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Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-0032, Japan
Correspondence
Cheng-Hui Xie
cxie{at}uams.edu
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), which comprises aerobic or facultatively anaerobic endospore-forming bacilli, is phenotypically related to other genera belonging to the family Bacillaceae. Phylogenetic analysis based on 16S rRNA gene sequences is the most useful method for identifying Paenibacillus species and differentiating them from other members of the family Bacillaceae. The genus currently contains 69 recognized species and the type species is Paenibacillus polymyxa. During the screening of nitrogen-fixing bacteria in a coastal soil sample from Chiba, Japan, a novel Gram-positive bacterium, designated strain A35T, was isolated. Strain A35T neither fixes nitrogen nor possesses the nifH gene (Xie & Yokota, 2006). On the basis of 16S rRNA gene sequence analysis, strain A35T showed a very low level of similarity (92 %) to the type species of the genus Paenibacillus, P. polymyxa. The next most closely related species were Paenibacillus koreensis (GenBank accession no. AF130254), Paenibacillus larvae (X60619) and Paenibacillus validus (AB073203), which showed approximately 94 % 16S rRNA gene sequence similarity to strain A35T. In comparisons with other recognized species of the genus Paenibacillus, the novel strain showed less than 94 % similarity. To elucidate the taxonomic position of strain A35T, we used a polyphasic taxonomic approach that included physiological tests and analyses of the fatty acid content, quinone system, DNA G+C content and 16S rRNA gene sequence. On the basis of the phenotypic and phylogenetic analyses, it is proposed that strain A35T represents a novel species of the genus Paenibacillus.
Strain A35T was incubated in nutrient broth (NB; Difco) at 29 °C. Cell morphology and motility were examined by phase-contrast microscopy (BX60; Olympus). Growth under anaerobic growth conditions was determined after 2 weeks incubation in an AnaeroPack (Mitsubishi Gas Chemical Co.). Gram staining was performed using 3 % KOH. Catalase activity was determined using 3 % H2O2 with the production of bubbles indicating a positive result. Oxidase was determined using cytochrome oxidase paper (Nissui Pharmaceutical Co.). API 20E and API 50 CH microtest galleries (bioMérieux) were used to determine physiological and biochemical characteristics. API strips were incubated for 2 days at 30 °C. Tolerance of salinity was determined by inoculating cells into NB supplemented with 0–4.0 % NaCl (w/v). For fatty acid analysis, cells were incubated on trypticase soy agar medium (Becton Dickinson) for 3 days at 29 °C. Cellular fatty acid methyl esters were prepared, separated and identified by using the Microbial Identification System (Microbial ID). Cells used to determine the respiratory quinone system and the DNA G+C content were incubated in NB for 5 days at 29 °C and then characterized by HPLC (Shimadzu) as described by Xie & Yokota (2003). Genomic DNA was isolated according to the method of Marmur (1961) using cells cultured for 2 days in NB. An acetylene-reduction assay and growth in nitrogen-free medium were used to determine nitrogenase activity (Xie & Yokota, 2006).
A fragment (approx. 1500 bp) of the 16S rRNA gene sequence was amplified by a PCR and sequenced as described by Xie & Yokota (2003). The 16S rRNA gene sequences obtained in this study and from GenBank were aligned using the CLUSTAL W software package (Thompson et al., 1994), and the evolutionary distances and the Knuc value (Kimura, 1980) were generated. Alignment gaps and ambiguous bases were excluded from the calculation. A phylogenetic tree based on a comparison of 1189 bases was constructed using the neighbour-joining method (Saitou & Nei, 1987). The topology of the tree was evaluated by using the bootstrap resampling method of Felsenstein (1985) with 1000 replicates. Similarity values were calculated using PAUP, version 4.0b1 (Swofford, 1998).
The 16S rRNA gene sequence search using BLAST revealed that strain A35T exhibited very low levels of similarity (not more than 94 %) with respect to the recognized species of Paenibacillus, which is sufficient to indicate that strain A35T represents a novel member of the genus (Stackebrandt & Goebel, 1994). The phylogenetic tree (Fig. 1) showed that strain A35T was affiliated to the genus Paenibacillus but was not closely related to any other species as poor bootstrap support (less than 60 %) was found at the branch node.
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). The presence of MK-7 as the major quinone system, the G+C DNA content (48.1 mol%) and the presence of anteiso-C15 : 0 as the major fatty acid (56.4–57.1 %) reflected the similarity of strain A35T to all members of the genus Paenibacillus (Shida et al., 1997). The fatty acid contents of the novel strain and the type strains of selected Paenibacillus species are shown in Table 1. Phenotypic differences between strain A35T and the reference strains are shown in Table 2.
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Description of Paenibacillus terrigena
Paenibacillus terrigena [ter.ri.ge'na. L. n. terra soil; L. v. gignere to bear; L. n. terrigena (nominative in apposition) born of, or from, the earth, earth-born].
Cells are Gram-positive. Vegetative cells are motile rods; terminal, oval spores can be observed within swollen sporangia. Facultatively anaerobic. Grows at 4–32 °C, pH 4.0–10.0 and 3.5 % NaCl. Shows poor growth in carbohydrate-containing media (determined on O/F basal medium; Xie & Yokota, 2006), but grows well in Luria–Bertani and trypticase soy agar media, producing irregular, smooth, cream-coloured colonies. Catalase-, oxidase- and 
-galactosidase-positive. Gives a positive result for nitrate reduction, aesculin hydrolysis and in the Voges–Proskauer test. Acid is produced from glucose, sucrose, melibiose and amygdalin. Glucose, glycerol, ribose, D-xylose, amygdalin, arbutin, methyl -D-xyloside, galactose, methyl 
-D-mannoside, methyl -D-glucoside, aesculin, salicin, cellobiose, lactose, melibiose, sucrose, raffinose, starch, gentiobiose, gluconate and D-turanose are assimilated. N-acetylglucosamine and maltose are poorly assimilated. Erythritol, arabinose, L-xylose, adonitol, fructose, mannose, sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, trehalose, inulin, melezitose, glycogen, xylitol, D-lyxose, tagatose, fucose, arabitol, caprate, adipate, malate, citrate, phenylacetate and 2-ketogluconate are not utilized. The major quinone system is MK-7. The DNA G+C content is 48.1 mol%. The fatty acids comprise anteiso-C15 : 0 (56.4–57.1 %), iso-C16 : 0 (13.0 %), anteiso-C17 : 0 (7.9–8.1 %), iso-C15 : 0 (5.7–5.8 %), C16 : 0 (4.0–4.4 %), C15 : 0 (3.8–4.1 %), iso-C14 : 0 (2.6 %), iso-C17 : 0 (1.9–2.1 %) and C14 : 0 (0.8 %).
The type strain, A35T (=IAM 15291T=CCTCC AB206026T), was isolated from coastal soil from Chiba, Japan.
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REFERENCES |
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Chung, Y. R., Kim, C. H., Hwang, I. & Chun, J. (2000). Paenibacillus koreensis sp. nov., a new species that produces an iturin-like antifungal compound. Int J Syst Evol Microbiol 50, 1495–1500.[Abstract]
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Shida, O., Takagi, H., Kadowaki, K., Nakamura, L. & Komagata, K. (1997). 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.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Swofford, D. L. (1998). PAUP*: Phylogenetic analysis using parsimony (* and other methods), version 4. Sunderland, MA: Sinauer Associates.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Xie, C.-H. & Yokota, A. (2003). Phylogenetic analyses of Lampropedia hyalina based on the 16S rRNA gene sequence. J Gen Appl Microbiol 49, 345–349.
Xie, C.-H. & Yokota, A. (2006). Reclassification of [Flavobacterium] ferrugineum as Terrimonas ferruginea gen. nov., comb. nov., and description of Terrimonas lutea sp. nov., isolated from soil. Int J Syst Evol Microbiol 56, 1117–1121.
Yoon, J.-H., Seo, W.-T., Shin, Y.-K., Kho, Y.-H., Kang, K.-H. & Park, Y.-H. (2002). Paenibacillus chinjuensis sp. nov., a novel exopolysaccharide-producing bacterium. Int J Syst Evol Microbiol 52, 415–421.[Abstract]
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