International Journal of Systematic and Evolutionary Microbiology (2015), 65, 1583–1586
DOI 10.1099/ijs.0.000141
Paenibacillus tibetensis sp. nov., a psychrophilic
bacterium isolated from alpine swamp meadow soil
Li-Li Han, Ji-Zheng He, Yuan-Ming Zheng, Jun Zeng and Li-Mei Zhang
Correspondence
Li-Mei Zhang
State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental
Sciences, Chinese Academy of Sciences, Beijing 100085, PR China
[email protected]
A novel psychrophilic strain, SSB001T, was isolated from an alpine swamp meadow soil in Tibet,
China, and identified as a representative of a novel phylogenetic subclade in the genus
Paenibacillus, with Paenibacillus antarcticus (96.2 %), Paenibacillus macquariensis (96.53 %)
and Paenibacillus glacialis (96.2 %) as the most closely related species on the basis of 16S rRNA
gene sequence analyses. The strain was distinguished from defined species of the genus
Paenibacillus by further study of rpoB gene sequences, phenotypic characterization, cellular fatty
acid composition, quinones, polar lipids and meso-diaminopimelic acid in the peptidoglycan.
Based upon these results, we propose the strain as a representative of a novel species named
Paenibacillus tibetensis sp. nov., with SSB001T (5ACCC 19728T5DSM 29321T) as the type
strain. The DNA G+C content (mol%) of strain SSB001T was 40.18 mol% (HPLC).
The Qinghai–Tibet plateau is the highest region on the
Earth, known as the ‘roof of the world’, with an average
altitude of more than 4500 m (http://www.china.org.cn/
english/scitech/104358.htm). The climate of the area is high
mountain cold zone with the mean annual temperature
varying from 22 uC to 8 uC and the lowest temperature being
250 uC. The mean annual precipitation is 1947.4 mm
(Falandysz et al., 2014). The air pressure and concentration
of oxygen in the air are 55–70 % less than those at sea level,
while the solar radiation is much stronger than in other
regions (Hou et al., 2009; Ni, 2000). These extreme
conditions lead to a unique composition of species in Tibet.
The novel strain SSB001T was isolated from an alpine swamp
meadow soil, at an altitude of 4185 m, located at
29u 369 210 N 94u 369 230 E in Shegyla Mountain, China.
According to Ash et al. (1993), members of ‘group 3’ within
the genus Bacillus can be distinguished from members of
other Bacillus groups using a battery of phenotypic characteristics and 16S rRNA gene sequences. A new genus
accommodating these bacteria was named Paenibacillus,
and Paenibacillus polymyxa was proposed as the type species
(Ash et al., 1993; Judicial Commission of the International
Committee for Systematics of Prokaryotes, 2005). At the time
of writing, 154 species of the genus and four subspecies
have been described. (http://www.bacterio.net/paenibacillus.
html). Among all the species, most species are either
mesophilic (Enright et al., 2003; Kim et al., 2014; Lim et al.,
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and
rpoB gene sequences of strain SSB001T are KM522843 and
KM522842, respectively.
Four supplementary figures are available with the online Supplementary
Material.
000141 G 2015 IUMS
2006; Saha et al., 2005) or thermophilic (Li et al., 2014;
Shimoyama et al., 2014; Yao et al., 2014), with the exception
of three psychrophilic species, Paenibacillus macquariensis
(Marshall & Ohye, 1966), Paenibacillus glacialis (Kishore
et al., 2010) and Paenibacillus darwinianus (Dsouza et al.,
2014). In this study, we isolated a psychrophilic strain,
SSB001T, closely related to these species, which belongs to a
novel species of genus Paenibacillus for which we propose the
name Paenibacillus tibetensis sp. nov.
Genomic DNA was extracted from strain SSB001T using a
MoBio UltraClean microbial DNA isolation kit according
to the manufacturer’s protocol. For phylogenetic analysis,
the 16S rRNA gene was amplified by PCR with primers 27f
(59-GAGTTTGATCCTGGCTCAG-39) and 1525r (59-AGAAAGGAGGTGATCCAGCC-39) (Rainey et al., 1996) and
the purified PCR product was sequenced. The sequences
acquired, together with related sequences obtained from
the GenBank database, were aligned using the CLUSTAL W
program in the MEGA 6.06 software (Tamura et al., 2013).
Phylogenetic trees were reconstructed with the neighbourjoining (Saitou & Nei, 1987) and maximum-likelihood
(Felsenstein, 1981) methods based upon the model of Jukes
& Cantor (1969). Phylogenetic trees were bootstrapped
with 1000 replicates. Filled circles in Figs S1 and S2 (available in the online Supplementary Material) indicate the
generic branches present in the phylogenetic trees generated by the neighbour-joining and maximum-likelihood
methods. Both of the trees shared similar topologies with
high bootstrap support, and only the smaller tree generated
by neighbour-joining method was shown in Fig. 1. Sequence
similarities were calculated using the database EzTaxon-e
(Ezbiocloud) to compare the sequences of the 16S rRNA
genes (Kim et al., 2012). The results showed that strain
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L.-L. Han and others
0.02
99
90 P. macquariensis subsp. macquariensis DSM 2T (X60625)
78 P. macquariensis subsp. defensor M4-1 (AB360547)
100
P. antarcticus LMG 22078T (AJ605292)
P. glacialis KFC91T (EU815300)
P. tibetensis SSB001T (KM522843)
P. stellifer IS 1T (AJ316013)
84
P. wynnii LMG 22176T (AJ633647)
P. anaericanus MH21T (AJ318909)
P. chibensis JCM 9905T (AB073194)
Bacillus subtilis DSM 10T (AJ276351)
Fig. 1. Neighbour-joining tree of 16S rRNA gene sequences showing the phylogenetic relationships among the test and close
reference strains of the genus Paenibacillus. Bootstrap values are based on 1000 resamplings and confidence levels greater
than 70 % are indicated at the internodes. Bacillus subtilis DSM 10T was used as an outgroup. Bar, 2 % substitution per site.
SSB001T was the closest match (96.53 % 16S rRNA gene
sequence similarity) to P. macquariensis subsp. macquariensis
NCTC 10419T, then P. macquariensis subsp. defensor M4-2T
(96.47 % similarity), P. glacialis KFC91T (96.2 %) and
Paenibacillus antarcticus LMG 22078T (96.2 %) in that order.
To further confirm the relationship of the four strains, the
rpoB gene encoding the b-subunit of RNA polymerase was
amplified from strain SSB001T using primers rpoB1698f (59-AACATCGGTTTGATCAAC-39, corresponding to Escherichia
coli position 1643) and rpoB2041r (59-CGTTGCATGTTGGTACCCAT-39, corresponding to E. coli position 2041)
(Dahllöf et al., 2000) and sequenced. The phylogenetic
analysis of rpoB gene sequences showed that the strain shared
43.4 %, 44.7 % and 73.7 % similarity with P. glacialis KFC91T,
P. antarcticus LMG 22078T and P. macquariensis LMG 6935T,
respectively (Fig. S3).
According to the phenotypic characterization partially
fulfilling the minimal standards of Logan et al. (2009), a
total of 153 features were analysed. P. antarcticus LMG
22078T and P. macquariensis LMG 6935T, used as the
reference strains, were purchased from the Belgian Coordinated Collections of Micro-organisms (BCCM/LMG),
and another reference strain, P. glacialis DSM 22343T
(5KFC91T) was purchased from the Deutsche Sammlung
von Mikroorganismen und Zellkulturen (DSMZ). Utilization
of 95 carbon sources was tested using a GP2 Microplate
(Biolog) following the manufacturer’s instructions. After
incubation for 24 h at 20 uC, strain SSB001T was able to utilize
dextrin, N-acetyl-D-glucosamine, L-arabinose, D-fructose, aD-glucose, maltose, maltotriose, D-mannose, methyl b-Dglucoside, D-ribose, trehalose, D-xylose, acetic acid, succinic
acid monomethyl ester, glycerol, adenosine, 29-deoxyadenosine, inosine, thymidine, uridine, 3-methyl-D-glucose
(weakly), palatinose (weakly), salicin (weakly), pyruvic acid
methyl ester (weakly) and pyruvic acid (weakly) as sole carbon
sources, but was not able to utilize a-cyclodextrin, bcyclodextrin, glycogen, inulin, mannan, Tween 40, Tween
80, N-acetyl-b-D-mannosamine, amygdalin, D-arabitol, arbutin, cellobiose, L-fucose, D-galactose, D-galacturonic acid,
1584
gentiobiose, D-gluconic acid, myo-inositol, a-lactose, lactulose, D-mannitol, melezitose, melibiose, methyl a-D-galactoside, methyl b-D-galactoside, methyl a-D-glucoside, methyl
a-D-mannoside, D-psicose, raffinose, L-rhamnose, sedoheptulosan, D-sorbitol, stachyose, sucrose, tagatose, turanose,
xylitol, a-hydroxybutyric acid, b-hydroxybutyric acid, chydroxybutyric acid, p-hydroxyphenylacetic acid, a-ketoglutaric acid, a-ketovaleric acid, lactamide, D-lactic acid methyl
ester, L-lactic acid, D-malic acid, L-malic acid, propionic acid,
succinamic acid, succinic acid, N-acetyl-L-glutamic acid, Lalaninamide, D-alanine, L-alanine, L-alanyl-glycine, L-asparagine, L-glutamic acid, glycyl-L-glutamic acid, L-pyroglutamic
acid, L-serine, putrescine, 2,3-butanediol, adenosine-59monophosphate, thymidine-59-monophosphate, uridine-59monophosphate, D-fructose 6-phosphate, a-D-glucose 1phosphate, D-glucose 6-phosphate or DL-a-glycerol phosphate.
Acid production and additional physiological tests were
performed using API 20E strips, API 50 CH strips and API 50
CHB/E medium (BioMérieux) according to the manufacturer’s protocol over 2 days at 20 uC (Table 1). In the API 20E
system, strain SSB001T was positive for hydrolysis of arginine
and starch, but negative for hydrolysis of ONPG, lysine,
ornithine and tryptophan, production of indole and H2S,
reduction of nitrate and the Voges–Proskauer test. In the API
50CH system, acid was produced from ribose, D-xylose,
fructose, mannose, glycogen and gluconate. Acid was not
produced from L-arabinose, methyl b-D-xylosidase, galactose, mannitol, methyl a-D-mannoside, methyl a-D-glucoside, arbutin, salicin, lactose, melibiose, sucrose, raffinose,
gentiobiose, turanose or 5-ketogluconate.
The novel strain also showed a fatty acid composition
different from those of the reference strains of related
species using a previously described method (Athalye et al.,
1985; Reddy et al., 2008). In our study, the novel strain and
three reference type strains were grown on TSA medium
for 4 days at 20 uC, then the cells were harvested and the
fatty acids were prepared and identified by the standard
method of the MIDI Sherlock Microbial Identification
System (Library RTSA6 6.0, MIDI Sherlock software
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Paenibacillus tibetensis sp. nov.
Table 1. Distinguishing characteristics among strain SSB001T
and representatives of P. glacialis, P. antarcticus and P.
macquariensis
Strains: 1, Paenibacillus tibetensis sp. nov. SSB001T; 2, P. glacialis DSM
22343T; 3, P. antarcticus LMG 22078T; 4, P. macquariensis LMG
6935T. All strains were negative for the Voges–Proskauer test.
Characteristic
Habitat
Temperature range for growth (uC)
Oxidase
Nitrate reduction
Urease
Arginine dihydrolase
Starch hydrolysis
Acid production from:
L-Arabinose
D-Xylose
Mannitol
Sucrose
Raffinose
Glycogen
Gentiobiose
Turanose
DNA G+C content (mol%)
1
2
3
4
Soil Soil Sediment Soil
4–25 4–30
4–31 0–25
2
+
+
2
2
+
2
2
2
2
+
2
+
+
2
2
+
2
+
+
2
2
+
2
2
2
2
+
2
2
+
+
2
2
2
+
40.18 42.0
+
+
2
2
+
2
+
+
40.7
2
+
+
+
+
2
+
+
39
package version 6.0). C16 : 0, iso-C15 : 0 and anteiso-C15 : 0 were
common among the novel strain and the three reference
strains, demonstrating their close relationship, but some
minor compounds were different among the novel strain and
the reference strains. The analysis of quinones, polar lipids
and meso-diaminopimelic acid in the peptidoglycan of
strain SSB001T were performed by CGMCC (China General
Microbiological Culture Collection Center). The diamino
acid in the peptidoglycan was meso-diaminopimelic acid,
MK-7 was the major isoprenoid quinone and phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol were the main polar lipids (Fig. S4). These peptidoglycan
characteristics are typical of the genus Paenibacillus.
Based upon all of the characteristics determined for strain
SSB001T, it is clear that these are in accordance with those
described for the genus Paenibacillus. The results of phenotypic
and chemotaxonomic tests were obviously difference from
those of related reference strains. Phylogenetic analysis of the
16S rRNA gene and rpoB gene sequences indicated that
the strain was very similar to P. macquariensis subsp.
macquariensis, but the similarity was lower than the standard
threshold for species delineation (97 % for 16S rRNA gene).
Therefore, we propose that strain SSB001T represents a novel
species within the genus Paenibacillus.
Description of Paenibacillus tibetensis sp. nov.
Paenibacillus tibetensis (ti.bet.en9sis. N.L. masc. adj. tibetensis of or pertaining to Tibet, where the organism was
isolated).
http://ijs.sgmjournals.org
Cells are Gram-stain-positive, aerobic, motile with a polar
flagellum, rod-shaped, 0.6 mm wide by 2.5 mm long.
Terminal ellipsoidal spores are formed in swollen sporangia.
Colonies are circular, slightly convex, shallow and yellow on
TSA under optimal growth temperature (20 uC) and pH (7).
Can grow at temperatures between 4 uC and 25 uC, between
pH 6 and 7.5, and weakly with up to 4 % (w/v) NaCl. Utilizes
dextrin, N-acetyl-D-glucosamine, L-arabinose, D-fructose, aD-glucose, maltose, maltotriose, D-mannose, methyl b-Dglucoside, D-ribose, trehalose, D-xylose, acetic acid, succinic
acid monomethyl ester, glycerol, adenosine, 29-deoxyadenosine, inosine, thymidine, uridine, 3-methyl-D-glucose
(weakly), palatinose (weakly), salicin (weakly), pyruvic acid
methyl ester (weakly) and pyruvic acid (weakly) as sole
carbon sources. Positive for hydrolysis of arginine and starch.
Acid is produced from ribose, D-xylose, fructose, mannose,
glycogen and gluconate. The fatty acid is anteiso-C15 : 0. The
diamino acid in the peptidoglycan is meso-diaminopimelic
acid, MK-7 is the major isoprenoid quinone and phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol are the main polar lipids.
The type strain is SSB001T (5ACCC 19728T5DSM
29321T), isolated from alpine swamp meadow soil in
Tibet, China. Its DNA G+C content is 40.18 mol%
(HPLC).
Acknowledgements
This work was supported financially by the National Natural Science
Foundation of China (41301265, 51221892), the Ministry of Science
and Technology of China (2013CB956300) and STSN-21-02.
References
Ash, C., Priest, F. G. & Collins, M. D. (1993). Molecular identification
of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using
a PCR probe test. Proposal for the creation of a new genus
Paenibacillus. Antonie van Leeuwenhoek 64, 253–260.
Athalye, M., Noble, W. C. & Minnikin, D. E. (1985). Analysis of cellular
fatty acids by gas chromatography as a tool in the identification of
medically important coryneform bacteria. J Appl Bacteriol 58, 507–
512.
Dahllöf, I., Baillie, H. & Kjelleberg, S. (2000). rpoB-based microbial
community analysis avoids limitations inherent in 16S rRNA gene
intraspecies heterogeneity. Appl Environ Microbiol 66, 3376–
3380.
Dsouza, M., Taylor, M. W., Ryan, J., MacKenzie, A., Lagutin, K.,
Anderson, R. F., Turner, S. J. & Aislabie, J. (2014). Paenibacillus
darwinianus sp. nov., isolated from gamma-irradiated Antarctic soil.
Int J Syst Evol Microbiol 64, 1406–1411.
Enright, M. R., McInerney, J. O. & Griffin, C. T. (2003).
Characterization of endospore-forming bacteria associated with
entomopathogenic nematodes, Heterorhabditis spp., and description
of Paenibacillus nematophilus sp. nov. Int J Syst Evol Microbiol 53,
435–441.
Falandysz, J., Dryżałowska, A., Saba, M., Wang, J. & Zhang, D.
(2014). Mercury in the fairy-ring of Gymnopus erythropus (Pers.) and
Marasmius dryophilus (Bull.) P. Karst. mushrooms from the Gongga
Mountain, Eastern Tibetan Plateau. Ecotoxicol Environ Saf 104, 18–22.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 00:09:47
1585
L.-L. Han and others
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a
maximum likelihood approach. J Mol Evol 17, 368–376.
Hou, B. C., Wang, E. T., Li, Y., Jia, R. Z., Chen, W. F., Man, C. X., Sui,
X. H. & Chen, W. X. (2009). Rhizobial resource associated with
aerobic, endospore-forming bacteria. Int J Syst Evol Microbiol 59,
2114–2121.
Marshall, B. J. & Ohye, D. F. (1966). Bacillus macquariensis n.sp., a
epidemic legumes in Tibet. Microb Ecol 57, 69–81.
psychrotrophic bacterium from sub-antarctic soil. J Gen Microbiol 44,
41–46.
Judicial Commission of the International Committee for
Systematics of Prokaryotes (2005). The type species of the genus
Ni, J. (2000). A simulation of biomes on the Tibetan Plateau and their
responses to global climate change. Mt Res Dev 20, 80–89.
Paenibacillus Ash et al. 1994 is Paenibacillus polymyxa. Opinion 77.
Int J Syst Evol Microbiol 55, 513.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules.
In Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by
H. N. Munro. New York: Academic Press.
Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C.,
Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-
e: a prokaryotic 16S rRNA gene sequence database with phylotypes
that represent uncultured species. Int J Syst Evol Microbiol 62, 716–
721.
Kim, J.-H., Kang, H. & Kim, W. (2014). Paenibacillus doosanensis sp.
nov., isolated from soil. Int J Syst Evol Microbiol 64, 1271–1277.
Kishore, K. H., Begum, Z., Pathan, A. A. K. & Shivaji, S. (2010).
Paenibacillus glacialis sp. nov., isolated from the Kafni glacier of the
Himalayas, India. Int J Syst Evol Microbiol 60, 1909–1913.
Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M. & Stackebrandt,
E. (1996). The genus Nocardiopsis represents a phylogenetically
coherent taxon and a distinct actinomycete lineage: proposal of
Nocardiopsaceae fam. nov. Int J Syst Bacteriol 46, 1088–1092.
Reddy, G. S., Prabagaran, S. R. & Shivaji, S. (2008). Leifsonia
pindariensis sp. nov., isolated from the Pindari glacier of the Indian
Himalayas, and emended description of the genus Leifsonia. Int J Syst
Evol Microbiol 58, 2229–2234.
Saha, P., Mondal, A. K., Mayilraj, S., Krishnamurthi, S., Bhattacharya,
A. & Chakrabarti, T. (2005). Paenibacillus assamensis sp. nov., a novel
bacterium isolated from a warm spring in Assam, India. Int J Syst Evol
Microbiol 55, 2577–2581.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
Li, Y.-F., Calley, J. N., Ebert, P. J. & Helmes, E. B. (2014). Paenibacillus
lentus sp. nov., a b-mannanolytic bacterium isolated from mixed soil
Shimoyama, T., Johari, N. B., Tsuruya, A., Nair, A. & Nakayama, T.
(2014). Paenibacillus relictisesami sp. nov., isolated from sesame oil
samples in a selective enrichment using guar gum as the sole carbon
source. Int J Syst Evol Microbiol 64, 1166–1172.
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013).
Lim, J.-M., Jeon, C. O., Lee, J.-C., Xu, L.-H., Jiang, C.-L. & Kim, C.-J.
(2006). Paenibacillus gansuensis sp. nov., isolated from desert soil of
MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol
Evol 30, 2725–2729.
Gansu Province in China. Int J Syst Evol Microbiol 56, 2131–2134.
Yao, R., Wang, R., Wang, D., Su, J., Zheng, S. & Wang, G. (2014).
Logan, N. A., Berge, O., Bishop, A. H., Busse, H. J., De Vos, P., Fritze,
D., Heyndrickx, M., Kämpfer, P., Rabinovitch, L. & other authors
(2009). Proposed minimal standards for describing new taxa of
Paenibacillus selenitireducens sp. nov., a selenite-reducing bacterium
isolated from a selenium mineral soil. Int J Syst Evol Microbiol 64,
805–811.
1586
cake. Int J Syst Evol Microbiol 64, 1534–1539.
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