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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 00:09:47 Printed in Great Britain 1583 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 Downloaded from www.microbiologyresearch.org by International Journal of Systematic and Evolutionary Microbiology 65 IP: 88.99.165.207 On: Sat, 17 Jun 2017 00:09:47 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. 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