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1 Korea Research Institute of Bioscience and Biotechnology, 52 Oeundong, Yusong, Daejeon 305-333, Republic of Korea
2 Division of Applied Life Science, EB-NCRC, PMBBRC, Gyeongsang National University, Jinju, 660-701, Republic of Korea
3 Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan, 650091, PR China
Correspondence
Chang-Jin Kim
changjin{at}kribb.re.kr
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ABSTRACT |
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meso-diaminopimelic acid; major fatty acids, anteiso-C15 : 0 and C16 : 0) supported affiliation of the isolate with the genus Paenibacillus. Comparative 16S rRNA gene sequence analyses showed that the isolate was most closely related to Paenibacillus glycanilyticus DS-1T, with 16S rRNA gene sequence similarity of 98.1 %; sequence similarities to other members of the genus Paenibacillus used in the phylogenetic tree were less than 96.5 %. The DNA–DNA relatedness between strain B538T and P. glycanilyticus DS-1T was about 8.0 %. On the basis of physiological and molecular properties, strain B538T (=KCTC 3952T=DSM 16970T) is proposed as the type strain of a novel species within the genus Paenibacillus, for which the name Paenibacillus xinjiangensis sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain B538T is AY839868.
A table giving the fatty acid composition of strain B538T and related strains and a TEM showing the general morphology of strain B538T are available as supplementary material in IJSEM Online.
These authors contributed equally to this work. 
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MAIN TEXT |
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. Since the description of the genus, quite a few species belonging to the genus Paenibacillus have been isolated from various ecological habitats including warm springs (Saha et al., 2005), rice fields (Sánchez et al., 2005), alkaline soils (Yoon et al., 2005), petroleum-hydrocarbon-contaminated sediment (Daane et al., 2002), the surface of sterilized seeds of garden peas (
merda et al., 2005), Antarctic sediments (Montes et al., 2004), the rhizosphere of trees (Rivas et al., 2005c) and air (Rivas et al., 2005a). These species also differ physiologically and produce diverse degradation enzymes (Rivas et al., 2005b; Takeda et al., 2005; Shida et al., 1997). These findings suggest that the members of the genus Paenibacillus may have important roles in environmental habitats. In this report, the taxonomic characterization is described of a novel aerobic, mesophilic, Gram-positive Paenibacillus species, which produces a spherical terminal endospore in a swollen sporangium.
Strain B538T was isolated from alkaline soil (pH 9.0, 1000 m above sea-level) collected in northern China (Heshuo area of Xinjiang province) by the serial dilution plating method with R2A agar (Difco) medium at 35 °C for 3 days. The isolate was routinely grown aerobically on TSB (tryptic soy broth; Difco) agar for 2 days at 32 °C, except where indicated otherwise. Gram staining was performed using the bioMérieux Gram Stain kit according to the manufacturer's instructions. Cell morphology was studied using TEM (JEM-1010; JEOL) as described by Jeon et al. (2005a). Motility was observed at 12 and 36 h with a light microscope (Nikon E600) according to a method described previously (Jeon et al., 2005b). Endospores were stained according to the method of Schaeffer-Fulton (Smibert & Krieg, 1981). Growth was tested at different temperatures (4–55 °C) on TSB agar and at different pH values in TSB. Media with different pH values were prepared using appropriate biological buffers; Na2HPO4/NaH2PO4 buffer, Na2CO3/NaHCO3 buffer and Na2HPO4/NaOH buffer were used for pH below 8.0, pH 8.0–10.0 and pH 11.0, respectively (Gomori, 1955). Oxidase activity was tested using Bactident Oxidase strips (Merck) and catalase activity was evaluated by production of oxygen bubbles in 3 % (v/v) aqueous hydrogen peroxide solution. Nitrate reduction and the hydrolysis of compounds were checked on TSB agar according to methods described previously (Lanyi, 1987; Smibert & Krieg, 1994). Acid production from D-glucose, lactose, glycerol, L-rhamnose, D-xylose, sucrose, D-melibiose, D-trehalose, maltose, D-raffinose, D-ribose, D-mannose, L-arabinose, D-fructose, D-mannitol, adonitol and salicin was evaluated in TSB containing 0.001 % (w/v) phenol red, 0.3 % (w/v) agar and 1 % (w/v) each carbohydrate according to the modified method of Leifson (1963) after 3 days cultivation at 32 °C. Strain B538T formed creamy, circular/slightly irregular and slightly convex colonies on TSB agar when grown at 32 °C for 2 days. The strain grew optimally on TSB agar without added NaCl and TSB agar supplemented with 1 % (w/v) NaCl, but did not grow on TSB agar with the addition of 4 % (w/v) NaCl or more. Growth occurred between 10 and 40 °C (optimum of 30–35 °C) and at pH 6.5–9.8 (optimum of pH 8.0–8.5). Cells of the isolate were motile short rods (0.8–1.2 µm wide and 2.0–3.2 µm long) with peritrichous flagella (see Supplementary Fig. S1 available in IJSEM Online). Strain B538T was Gram-positive, oxidase-negative, catalase-positive and did not reduce nitrate to nitrite. A spherical endospore was formed at a terminal position in a swollen sporangium. Anaerobic growth was not observed during anaerobic cultivation over 5 days at 32 °C on TSA.
Whole-cell fatty acids of strain B538T were analysed according to the Microbial Identification System (MIDI; Microbial ID) after cultivation on R2A, TSB agar or twofold-diluted nutrient agar (Difco) for 2 days at 32 °C. Analysis of peptidoglycan and isoprenoid quinones was carried out as described by Komagata & Suzuki (1987). The genomic DNA G+C content of strain B538T was determined by HPLC fitted with a reversed-phase column (GROM-SIL 100 ODS-2FE; GROM) according to the method of Tamaoka & Komagata (1984). The major isoprenoid quinone was menaquinone-7 (MK-7). The predominant cellular fatty acids of strain B538T when grown on TSB agar were anteiso-C15 : 0 (48.61 %), C16 : 0 (14.43 %), iso-C16 : 0 (9.65 %) and iso-C15 : 0 (9.32 %); no significant differences in the fatty acid profile were found when the isolate was grown on R2A. The fatty acid profile was similar to those reported for other related type strains of the genus Paenibacillus (Supplementary Table S1 in IJSEM Online). Analysis of cell wall peptidoglycan showed that strain B538T possessed type A1 with meso-diaminopimelic acid as the diagnostic diamino acid. The genomic DNA G+C content of strain B538T was 47.0 mol%. Typical phenotypic properties of strain B538T are summarized in Table 1 along with those of the type strains of closely related taxa. The characteristics of strain B538T are consistent with those described for members of the genus Paenibacillus (Table 1), i.e. they produce a terminal endospore in a swollen sporangium, possess anteiso-C15 : 0 as the major fatty acid and have a DNA G+C content in the range 39–55 mol% (Shida et al., 1997; Montes et al., 2004; Takeda et al., 2005).
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). The resultant 16S rRNA gene sequence of strain B538T was compared with available 16S rRNA gene sequences from GenBank using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/) to determine an approximate phylogenetic affiliation and the gene sequences were aligned with closely related members using CLUSTAL W software (Thompson et al., 1994). Phylogenetic trees were constructed using three different methods, neighbour-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP) algorithms, which are available in the PHYLIP software, version 3.6 (Felsenstein, 2002). Sequence similarity values were computed using Similarity Matrix version 1.1 (Ribosomal Database Project II; http://35.8.164.52/html/; Cole et al., 2003). Bootstrap analysis (1000 replications) was performed to evaluate the stability of the phylogenetic tree according to the algorithm of the Kimura two-parameter model (Kimura, 1980) of the NJ method in the PHYLIP package.
DNA–DNA hybridization was carried out to evaluate the genomic DNA relatedness between strain B538T and Paenibacillus glycanilyticus DS-1T. Extracted genomic DNAs were fragmented with HaeIII for slot hybridization. Digested DNAs were diluted serially and loaded into slots with three replications for each sample and their DNAs were used individually as labelled DNA probes for cross-hybridization. Random primed DNA labelling with digoxigenin-dUTP and hybridization (hybridization temperature, 46 °C; washing temperature, 65 °C) were performed using a DIG High Prime DNA labelling kit (Roche Applied Science) according to the manufacturer's instructions and standard procedures (Sambrook & Russell, 2001; Lim et al., 2005). The signals of the series of dilutions were quantified using Bio-Rad GelDoc scanning software. The signals produced by self-hybridization were inferred as 100 % and hybridization values (%) were calculated from results of three samples. Phylogenetic analysis using a nearly complete 16S rRNA gene sequence (1521 nt) of strain B538T indicated that the isolate formed a phylogenetic cluster with P. glycanilyticus DS-1T with a 99.7 % bootstrap value within the genus Paenibacillus (Fig. 1). Topologies of phylogenetic trees built using the ML and MP algorithms were similar to that of the tree constructed by NJ analysis (data not shown). Comparative 16S rRNA gene sequence analyses showed that the isolate was most closely related to P. glycanilyticus DS-1T, with a 16S rRNA gene sequence similarity of 98.1 %; sequence similarities to other members of the genus Paenibacillus used in the phylogenetic tree were less than 96.5 %. The DNA–DNA relatedness between strain B538T and P. glycanilyticus DS-1T was about 8 %, which is sufficient for strain B538T to be classified within a different species (Stackebrandt et al., 2002). Therefore, on the basis of physiological, biochemical and phylogenetic properties, strain B538T should be considered as representing a novel species within the genus Paenibacillus, for which the name Paenibacillus xinjiangensis sp. nov. is proposed.
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Cells are approximately 0.8–1.2 µm wide and 2.0–3.2 µm long. Strictly aerobic, spore-forming, motile rods. Catalase-positive and oxidase-negative. Nitrate is not reduced to nitrite. Colonies are smooth, circular to slightly irregular, slightly convex and cream coloured on TSB agar. Growth occurs at 10–40 °C (optimum, 30–35 °C), 0–3 % (w/v) NaCl (optimum, 0–1 %) and pH 6.5–9.8 (optimum, pH 8.0–8.5). Hydrolyses aesculin. Does not hydrolyse casein, starch, Tweens 20 or 80, L-tyrosine, hypoxanthine, xanthine or urea. Acids are produced from D-glucose, lactose, glycerol, L-rhamnose, D-xylose, sucrose, D-melibiose, D-trehalose, maltose, D-raffinose, D-ribose, D-mannose, L-arabinose and D-fructose, but not from D-mannitol, adonitol or salicin. Cell wall contains meso-diaminopimelic acid (A1 type). The predominant menaquinone is MK-7. The major cellular fatty acids on TSB agar are anteiso-C15 : 0 (48.61 %), C16 : 0 (14.43 %), iso-C16 : 0 (9.65 %) and iso-C15 : 0 (9.32 %). DNA G+C content is 47.0 mol% (HPLC).
The type strain is B538T (=KCTC 3952T=DSM 16970T), isolated from Xinjiang province in China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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