International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1271–1277
DOI 10.1099/ijs.0.050716-0
Paenibacillus doosanensis sp. nov., isolated from soil Jong-Hwa Kim, Hyeonji Kang and Wonyong Kim Correspondence
Department of Microbiology, Chung-Ang University College of Medicine, Seoul, Republic of Korea.
Wonyong Kim [email protected]
A Gram-stain-positive, aerobic, endospore-forming bacterium, designated CAU 1055T, was isolated from soil and its taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that the strain formed a distinct lineage within the genus Paenibacillus and was most closely related to Paenibacillus contaminans CKOBP-6T (similarity, 95.2 %) and Paenibacillus terrigena A35T (similarity, 95.2 %). The levels of 16S rRNA gene sequence similarity with other species of the genus Paenibacillus, including the type species of the genus, Paenibacillus polymyxa IAM 13419T (similarity, 91.7 %), were all ,94.6 %. Strain CAU 1055T contained MK-7 as the only isoprenoid quinone and anteiso-C15 : 0 and iso-C16 : 0 as the major fatty acids. The cell-wall peptidoglycan of strain CAU 1055T contained meso-diaminopimelic acid. The polar lipids were composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, lysyl-phospatidylglycerol and three unidentified aminophospholipids. The DNA G+C content was 48.3 mol%. The results of physiological and biochemical tests allowed phenotypic differentiation of strain CAU 1055T from closely related recognized species. On the basis of phenotypic data and phylogenetic inference, strain CAU 1055T should be classified in the genus Paenibacillus, as a member of a novel species, for which the name Paenibacillus doosanensis sp. nov. is proposed. The type strain is CAU 1055T (5KCTC 33036T5CCUG 63270T).
The genus Paenibacillus, a member of the family Paenibacillaceae (De Vos et al. 2010) was proposed by Ash et al. (1993-1994) and accommodates members of ‘16S rRNA group 3’ bacilli. This genus now comprises more than 100 species isolated from various ecological niches (http:// www.bacterio.net/paenibacillus.html). The type species of the genus is Paenibacillus polymyxa (Ash et al. 1994). The genus Paenibacillus comprises aerobic, Gram-stainingpositive, motile, rod-shaped bacteria that are characterized chemotaxonomically by the presence of MK-7 as the major isoprenoid quinone, meso-diaminopimelic acid as the cellwall peptidoglycan type, and anteiso-C15 : 0 as the predominant cellular fatty acid (Shida et al. 1997a, b). Recently several species of the genus Paenibacillus have been isolated from various habitats including glacier (Paenibacillus glacialis; Kishore et al., 2010), wetland (Paenibacillus rigui; Baik et al., 2011), sandbank (Paenibacillus puldeungensis; Traiwan et al., 2011), a molybdenum mine (Paenibacillus phoenicis; Benardini et al., 2011), a cold spring (Paenibacillus algorifonticola; Tang et al., 2011), tidal flat (Paenibacillus sediminis; Wang et al., 2012), sediment of a hot spring (Paenibacillus thermophilus; Zhou et al., 2012), naked barley The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CAU 1055T is JX233493. Three supplementary figures and one supplementary table are available with the online version of this paper.
050716 G 2014 IUMS
Printed in Great Britain
(Paenibacillus hordei; Kim et al., 2013), marine sediment (Paenibacillus oceanisediminis; Lee et al., 2013), a necrotic wound (Paenibacillus vulneris; Glaeser et al., 2013), roots (Paenibacillus typhae; Kong et al., 2013) and rhizosphere soil (Paenibacillus catalpae; Zhang et al., 2013). In the course of the screening of bacteria with biotechnological potential from environmental samples, a bacterial strain, designated CAU 1055T, was isolated from a soil sample with a neutral pH collected on Jeju Island (33u 209 58.910 N 126u 299 40.100 E) in the Republic of Korea. The purpose of the present study was to establish the taxonomic position of this bacterial strain by using a polyphasic characterization that included the determination of phenotypic (including chemotaxonomic) properties, a detailed phylogenetic investigation based on 16S rRNA gene sequences and genetic analysis. Isolation was performed according to the protocol of Gordon & Mihm (1962) using nutrient agar (NA; Difco), supplemented with cycloheximide (50 mg l21) and nalidixic acid (20 mg l21). The crushed sample was diluted with sterile saline solution. Two dilution series were made of the sample; one series was plated directly on NA and the other was plated after heating for 15 min at 80 uC to select for endospores. The agar plates were incubated under aerobic conditions at 30 uC for 7 days. Pure single colonies were purified by subculturing. Strain CAU 1055T was one 1271
J.-H. Kim, H. Kang and W. Kim
of the isolates that appeared by direct plating on NA. Pure cultures were preserved at 270 uC in nutrient broth (NB; Difco) supplemented with 25 % (v/v) glycerol. The type strains of two closely related species and a strain representing the type species of the genus, P. polymyxa KCTC 1099, were used as reference strains in most analyses. Paenibacillus contaminans KCTC 13623T, Paenibacillus terrigena KCTC 13715T and P. polymyxa KCTC 1099 were obtained from the Korean Collection for Type Cultures (KCTC; Taejon, Korea). The taxonomic study of strain CAU 1055T followed the recommendations for the characterization and description of new taxa of aerobic, endospore-forming bacteria as recommended by Logan et al. (2009). Genomic DNA of strain CAU 1055T was isolated by the method of Marmur (1961). The 16S rRNA gene was amplified by PCR following established procedures (Nam et al., 2004). The amplified 16S rRNA gene was sequenced directly using a BigDye Terminator Cycle Sequencing kit (Applied Biosystems) and an automatic DNA sequencer (model 3730; Applied Biosystems). Multiple alignments with sequences of a broad selection of species of the genus Paenibacillus and calculation of sequence similarity levels were carried out by using the EzTaxon-e server (Kim et al., 2012; http://eztaxon-e.ezbiocloud.net/) and CLUSTAL X (Thompson et al., 1997). Evolutionary distance matrices were generated by the neighbour-joining method (Jukes & Cantor, 1969). Phylogenetic trees were reconstructed using the neighbour-joining (Saitou & Nei, 1987), least-squares (Fitch & Margoliash, 1967) and maximum-likelihood (Felsenstein, 1981) algorithms in the PHYLIP package (Felsenstein, 1989), and tree topology was evaluated by the bootstrap resampling method (Felsenstein, 1985) with 1000 replicates of the neighbour-joining dataset with the SEQBOOT and CONSENSE programs from the PHYLIP package.
0.1
Strain CAU 1055T was cultivated routinely on NA at 30 uC to investigate all morphological, physiological and biochemical characteristics, except for endospore formation that was assessed on nutrient sporulation medium (Nicholson & Setlow, 1990). Cell morphology was observed by light (DM 1000; Leica), phase-contrast (BX51; Olympus) and transmission electron (JEM 1010; JEOL) microscopy using cells from an exponentially growing culture. For transmission electron microscopy, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after being air-dried. Gram staining was carried out using the bioMe´rieux Gram staining kit according to the manufacturer’s instructions. Motility was assessed using the hanging-drop method. After 7 days of growth, endospore formation was determined by staining with malachite green as described by Conn et al. (1957).
T 80Paenibacillus cookii LMG 18419 (AJ250317) T 99 Paenibacillus chibensis JCM 9905 (AB073194) Paenibacillus cineris LMG 18439T (AJ575658)
Paenibacillus polymyxa IAM 13419T (D16276) Paenibacillus turicensis MOL722T (AF378694) Paenibacillus pini S22T (GQ423056) T 100 Paenibacillus alkaliterrae KSL-134 (AY960748) T (AY839867) Paenibacillus harenae B519 100 Paenibacillus thailandensis S3-4AT (AB265205) 88 Paenibacillus agaridevorans DSM 1355T (AJ345023) 88 Paenibacillus nanensis MX2-3T (AB265206) Paenibacillus contaminans CKOBP-6T (EF626690) 75 Paenibacillus doosanensis CAU 1055T (JX233493) Paenibacillus terrigena A35T (AB248087) T 70 Paenibacillus chitinolyticus NBRC15660 (AB021183) Paenibacillus gansuensis B518T (AY839866) 100 T 95 Paenibacillus rigui WPCB173 (EU939688) Paenibacillus chinjuensis WN9T (AF164345) 83 Paenibacillus elgii SD17T (AY090110) 100 Paenibacillus tianmuensis B27T rrnC (FJ719492) 100 Paenibacillus tianmuensis B27T rrnB (FJ719491) Paenibacillus larvae DSM 7030T (AY530294) Alicyclobacillus acidocaldarius ATCC 27009T (AB042056)
1272
The nearly complete 16S rRNA gene sequence of strain CAU 1055T (1519 bp) was determined and compared with the corresponding sequences of other bacterial strains in the GenBank database. Phylogenetic analysis indicated that the strain fell into the genus Paenibacillus. The neighbourjoining tree is shown in Fig. 1. The trees obtained with the two other treeing methods used showed essentially the same topology (Fig. 1). Pairwise analysis showed that the most closely related strains were P. contaminans CKOBP-6T (EF626690; 16S rRNA gene similarity, 95.2 %), P. terrigena A35T (AB248087; similarity, 95.2 %), Paenibacillus chinjuensis WN9T (AF164345; similarity, 94.6 %), Paenibacillus pini S22T (GQ423056; similarity, 94.5 %), Paenibacillus thailandensis S3-4AT (AB265205; similarity, 94.3 %) and P. rigui WPCB173T (EU939688; similarity, 94.2 %,). By contrast, 16S rRNA gene similarity between strain CAU 1055T and the type species of the genus Paenibacillus, P. polymyxa IAM 13419T was 91.7 %.
Fig. 1. Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences showing the relationships between strain CAU 1055T and the type strains of recognized species of the genus Paenibacillus. Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and least-squares algorithms. Numbers at nodes indicate levels of bootstrap support based on a neighbour-joining analysis of 1000 resampled datasets; only values .70 % are given. Bar, 0.1 substitutions per nucleotide position. Alicyclobacillus acidocaldarius ATCC 27009T (AB042056) is used as an outgroup organism. International Journal of Systematic and Evolutionary Microbiology 64
Paenibacillus doosanensis sp. nov.
Growth in NA at 4, 10, 20, 30, 37 and 45 uC in an aerobic incubator (MIR-253; Sanyo) and in an anaerobic chamber (Bactron; Sheldon) was evaluated by measuring the turbidity of the broth after 7 days. Growth was tested at 30 uC in NA adjusted to pH 4.5–10.0 in increments of 0.5 pH unit by using sodium acetate/acetic acid and Na2CO3 buffers. The pH was readjusted again after autoclaving. Growth in the absence of NaCl and in the presence of 0–15.0 % (w/v) NaCl at 1 % intervals was investigated at 30 uC in NA prepared according to the formula of the Difco medium except that NaCl was excluded and 0.45 % (w/v) MgCl2 . 6H2O and 0.06 % (w/v) KCl were added. Catalase, oxidase and urease activities, nitrate and nitrite reduction, hydrolysis of aesculin, production of indole, and Methyl red and Voges-Proskauer tests were determined as recommended by Smibert & Krieg (1994). Hydrolysis of casein, gelatin, starch, aesculin citrate and Tween 80 were examined as described by La´nyı´ (1987) and Smibert & Krieg (1994). Acid production from carbohydrates, as well as utilization of carbon and energy sources, was performed as recommended by Ventosa et al. (1982). Other physiological and biochemical properties were tested with API 20E and API 50CH kits (bioMe´rieux) according to the manufacturer’s instructions. The morphological, cultural, physiological and biochemical characteristics of strain CAU 1055T are given in Table 1 and in the species description. Overall, the results obtained in this study are in agreement with previously published data for species of the genus Paenibacillus (Ash et al., 1994; Shida et al., 1997a; Xie & Yokota 2007; Chou et al., 2009) and the type species of the genus, P. polymyxa IAM 13419T (Ka¨mpfer et al., 2006; Kim et al., 2013; Zhang et al., 2013). Strain CAU 1055T was found to consist of Gram-staining-positive, strictly aerobic, motile, rod-shaped cells. Subterminal ellipsoidal endospores were observed in swollen sporangia (Fig. S1, available in the online Supplementary Material). Cells are rods approximately 0.5–0.7 mm in diameter and 2.2–3.7 mm in length. The isolate was observed to be motile and transmission electron microscopy demonstrated the presence of peritrichous flagella (Fig. S2). Despite the fact that CAU 1055T was isolated from a non-saline environment, it was shown to tolerate NaCl concentrations of up to 4 % (w/v), although it did not require NaCl for growth. Strain CAU 1055T produced acids from amygdalin and Dglucose, hydrolysed aesculin, 2-nitrophenyl b-D-galactopyranoside and Tween 80, and utilized glycerol, D-ribose, D-galactose, D-glucose, methyl a-D-glucopyranoside, amygdalin, arbutin, maltose, lactose, melibiose, trehalose, inulin, raffinose and gentiobiose as sole carbon sources. However, strain CAU 1055T differed from its closest phylogenetic relatives, P. contaminans KCTC 13623T (Chou et al., 2009) and P. terrigena KCTC 13715T (Wang et al., 2012), and from a strain of the type species of the genus, P. polymyxa KCTC 1099 (Ash et al., 19931994, 1994), by its ability to utilize inulin and disability to utilize cellobiose and turanose, and its optimum pH range and optimum NaCl tolerance. http://ijs.sgmjournals.org
For fatty acid analysis, cell mass of strains CAU 1055T, P. contaminans KCTC 13623T, P. terrigena KCTC 13715T and P. polymyxa KCTC 1099 was harvested from tryptic soy agar (TSA; Difco) after cultivation for 3 days at 30 uC. The physiological age of the biomass harvested for fatty acid analysis was standardized by observing growth development during incubation of the three different cultures and choosing the moment of harvesting according to the standard MIDI protocol (Sherlock Microbial Identification System version 6.1). Cellular fatty acid methyl esters were obtained as described by Minnikin et al. (1980) and separated by an automated GC system (model 6890N and 7683 autosampler; Agilent). Peaks were identified by using the Microbial Identification software package (MOORE library version 5.0; MIDI database TSBA6). The polar lipids of strain CAU 1055T were identified using twodimensional TLC by the method of Minnikin et al. (1984). The plate was sprayed with 10 % ethanolic molybdatophosphoric acid (for the total lipids), molybdenum blue (for phospholipids), ninhydrin (for aminolipids), a-naphthol/ sulphuric acid reagent (for glycolipids) (Sigma-Aldrich) and 100 mg CuSO4 ml21 containing 8 % phosphoric acid (for lysyl-phosphatidylglycerol) as described by Oku et al. (2004). The following analyses were performed on strain CAU 1055T. Isoprenoid quinones were separated by HPLC using an isocratic solvent system [methanol/isopropyl ether (3 : 1, v/v)] and a flow rate of 1 ml min21 (Komagata & Suzuki, 1987). Peptidoglycan was analysed as described by Schleifer & Seidl (1985), with the modification that a cellulose sheet was substituted for chromatography paper. The mol% G+C content of the genomic DNA was determined using HPLC by the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. The peptidoglycan of strain CAU 1055T contained mesodiaminopimelic acid, and menaquinone 7 (MK-7) was the only respiratory quinone. These characteristics are in agreement with those of numerous species of the genus Paenibacillus, including the type species of the genus, P. polymyxa (Ka¨mpfer et al., 2006). The fatty acid profile was very similar to those of type strains of species of the genus Paenibacillus. The strain contained saturated, unsaturated and branched-chain fatty acids (Table S1). The major fatty acids were anteiso-C15 : 0 (52.5 %) and iso-C16 : 0 (16.1 %), which are characteristic of numerous taxa within the paenibacilli (Ka¨mpfer, 1994). The following fatty acids were present to at least 1 %: iso-C15 : 0, anteiso-C17 : 0, isoC14 : 0, C16 : 0, iso-C17 : 0, C16 : 1v7c alcohol, C16 : 1v11c and iso-C17 : 1v10c. However, some qualitative differences in fatty acid content could be observed between strain CAU 1055T and its phylogenetically closest relatives. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and lysyl-phosphatidylglycerol were the polar lipids identified in strain CAU 1055T. The other unidentified polar lipids were three aminophospholipids. (Fig. S3). The polar lipid profile of strain CAU 1055T 1273
J.-H. Kim, H. Kang and W. Kim
Table 1. Differential properties of strain CAU 1055T and strains of the most closely related species of the genus Paenibacillus and the type species of the genus Paenibacillus Strains: 1, CAU 1055T; 2, P. contaminans KCTC 13623T; 3, P. terrigena KCTC 13715T; 4, P. polymyxa KCTC 1099. Data were obtained in this study unless indicated. +, Positive; 2, negative; V, variable; ND, not determined. Characteristic Source Gram reaction Anaerobic growth Spore position Temperature (uC) Range Optimum pH Range Optimum NaCl tolerance (%) Maximum Optimum Catalase Nitrate reduction Hydrolysis of: Casein Gelatin Starch Urea 2-Nitrophenyl b-D-galactopyranoside Tween 80 Acid production from: Sucrose Amygdalin Utilization of: Glycerol D-Ribose D-Xylose Methyl b-D-xylopyranoside D-Galactose D-Fructose D-Mannose Inositol D-Sorbitol Methyl a-D-mannopyranoside Methyl a-D-glucopyranoside N-Acetylglucosamine Amygdalin Salicin Cellobiose Maltose Lactose Melibiose Sucrose Trehalose Inulin Raffinose Glycogen Xylitol Gentiobiose
1274
1
2
3 a
4 b
Soild +c +b Subterminale
Soil + 2 Subterminal
Contaminated laboratory plate *
20–37 30
10–37a 30a
4–32b 29b
10-40e 29e
4.5–7.5 6.5
6.5–8.0a 7a
4.0–10.0b ND
4.5–6.8e 7e
4.0 2 + 2
2.0a 0.5a +a 2
4.0b 3.5b +b +
3.0e 0.5e 2b +
2 2 2 2 + +
2a 2 2 2 2 +a
+a 2 + 2 + ND
2e + + + 2 +f
2 +
2 2
2 +
+ +
+ + 2 2 + 2 2 2 2 2 + 2 + 2 2 + + + 2 + + + 2 2 +
+ + 2 + 2 2 2 2 2 2 + 2 2 + + + 2 + + + 2 2 2 2 +
+ + + + + + + 2 2 + + + + + + + + + + + 2 + + 2 +
2 2 2 2 2 + + + + 2 2 2 + 2 + 2 2 2 2 2 2 2 2 + 2
a
V
+a ND
Coastal soil +b +a Terminalb
International Journal of Systematic and Evolutionary Microbiology 64
Paenibacillus doosanensis sp. nov.
Table 1. cont. Characteristic Turanose DNA G+C content (mol%)
1
2
3
4
2 48.3
+ 51.2a
+ 48.1b
+ 43–46b
*Data taken from: a, Chou et al. (2009); b, Xie & Yokota (2007); c, Kim et al. (2010); d, Shida et al. (1997a); e, Zhang et al. (2013); f, Kim et al. (2013).
shared the major compounds diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol with its closest relatives, P. contaminans KCTC 13623T and P. terrigena KCTC 13715T, and a strain representing the type species of the genus, P. polymyxa KCTC 1099. However, the presence of lysyl-phosphatidylglycerol and three aminophospholipids is clearly different from the polar lipid profiles observed in the type strains of two closely related species and the type species of the genus Paenibacillus. The genomic DNA of strain CAU 1055T had a G+C content of 48.3 mol%. These data provide sufficient evidence to support the proposal to recognize strain CAU 1055T as a representative of a novel species, as recommended by Logan et al. (2009). The strain should be assigned to a novel species of the genus Paenibacillus, for which the name Paenibacillus doosanensis sp. nov. is proposed. Description of Paenibacillus doosanensis sp. nov. Paenibacillus doosanensis sp. nov. (doo.san.en9sis. N.L. masc. adj. doosanensis belonging to Doosan, named after the Doosan group, the Foundation of Chung-Ang University where the taxonomic studies on this species were performed). Cells are Gram-staining-positive, motile, strictly aerobic rods approximately 0.5–0.7 mm in diameter and 2.2– 3.7 mm in length. Subterminal ellipsoidal endospores are observed in the swollen sporangia. Colonies on NA are cream-coloured, circular and convex with entire margins after 3 days of incubation at 30 uC. Growth occurs at 4–45 uC (optimum, 30 uC) and at pH 4.5–7.5 (optimum, pH 6.5). NaCl is not required for growth but up to 4.0 % (w/v) NaCl is tolerated. Catalase and oxidase are positive. Aesculin and Tween 80 are hydrolysed but casein, gelatin, starch and urea are not hydrolysed. Nitrate is not reduced. Citrate is not utilized. Indole and H2S are not produced. bGalactosidase and Voges–Proskauer tests are positive but methyl red, urease, lysine and ornithine decarboxylases, and arginine dihydrolase tests are negative. Acid production from amygdalin and D-glucose are positive but Dmannitol, inositol, D-sorbitol, L-rhamnose, sucrose, melibiose and L-arabinose are negative. The following carbohydrates are utilized as sole carbon source: glycerol, D-ribose, D-galactose, D-glucose, methyl a-D-glucopyranoside, amygdalin, arbutin, maltose, lactose, melibiose, trehalose, inulin, raffinose and gentiobiose. The following compounds are http://ijs.sgmjournals.org
not utilized: erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, D-fructose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, Dmannitol, D-sorbitol, methyl a-D-mannopyranoside, Nacetylglucosamine, salicin, cellobiose, sucrose, melezitose, glycogen, xylitol, turanose, D-lyxose, D-tagatose, D-fucose, Lfucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketagluconate. The cell-wall peptidoglycan contains meso-diaminopimelic acid. The only isoprenoid quinone is MK-7. The whole-cell hydrolysate contains glucose and ribose. The polar lipid pattern consists of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, lysyl-phosphatidylglycerol and three unidentified aminophospholipids. The predominant cellular fatty acids are anteiso-C15 : 0 and iso-C16 : 0. The type strain is CAU 1055T (5KCTC 33036T5CCUG 63270T), isolated from soil collected from Jeju Island in the Republic of Korea. The DNA G+C content of the type strain is 48.3 mol%.
Acknowledgements This research was supported by the Biomedical Science Scholarship Grants, Department of Medicine, Chung-Ang University, in 2012.
References Ash, C., Priest, F. G. & Collins, M. D. (1993-1994). Molecular
identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test. Antonie van Leeuwenhoek 64, 253– 260. Ash, C., Priest, F. G. & Collins, M. D. (1994). Paenibacillus gen. nov.
and Paenibacillus polymyxa comb. nov. In Validation of the Publication of New Names and New Combinations Previously Effectively Published Outside the IJSB, List no. 51. Int J Syst Bacteriol 44, 852. Baik, K. S., Lim, C. H., Choe, H. N., Kim, E. M. & Seong, C. N. (2011).
Paenibacillus rigui sp. nov., isolated from a freshwater wetland. Int J Syst Evol Microbiol 61, 529–534. Benardini, J. N., Vaishampayan, P. A., Schwendner, P., Swanner, E., Fukui, Y., Osman, S., Satomi, M. & Venkateswaran, K. (2011).
Paenibacillus phoenicis sp. nov., isolated from the Phoenix Lander assembly facility and a subsurface molybdenum mine. Int J Syst Evol Microbiol 61, 1338–1343. Chou, J. H., Lee, J. H., Lin, M. C., Chang, P. S., Arun, A. B., Young, C. C. & Chen, W.-M. (2009). Paenibacillus contaminans sp. nov., isolated
from a contaminated laboratory plate. Int J Syst Evol Microbiol 59, 125–129. 1275
J.-H. Kim, H. Kang and W. Kim Conn, H. J., Bartholomew, J. W. & Jennison, M. W. (1957). Staining
methods. In Manual of Microbiological Methods, pp. 10–36. Edited by Society of American Bacteriologists. New York: McGraw-Hill.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
De Vos, P., Ludwig, W., Schleifer, K.-H. & Whitman, W. B. (2010).
Minnikin, D. E., Hutchinson, I. G., Caldicott, A. B. & Goodfellow, M. (1980). Thin-layer chromatography of methanolysates of mycolic
Paenibacillaceae fam. nov. In List of New Names and New Combinations Previously Effectively, but not Validly, Published, Validation List no. 132. Int J Syst Evol Microbiol 60, 469–472.
Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.
procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach
Nam, S. W., Kim, W., Chun, J. & Goodfellow, M. (2004). Tsukamurella
using the bootstrap. Evolution 39, 783–791. Felsenstein, J. (1989). PHYLIP – phylogeny inference package (version
pseudospumae sp. nov., a novel actinomycete isolated from activated sludge foam. Int J Syst Evol Microbiol 54, 1209–1212.
3.2). Cladistics 5, 164–166.
Nicholson, W. L. & Setlow, P. (1990). Sporulation, germination
Fitch, W. M. & Margoliash, E. (1967). Construction of phylogenetic
and outgrowth. In Molecular Biological Methods for Bacillus, pp. 391–450. Edited by C. R. Harwood & S. M. Cutting. Chichester: Wiley.
trees. Science 155, 279–284. Glaeser, S. P., Falsen, E., Busse, H. J. & Ka¨mpfer, P. (2013).
Paenibacillus vulneris sp. nov., isolated from a necrotic wound. Int J Syst Evol Microbiol 63, 777–782. Gordon, R. E. & Mihm, J. M. (1962). Identification of Nocardia caviae
(Erikson) nov. comb. Ann N Y Acad Sci 98, 628–636. Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In
Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by H. H. Munro. New York: Academic Press. Ka¨mpfer, P. (1994). Limits and possibilities of total fatty acid analysis
for classification and identification of Bacillus species. Syst Appl Microbiol 17, 86–98. Ka¨mpfer, P., Rossello´-Mora, R., Falsen, E., Busse, H.-J. & Tindall, B. J. (2006). Cohnella thermotolerans gen. nov., sp. nov., and classification
of ‘Paenibacillus hongkongensis’ as Cohnella hongkongensis sp. nov. Int J Syst Evol Microbiol 56, 781–786. Kim, J. F., Jeong, H., Park, S. Y., Kim, S. B., Park, Y. K., Choi, S. K., Ryu, C. M., Hur, C. G., Ghim, S. Y. & other authors (2010). Genome
sequence of the polymyxin-producing plant-probiotic rhizobacterium Paenibacillus polymyxa E681. J Bacteriol 192, 6103–6104. 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. M., Lee, S. H., Lee, S. H., Choi, E. J. & Jeon, C. O. (2013).
Paenibacillus hordei sp. nov., isolated from naked barley in Korea. Antonie van Leeuwenhoek 103, 3–9.
acid-containing bacteria. J Chromatogr A 188, 221–233.
Oku, Y., Kurokawa, K., Ichihashi, N. & Sekimizu, K. (2004).
Characterization of the Staphylococcus aureus mprF gene, involved in lysinylation of phosphatidylglycerol. Microbiology 150, 45–51. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406– 425. Schleifer, K. H. & Seidl, P. H. (1985). Chemical composition and
structure of murein. In Chemical Methods in Bacterial Systematics, pp. 201–219. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press. Shida, O., Takagi, H., Kadowaki, K., Nakamura, L. K. & Komagata, K. (1997a). Transfer of Bacillus alginolyticus, Bacillus chondroitinus,
Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus to the genus Paenibacillus and emended description of the genus Paenibacillus. Int J Syst Bacteriol 47, 289– 298. Shida, O., Takagi, H., Kadowaki, K., Nakamura, L. K. & Komagata, K. (1997b). Emended description of Paenibacillus amylolyticus and
description of Paenibacillus illinoisensis sp. nov. and Paenibacillus chibensis sp. nov. Int J Syst Bacteriol 47, 299–306. Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In
Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology. Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
Kishore, K. H., Begum, Z., Pathan, A. A. K. & Shivaji, S. (2010).
composition by reverse-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Paenibacillus glacialis sp. nov., isolated from the Kafni glacier of the Himalayas, India. Int J Syst Evol Microbiol 60, 1909–1913.
Tang, Q. Y., Yang, N., Wang, J., Xie, Y. Q., Ren, B., Zhou, Y. G., Gu, M. Y., Mao, J., Li, W. J. & other authors (2011). Paenibacillus
Komagata, K. & Suzuki, K. (1987). Lipid and cell wall analysis in
bacterial systematics. Methods Microbiol 19, 161–207.
algorifonticola sp. nov., isolated from a cold spring. Int J Syst Evol Microbiol 61, 2167–2172.
Kong, B. H., Liu, Q. F., Liu, M., Liu, Y., Liu, L., Li, C. L., Yu, R. & Li, Y. H. (2013). Paenibacillus typhae sp. nov., isolated from roots of Typha
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
angustifolia L. Int J Syst Evol Microbiol 63, 1037–1044.
strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
La´nyı´, B. (1987). Classical and rapid identification methods for
medically important bacteria. Methods Microbiol 19, 1–67. Lee, J., Shin, N. R., Jung, M. J., Roh, S. W., Kim, M. S., Lee, J. S., Lee, K. C., Kim, Y. O. & Bae, J. W. (2013). Paenibacillus oceanisediminis sp.
Traiwan, J., Park, M. H. & Kim, W. (2011). Paenibacillus puldeungensis sp. nov., isolated from a grassy sandbank. Int J Syst Evol Microbiol 61, 670–673.
nov. isolated from marine sediment. Int J Syst Evol Microbiol 63, 428– 434.
Ventosa, A., Quesada, E., Rodrı´guez-Valera, F., Ruiz-Berraquero, F. & Ramos-Cormenzana, A. (1982). Numerical taxonomy of moder-
Logan, N. A., Berge, O., Bishop, A. H., Busse, H.-J., De Vos, P., Fritze, D., Heyndrickx, M., Ka¨mpfer, P., Rabinovitch, L. & other authors (2009). Proposed minimal standards for describing new taxa of
ately halophilic Gram-negative rods. J Gen Microbiol 128, 1959– 1968.
aerobic, endospore-forming bacteria. Int J Syst Evol Microbiol 59, 2114–2121.
Paenibacillus sediminis sp. nov., a xylanolytic bacterium isolated from a tidal flat. Int J Syst Evol Microbiol 62, 1284–1288.
1276
International Journal of Systematic and Evolutionary Microbiology 64
Wang, L., Baek, S.-H., Cui, Y., Lee, H.-G. & Lee, S.-T. (2012).
Paenibacillus doosanensis sp. nov. Xie, C. H. & Yokota, A. (2007). Paenibacillus terrigena sp. nov., isolated
from soil. Int J Syst Evol Microbiol 57, 70–72. Zhang, J., Wang, Z. T., Yu, H. M. & Ma, Y. (2013). Paenibacillus catalpae
sp. nov., isolated from the rhizosphere soil of Catalpa speciosa. Int J Syst Evol Microbiol 63, 1776–1781.
http://ijs.sgmjournals.org
Zhou, Y., Gao, S., Wei, D. Q., Yang, L. L., Huang, X., He, J., Zhang, Y. J., Tang, S. K. & Li, W. J. (2012). Paenibacillus thermophilus
sp. nov., a novel bacterium isolated from a sediment of hot spring in Fujian province, China. Antonie van Leeuwenhoek 102, 601– 609.
1277
DOI 10.1099/ijs.0.050716-0
Paenibacillus doosanensis sp. nov., isolated from soil Jong-Hwa Kim, Hyeonji Kang and Wonyong Kim Correspondence
Department of Microbiology, Chung-Ang University College of Medicine, Seoul, Republic of Korea.
Wonyong Kim [email protected]
A Gram-stain-positive, aerobic, endospore-forming bacterium, designated CAU 1055T, was isolated from soil and its taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that the strain formed a distinct lineage within the genus Paenibacillus and was most closely related to Paenibacillus contaminans CKOBP-6T (similarity, 95.2 %) and Paenibacillus terrigena A35T (similarity, 95.2 %). The levels of 16S rRNA gene sequence similarity with other species of the genus Paenibacillus, including the type species of the genus, Paenibacillus polymyxa IAM 13419T (similarity, 91.7 %), were all ,94.6 %. Strain CAU 1055T contained MK-7 as the only isoprenoid quinone and anteiso-C15 : 0 and iso-C16 : 0 as the major fatty acids. The cell-wall peptidoglycan of strain CAU 1055T contained meso-diaminopimelic acid. The polar lipids were composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, lysyl-phospatidylglycerol and three unidentified aminophospholipids. The DNA G+C content was 48.3 mol%. The results of physiological and biochemical tests allowed phenotypic differentiation of strain CAU 1055T from closely related recognized species. On the basis of phenotypic data and phylogenetic inference, strain CAU 1055T should be classified in the genus Paenibacillus, as a member of a novel species, for which the name Paenibacillus doosanensis sp. nov. is proposed. The type strain is CAU 1055T (5KCTC 33036T5CCUG 63270T).
The genus Paenibacillus, a member of the family Paenibacillaceae (De Vos et al. 2010) was proposed by Ash et al. (1993-1994) and accommodates members of ‘16S rRNA group 3’ bacilli. This genus now comprises more than 100 species isolated from various ecological niches (http:// www.bacterio.net/paenibacillus.html). The type species of the genus is Paenibacillus polymyxa (Ash et al. 1994). The genus Paenibacillus comprises aerobic, Gram-stainingpositive, motile, rod-shaped bacteria that are characterized chemotaxonomically by the presence of MK-7 as the major isoprenoid quinone, meso-diaminopimelic acid as the cellwall peptidoglycan type, and anteiso-C15 : 0 as the predominant cellular fatty acid (Shida et al. 1997a, b). Recently several species of the genus Paenibacillus have been isolated from various habitats including glacier (Paenibacillus glacialis; Kishore et al., 2010), wetland (Paenibacillus rigui; Baik et al., 2011), sandbank (Paenibacillus puldeungensis; Traiwan et al., 2011), a molybdenum mine (Paenibacillus phoenicis; Benardini et al., 2011), a cold spring (Paenibacillus algorifonticola; Tang et al., 2011), tidal flat (Paenibacillus sediminis; Wang et al., 2012), sediment of a hot spring (Paenibacillus thermophilus; Zhou et al., 2012), naked barley The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CAU 1055T is JX233493. Three supplementary figures and one supplementary table are available with the online version of this paper.
050716 G 2014 IUMS
Printed in Great Britain
(Paenibacillus hordei; Kim et al., 2013), marine sediment (Paenibacillus oceanisediminis; Lee et al., 2013), a necrotic wound (Paenibacillus vulneris; Glaeser et al., 2013), roots (Paenibacillus typhae; Kong et al., 2013) and rhizosphere soil (Paenibacillus catalpae; Zhang et al., 2013). In the course of the screening of bacteria with biotechnological potential from environmental samples, a bacterial strain, designated CAU 1055T, was isolated from a soil sample with a neutral pH collected on Jeju Island (33u 209 58.910 N 126u 299 40.100 E) in the Republic of Korea. The purpose of the present study was to establish the taxonomic position of this bacterial strain by using a polyphasic characterization that included the determination of phenotypic (including chemotaxonomic) properties, a detailed phylogenetic investigation based on 16S rRNA gene sequences and genetic analysis. Isolation was performed according to the protocol of Gordon & Mihm (1962) using nutrient agar (NA; Difco), supplemented with cycloheximide (50 mg l21) and nalidixic acid (20 mg l21). The crushed sample was diluted with sterile saline solution. Two dilution series were made of the sample; one series was plated directly on NA and the other was plated after heating for 15 min at 80 uC to select for endospores. The agar plates were incubated under aerobic conditions at 30 uC for 7 days. Pure single colonies were purified by subculturing. Strain CAU 1055T was one 1271
J.-H. Kim, H. Kang and W. Kim
of the isolates that appeared by direct plating on NA. Pure cultures were preserved at 270 uC in nutrient broth (NB; Difco) supplemented with 25 % (v/v) glycerol. The type strains of two closely related species and a strain representing the type species of the genus, P. polymyxa KCTC 1099, were used as reference strains in most analyses. Paenibacillus contaminans KCTC 13623T, Paenibacillus terrigena KCTC 13715T and P. polymyxa KCTC 1099 were obtained from the Korean Collection for Type Cultures (KCTC; Taejon, Korea). The taxonomic study of strain CAU 1055T followed the recommendations for the characterization and description of new taxa of aerobic, endospore-forming bacteria as recommended by Logan et al. (2009). Genomic DNA of strain CAU 1055T was isolated by the method of Marmur (1961). The 16S rRNA gene was amplified by PCR following established procedures (Nam et al., 2004). The amplified 16S rRNA gene was sequenced directly using a BigDye Terminator Cycle Sequencing kit (Applied Biosystems) and an automatic DNA sequencer (model 3730; Applied Biosystems). Multiple alignments with sequences of a broad selection of species of the genus Paenibacillus and calculation of sequence similarity levels were carried out by using the EzTaxon-e server (Kim et al., 2012; http://eztaxon-e.ezbiocloud.net/) and CLUSTAL X (Thompson et al., 1997). Evolutionary distance matrices were generated by the neighbour-joining method (Jukes & Cantor, 1969). Phylogenetic trees were reconstructed using the neighbour-joining (Saitou & Nei, 1987), least-squares (Fitch & Margoliash, 1967) and maximum-likelihood (Felsenstein, 1981) algorithms in the PHYLIP package (Felsenstein, 1989), and tree topology was evaluated by the bootstrap resampling method (Felsenstein, 1985) with 1000 replicates of the neighbour-joining dataset with the SEQBOOT and CONSENSE programs from the PHYLIP package.
0.1
Strain CAU 1055T was cultivated routinely on NA at 30 uC to investigate all morphological, physiological and biochemical characteristics, except for endospore formation that was assessed on nutrient sporulation medium (Nicholson & Setlow, 1990). Cell morphology was observed by light (DM 1000; Leica), phase-contrast (BX51; Olympus) and transmission electron (JEM 1010; JEOL) microscopy using cells from an exponentially growing culture. For transmission electron microscopy, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after being air-dried. Gram staining was carried out using the bioMe´rieux Gram staining kit according to the manufacturer’s instructions. Motility was assessed using the hanging-drop method. After 7 days of growth, endospore formation was determined by staining with malachite green as described by Conn et al. (1957).
T 80Paenibacillus cookii LMG 18419 (AJ250317) T 99 Paenibacillus chibensis JCM 9905 (AB073194) Paenibacillus cineris LMG 18439T (AJ575658)
Paenibacillus polymyxa IAM 13419T (D16276) Paenibacillus turicensis MOL722T (AF378694) Paenibacillus pini S22T (GQ423056) T 100 Paenibacillus alkaliterrae KSL-134 (AY960748) T (AY839867) Paenibacillus harenae B519 100 Paenibacillus thailandensis S3-4AT (AB265205) 88 Paenibacillus agaridevorans DSM 1355T (AJ345023) 88 Paenibacillus nanensis MX2-3T (AB265206) Paenibacillus contaminans CKOBP-6T (EF626690) 75 Paenibacillus doosanensis CAU 1055T (JX233493) Paenibacillus terrigena A35T (AB248087) T 70 Paenibacillus chitinolyticus NBRC15660 (AB021183) Paenibacillus gansuensis B518T (AY839866) 100 T 95 Paenibacillus rigui WPCB173 (EU939688) Paenibacillus chinjuensis WN9T (AF164345) 83 Paenibacillus elgii SD17T (AY090110) 100 Paenibacillus tianmuensis B27T rrnC (FJ719492) 100 Paenibacillus tianmuensis B27T rrnB (FJ719491) Paenibacillus larvae DSM 7030T (AY530294) Alicyclobacillus acidocaldarius ATCC 27009T (AB042056)
1272
The nearly complete 16S rRNA gene sequence of strain CAU 1055T (1519 bp) was determined and compared with the corresponding sequences of other bacterial strains in the GenBank database. Phylogenetic analysis indicated that the strain fell into the genus Paenibacillus. The neighbourjoining tree is shown in Fig. 1. The trees obtained with the two other treeing methods used showed essentially the same topology (Fig. 1). Pairwise analysis showed that the most closely related strains were P. contaminans CKOBP-6T (EF626690; 16S rRNA gene similarity, 95.2 %), P. terrigena A35T (AB248087; similarity, 95.2 %), Paenibacillus chinjuensis WN9T (AF164345; similarity, 94.6 %), Paenibacillus pini S22T (GQ423056; similarity, 94.5 %), Paenibacillus thailandensis S3-4AT (AB265205; similarity, 94.3 %) and P. rigui WPCB173T (EU939688; similarity, 94.2 %,). By contrast, 16S rRNA gene similarity between strain CAU 1055T and the type species of the genus Paenibacillus, P. polymyxa IAM 13419T was 91.7 %.
Fig. 1. Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences showing the relationships between strain CAU 1055T and the type strains of recognized species of the genus Paenibacillus. Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and least-squares algorithms. Numbers at nodes indicate levels of bootstrap support based on a neighbour-joining analysis of 1000 resampled datasets; only values .70 % are given. Bar, 0.1 substitutions per nucleotide position. Alicyclobacillus acidocaldarius ATCC 27009T (AB042056) is used as an outgroup organism. International Journal of Systematic and Evolutionary Microbiology 64
Paenibacillus doosanensis sp. nov.
Growth in NA at 4, 10, 20, 30, 37 and 45 uC in an aerobic incubator (MIR-253; Sanyo) and in an anaerobic chamber (Bactron; Sheldon) was evaluated by measuring the turbidity of the broth after 7 days. Growth was tested at 30 uC in NA adjusted to pH 4.5–10.0 in increments of 0.5 pH unit by using sodium acetate/acetic acid and Na2CO3 buffers. The pH was readjusted again after autoclaving. Growth in the absence of NaCl and in the presence of 0–15.0 % (w/v) NaCl at 1 % intervals was investigated at 30 uC in NA prepared according to the formula of the Difco medium except that NaCl was excluded and 0.45 % (w/v) MgCl2 . 6H2O and 0.06 % (w/v) KCl were added. Catalase, oxidase and urease activities, nitrate and nitrite reduction, hydrolysis of aesculin, production of indole, and Methyl red and Voges-Proskauer tests were determined as recommended by Smibert & Krieg (1994). Hydrolysis of casein, gelatin, starch, aesculin citrate and Tween 80 were examined as described by La´nyı´ (1987) and Smibert & Krieg (1994). Acid production from carbohydrates, as well as utilization of carbon and energy sources, was performed as recommended by Ventosa et al. (1982). Other physiological and biochemical properties were tested with API 20E and API 50CH kits (bioMe´rieux) according to the manufacturer’s instructions. The morphological, cultural, physiological and biochemical characteristics of strain CAU 1055T are given in Table 1 and in the species description. Overall, the results obtained in this study are in agreement with previously published data for species of the genus Paenibacillus (Ash et al., 1994; Shida et al., 1997a; Xie & Yokota 2007; Chou et al., 2009) and the type species of the genus, P. polymyxa IAM 13419T (Ka¨mpfer et al., 2006; Kim et al., 2013; Zhang et al., 2013). Strain CAU 1055T was found to consist of Gram-staining-positive, strictly aerobic, motile, rod-shaped cells. Subterminal ellipsoidal endospores were observed in swollen sporangia (Fig. S1, available in the online Supplementary Material). Cells are rods approximately 0.5–0.7 mm in diameter and 2.2–3.7 mm in length. The isolate was observed to be motile and transmission electron microscopy demonstrated the presence of peritrichous flagella (Fig. S2). Despite the fact that CAU 1055T was isolated from a non-saline environment, it was shown to tolerate NaCl concentrations of up to 4 % (w/v), although it did not require NaCl for growth. Strain CAU 1055T produced acids from amygdalin and Dglucose, hydrolysed aesculin, 2-nitrophenyl b-D-galactopyranoside and Tween 80, and utilized glycerol, D-ribose, D-galactose, D-glucose, methyl a-D-glucopyranoside, amygdalin, arbutin, maltose, lactose, melibiose, trehalose, inulin, raffinose and gentiobiose as sole carbon sources. However, strain CAU 1055T differed from its closest phylogenetic relatives, P. contaminans KCTC 13623T (Chou et al., 2009) and P. terrigena KCTC 13715T (Wang et al., 2012), and from a strain of the type species of the genus, P. polymyxa KCTC 1099 (Ash et al., 19931994, 1994), by its ability to utilize inulin and disability to utilize cellobiose and turanose, and its optimum pH range and optimum NaCl tolerance. http://ijs.sgmjournals.org
For fatty acid analysis, cell mass of strains CAU 1055T, P. contaminans KCTC 13623T, P. terrigena KCTC 13715T and P. polymyxa KCTC 1099 was harvested from tryptic soy agar (TSA; Difco) after cultivation for 3 days at 30 uC. The physiological age of the biomass harvested for fatty acid analysis was standardized by observing growth development during incubation of the three different cultures and choosing the moment of harvesting according to the standard MIDI protocol (Sherlock Microbial Identification System version 6.1). Cellular fatty acid methyl esters were obtained as described by Minnikin et al. (1980) and separated by an automated GC system (model 6890N and 7683 autosampler; Agilent). Peaks were identified by using the Microbial Identification software package (MOORE library version 5.0; MIDI database TSBA6). The polar lipids of strain CAU 1055T were identified using twodimensional TLC by the method of Minnikin et al. (1984). The plate was sprayed with 10 % ethanolic molybdatophosphoric acid (for the total lipids), molybdenum blue (for phospholipids), ninhydrin (for aminolipids), a-naphthol/ sulphuric acid reagent (for glycolipids) (Sigma-Aldrich) and 100 mg CuSO4 ml21 containing 8 % phosphoric acid (for lysyl-phosphatidylglycerol) as described by Oku et al. (2004). The following analyses were performed on strain CAU 1055T. Isoprenoid quinones were separated by HPLC using an isocratic solvent system [methanol/isopropyl ether (3 : 1, v/v)] and a flow rate of 1 ml min21 (Komagata & Suzuki, 1987). Peptidoglycan was analysed as described by Schleifer & Seidl (1985), with the modification that a cellulose sheet was substituted for chromatography paper. The mol% G+C content of the genomic DNA was determined using HPLC by the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. The peptidoglycan of strain CAU 1055T contained mesodiaminopimelic acid, and menaquinone 7 (MK-7) was the only respiratory quinone. These characteristics are in agreement with those of numerous species of the genus Paenibacillus, including the type species of the genus, P. polymyxa (Ka¨mpfer et al., 2006). The fatty acid profile was very similar to those of type strains of species of the genus Paenibacillus. The strain contained saturated, unsaturated and branched-chain fatty acids (Table S1). The major fatty acids were anteiso-C15 : 0 (52.5 %) and iso-C16 : 0 (16.1 %), which are characteristic of numerous taxa within the paenibacilli (Ka¨mpfer, 1994). The following fatty acids were present to at least 1 %: iso-C15 : 0, anteiso-C17 : 0, isoC14 : 0, C16 : 0, iso-C17 : 0, C16 : 1v7c alcohol, C16 : 1v11c and iso-C17 : 1v10c. However, some qualitative differences in fatty acid content could be observed between strain CAU 1055T and its phylogenetically closest relatives. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and lysyl-phosphatidylglycerol were the polar lipids identified in strain CAU 1055T. The other unidentified polar lipids were three aminophospholipids. (Fig. S3). The polar lipid profile of strain CAU 1055T 1273
J.-H. Kim, H. Kang and W. Kim
Table 1. Differential properties of strain CAU 1055T and strains of the most closely related species of the genus Paenibacillus and the type species of the genus Paenibacillus Strains: 1, CAU 1055T; 2, P. contaminans KCTC 13623T; 3, P. terrigena KCTC 13715T; 4, P. polymyxa KCTC 1099. Data were obtained in this study unless indicated. +, Positive; 2, negative; V, variable; ND, not determined. Characteristic Source Gram reaction Anaerobic growth Spore position Temperature (uC) Range Optimum pH Range Optimum NaCl tolerance (%) Maximum Optimum Catalase Nitrate reduction Hydrolysis of: Casein Gelatin Starch Urea 2-Nitrophenyl b-D-galactopyranoside Tween 80 Acid production from: Sucrose Amygdalin Utilization of: Glycerol D-Ribose D-Xylose Methyl b-D-xylopyranoside D-Galactose D-Fructose D-Mannose Inositol D-Sorbitol Methyl a-D-mannopyranoside Methyl a-D-glucopyranoside N-Acetylglucosamine Amygdalin Salicin Cellobiose Maltose Lactose Melibiose Sucrose Trehalose Inulin Raffinose Glycogen Xylitol Gentiobiose
1274
1
2
3 a
4 b
Soild +c +b Subterminale
Soil + 2 Subterminal
Contaminated laboratory plate *
20–37 30
10–37a 30a
4–32b 29b
10-40e 29e
4.5–7.5 6.5
6.5–8.0a 7a
4.0–10.0b ND
4.5–6.8e 7e
4.0 2 + 2
2.0a 0.5a +a 2
4.0b 3.5b +b +
3.0e 0.5e 2b +
2 2 2 2 + +
2a 2 2 2 2 +a
+a 2 + 2 + ND
2e + + + 2 +f
2 +
2 2
2 +
+ +
+ + 2 2 + 2 2 2 2 2 + 2 + 2 2 + + + 2 + + + 2 2 +
+ + 2 + 2 2 2 2 2 2 + 2 2 + + + 2 + + + 2 2 2 2 +
+ + + + + + + 2 2 + + + + + + + + + + + 2 + + 2 +
2 2 2 2 2 + + + + 2 2 2 + 2 + 2 2 2 2 2 2 2 2 + 2
a
V
+a ND
Coastal soil +b +a Terminalb
International Journal of Systematic and Evolutionary Microbiology 64
Paenibacillus doosanensis sp. nov.
Table 1. cont. Characteristic Turanose DNA G+C content (mol%)
1
2
3
4
2 48.3
+ 51.2a
+ 48.1b
+ 43–46b
*Data taken from: a, Chou et al. (2009); b, Xie & Yokota (2007); c, Kim et al. (2010); d, Shida et al. (1997a); e, Zhang et al. (2013); f, Kim et al. (2013).
shared the major compounds diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol with its closest relatives, P. contaminans KCTC 13623T and P. terrigena KCTC 13715T, and a strain representing the type species of the genus, P. polymyxa KCTC 1099. However, the presence of lysyl-phosphatidylglycerol and three aminophospholipids is clearly different from the polar lipid profiles observed in the type strains of two closely related species and the type species of the genus Paenibacillus. The genomic DNA of strain CAU 1055T had a G+C content of 48.3 mol%. These data provide sufficient evidence to support the proposal to recognize strain CAU 1055T as a representative of a novel species, as recommended by Logan et al. (2009). The strain should be assigned to a novel species of the genus Paenibacillus, for which the name Paenibacillus doosanensis sp. nov. is proposed. Description of Paenibacillus doosanensis sp. nov. Paenibacillus doosanensis sp. nov. (doo.san.en9sis. N.L. masc. adj. doosanensis belonging to Doosan, named after the Doosan group, the Foundation of Chung-Ang University where the taxonomic studies on this species were performed). Cells are Gram-staining-positive, motile, strictly aerobic rods approximately 0.5–0.7 mm in diameter and 2.2– 3.7 mm in length. Subterminal ellipsoidal endospores are observed in the swollen sporangia. Colonies on NA are cream-coloured, circular and convex with entire margins after 3 days of incubation at 30 uC. Growth occurs at 4–45 uC (optimum, 30 uC) and at pH 4.5–7.5 (optimum, pH 6.5). NaCl is not required for growth but up to 4.0 % (w/v) NaCl is tolerated. Catalase and oxidase are positive. Aesculin and Tween 80 are hydrolysed but casein, gelatin, starch and urea are not hydrolysed. Nitrate is not reduced. Citrate is not utilized. Indole and H2S are not produced. bGalactosidase and Voges–Proskauer tests are positive but methyl red, urease, lysine and ornithine decarboxylases, and arginine dihydrolase tests are negative. Acid production from amygdalin and D-glucose are positive but Dmannitol, inositol, D-sorbitol, L-rhamnose, sucrose, melibiose and L-arabinose are negative. The following carbohydrates are utilized as sole carbon source: glycerol, D-ribose, D-galactose, D-glucose, methyl a-D-glucopyranoside, amygdalin, arbutin, maltose, lactose, melibiose, trehalose, inulin, raffinose and gentiobiose. The following compounds are http://ijs.sgmjournals.org
not utilized: erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, D-fructose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, Dmannitol, D-sorbitol, methyl a-D-mannopyranoside, Nacetylglucosamine, salicin, cellobiose, sucrose, melezitose, glycogen, xylitol, turanose, D-lyxose, D-tagatose, D-fucose, Lfucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketagluconate. The cell-wall peptidoglycan contains meso-diaminopimelic acid. The only isoprenoid quinone is MK-7. The whole-cell hydrolysate contains glucose and ribose. The polar lipid pattern consists of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, lysyl-phosphatidylglycerol and three unidentified aminophospholipids. The predominant cellular fatty acids are anteiso-C15 : 0 and iso-C16 : 0. The type strain is CAU 1055T (5KCTC 33036T5CCUG 63270T), isolated from soil collected from Jeju Island in the Republic of Korea. The DNA G+C content of the type strain is 48.3 mol%.
Acknowledgements This research was supported by the Biomedical Science Scholarship Grants, Department of Medicine, Chung-Ang University, in 2012.
References Ash, C., Priest, F. G. & Collins, M. D. (1993-1994). Molecular
identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test. Antonie van Leeuwenhoek 64, 253– 260. Ash, C., Priest, F. G. & Collins, M. D. (1994). Paenibacillus gen. nov.
and Paenibacillus polymyxa comb. nov. In Validation of the Publication of New Names and New Combinations Previously Effectively Published Outside the IJSB, List no. 51. Int J Syst Bacteriol 44, 852. Baik, K. S., Lim, C. H., Choe, H. N., Kim, E. M. & Seong, C. N. (2011).
Paenibacillus rigui sp. nov., isolated from a freshwater wetland. Int J Syst Evol Microbiol 61, 529–534. Benardini, J. N., Vaishampayan, P. A., Schwendner, P., Swanner, E., Fukui, Y., Osman, S., Satomi, M. & Venkateswaran, K. (2011).
Paenibacillus phoenicis sp. nov., isolated from the Phoenix Lander assembly facility and a subsurface molybdenum mine. Int J Syst Evol Microbiol 61, 1338–1343. Chou, J. H., Lee, J. H., Lin, M. C., Chang, P. S., Arun, A. B., Young, C. C. & Chen, W.-M. (2009). Paenibacillus contaminans sp. nov., isolated
from a contaminated laboratory plate. Int J Syst Evol Microbiol 59, 125–129. 1275
J.-H. Kim, H. Kang and W. Kim Conn, H. J., Bartholomew, J. W. & Jennison, M. W. (1957). Staining
methods. In Manual of Microbiological Methods, pp. 10–36. Edited by Society of American Bacteriologists. New York: McGraw-Hill.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
De Vos, P., Ludwig, W., Schleifer, K.-H. & Whitman, W. B. (2010).
Minnikin, D. E., Hutchinson, I. G., Caldicott, A. B. & Goodfellow, M. (1980). Thin-layer chromatography of methanolysates of mycolic
Paenibacillaceae fam. nov. In List of New Names and New Combinations Previously Effectively, but not Validly, Published, Validation List no. 132. Int J Syst Evol Microbiol 60, 469–472.
Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.
procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach
Nam, S. W., Kim, W., Chun, J. & Goodfellow, M. (2004). Tsukamurella
using the bootstrap. Evolution 39, 783–791. Felsenstein, J. (1989). PHYLIP – phylogeny inference package (version
pseudospumae sp. nov., a novel actinomycete isolated from activated sludge foam. Int J Syst Evol Microbiol 54, 1209–1212.
3.2). Cladistics 5, 164–166.
Nicholson, W. L. & Setlow, P. (1990). Sporulation, germination
Fitch, W. M. & Margoliash, E. (1967). Construction of phylogenetic
and outgrowth. In Molecular Biological Methods for Bacillus, pp. 391–450. Edited by C. R. Harwood & S. M. Cutting. Chichester: Wiley.
trees. Science 155, 279–284. Glaeser, S. P., Falsen, E., Busse, H. J. & Ka¨mpfer, P. (2013).
Paenibacillus vulneris sp. nov., isolated from a necrotic wound. Int J Syst Evol Microbiol 63, 777–782. Gordon, R. E. & Mihm, J. M. (1962). Identification of Nocardia caviae
(Erikson) nov. comb. Ann N Y Acad Sci 98, 628–636. Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In
Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by H. H. Munro. New York: Academic Press. Ka¨mpfer, P. (1994). Limits and possibilities of total fatty acid analysis
for classification and identification of Bacillus species. Syst Appl Microbiol 17, 86–98. Ka¨mpfer, P., Rossello´-Mora, R., Falsen, E., Busse, H.-J. & Tindall, B. J. (2006). Cohnella thermotolerans gen. nov., sp. nov., and classification
of ‘Paenibacillus hongkongensis’ as Cohnella hongkongensis sp. nov. Int J Syst Evol Microbiol 56, 781–786. Kim, J. F., Jeong, H., Park, S. Y., Kim, S. B., Park, Y. K., Choi, S. K., Ryu, C. M., Hur, C. G., Ghim, S. Y. & other authors (2010). Genome
sequence of the polymyxin-producing plant-probiotic rhizobacterium Paenibacillus polymyxa E681. J Bacteriol 192, 6103–6104. 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. M., Lee, S. H., Lee, S. H., Choi, E. J. & Jeon, C. O. (2013).
Paenibacillus hordei sp. nov., isolated from naked barley in Korea. Antonie van Leeuwenhoek 103, 3–9.
acid-containing bacteria. J Chromatogr A 188, 221–233.
Oku, Y., Kurokawa, K., Ichihashi, N. & Sekimizu, K. (2004).
Characterization of the Staphylococcus aureus mprF gene, involved in lysinylation of phosphatidylglycerol. Microbiology 150, 45–51. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406– 425. Schleifer, K. H. & Seidl, P. H. (1985). Chemical composition and
structure of murein. In Chemical Methods in Bacterial Systematics, pp. 201–219. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press. Shida, O., Takagi, H., Kadowaki, K., Nakamura, L. K. & Komagata, K. (1997a). Transfer of Bacillus alginolyticus, Bacillus chondroitinus,
Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus to the genus Paenibacillus and emended description of the genus Paenibacillus. Int J Syst Bacteriol 47, 289– 298. Shida, O., Takagi, H., Kadowaki, K., Nakamura, L. K. & Komagata, K. (1997b). Emended description of Paenibacillus amylolyticus and
description of Paenibacillus illinoisensis sp. nov. and Paenibacillus chibensis sp. nov. Int J Syst Bacteriol 47, 299–306. Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In
Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology. Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
Kishore, K. H., Begum, Z., Pathan, A. A. K. & Shivaji, S. (2010).
composition by reverse-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Paenibacillus glacialis sp. nov., isolated from the Kafni glacier of the Himalayas, India. Int J Syst Evol Microbiol 60, 1909–1913.
Tang, Q. Y., Yang, N., Wang, J., Xie, Y. Q., Ren, B., Zhou, Y. G., Gu, M. Y., Mao, J., Li, W. J. & other authors (2011). Paenibacillus
Komagata, K. & Suzuki, K. (1987). Lipid and cell wall analysis in
bacterial systematics. Methods Microbiol 19, 161–207.
algorifonticola sp. nov., isolated from a cold spring. Int J Syst Evol Microbiol 61, 2167–2172.
Kong, B. H., Liu, Q. F., Liu, M., Liu, Y., Liu, L., Li, C. L., Yu, R. & Li, Y. H. (2013). Paenibacillus typhae sp. nov., isolated from roots of Typha
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
angustifolia L. Int J Syst Evol Microbiol 63, 1037–1044.
strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
La´nyı´, B. (1987). Classical and rapid identification methods for
medically important bacteria. Methods Microbiol 19, 1–67. Lee, J., Shin, N. R., Jung, M. J., Roh, S. W., Kim, M. S., Lee, J. S., Lee, K. C., Kim, Y. O. & Bae, J. W. (2013). Paenibacillus oceanisediminis sp.
Traiwan, J., Park, M. H. & Kim, W. (2011). Paenibacillus puldeungensis sp. nov., isolated from a grassy sandbank. Int J Syst Evol Microbiol 61, 670–673.
nov. isolated from marine sediment. Int J Syst Evol Microbiol 63, 428– 434.
Ventosa, A., Quesada, E., Rodrı´guez-Valera, F., Ruiz-Berraquero, F. & Ramos-Cormenzana, A. (1982). Numerical taxonomy of moder-
Logan, N. A., Berge, O., Bishop, A. H., Busse, H.-J., De Vos, P., Fritze, D., Heyndrickx, M., Ka¨mpfer, P., Rabinovitch, L. & other authors (2009). Proposed minimal standards for describing new taxa of
ately halophilic Gram-negative rods. J Gen Microbiol 128, 1959– 1968.
aerobic, endospore-forming bacteria. Int J Syst Evol Microbiol 59, 2114–2121.
Paenibacillus sediminis sp. nov., a xylanolytic bacterium isolated from a tidal flat. Int J Syst Evol Microbiol 62, 1284–1288.
1276
International Journal of Systematic and Evolutionary Microbiology 64
Wang, L., Baek, S.-H., Cui, Y., Lee, H.-G. & Lee, S.-T. (2012).
Paenibacillus doosanensis sp. nov. Xie, C. H. & Yokota, A. (2007). Paenibacillus terrigena sp. nov., isolated
from soil. Int J Syst Evol Microbiol 57, 70–72. Zhang, J., Wang, Z. T., Yu, H. M. & Ma, Y. (2013). Paenibacillus catalpae
sp. nov., isolated from the rhizosphere soil of Catalpa speciosa. Int J Syst Evol Microbiol 63, 1776–1781.
http://ijs.sgmjournals.org
Zhou, Y., Gao, S., Wei, D. Q., Yang, L. L., Huang, X., He, J., Zhang, Y. J., Tang, S. K. & Li, W. J. (2012). Paenibacillus thermophilus
sp. nov., a novel bacterium isolated from a sediment of hot spring in Fujian province, China. Antonie van Leeuwenhoek 102, 601– 609.
1277
