Major polar lipids of P. methylaliphato-
major fatty acid iso-C reaching abundances that are genes K22T are PC, PE and an unknown phospholipid
15:0
remarkably higher (i.e. above 40% of the total fatty acid (Croweetal.,2014).
3360 InternationalJournalofSystematicandEvolutionaryMicrobiology66
Brevitaleaaridisoli,gen.nov.,sp.nov.,B.deliciosasp.nov.andArenimicrobiumluteum,gen.nov.,sp.nov.
Growth range and optima of temperature and pH were better in the dark, under microaerobic conditions no
determined in duplicates and triplicates, respectively, growth was observed under any conditions. This is in con-
under oxic conditions in liquid medium SSE/HD1:10 gruence with the findings for P. methylaliphatogenes K22T
(Supplementary Materials and Methods). Depending on which, in contrast to the photoheterotroph C. thermophi-
the desired pH value, MES, HEPES, HEPPS or CHES lum BT, lacks all indications of phototrophy (Crowe et al.,
(Sigma-Aldrich or Applichem) were used as buffers at a 2014;Leeetal.,2015).
final concentration of 10 mM. Salt tolerance was deter- Growth substrates utilized by Ac_11_E3T, Ac_12_G8T and
mined in triplicates in oxic liquid HD 1:10 (DSMZ Ac_16_C4T were determined in microtiter plates in dupli-
medium 1124; https://www.dsmz.de/microorganisms/ cates (Ac_11_E3T and Ac_12_G8T) and triplicates
medium/pdf/DSMZ_Medium1124.pdf) amended with (Ac_16_C4T), respectively, using oxic liquid medium SSE/
NaCl at concentrations ranging from 0 to 10% (w/v)
HD 1:10 (Supplementary Materials and Methods) without
since medium SSE/HD1 :10 already contains high
peptone,glucoseandyeastextract(Huberetal.,2014).Sub-
amounts of salts. Growth was measured by recording the
strates were scored as growth-supporting when a final
OD . All three strains were mesophiles with minimum
660 OD (mean of the parallels) of 1.5(cid:2) the control value
660
doubling times ranging between 4.4 and 9.9 h (Table 1).
(without addition of substrate) was reached. Weak growth
Ac_11_E3T and Ac_12_G8T grew at broad temperature
was recorded when the final OD of one of the parallels
ranges (11.0–52.5(cid:14)C and 8.0–45.0(cid:14)C, respectively) and washigherthan1.5(cid:2)thecontrolv66a0lue.Allthreestrainsuti-
had similar growth optima (defined as (cid:21)75% of the high-
lized the complex protein-containing substrates casamino
est growth rate) for temperature (35.0–45.0(cid:14)C and 28.0–
acids,caseinhydrolysate,peptoneandyeastextract(Table1)
45.0(cid:14)C, respectively). In contrast, Ac_16_C4T grew at a
which is a common trait among described members of the
narrower temperature range (25.0–37.0(cid:14)C) with optimal
class Blastocatellia (e.g. Foesel et al., 2013; Huber et al.,
growth occurring at between 33.0 and 37.0(cid:14)C. The high- 2014;Pascualetal.,2015).Inaddition,Ac_12_G8Twasable
est growth rates of Ac_11_E3T, Ac_12_G8T and
to utilize maltose and protocatechuate (Table 1). Weak
Ac_16_C4T were observed at 42(cid:14)C, 41(cid:14)C and 37(cid:14)C, growth of Ac_11_E3T, Ac_12_G8T and Ac_16_C4T was
respectively. All three strains tolerated acidic to basic pH
observedonfive,fourandtwoadditionalsubstrates,respec-
values with Ac_12_G8T showing the broadest growth
tively(Table1).
range (pH 3.5–9.5). Growth optima for pH were similar
Growth on polymers (i.e. cellulose, chitin, lignin, pectin,
for all three strains and ranged between slightly acidic and
neutral pH values (pH 5.4–7.0). Ac_11_E3T, Ac_12_G8T starchand xylan) andlaccaseactivityweretestedonsolidi-
and Ac_16_C4T grew best at pH 5.6, pH 6.0 and pH 6.1, fied media as described previously (Foesel et al., 2015).
Ac_11_E3T hydrolyzed cellulose and xylan, while
respectively. In contrast, the moderate extremophile
P. methylaliphatogenes K22T grows at a minimum temper- Ac_12_G8T and Ac_16_C4T were able to degrade starch
ature of 50(cid:14)C, but its growth range and optimum pH are (Table 1). A genome analysis indicated the capability of
P. methylaliphatogenes K22T to degrade cellulose and xylan
similar to those observed for the three novel strains. All
(Leeetal.,2015).
three strains showed a low tolerance towards NaCl
(Table 1) which has also been shown for species of the ThesedataindicatethatstrainsAc_11_E3T,Ac_12_G8Tand
genera Aridibacter and Blastocatella (Foesel et al., 2013; Ac_16_C4T are obligate aerobic chemoorganoheterotrophs
Huber et al., 2014). The highest growth rates of thatuseaverylimitedspectrumofcarbonandenergysour-
Ac_11_E3T and Ac_12_G8T were observed with 0% (w/v) ces for growth. Like Blastocatella fastidiosa (Foesel et al.,
NaCl; Ac_16_C4T grew best at 0.25% (w/v) NaCl. While 2013) Ac_16_C4T grew on even fewer substrates than the
Ac_11_E3T tolerated up to 1% (w/v) NaCl, Ac_16_C4T relatedandrecentlydescribedspeciesofthegenusStenotro-
grew only at 0–0.5% (w/v) NaCl, and the most salt- phobacter (Pascual et al., 2015). All three novel strains
sensitive strain Ac_12_G8T tolerated not more than showed a preference for complex proteinaceous substrates
0.25% (w/v) NaCl. which was also observed for their next most closely related
Phototrophic growth of Ac_11_E3T, Ac_12_G8T and speciesP.methylaliphatogenesK22T(Croweetal.,2014)and
Ac_16_C4Twasassessedintwodifferentmedia,bothtested many other members of the class Blastocatellia (Pascual
etal.,2015).Thecapabilitytohydrolyzethepolymerscellu-
underaerobicandmicroaerobicconditionsandunderincu-
lose, starch and/or xylan was present in one or two of the
bation in the light versus in the dark. Media were (i) SSE/
threenovelstrains.
HD1 :10 (Supplementary Materials and Methods) without
glucose and peptone and yeast extract content reduced to Catalase and cytochrome-c oxidase were determined by
50 mg l(cid:0)1 and (ii) the CTM-Medium used to cultivate established protocols (Barrow & Feltham, 1993; Gerhardt
C.thermophilum(Tank&Bryant,2015),butwiththeusual et al., 1994). Cytochrome-c oxidase was additionally tested
vitamin solution (Supplementary Materials and Methods) byBactidentOxidaseteststrips(Merck).Furtherenzymatic
instead of the vitamins given in the reference. Growth was activities were determined using API 20NE and API ZYM
measured by recording OD over time in three test systems (bioM(cid:19)erieux), according to the instructions of
660
parallel cultures per strain and condition. Under aerobic themanufacturer.LikemostmembersoftheclassBlastoca-
conditions all three strains grew equally well to slightly tellia,allthreenovelstrainswerenegativeforcytochrome-c
http://ijs.microbiologyresearch.org 3361
P.K.Wüstandothers
oxidase (Table 1). Among members of the class derivative of iso-diabolic acid like the other species do
Blastocatellia only Tellurimicrobium multivorans Ac_18_E7T (SinningheDamst(cid:19)eetal.,2014).
and P. methylaliphatogenes K22T are cytochrome-c oxidase-
The lowest pairwise 16S rRNA gene sequence similarity of
positive (Crowe et al., 2014; Pascual et al., 2015).
the three novel strains to their closest relative P. methylali-
Ac_12_G8T was catalase-positive while Ac_11_D3T and phatogenesK22Tis88.7%.Thisvalueisabovethethreshold
Ac_16_C4T did not show any catalase activity. With the
value of 86.5% which would be indicative of an affiliation
exceptionofStenotrophobacterterraeAc_28_D10T,allother
with different families (Yarza et al., 2014). Based on the
described representatives of the class Blastocatellia are
phylogenetic position and several morphological and che-
catalase-positive (Pascual et al., 2015). All three novel
motaxonomic characteristics of the two novel genera, we
strains were able to hydrolyze aesculin, which is indicative
proposetheformaldescriptionofthefamilyPyrinomonada-
of b-glucosidase activity (Table S2). Like Aridibacter fami-
ceaefam.nov.thataccommodatestheaboveproposedgen-
durans A22_HD_4HT and A. kavangonensis Ac_23_E3T
era and the genus Pyrinomonas. Signature nucleotide
(Huber et al., 2014), Ac_12_G8T hydrolyzed 4-nitrophenyl
patterns in the 16S rRNA gene sequence (Escherichia coli
b-D-galactopyranoside, indicating b-galactosidase activity. referencegene;Brosiusetal.,1978)wereadoptedfrompre-
The three novel strains were unable to reduce nitrate, pro-
vious work (Pascual et al., 2015) and updated considering
duce indole from the degradation of tryptophan, ferment
newly added species (Supplementary Table S3). The signa-
glucose or anaerobically hydrolyze arginine, urea or gela-
ture nucleotides of the family Pyrinomonadaceae fam. nov.
tine. Together with P. methylaliphatogenes K22T, the three are:69:99(G–A),326(C),327(A),360(G),371:390(G–
strains share the enzymes valine arylamidase, leucine aryla- C), 605:633 (U–C), 725:732 (G–C), 726:731 (C–G),
midase, alkaline phosphatase, acid phosphatase, naphthol- 772:807 (A–U), 861:868 (G–C), 895:904 (G–C),
AS-BI-phosphohydrolase and N-acetyl-b-glucosaminidase. 929:1388 (A–U), 1064:1192 (G–C), 1165:1171 (U–A),
In summary, the enzymatic equipment of Ac_12_G8T sur- 1201 (C) and 1313:1324 (U–A) with positions 326 and
passed that of Ac_11_E3T and Ac_16_C4T. Detailed enzy- 895:904beinguniqueforthisnovelfamily.
matic activities are listed in Table S2 and in the species
descriptionsbelow.
Description of Arenimicrobium gen. nov.
Based on these collective findings, Ac_11_E3T, Ac_12_G8T
andAc_16_C4Trepresentthreenovelspeciesthatareaffili- Arenimicrobium (A.re.ni.mi.cro¢bi.um. L. fem. n. arena,
ated with the order Blastocatellales, but are only distantly sand;N.L.neut.n.microbium,microbe;N.L.neut.n.Areni-
related to other described members of this order. Conse- microbium,abacteriumisolatedfromsandysoil).
quently, the two novel genera Brevitalea gen. nov. and Are-
Gram-staining negative, non-spore-forming, non-capsule-
nimicrobium gen. nov. are proposed, comprising the novel
forming, non-motile, ovoid to rod-shaped cells that divide
speciesBrevitaleaaridisolisp.nov.(Ac_11_E3T),B.deliciosa
by binary fission. Strictly aerobic, chemoorganoheterotro-
sp. nov. (Ac_16_C4T) and Arenimicrobium luteum sp. nov.
phic mesophiles. Catalase-positive, cytochrome-c oxidase-
(Ac_12_G8T), respectively. Like their closest phylogenetic
negative. Complex protein-containing substrates are the
relative Pyrinomonas methylaliphatogenes K22T the three
preferred carbon and energy sources. Major fatty acids
novel strains did not show phototrophic growth. This is a detected afterBlight–Dyer extractionareiso-C andiso-
15:0
central trait that distinguishes them from the photohetero-
C H/C 3-OH.ContainDPG,PE,PC,andPIasmajor
trophChloracidobacteriumthermophilumBT.Anotherchar- 15:1 13:0
polar lipids. Contain MK-8 as major respiratory
acteristic feature that differentiates C. thermophilum BT
quinoneandminoramountsofMK-7.Thegenusisamem-
fromthenovelstrains andotherspecies oftheclassBlasto-
ber of the order Blastocatellales within the class Blastocatel-
catellia is the high abundance of the polar lipids diacylgly-
lia.ThetypespeciesisArenimicrobiumluteum.
cerylhydroxymethyl-N,N,N-trimethyl-b-alanine (DGTA)
and phosphatidylmonomethylethanolamine (PME) (Garcia
Costas et al., 2012). All members of the class Blastocatellia Description of Arenimicrobium luteum
share the presence of iso-C as a major fatty acid. The sp. nov.
15:0
abundances of this iso-branched fatty acid are remarkably
higher in the three novel strains (43.7–59.8%; this study) Arenimicrobium luteum (lu¢te.um. L. neut. adj. luteum,
and P. methylaliphatogenes K22T (40.8%; Crowe et al., golden-yellow/orangecolored).
2014)thaninmembersofthefamilyBlastocatellaceae(5.0– Displays the following characteristics in addition to those
38.0%; Pascual et al., 2015). Previous studies indicate that given in the genus description. On solid media forms soft
the presence of iso-diabolic acid is a characteristic feature and slimy colonies of less than 1 mm in diameter that are
of members of the class Blastocatellia (Sinninghe Damst(cid:19)e smooth, shiny, circular, and convex with entire margins
et al., 2014). However, C. thermophilum BT contains iso- and intense orange-yellowish in color. Cells are ovoid to
diabolic acid in significantly higher amounts than strain rod-shaped with an average size of 1.6(cid:2)0.8 µm (1.0–
Ac_11_E3T, P. methylaliphatogenes K22T and members of 2.3(cid:2)0.7–1.0 µm). Grows at 8.0–44.6(cid:14)C and pH 3.5–9.5;
the family Blastocatellaceae. Moreover C. thermophilum BT optimal growth at 28.0–44.6(cid:14)C and pH 5.5–7.0. Minimal
does not contain the monoalkyl glycerol ether (MGE) doubling time is 9.9h. Tolerates NaCl concentrations of
3362 InternationalJournalofSystematicandEvolutionaryMicrobiology66
Brevitaleaaridisoli,gen.nov.,sp.nov.,B.deliciosasp.nov.andArenimicrobiumluteum,gen.nov.,sp.nov.
up to 0.25% (w/v); optimal growth at 0% NaCl. Grows rangesfrom54.7to55.9%.Thegenusbelongstotheorder
on maltose, casamino acids, casein hydrolysate, peptone, Blastocatellales within the class Blastocatellia. The type spe-
yeast extract, protocatechuate and starch. Weak growth is ciesisBrevitaleaaridisoli.
observed on fructose, mannose, ribitol and sucrose. No
growth is observed on arabinose, cellobiose, erythrose,
erythrulose, fucose, galactose, glucose, lactose, lyxose, mele-
Description of Brevitalea aridisoli sp. nov.
zitose, raffinose, rhamnose, sorbose, trehalose, xylose, glu-
cosamine, N-acetylglucosamine, N-acetylgalactosamine, Brevitalea aridisoli (a.ri.di.so¢li. L. masc. adj. aridus dry;
acetoin, arabitol, dulcitol, lyxitol, mannitol, myo-inositol, L. neut. gen. n. soli of soil; N.L. gen. n. aridisoli, isolated
sorbitol, xylitol, alanine, arginine, asparagine, cysteine, glu- fromdrysoil).
tamine, glycine, histidine, isoleucine, leucine, lysine, methi-
Displays the following characteristics in addition to those
onine, ornithine, phenylalanine, proline, hydroxy-proline,
given in the genus description. On solid media forms soft
serine, threonine, tryptophan, tyrosine, valine, adipate, ace-
and slimy colonies of up to 1 mm in diameter that are
tate, ascorbate, aspartate, benzoate, trimethoxybenzoate,
smooth, shiny, circular and convex with entire margins
butyrate, a-hydroxybutyrate, b-hydroxybutyrate, g-hydrox-
and white. Cells are rod-shaped with an average size of
ybutyrate, isobutyrate, caproate, caprylate, citrate, isocitrate,
1.6(cid:2)0.8 µm (1.0–2.2(cid:2)0.7–0.9 µm). Grows at 11.0–52.5(cid:14)C
crotonate, formate, fumarate, gluconate, 2-oxogluconate,
and pH 4.7–8.1; optimal growth at 35.4–45.0(cid:14)C and pH
glucuronate, 2-oxoglutarate, glutamate, glycolate, glyoxylate,
5.4–7.0. Minimal doubling time is 4.4h. Tolerates NaCl
heptanoate, isovalerate, laevulinate, lactate, malate, maleic
concentrations of up to 1.0% (w/v); optimal growth at
acid, malonate, nicotinic acid, oxaloacetate, propionate,
0% NaCl. Grows on casamino acids, casein hydrolysate,
pyruvate, shikimate, succinate, tartrate, 2-oxovalerate, buta-
peptone, yeast extract, cellulose and xylan. Weak growth
nol, 1,2-butandiol, 2,3-butandiol, ethanol, ethylene glycol,
is observed on lyxose, maltose, sucrose, mannitol and
glycerol, methanol, propanol, 1,2-propandiol, carboxy-
succinate. No growth is observed on arabinose, cellobiose,
methyl cellulose, chitin, cellulose, fermented rumen extract,
erythrose, erythrulose, fructose, fucose, galactose, glucose,
laminarin, lignin, pectin, Tween 80 and xylan. Aesculin
lactose, mannose, melezitose, raffinose, rhamnose, sor-
and 4-nitrophenyl b-D-galactopyranoside are hydrolyzed
bose, trehalose, xylose, glucosamine, N-acetylglucosamine,
(API 20NE). Unable to reduce nitrate, produce indole
N-acetylgalactosamine, acetoin, arabitol, dulcitol, lyxitol,
from the degradation of tryptophan, ferment glucose,
myo-inositol, ribitol, sorbitol, xylitol, alanine, arginine,
anaerobically hydrolyze arginine, hydrolyze urea or hydro-
asparagine, cysteine, glutamine, glycine, histidine, isoleu-
lyze gelatine. The following additional enzyme activities
cine, leucine, lysine, methionine, ornithine, phenylalanine,
(API ZYM) are present: acid and alkaline phosphatase,
proline, hydroxy-proline, serine, threonine, tryptophan,
naphtol-AS-BI-phosphohydrolase, esterase (C4), esterase
tyrosine, valine, adipate, acetate, ascorbate, aspartate, ben-
lipase (C8), leucine arylamidase, valine arylamidase, cystine
zoate, trimethoxybenzoate, butyrate, a-hydroxybutyrate,
arylamidase, trypsin (weak), a-chymotrypsin, a- and b-
b-hydroxybutyrate, g-hydroxybutyrate, isobutyrate, cap-
galactosidase, a- and b-glucosidase, N-acetyl-b-glucosami-
roate, caprylate, citrate, isocitrate, crotonate, formate,
nidase and a-fucosidase (weak). Major polar lipids of
fumarate, gluconate, 2-oxogluconate, glucuronate, 2-oxo-
strains are DPG, PE, PC and PI.
glutarate, glutamate, glycolate, glyoxylate, heptanoate, iso-
The type strain is Ac_12_G8T (=DSM 26556T=LMG valerate, laevulinate, lactate, malate, maleic acid,
29166T), isolated from a sandy agriculturally used old malonate, nicotinic acid, oxaloacetate, propionate, proto-
flood plain soil in Mashare, Namibia (17(cid:14)53¢32.4† S, catechuate, pyruvate, shikimate, tartrate, 2-oxovalerate,
20(cid:14)11¢15.4† E). The DNA G+C content of the type strain butanol, 1,2-butandiol, 2,3-butandiol, ethanol, ethylene
is 66.9 mol%. glycol, glycerol, methanol, propanol, 1,2-propandiol, car-
boxymethyl cellulose, chitin, fermented rumen extract,
laminarin, lignin, pectin, Tween 80 and starch. Aesculin
Description of Brevitalea gen. nov.
is hydrolyzed (API 20NE). Unable to reduce nitrate, pro-
Brevitalea(Bre.vi.ta¢le.a.L.adj.brevis,short;L.fem.n.talea, duce indole from the degradation of tryptophan, ferment
arod,astick;N.L.fem.n.Brevitalea,ashortrod). glucose, anaerobically hydrolyze arginine, hydrolyze 4-
nitrophenyl b-D-galactopyranoside, urea or gelatine. The
Gram-staining negative, non-spore-forming, non-capsule-
following additional enzyme activities (API ZYM) are
forming,non-motile,rod-shapedcellsthatdividebybinary
present: acid and alkaline phosphatase, naphtol-AS-BI-
fission. Strictly aerobic, chemoorganoheterotrophic meso-
phosphohydrolase, esterase lipase (C8) (weak), leucine
philes. Catalase-negative, cytochrome-c oxidase-negative.
arylamidase, valine arylamidase, cystine arylamidase
Complex protein-containing substrates are the preferred
(weak), trypsin, a- and b-glucosidase and N-acetyl-b-
carbonandenergysource.Theabilitytohydrolyzepolymers
glucosaminidase. Major polar lipids are DPG, PE, PC
is variable. Major fatty acids detected after Blight–Dyer
and PI.
extraction are iso-C anteiso-C and iso-C H/
15:0 17:0, 15:1
C 3-OH. Contain DPG and PE as major polar lipids. The type strain is Ac_11_E3T (=DSM 27934T= LMG
13:0
Sole respiratory quinone is MK-8. The DNA G+C content 28618T), isolated from a sandy old flood plain bush veld
http://ijs.microbiologyresearch.org 3363
P.K.Wüstandothers
soil in Mashare, Namibia (17(cid:14)53¢39.3† S, 20(cid:14)13¢39.5† E). Description of Pyrinomonadaceae
The DNA G+C content of the type strain is 55.9 mol%. fam. nov.
Pyrinomonadaceae (Py.ri.no.mo.na.da.ce¢ae. N.L. fem. n.
Pyrinomonas,abacteriagenus;suff.-aceaeendingtodenote
Description of Brevitalea deliciosa sp. nov.
a family; N.L. fem. pl. n. Pyrinomonadaceae, the Pyrinomo-
Brevitaleadeliciosa(de.li.ci.o¢sa.L.fem.adj.deliciosa,fastidi- nasfamily).
ous, referring to the limited number of substrates utilized Gram-staining-negative, non-spore-forming and non-cap-
bythestrain). sule-forming bacteria. Cells are ovoid or rod-shaped and
dividebybinaryfission.Aerobicchemoorganoheterotrophs,
Displays the following characteristics in addition to those
unable to grow phototrophically, unable to reduce nitrate
given in the genus description. On solid media forms soft
or ferment glucose. Cytochrome-c oxidase and catalase
and slimy colonies of less than 1 mm in diameter that are
activities variable. Mesophiles or thermophiles that tolerate
smooth,shiny,circularandconvexwithentiremarginsand
a broad pH range. Limited substrate-utilization spectrum
white. Cellsarerod-shapedwithanaveragesize of1.4(cid:2)0.7
µm (0.9–1.8(cid:2)0.6–0.8 µm). Grows at 24.6–34.2(cid:14)C and pH with preference for complex proteinaceous growth sub-
4.7–9.0; optimal growth at 32.9–37.2(cid:14)C and pH 5.4–6.5. strates. The ability to hydrolyze polymers is variable. Con-
tain MK-8 as major respiratory quinone and in some cases
Minimal doubling time is 7.6h. Tolerates NaCl concentra-
minor amounts of MK-7. The major shared polar lipid is
tions ofupto0.5%(w/v); optimalgrowthat0.25%NaCl.
PE. The major shared fatty acid is iso-C . Signature
Growsoncasaminoacids,caseinhydrolysate,peptone,yeast 15:0
nucleotides in the 16S rRNA gene sequence (E. coli refer-
extract and starch. Weak growth is observed on tyrosine
ence gene; Brosius et al., 1978) are: 69:99 (G–A), 326 (C),
and protocatechuate. No growth is observed on arabinose,
327 (A), 360 (G), 371:390 (G–C), 605:633 (U–C),
cellobiose, erythrose, erythrulose, fructose, fucose, galac-
725:732(G–C),726:731(C–G),772:807(A–U),861:868
tose,glucose,lactose,lyxose,maltose,mannose,melezitose,
(G–C), 895:904 (G–C), 929:1388 (A–U), 1064:1192 (G–
raffinose,rhamnose,sorbose,sucrose,trehalose,xylose,glu-
C),1165:1171(U–A),1201(C)and1313:1324(U–A).
cosamine, N-acetylglucosamine, N-acetylgalactosamine,
acetoin, arabitol, dulcitol, lyxitol, mannitol, myo-inositol, The family accommodates the proposed genera Brevitalea
ribitol, sorbitol, xylitol, alanine, arginine,asparagine, cyste- gen.nov.andArenimicrobiumgen.nov.aswellasthegenus
ine,glutamine,glycine,histidine,isoleucine,leucine,lysine, Pyrinomonas.ThefamilyisamemberoftheorderBlastoca-
methionine, ornithine, phenylalanine, proline, hydroxy- tellaleswithintheclassBlastocatellia.ThetypegenusisPyri-
proline, serine, threonine, tryptophan, valine, adipate, ace- nomonas. The DNA G+C content ranges from 54.7 to
tate, ascorbate, aspartate, benzoate, trimethoxybenzoate, 66.9mol%.
butyrate, a-hydroxybutyrate, b-hydroxybutyrate, g-
hydroxybutyrate, isobutyrate, caproate, caprylate, citrate,
isocitrate, crotonate, formate, fumarate, gluconate, 2-oxo-
Acknowledgements
gluconate,glucuronate,2-oxoglutarate,glutamate,glycolate,
glyoxylate, heptanoate, isovalerate, laevulinate, We thank Gabriele Pötter for analysis of fatty acids, polar lipids and
lactate,malate,maleicacid,malonate,nicotinicacid,oxalo- quinones, Dr Cathrin Spröer, Dr Peter Schumann, Bettina Str€aubler
acetate,propionate,pyruvate,shikimate,succinate,tartrate, andBirgitGrünfordeterminingDNAG+C-contentandcarryingout
DNA–DNA hybridization and Caroline Pilke for preparing cultiva-
2-oxovalerate, butanol, 1,2-butandiol, 2,3-butandiol, etha-
tion media (all DSMZ). We also thank Silvia Dobler (Ludwig-Maxi-
nol,ethyleneglycol,glycerol,methanol,propanol,1,2-prop- milians-Universit€atMünchen)forexcellenttechnicalassistance.Soils
andiol,carboxymethylcellulose,chitin,cellulose,fermented in Namibia were sampled under collection permits 1569/2011 and
rumen extract, laminarin, lignin, pectin, Tween 80, and 1635/2011andexportedunderpermitsES25691(ofApril12,2011)
xylan.Aesculinishydrolyzed(API20NE).Unabletoreduce and ES 27282 (of April 5, 2012). Isolation and deposit of strains
nitrate,produceindolefromthedegradationoftryptophan, Ac_11_E3TandAc_12_G8TisundertheMTAoftheNBRI(National
Botanical Research Institute, Namibia) of April 04, 2011. Isolation
ferment glucose, anaerobically hydrolyze arginine, 4-nitro-
and deposit of strain Ac_16_C4T is under the MTA of the NBRI of
phenylb-D-galactopyranoside,ureaorgelatine.Thefollow-
April05,2012.ThisworkwassupportedbygrantsfromtheGerman
ing additional enzyme activities (API ZYM) are present:
Federal Ministry of Science and Education to Jörg Overmann (TFO
acid and alkaline phosphatase, naphtol-AS-BI-phosphohy- project01LL0912M).
drolase,esterase(C4),esteraselipase(C8),leucinearylami-
dase, valine arylamidase, cystine arylamidase (weak),
trypsin, a-galactosidase (weak), a- and b-glucosidase and
References
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The type strain is Ac_16_C4T (=DSM 29892T=LMG ronment.ApplEnvironMicrobiol65,1731–1737.
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DNAG+Ccontentofthetypestrainis54.7mol%. CambridgeUniversityPress.
3364 InternationalJournalofSystematicandEvolutionaryMicrobiology66