International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1406–1411
DOI 10.1099/ijs.0.056697-0
Paenibacillus darwinianus sp. nov., isolated from gamma-irradiated Antarctic soil Melissa Dsouza,1 Michael W. Taylor,1 Jason Ryan,2 Andrew MacKenzie,2 Kirill Lagutin,2 Robert F. Anderson,3 Susan J. Turner1,4 and Jackie Aislabie5 Correspondence Melissa Dsouza [email protected]
1
Centre for Microbial Innovation, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
2
Callaghan Innovation Limited, PO Box 31310, Lower Hutt 5040, New Zealand
3
School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
4
BioDiscovery New Zealand Limited, Parnell, Auckland, New Zealand
5
Landcare Research, Private Bag 3127, Hamilton, New Zealand
A novel bacterium, strain BrT, was isolated from gamma-irradiated soils of the Britannia drift, Lake Wellman Region, Antarctica. This isolate was rod-shaped, endospore forming, Gram-stainvariable, catalase-positive, oxidase-negative and strictly aerobic. Cells possessed a monotrichous flagellum. Optimal growth was observed at 18 6C, pH 7.0 in PYGV or R2A broth. The major cellular fatty acid was anteiso-C15 : 0 (63.4 %). Primary identified lipids included phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. Total phospholipid was 60 % (w/w) of the total lipid extract. MK-7 was the dominant isoprenoid quinone. The genomic DNA G+C content was 55.6 mol%. Based on 16S rRNA gene sequence similarity, strain BrT clusters within the genus Paenibacillus with similarity values ranging from 93.9 to 95.1 %. Phylogenetic analyses by maximum-likelihood, maximum-parsimony and neighbourjoining methods revealed that strain BrT clusters with Paenibacillus daejeonensis (AF290916), Paenibacillus tarimensis (EF125184) and Paenibacillus pinihumi (GQ423057), albeit with weak bootstrap support. On the basis of phenotypic, chemotaxonomic and phylogenetic characteristics, we propose that strain BrT represents a novel species, Paenibacillus darwinianus sp. nov. The type strain is BrT (5DSM 27245T5ICMP 19912T).
The genus Paenibacillus belonging to family Paenibacillaceae was first resolved from members of rRNA group 3 bacilli by Ash et al. (1993) based on comparative 16S rRNA gene sequence analysis. Members of the genus Paenibacillus are characterized as rod-shaped, Gram-positive or variable (depending on growth phase), endospore-forming, aerobic or facultatively anaerobic bacteria, possessing anteiso-C15 : 0 as the major cellular fatty acid and unsaturated menaquinone with seven units (MK-7) as the predominant isoprenoid quinone, and having a genomic DNA G+C content ranging between 40 and 54 mol% (Ash et al., 1993; Shida et al., 1997). The genus Paenibacillus contains several species that have been isolated from a wide range of cold The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and rpoB gene sequences of Paenibacillus darwinianus BrT are KF264455 and KF819812, respectively. Four supplementary figures are available with the online version of this paper.
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environments including sediment (Montes et al., 2004) and soil (Marshall & Ohye, 1966; Rodrı´guez-Dı´az et al., 2005) from the Antarctic Peninsula, a cold spring in China (Tang et al., 2011), soil on the Kafni glacier, Himalayas (Kishore et al., 2010), Alaskan soil (Nelson et al., 2009) and a peat bog in Northern China (Ming et al., 2012). In this study we investigated the taxonomic status of strain BrT isolated from Antarctic soils which were exposed to gamma irradiation, using a combination of physiological, biochemical, chemotaxonomic and phylogenetic techniques. Based on these analyses we propose strain BrT as a representative of a novel species, Paenibacillus darwinianus sp. nov. Soil samples on the Britannia drift, Lake Wellman Region (79u 559 16.20 S 156u 559 30.70 E) in the south-eastern Darwin Mountains, Antarctica, were collected for the isolation of bacteria (Aislabie et al., 2012). The soil samples were irradiated with 60Co c-rays at a dose of 288 Gy h21 for 80 h and plated onto PYGV (peptone yeast extract 056697 G 2014 IUMS Printed in Great Britain
Paenibacillus darwinianus sp. nov.
glucose vitamin) gellan gum-based solid medium at 15 uC for up to two months (Hirsch et al., 2004), to isolate and identify novel, fastidious bacterial species that are exposed to high levels of ionizing radiation and desiccation in the nutrient-poor Antarctic soil environment. Colonies with different morphologies were restreaked onto PYGV gellan gum plates for primary isolation and purification. Among the bacteria, strain BrT was isolated and subsequently routinely cultured on PYGV gellan gum plates at 15–18 uC and maintained as a glycerol suspension (15 %, v/v) at 280 uC. Following purification, gamma radiation resistance was determined as described by Callegan et al. (2008). In contrast to the data reported for species of the genus Deinococcus that can exhibit up to 10 % survival following exposure to 10 kGy gamma radiation (Ekman et al., 2011), strain BrT showed sensitivity to gamma radiation by exhibiting only 18 % survival following exposure to 2 kGy, and eradication upon exposure to 5 kGy gamma radiation. Closely related type strains of species of the genus Paenibacillus, Paenibacillus daejeonensis DSM 15491T, Paenibacillus pinihumi DSM 23905T and Paenibacillus tarimensis DSM 19409T were obtained from DSMZ, Germany, for the concurrent comparison of fatty acid methyl esters and physiological characteristics. Colony morphology was observed on PYGV gellan gum plates after culturing for 4 days at 15–18 uC. Strain BrT formed circular, flat, opaque, glossy, white colonies, up to 0.3 mm in size with an entire margin. Cell morphology was examined by light microscopy (Nikon H550S). Spores and the flagellum of the cells were examined by scanning (Philips XL30 S-FEG) and transmission electron microscopy (Philips CM12), respectively. For scanning electron microscopy, cells from a PYGV gellan gum plate incubated for 10 days at 18 uC were collected on an Isopore membrane filter with pore size 0.2 mm (Millipore), fixed in 2.5 % (v/v) glutaraldehyde in 0.1 M Sorensens phosphate buffer (pH 7.2) for 3 h at room temperature and washed three times in the same buffer. The specimens were dehydrated in a graded series of ethanol, critical point dried with CO2 and coated with platinum for examination on a field emission scanning electron microscope at 5 kV. For transmission electron microscopy, cells were grown in PYGV broth for four days at 18 uC, negatively stained with 2 % (w/v) nitrilotriacetic acid and examined on carbon grids. Gram staining was performed on cells grown on PYGV gellan gum for four days at 18 uC using the 4-Step Gram Stain kit (BD). Motility was tested using liquid cultures grown in R2A broth (Lab M) and PYGV broth at 18 uC for 4 days by the wet mount and hanging drop techniques as described by Murray & Robinow (1994), and by stab inoculating PYGV gellan gum slants at 18 uC for 7 days. Oxidase activity was colorimetrically assessed using a Bactident Oxidase kit (Merck) and catalase activity was determined by bubble production using 3 % (v/v) H2O2 (Smibert & Krieg, 1994). Anaerobic growth was tested on R2A gellan gum and PYGV gellan gum plates. Anaerobicity was maintained by using a GasPak EZ Anaerobe Container http://ijs.sgmjournals.org
system (BD). Cells were strictly aerobic, Gram-stain-variable, non-motile, catalase-positive, oxidase-negative and spore forming. Ellipsoidal spores (1.3–2 mm in length, 0.63– 1.25 mm in width) were observed in swollen sporangia (Fig. S1a, available in the online Supplementary Material). Cells were rod-shaped and measured 3.1–6.8 mm in length and 0.67–1.3 mm in width (Fig. S1b). Cells possessed a monotrichous flagellum (Fig. 1), despite absence of visible motility as tested by hanging drop, wet mount and media stabbing techniques. Growth ability in tryptic soy broth (TSB; Difco), nutrient broth (NB; Difco) and R2A broth was determined at 18 uC over a period of 5 days. Growth at various temperatures including 4, 10, 15, 18, 28 and 37 uC was tested using TSB, NB, R2A and PYGV broth over 5 days. The effects of pH were tested using pH-adjusted R2A broth ranging between pH 5.5 and 12. The effects of salinity were tested using R2A broth supplemented with 1–5 % (w/v) NaCl. For these experiments growth was estimated over 7 days using optical density (OD650 nm) as described by Lee et al. (2002). Growth was observed between 15 and 37 uC, with optimal growth occurring at 18 uC in R2A and PYGV broth. Poor growth was observed in nutrient and tryptic soy broth between 15 and 28 uC, with no growth occurring below 15 uC or above 28 uC. Growth was observed between pH 6.0 and 10.0 with the optimal growth at pH 7.0. Poor growth was observed in R2A media containing ¡4 % (w/v) NaCl and no growth was observed in media containing .4 % (w/v) NaCl.
Fig. 1. Transmission electron micrograph of strain BrT grown in R2A broth showing monotrichous flagellum. Magnification, ¾11 500. Bar, 1 mm. 1407
M. Dsouza and others
Growth ability on MacConkey agar plates was tested using standard MacConkey agar (Scharlau Microbiology) at 18 uC for seven days. Hydrolysis of casein, gelatin and starch was determined as described by Smibert and Krieg (1994) and hydrolysis of Tween 80 was determined as described by Kilburn et al. (1973). Citrate utilization was tested at 18 uC for up to 7 days (Smibert & Krieg, 1994). No growth was observed on MacConkey agar. Strain BrT tested positive for the hydrolysis of starch and negative for citrate utilization and for the hydrolysis of Tween 80, casein and gelatin. Carbohydrate fermentation was tested using API 50 CH strips and API 50 CHB/E medium (BioMe´rieux) as per the manufacturer’s protocol over 2 days at 28 uC. Utilization of 95 carbon sources was tested using a GP2 Microplate (Biolog) following the manufacturer’s instructions. In addition to the GN/GP inoculating fluid, PYGV medium (without glucose, yeast extract and peptone) was utilized as an inoculating fluid. No growth was observed on GP2 plates over 3 weeks. As a result, carbon utilization was also tested by Phenotype Microarray (PM) technology (Biolog) using the PM1 plate, prepared as per the manufacturer’s instructions using PYGV medium (without glucose, yeast extract and peptone) as the inoculating fluid, with and without Biolog’s proprietary reducing dye. No growth was observed in PM1 C plates inoculated with dye. Therefore carbon utilization was measured as a function of cell turbidity on PM1 plates without dye. Differential morphological and physiological characteristics of strain BrT with closely related and concurrently tested type strains of species of the genus Paenibacillus are shown in Table 1. A complete description of the physiological and biochemical properties of strain BrT is given in the species description.
Table 1. Distinctive phenotypic characteristics of strain BrT and closely related species of the genus Paenibacillus Strains: 1, BrT (P. darwinianus sp. nov.); 2, P. daejeonensis DSM 15491T (Lee et al., 2002); 3, P. tarimensis DSM 19409T (Wang et al., 2008); 4, P. pinihumi DSM 23905T (Kim et al., 2009). All species were negative for oxidase and the hydrolysis of gelatin and casein. All species were positive for the hydrolysis of starch. All species produced acid from aesculin. None of the species produced acid from glycerol, erythritol, D-ribose, L-xylose, adonitol, methyl b-D-xyloside, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, methyl aD-mannoside, melezitose, D-tagatose, D-fucose, D-arabitol, L-arabitol, potassium gluconate, 2-ketogluconate, D-arabinose, L-arabinose, D-mannose, methyl a-D-glucopyranoside, inulin, xylitol, turanose, D-lyxose, L-fucose or 5-ketogluconate. All strains were tested concurrently in this study. +, positive; 2, negative; V, variable; m, monotrichous; p, peritrichous. Characteristic Gram stain Flagella arrangement Catalase Optimum growth temperature (uC) Hydrolysis of Tween 80 Acid Production from: D-Galactose, salicin D-Fructose D-Xylose, glucose, sucrose D-Mannitol, N-acetylglucosamine Amygdalin, glycogen, maltose, starch Arbutin, cellobiose, lactose, raffinose Trehalose DNA G+C content (mol%)
1
2
3
4
V
m + 18 2
2 p + 30 +
2 p 2 37 +
2 p + 25–30 2
+ + + 2 2
+ 2 2 + +
+ 2 + 2 +
2 2 2 2 +
2 2 55.6
+ + 53.0
+ 2 + + 53.7 49.5
For chemotaxonomic analyses, strain BrT and closely related type strains of species of the genus Paenibacillus were grown in R2A broth at 18 uC for 4 days. Isoprenoid quinone and DNA G+C contents were determined by the micro-organism identification service offered by DSMZ, Germany. The genomic DNA G+C content of strain BrT was 55.6 mol% and MK-7 (100 %) was the major respiratory quinone, which is in accordance with other members of the genus Paenibacillus. Polar lipids were analysed by TLC (Tindall et al., 2007) and phospholipids were quantified by 31P-NMR (MacKenzie et al., 2009). Strain BrT displayed a lipid profile comprising five phospholipids, two unidentified glycolipids and three other unidentified polar lipids. Phospholipids were phosphatidylethanolamine (39 mol%), diphosphatidylglycerol (35 mol%), phosphatidylglycerol (17 mol%), and two unidentified minor phospholipids (6 mol% and 3 mol%) (Fig. S2). This profile was similar to that of Paenibacillus polymyxa DSM 36T with diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol identified as major polar lipids (Ka¨mpfer et al., 2006). Fatty acid methyl esters were prepared and analysed without prior extraction as described by Vyssotski et al. (2012). Fatty acid analysis of
strain BrT revealed that anteiso-C15 : 0 (63.4 %), C16 : 1v11c (8.1 %) and C16 : 0 (5.7 %) were the major cellular fatty acids. These fatty acids are common to described species in the genus Paenibacillus (Kaneda, 1991). anteiso-C17 : 0 was the major cellular fatty acid observed in the closely related type strains (Table 2). However, cellular fatty acid profiles of P. daejeonensis (Lee et al., 2002), P. tarimensis (Wang et al., 2008) and P. pinihumi (Kim et al., 2009) determined following growth on TSA at 30 uC for 48 h revealed that anteiso-C15 : 0 was the major cellular fatty acid. In response to a reduction in growth temperature to 18 uC, these mesophilic strains showed a significant decrease in anteiso-C15 : 0 and an increase in straight-chain fatty acids. This may be compensated by the significant increase in anteiso-C17 : 0 and unsaturated fatty acid content, allowing maintenance of optimal membrane fluidity. In species of the genus Bacillus a reduction in growth temperature can induce significant changes in fatty acid branching, chain length and unsaturation. Following a drop in temperature from 40 to 15 uC, Bacillus subtilis showed a decrease in isobranched acids and an increase in anteiso-branched and unsaturated fatty acids (Suutari & Laakso, 1992).
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Paenibacillus darwinianus sp. nov.
Table 2. Cellular fatty acid contents of strain BrT and closely related species of the genus Paenibacillus Strains: 1, BrT (P. darwinianus sp. nov.); 2, P. daejeonensis DSM 15491T (Lee et al., 2002); 3, P. tarimensis DSM 19409T (Wang et al., 2008); 4, P. pinihumi DSM 23905T (Kim et al., 2009). All strains were tested under identical conditions, namely by growth in R2A broth at 18 uC for four days. –, Not determined/not measured; TR, trace amount (,1 %). Fatty acid Straight-chained saturated C14 : 0 C15 : 0 C16 : 0 C17 : 0 C18 : 0 Branched saturated iso-C14 : 0 iso-C15 : 0 iso-C16 : 0 iso-C17 : 0 anteiso-C15 : 0 anteiso-C16 : 0 anteiso-C17 : 0 Unsaturated C16 : 1v11c C17 : 1v12c anteiso-C17 : 1v12 C18 : 1v9c Total
1
1.0 0.6 5.7 – 1.7 1.2 1.4 4.8 TR
2
3
4
–
– – 8.4 2.1 46.6
– 2.2 12.4 2.5 5.3 – 5.9 12.2 20.5 13.4 1.1 17.6
TR
11.7 16.1 16.8 –
–
TR
TR
3.6 8.1
– 3.3
63.4 2.6 4.5
TR
TR
– 32.3
– 30.5
8.1 – 3.1 – 98.1
2.3 3.0 3.5 2.5 99.9
1.6 – 3.2 4.3 100
5.3 TR TR TR
98.4
& Miller, 1988). In the 16S rRNA gene-based ML tree, strain BrT clusters with P. daejeonensis AP-20T (AF290916), P. tarimensis SA-7-6T (EF125184) and P. pinihumi S23T (GQ423057), albeit with weak bootstrap support (Fig. 2). However, this clustering pattern was also observed in 16S rRNA gene-based phylogenetic trees constructed by MP and NJ methods (Fig. S3). In addition, phylogenetic trees based on RpoB protein sequences exhibited similar clustering of strain BrT with P. daejeonensis and P. pinihumi (Fig. S4). 16S rRNA gene sequence similarity values revealed that strain BrT shares 95.1 % 16S rRNA gene sequence similarity with P. tarimensis SA-7-6T, 94.5 % similarity with P. pinihumi S23T, and 93.9 % similarity with P. daejeonensis AP-20T. Overall the highest 16S rRNA gene sequence similarity shown by strain BrT as compared with other recognized species of the genus Paenibacillus was 95.1 %. According to Stackebrandt & Goebel (1994) an isolate with less than 97 % 16S rRNA gene sequence similarity to any known microbial strain represents a novel species, thereby providing evidence for the distinction of strain BrT as a representative of a novel species within genus Paenibacillus. In conclusion, the morphological, physiological, chemotaxonomic and phylogenetic data for strain BrT are in accordance with characteristics of members of genus Paenibacillus. However, the differences in the cellular fatty acid content and in the 16S rRNA gene similarity clearly distinguish strain BrT as a representative of a novel species within the genus Paenibacillus, for which the name Paenibacillus darwinianus sp. nov. is proposed. Description of Paenibacillus darwinianus sp. nov.
DNA was extracted from strain BrT by mechanical cell disruption (bead-beating) as described by Foght et al. (2004). The nearly complete 16S rRNA gene sequence was obtained by PCR amplification using universal primers, PB36 and PB38 (Aislabie et al., 2006). The nearly complete 16S rRNA gene sequence was assembled in Geneious version 5.5.6 (Biomatters) (Kearse et al., 2012), aligned via the SINA web aligner and imported into the ARB phylogenetic package using the SILVA 108 database (Ludwig et al., 2004). The RNA polymerase b-subunit (rpoB) protein sequence was extracted from the whole genome sequence available for strain BrT (Dsouza and others, unpublished). In addition, RpoB protein sequences of related species of the genus Paenibacillus were obtained from the Integrated Microbial Genome (IMG) database (Markowitz et al., 2012) and aligned by the CLUSTAL W method (Larkin et al., 2007). Phylogenetic trees based on the nearly complete 16S rRNA gene and RpoB protein sequences from strain BrT and closely related species of the genus Paenibacillus were reconstructed by neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) methods. The topology of each tree was tested in the MEGA 5.2 software by bootstrap analysis based on 1000 resamplings (Tamura et al., 2011). 16S rRNA gene sequence similarity values were obtained using the EzTaxon Server 2.1 (Myers http://ijs.sgmjournals.org
Paenibacillus darwinianus (dar.wi.ni.a9nus. N.L. masc. adj. darwinianus referring to Darwin Mountains, Antarctica, the geographical origin of the type strain). Cells are Gram-stain-variable, rod-shaped (3.1–6.8 mm length; 0.67–1.3 mm width), strictly aerobic and possess a monotrichous flagellum. Ellipsoidal spores (1.3–2 mm) are produced in swollen sporangia. Colonies are circular, flat, opaque and white with an entire margin on PYGV gellan gum plates. Growth is observed between 15 and 37 uC, with ¡4 % (w/v) NaCl and at pH 6–10 in R2A or PYGV media (broth or gellan gum plates). Optimal growth occurs between 18 and 28 uC at pH 7.0 in PYGV or R2A broth. Poor growth is observed in TSB and NB. No growth is observed on MacConkey agar. Positive reactions are observed for catalase, hydrolysis of starch and acid production from D-xylose, galactose, glucose, salicin, sucrose, D-fructose and aesculin. Negative reactions were observed for oxidase, hydrolysis of casein, Tween 80 and gelatin, citrate utilization, acid production from glycerol, erythritol, ribose, L-xylose, D-adonitol, methyl b-D-xyloside, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannoside, N-acetylglucosamine, amygdalin, arbutin, maltose, melezitose, starch, glycogen, D-tagatose, D-fucose, D-arabitol, L-arabitol, gluconate, 1409
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Paenibacillus humicus PC-147T, AM411528 Paenibacillus pasadenensis SAFN-007T, AY167820 Paenibacillus phyllosphaerae PALXIL04T, AY598818 Paenibacillus cellulosilyticus PALXIL 08T, DQ407282 99 Paenibacillus kobensis DSM 10249T, AB073363 Paenibacillus mendelii C/2T, AF537343 72 Paenibacillus sepulcri CCM 7311T, DQ291142 Paenibacillus darwinianus BrT, KF264455 30 Paenibacillus daejeonensis AP-20T, AF290916 54 Paenibacillus pinihumi S23T, GQ423057 Paenibacillus tarimensis SA-7-6T, EF125184 Paenibacillus agarexedens DSM 1327T, AJ345020 94 56 Paenibacillus alkaliterrae KSL-134T, AY960748 Paenibacillus glycanilyticus DS-1T, AB042938 Paenibacillus thailandensis S3-4AT, AB265205 Paenibacillus tundrae A10bT, EU558284 100 97 Paenibacillus xylanilyticus XIL14T, AY427832 Paenibacillus xylanexedens B22aT, EU558281 Paenibacillus wynnii LMG 22176T, AJ633647 Paenibacillus macquariensis DSM 2T, AB073193 97 87 Paenibacillus glacialis KFC91T, EU815294 Paenibacillus antarcticus LMG 22078T, AJ605292 Paenibacillus cookii LMG 18419T, AJ250317 Bacillus circulans NBRC 13626T, AB271747 97 78 Bacillus oceanisediminis H2T, GQ292772 100 Bacillus koreensis BR030T, AY667496 Bacillus cereus WSBC 10204T, Z84578 100
0.11
Fig. 2. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences from strain BrT (highlighted in bold type) and closely related species of the genus Paenibacillus. Bootstrap values (%) for 1000 resamplings are indicated for each node. Nodes with no bootstrap values were not represented in the consensus bootstrap tree. Bar, 0.11 substitutions per compared nucleotide site.
2-ketogluconate, D-arabinose, L-arabinose, D-mannose, methyl a-D-glucoside, cellobiose, lactose, melibiose, trehalose, inulin, raffinose, xylitol, gentiobiose, turanose, D-lyxose, L-fucose and 5-ketogluconate. For PM1 Biolog plates, none of the carbon sources except acetoacetic acid, phenylethylamine, glycl-L-glutamic acid, D-glucose 6-phosphate, D-galactonic acid c-lactone and mucic acid were utilised. anteiso-C15 : 0 is the major cellular fatty acid. Primary lipids are phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. Total phospholipid is 60 % (w/w) of the total lipid. Unidentified lipids include two glycolipids, two phospholipids and three other lipids. MK-7 is the dominant isoprenoid quinone. The type strain, BrT (5DSM 27245T5ICMP 19883T), was isolated from gamma-irradiated soil of the Britannia drift, Darwin Mountains, Antarctica. The genomic DNA G+C content of the type strain is 55.6 mol%.
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23, 2947–2948. Lee, J.-S., Lee, K. C., Chang, Y.-H., Hong, S. G., Oh, H. W., Pyun, Y.-R. & Bae, K. S. (2002). Paenibacillus daejeonensis sp. nov., a novel
alkaliphilic bacterium from soil. Int J Syst Evol Microbiol 52, 2107– 2111. Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai, T., Steppi, S. & other authors (2004). ARB: a software environment for sequence data. Nucleic Acids
Res 32, 1363–1371. MacKenzie, A., Vyssotski, M. & Nekrasov, E. (2009). Quantitative
analysis of dairy phospholipids by 757–763.
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Markowitz, V. M., Chen, I.-M. A., Palaniappan, K., Chu, K., Szeto, E., Grechkin, Y., Ratner, A., Jacob, B., Huang, J. & other authors (2012).
IMG: the integrated microbial genomes database and comparative analysis system. Nucleic Acids Res 40 (D1), D115–D122. Marshall, B. J. & Ohye, D. F. (1966). Bacillus macquariensis n.sp., a
psychrotrophic bacterium from sub-antarctic soil. J Gen Microbiol 44, 41–46. Ming, H., Nie, G.-X., Jiang, H.-C., Yu, T.-T., Zhou, E.-M., Feng, H.-G., Tang, S. K. & Li, W. J. (2012). Paenibacillus frigoriresistens sp. nov.,
a novel psychrotroph isolated from a peat bog in Heilongjiang, Northern China. Antonie van Leeuwenhoek 102, 297–305.
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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. Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In
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algorifonticola sp. nov., isolated from a cold spring. Int J Syst Evol Microbiol 61, 2167–2172. Tindall, B., Sikorski, J., Smibert, R. & Krieg, N. (2007). Phenotypic
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DOI 10.1099/ijs.0.056697-0
Paenibacillus darwinianus sp. nov., isolated from gamma-irradiated Antarctic soil Melissa Dsouza,1 Michael W. Taylor,1 Jason Ryan,2 Andrew MacKenzie,2 Kirill Lagutin,2 Robert F. Anderson,3 Susan J. Turner1,4 and Jackie Aislabie5 Correspondence Melissa Dsouza [email protected]
1
Centre for Microbial Innovation, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
2
Callaghan Innovation Limited, PO Box 31310, Lower Hutt 5040, New Zealand
3
School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
4
BioDiscovery New Zealand Limited, Parnell, Auckland, New Zealand
5
Landcare Research, Private Bag 3127, Hamilton, New Zealand
A novel bacterium, strain BrT, was isolated from gamma-irradiated soils of the Britannia drift, Lake Wellman Region, Antarctica. This isolate was rod-shaped, endospore forming, Gram-stainvariable, catalase-positive, oxidase-negative and strictly aerobic. Cells possessed a monotrichous flagellum. Optimal growth was observed at 18 6C, pH 7.0 in PYGV or R2A broth. The major cellular fatty acid was anteiso-C15 : 0 (63.4 %). Primary identified lipids included phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. Total phospholipid was 60 % (w/w) of the total lipid extract. MK-7 was the dominant isoprenoid quinone. The genomic DNA G+C content was 55.6 mol%. Based on 16S rRNA gene sequence similarity, strain BrT clusters within the genus Paenibacillus with similarity values ranging from 93.9 to 95.1 %. Phylogenetic analyses by maximum-likelihood, maximum-parsimony and neighbourjoining methods revealed that strain BrT clusters with Paenibacillus daejeonensis (AF290916), Paenibacillus tarimensis (EF125184) and Paenibacillus pinihumi (GQ423057), albeit with weak bootstrap support. On the basis of phenotypic, chemotaxonomic and phylogenetic characteristics, we propose that strain BrT represents a novel species, Paenibacillus darwinianus sp. nov. The type strain is BrT (5DSM 27245T5ICMP 19912T).
The genus Paenibacillus belonging to family Paenibacillaceae was first resolved from members of rRNA group 3 bacilli by Ash et al. (1993) based on comparative 16S rRNA gene sequence analysis. Members of the genus Paenibacillus are characterized as rod-shaped, Gram-positive or variable (depending on growth phase), endospore-forming, aerobic or facultatively anaerobic bacteria, possessing anteiso-C15 : 0 as the major cellular fatty acid and unsaturated menaquinone with seven units (MK-7) as the predominant isoprenoid quinone, and having a genomic DNA G+C content ranging between 40 and 54 mol% (Ash et al., 1993; Shida et al., 1997). The genus Paenibacillus contains several species that have been isolated from a wide range of cold The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and rpoB gene sequences of Paenibacillus darwinianus BrT are KF264455 and KF819812, respectively. Four supplementary figures are available with the online version of this paper.
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environments including sediment (Montes et al., 2004) and soil (Marshall & Ohye, 1966; Rodrı´guez-Dı´az et al., 2005) from the Antarctic Peninsula, a cold spring in China (Tang et al., 2011), soil on the Kafni glacier, Himalayas (Kishore et al., 2010), Alaskan soil (Nelson et al., 2009) and a peat bog in Northern China (Ming et al., 2012). In this study we investigated the taxonomic status of strain BrT isolated from Antarctic soils which were exposed to gamma irradiation, using a combination of physiological, biochemical, chemotaxonomic and phylogenetic techniques. Based on these analyses we propose strain BrT as a representative of a novel species, Paenibacillus darwinianus sp. nov. Soil samples on the Britannia drift, Lake Wellman Region (79u 559 16.20 S 156u 559 30.70 E) in the south-eastern Darwin Mountains, Antarctica, were collected for the isolation of bacteria (Aislabie et al., 2012). The soil samples were irradiated with 60Co c-rays at a dose of 288 Gy h21 for 80 h and plated onto PYGV (peptone yeast extract 056697 G 2014 IUMS Printed in Great Britain
Paenibacillus darwinianus sp. nov.
glucose vitamin) gellan gum-based solid medium at 15 uC for up to two months (Hirsch et al., 2004), to isolate and identify novel, fastidious bacterial species that are exposed to high levels of ionizing radiation and desiccation in the nutrient-poor Antarctic soil environment. Colonies with different morphologies were restreaked onto PYGV gellan gum plates for primary isolation and purification. Among the bacteria, strain BrT was isolated and subsequently routinely cultured on PYGV gellan gum plates at 15–18 uC and maintained as a glycerol suspension (15 %, v/v) at 280 uC. Following purification, gamma radiation resistance was determined as described by Callegan et al. (2008). In contrast to the data reported for species of the genus Deinococcus that can exhibit up to 10 % survival following exposure to 10 kGy gamma radiation (Ekman et al., 2011), strain BrT showed sensitivity to gamma radiation by exhibiting only 18 % survival following exposure to 2 kGy, and eradication upon exposure to 5 kGy gamma radiation. Closely related type strains of species of the genus Paenibacillus, Paenibacillus daejeonensis DSM 15491T, Paenibacillus pinihumi DSM 23905T and Paenibacillus tarimensis DSM 19409T were obtained from DSMZ, Germany, for the concurrent comparison of fatty acid methyl esters and physiological characteristics. Colony morphology was observed on PYGV gellan gum plates after culturing for 4 days at 15–18 uC. Strain BrT formed circular, flat, opaque, glossy, white colonies, up to 0.3 mm in size with an entire margin. Cell morphology was examined by light microscopy (Nikon H550S). Spores and the flagellum of the cells were examined by scanning (Philips XL30 S-FEG) and transmission electron microscopy (Philips CM12), respectively. For scanning electron microscopy, cells from a PYGV gellan gum plate incubated for 10 days at 18 uC were collected on an Isopore membrane filter with pore size 0.2 mm (Millipore), fixed in 2.5 % (v/v) glutaraldehyde in 0.1 M Sorensens phosphate buffer (pH 7.2) for 3 h at room temperature and washed three times in the same buffer. The specimens were dehydrated in a graded series of ethanol, critical point dried with CO2 and coated with platinum for examination on a field emission scanning electron microscope at 5 kV. For transmission electron microscopy, cells were grown in PYGV broth for four days at 18 uC, negatively stained with 2 % (w/v) nitrilotriacetic acid and examined on carbon grids. Gram staining was performed on cells grown on PYGV gellan gum for four days at 18 uC using the 4-Step Gram Stain kit (BD). Motility was tested using liquid cultures grown in R2A broth (Lab M) and PYGV broth at 18 uC for 4 days by the wet mount and hanging drop techniques as described by Murray & Robinow (1994), and by stab inoculating PYGV gellan gum slants at 18 uC for 7 days. Oxidase activity was colorimetrically assessed using a Bactident Oxidase kit (Merck) and catalase activity was determined by bubble production using 3 % (v/v) H2O2 (Smibert & Krieg, 1994). Anaerobic growth was tested on R2A gellan gum and PYGV gellan gum plates. Anaerobicity was maintained by using a GasPak EZ Anaerobe Container http://ijs.sgmjournals.org
system (BD). Cells were strictly aerobic, Gram-stain-variable, non-motile, catalase-positive, oxidase-negative and spore forming. Ellipsoidal spores (1.3–2 mm in length, 0.63– 1.25 mm in width) were observed in swollen sporangia (Fig. S1a, available in the online Supplementary Material). Cells were rod-shaped and measured 3.1–6.8 mm in length and 0.67–1.3 mm in width (Fig. S1b). Cells possessed a monotrichous flagellum (Fig. 1), despite absence of visible motility as tested by hanging drop, wet mount and media stabbing techniques. Growth ability in tryptic soy broth (TSB; Difco), nutrient broth (NB; Difco) and R2A broth was determined at 18 uC over a period of 5 days. Growth at various temperatures including 4, 10, 15, 18, 28 and 37 uC was tested using TSB, NB, R2A and PYGV broth over 5 days. The effects of pH were tested using pH-adjusted R2A broth ranging between pH 5.5 and 12. The effects of salinity were tested using R2A broth supplemented with 1–5 % (w/v) NaCl. For these experiments growth was estimated over 7 days using optical density (OD650 nm) as described by Lee et al. (2002). Growth was observed between 15 and 37 uC, with optimal growth occurring at 18 uC in R2A and PYGV broth. Poor growth was observed in nutrient and tryptic soy broth between 15 and 28 uC, with no growth occurring below 15 uC or above 28 uC. Growth was observed between pH 6.0 and 10.0 with the optimal growth at pH 7.0. Poor growth was observed in R2A media containing ¡4 % (w/v) NaCl and no growth was observed in media containing .4 % (w/v) NaCl.
Fig. 1. Transmission electron micrograph of strain BrT grown in R2A broth showing monotrichous flagellum. Magnification, ¾11 500. Bar, 1 mm. 1407
M. Dsouza and others
Growth ability on MacConkey agar plates was tested using standard MacConkey agar (Scharlau Microbiology) at 18 uC for seven days. Hydrolysis of casein, gelatin and starch was determined as described by Smibert and Krieg (1994) and hydrolysis of Tween 80 was determined as described by Kilburn et al. (1973). Citrate utilization was tested at 18 uC for up to 7 days (Smibert & Krieg, 1994). No growth was observed on MacConkey agar. Strain BrT tested positive for the hydrolysis of starch and negative for citrate utilization and for the hydrolysis of Tween 80, casein and gelatin. Carbohydrate fermentation was tested using API 50 CH strips and API 50 CHB/E medium (BioMe´rieux) as per the manufacturer’s protocol over 2 days at 28 uC. Utilization of 95 carbon sources was tested using a GP2 Microplate (Biolog) following the manufacturer’s instructions. In addition to the GN/GP inoculating fluid, PYGV medium (without glucose, yeast extract and peptone) was utilized as an inoculating fluid. No growth was observed on GP2 plates over 3 weeks. As a result, carbon utilization was also tested by Phenotype Microarray (PM) technology (Biolog) using the PM1 plate, prepared as per the manufacturer’s instructions using PYGV medium (without glucose, yeast extract and peptone) as the inoculating fluid, with and without Biolog’s proprietary reducing dye. No growth was observed in PM1 C plates inoculated with dye. Therefore carbon utilization was measured as a function of cell turbidity on PM1 plates without dye. Differential morphological and physiological characteristics of strain BrT with closely related and concurrently tested type strains of species of the genus Paenibacillus are shown in Table 1. A complete description of the physiological and biochemical properties of strain BrT is given in the species description.
Table 1. Distinctive phenotypic characteristics of strain BrT and closely related species of the genus Paenibacillus Strains: 1, BrT (P. darwinianus sp. nov.); 2, P. daejeonensis DSM 15491T (Lee et al., 2002); 3, P. tarimensis DSM 19409T (Wang et al., 2008); 4, P. pinihumi DSM 23905T (Kim et al., 2009). All species were negative for oxidase and the hydrolysis of gelatin and casein. All species were positive for the hydrolysis of starch. All species produced acid from aesculin. None of the species produced acid from glycerol, erythritol, D-ribose, L-xylose, adonitol, methyl b-D-xyloside, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, methyl aD-mannoside, melezitose, D-tagatose, D-fucose, D-arabitol, L-arabitol, potassium gluconate, 2-ketogluconate, D-arabinose, L-arabinose, D-mannose, methyl a-D-glucopyranoside, inulin, xylitol, turanose, D-lyxose, L-fucose or 5-ketogluconate. All strains were tested concurrently in this study. +, positive; 2, negative; V, variable; m, monotrichous; p, peritrichous. Characteristic Gram stain Flagella arrangement Catalase Optimum growth temperature (uC) Hydrolysis of Tween 80 Acid Production from: D-Galactose, salicin D-Fructose D-Xylose, glucose, sucrose D-Mannitol, N-acetylglucosamine Amygdalin, glycogen, maltose, starch Arbutin, cellobiose, lactose, raffinose Trehalose DNA G+C content (mol%)
1
2
3
4
V
m + 18 2
2 p + 30 +
2 p 2 37 +
2 p + 25–30 2
+ + + 2 2
+ 2 2 + +
+ 2 + 2 +
2 2 2 2 +
2 2 55.6
+ + 53.0
+ 2 + + 53.7 49.5
For chemotaxonomic analyses, strain BrT and closely related type strains of species of the genus Paenibacillus were grown in R2A broth at 18 uC for 4 days. Isoprenoid quinone and DNA G+C contents were determined by the micro-organism identification service offered by DSMZ, Germany. The genomic DNA G+C content of strain BrT was 55.6 mol% and MK-7 (100 %) was the major respiratory quinone, which is in accordance with other members of the genus Paenibacillus. Polar lipids were analysed by TLC (Tindall et al., 2007) and phospholipids were quantified by 31P-NMR (MacKenzie et al., 2009). Strain BrT displayed a lipid profile comprising five phospholipids, two unidentified glycolipids and three other unidentified polar lipids. Phospholipids were phosphatidylethanolamine (39 mol%), diphosphatidylglycerol (35 mol%), phosphatidylglycerol (17 mol%), and two unidentified minor phospholipids (6 mol% and 3 mol%) (Fig. S2). This profile was similar to that of Paenibacillus polymyxa DSM 36T with diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol identified as major polar lipids (Ka¨mpfer et al., 2006). Fatty acid methyl esters were prepared and analysed without prior extraction as described by Vyssotski et al. (2012). Fatty acid analysis of
strain BrT revealed that anteiso-C15 : 0 (63.4 %), C16 : 1v11c (8.1 %) and C16 : 0 (5.7 %) were the major cellular fatty acids. These fatty acids are common to described species in the genus Paenibacillus (Kaneda, 1991). anteiso-C17 : 0 was the major cellular fatty acid observed in the closely related type strains (Table 2). However, cellular fatty acid profiles of P. daejeonensis (Lee et al., 2002), P. tarimensis (Wang et al., 2008) and P. pinihumi (Kim et al., 2009) determined following growth on TSA at 30 uC for 48 h revealed that anteiso-C15 : 0 was the major cellular fatty acid. In response to a reduction in growth temperature to 18 uC, these mesophilic strains showed a significant decrease in anteiso-C15 : 0 and an increase in straight-chain fatty acids. This may be compensated by the significant increase in anteiso-C17 : 0 and unsaturated fatty acid content, allowing maintenance of optimal membrane fluidity. In species of the genus Bacillus a reduction in growth temperature can induce significant changes in fatty acid branching, chain length and unsaturation. Following a drop in temperature from 40 to 15 uC, Bacillus subtilis showed a decrease in isobranched acids and an increase in anteiso-branched and unsaturated fatty acids (Suutari & Laakso, 1992).
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International Journal of Systematic and Evolutionary Microbiology 64
Paenibacillus darwinianus sp. nov.
Table 2. Cellular fatty acid contents of strain BrT and closely related species of the genus Paenibacillus Strains: 1, BrT (P. darwinianus sp. nov.); 2, P. daejeonensis DSM 15491T (Lee et al., 2002); 3, P. tarimensis DSM 19409T (Wang et al., 2008); 4, P. pinihumi DSM 23905T (Kim et al., 2009). All strains were tested under identical conditions, namely by growth in R2A broth at 18 uC for four days. –, Not determined/not measured; TR, trace amount (,1 %). Fatty acid Straight-chained saturated C14 : 0 C15 : 0 C16 : 0 C17 : 0 C18 : 0 Branched saturated iso-C14 : 0 iso-C15 : 0 iso-C16 : 0 iso-C17 : 0 anteiso-C15 : 0 anteiso-C16 : 0 anteiso-C17 : 0 Unsaturated C16 : 1v11c C17 : 1v12c anteiso-C17 : 1v12 C18 : 1v9c Total
1
1.0 0.6 5.7 – 1.7 1.2 1.4 4.8 TR
2
3
4
–
– – 8.4 2.1 46.6
– 2.2 12.4 2.5 5.3 – 5.9 12.2 20.5 13.4 1.1 17.6
TR
11.7 16.1 16.8 –
–
TR
TR
3.6 8.1
– 3.3
63.4 2.6 4.5
TR
TR
– 32.3
– 30.5
8.1 – 3.1 – 98.1
2.3 3.0 3.5 2.5 99.9
1.6 – 3.2 4.3 100
5.3 TR TR TR
98.4
& Miller, 1988). In the 16S rRNA gene-based ML tree, strain BrT clusters with P. daejeonensis AP-20T (AF290916), P. tarimensis SA-7-6T (EF125184) and P. pinihumi S23T (GQ423057), albeit with weak bootstrap support (Fig. 2). However, this clustering pattern was also observed in 16S rRNA gene-based phylogenetic trees constructed by MP and NJ methods (Fig. S3). In addition, phylogenetic trees based on RpoB protein sequences exhibited similar clustering of strain BrT with P. daejeonensis and P. pinihumi (Fig. S4). 16S rRNA gene sequence similarity values revealed that strain BrT shares 95.1 % 16S rRNA gene sequence similarity with P. tarimensis SA-7-6T, 94.5 % similarity with P. pinihumi S23T, and 93.9 % similarity with P. daejeonensis AP-20T. Overall the highest 16S rRNA gene sequence similarity shown by strain BrT as compared with other recognized species of the genus Paenibacillus was 95.1 %. According to Stackebrandt & Goebel (1994) an isolate with less than 97 % 16S rRNA gene sequence similarity to any known microbial strain represents a novel species, thereby providing evidence for the distinction of strain BrT as a representative of a novel species within genus Paenibacillus. In conclusion, the morphological, physiological, chemotaxonomic and phylogenetic data for strain BrT are in accordance with characteristics of members of genus Paenibacillus. However, the differences in the cellular fatty acid content and in the 16S rRNA gene similarity clearly distinguish strain BrT as a representative of a novel species within the genus Paenibacillus, for which the name Paenibacillus darwinianus sp. nov. is proposed. Description of Paenibacillus darwinianus sp. nov.
DNA was extracted from strain BrT by mechanical cell disruption (bead-beating) as described by Foght et al. (2004). The nearly complete 16S rRNA gene sequence was obtained by PCR amplification using universal primers, PB36 and PB38 (Aislabie et al., 2006). The nearly complete 16S rRNA gene sequence was assembled in Geneious version 5.5.6 (Biomatters) (Kearse et al., 2012), aligned via the SINA web aligner and imported into the ARB phylogenetic package using the SILVA 108 database (Ludwig et al., 2004). The RNA polymerase b-subunit (rpoB) protein sequence was extracted from the whole genome sequence available for strain BrT (Dsouza and others, unpublished). In addition, RpoB protein sequences of related species of the genus Paenibacillus were obtained from the Integrated Microbial Genome (IMG) database (Markowitz et al., 2012) and aligned by the CLUSTAL W method (Larkin et al., 2007). Phylogenetic trees based on the nearly complete 16S rRNA gene and RpoB protein sequences from strain BrT and closely related species of the genus Paenibacillus were reconstructed by neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) methods. The topology of each tree was tested in the MEGA 5.2 software by bootstrap analysis based on 1000 resamplings (Tamura et al., 2011). 16S rRNA gene sequence similarity values were obtained using the EzTaxon Server 2.1 (Myers http://ijs.sgmjournals.org
Paenibacillus darwinianus (dar.wi.ni.a9nus. N.L. masc. adj. darwinianus referring to Darwin Mountains, Antarctica, the geographical origin of the type strain). Cells are Gram-stain-variable, rod-shaped (3.1–6.8 mm length; 0.67–1.3 mm width), strictly aerobic and possess a monotrichous flagellum. Ellipsoidal spores (1.3–2 mm) are produced in swollen sporangia. Colonies are circular, flat, opaque and white with an entire margin on PYGV gellan gum plates. Growth is observed between 15 and 37 uC, with ¡4 % (w/v) NaCl and at pH 6–10 in R2A or PYGV media (broth or gellan gum plates). Optimal growth occurs between 18 and 28 uC at pH 7.0 in PYGV or R2A broth. Poor growth is observed in TSB and NB. No growth is observed on MacConkey agar. Positive reactions are observed for catalase, hydrolysis of starch and acid production from D-xylose, galactose, glucose, salicin, sucrose, D-fructose and aesculin. Negative reactions were observed for oxidase, hydrolysis of casein, Tween 80 and gelatin, citrate utilization, acid production from glycerol, erythritol, ribose, L-xylose, D-adonitol, methyl b-D-xyloside, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannoside, N-acetylglucosamine, amygdalin, arbutin, maltose, melezitose, starch, glycogen, D-tagatose, D-fucose, D-arabitol, L-arabitol, gluconate, 1409
M. Dsouza and others
Paenibacillus humicus PC-147T, AM411528 Paenibacillus pasadenensis SAFN-007T, AY167820 Paenibacillus phyllosphaerae PALXIL04T, AY598818 Paenibacillus cellulosilyticus PALXIL 08T, DQ407282 99 Paenibacillus kobensis DSM 10249T, AB073363 Paenibacillus mendelii C/2T, AF537343 72 Paenibacillus sepulcri CCM 7311T, DQ291142 Paenibacillus darwinianus BrT, KF264455 30 Paenibacillus daejeonensis AP-20T, AF290916 54 Paenibacillus pinihumi S23T, GQ423057 Paenibacillus tarimensis SA-7-6T, EF125184 Paenibacillus agarexedens DSM 1327T, AJ345020 94 56 Paenibacillus alkaliterrae KSL-134T, AY960748 Paenibacillus glycanilyticus DS-1T, AB042938 Paenibacillus thailandensis S3-4AT, AB265205 Paenibacillus tundrae A10bT, EU558284 100 97 Paenibacillus xylanilyticus XIL14T, AY427832 Paenibacillus xylanexedens B22aT, EU558281 Paenibacillus wynnii LMG 22176T, AJ633647 Paenibacillus macquariensis DSM 2T, AB073193 97 87 Paenibacillus glacialis KFC91T, EU815294 Paenibacillus antarcticus LMG 22078T, AJ605292 Paenibacillus cookii LMG 18419T, AJ250317 Bacillus circulans NBRC 13626T, AB271747 97 78 Bacillus oceanisediminis H2T, GQ292772 100 Bacillus koreensis BR030T, AY667496 Bacillus cereus WSBC 10204T, Z84578 100
0.11
Fig. 2. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences from strain BrT (highlighted in bold type) and closely related species of the genus Paenibacillus. Bootstrap values (%) for 1000 resamplings are indicated for each node. Nodes with no bootstrap values were not represented in the consensus bootstrap tree. Bar, 0.11 substitutions per compared nucleotide site.
2-ketogluconate, D-arabinose, L-arabinose, D-mannose, methyl a-D-glucoside, cellobiose, lactose, melibiose, trehalose, inulin, raffinose, xylitol, gentiobiose, turanose, D-lyxose, L-fucose and 5-ketogluconate. For PM1 Biolog plates, none of the carbon sources except acetoacetic acid, phenylethylamine, glycl-L-glutamic acid, D-glucose 6-phosphate, D-galactonic acid c-lactone and mucic acid were utilised. anteiso-C15 : 0 is the major cellular fatty acid. Primary lipids are phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. Total phospholipid is 60 % (w/w) of the total lipid. Unidentified lipids include two glycolipids, two phospholipids and three other lipids. MK-7 is the dominant isoprenoid quinone. The type strain, BrT (5DSM 27245T5ICMP 19883T), was isolated from gamma-irradiated soil of the Britannia drift, Darwin Mountains, Antarctica. The genomic DNA G+C content of the type strain is 55.6 mol%.
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