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1 National Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, People's Republic of China
2 College of Biological Science, China Agricultural University, Beijing 100094, People's Republic of China
3 College of Life Science and Engineering, Qiqihar University, Qiqihar 161006, People's Republic of China
4 Key Laboratory of Agro-Microbial and Application, China Agricultural University, Beijing 100094, People's Republic of China
Correspondence
Sanfeng Chen
chensf{at}cau.edu.cn
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ABSTRACT |
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A more detailed phylogenetic tree that includes additional recognized species of the genus Paenibacillus is available as a supplementary figure in IJSEM Online.
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MAIN TEXT |
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created the genus Paenibacillus from the genus Bacillus based on their 16S rRNA gene sequences. Members of the genus Paenibacillus are aerobic or facultatively anaerobic, rod-shaped, endospore-forming bacteria that are widely distributed in nature (Claus & Berkeley, 1986; Slepecky & Hemphill, 1991). Nitrogen fixation has been described in some species of Paenibacillus, such as Paenibacillus polymyxa (Grau & Wilson, 1962), Paenibacillus macerans (Witz et al., 1967), Paenibacillus azotofixans (Seldin et al., 1984), Paenibacillus peoriae (Elo et al., 2001), Paenibacillus graminis, Paenibacillus odorifer (Berge et al., 2002) and Paenibacillus brasilensis (von der Weid et al., 2002). It has been reported that P. azotofixans has the highest nitrogenase activity among these nitrogen-fixers (Achouak et al., 1999; Berge et al., 2002). P. azotofixans has been found in Brazilian and Hawaiian soils, but not in soils from temperate zones, while P. polymyxa has been isolated from various environments around the world on different plant roots (Berge et al., 2002). Here we report the isolation of five novel strains from the rhizosphere of Zanthoxylum simulans (prickly-ash). The five novel strains have nitrogenase activities higher than that of P. azotofixans and their 16S rRNA gene sequences have a high level of similarity to that of P. azotofixans. The strains were identified as a novel species of the genus Paenibacillus based on phenotypic properties, 16S rRNA gene sequences, DNA G+C content, DNA–DNA hybridization, chemotaxonomic properties and nifH gene sequence.
The bacterial strains examined were isolated from the rhizosphere soil of prickly-ash planted in Beijing, China. A 1 g sample of soil was placed in 9 ml sterile water and stirred for 50 min. Aqueous portions (100 µl) were heated at 80 °C for 10 min and then spread on nitrogen-free medium in triplicate and incubated at 30 °C. The nitrogen-free medium used for the isolation of bacterial strains contained (l–1): 20 g sucrose, 0.1 g K2HPO4, 0.4 g KH2PO4, 0.2 g MgSO4.7H2O, 0.1 g NaCl, 0.01 g FeCl3 and 0.002 g Na2MoO4. After 5 days incubation, five strains, designated JH8, JH29T, JH31, JT34 and JH95 were selected for further study.
To determine cell morphology, the five novel strains were grown on endospore-forming medium [0.07 % yeast extract, 0.1 % tryptone, 0.1 % glucose, 0.02 % (NH4)2SO4, 0.02 % MgSO4.7H2O, 0.1 % K2HPO4 (w/v), pH 7.2] for 72 h. Cell morphology was examined by light microscopy. Catalase activity was analysed by bubble formation in a 3 % (w/v) H2O2 solution. Hydrolysis of casein, gelatin and starch was determined as described by Cowan & Steel (1965). Utilization of various substrates as carbon and energy sources was determined as described by Shirling & Gottlieb (1966). Tolerance to NaCl was measured in a medium [0.5 % peptone, 0.3 % (w/v) beef extract] containing NaCl at concentrations of 3 and 5 % (w/v). Other physiological and biochemical tests were also performed using conventional methods (Priest et al., 1981; Rhodes-Roberts, 1981).
Analysis of cellular fatty acid contents was carried out as described by Komagata & Suzuki (1987) using the Sherlock Identification System (MIDI).
To determine the nitrogen-fixing capability of the five strains, an assay for nitrogenase activity and PCR amplification of the nifH gene were carried out. A 324 bp fragment of the nifH gene was isolated from the five novel strains as described by Ding et al. (2005). Nitrogenase activity was determined by the acetylene-reduction assay method (Berge et al., 2002).
Chromosomal DNA was extracted and purified according to standard method (Sambrook et al., 1989). The 16S rRNA gene sequence was amplified as described by Yanagi & Yamasato (1993) and sequenced (Ding et al., 2005). Sequences were aligned using CLUSTAL_X software (Thompson et al., 1997). Evolutionary distances were calculated using the DNADIST program from the TREECONW software package. A phylogenetic tree was generated by the neighbour-joining method using the TREECONW software package (Van der Peer & De Wachter, 1994).
DNA–DNA hybridization and DNA G+C content were determined as described by De Ley et al. (1970).
The nearly complete 16S rRNA gene sequences of the five novel strains were compared with sequences held in GenBank. This analysis indicated that the novel strains are phylogenetically related to members of the genus Paenibacillus. A neighbour-joining phylogenetic tree showing the position of the novel strains in relation to some other Paenibacillus species is shown in Fig. 1. A more detailed phylogenetic tree that includes additional recognized species of the genus Paenibacillus is available as Supplementary Fig. S1 in IJSEM Online. The five novel strains showed high levels of 16S rRNA gene sequence similarity with P. azotofixans ATCC 35681T (97.81–98.5 %) and Paenibacillus stellifer DSM 14472T (95.39–96.25 %). Levels of 16S rRNA gene sequence similarity between the five novel strains and other species of Paenibacillus were less than 95.0 %. Levels of 16S rRNA gene sequence similarity among the five novel strains were more than 98.0 %.
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. The five novel strains were found to have physiological properties that enabled them to be distinguished from these recognized Paenibacillus species. The DNA G+C contents of strains JH29, JH8, JH31, JH34 and JH95 were 52.3–54.4 mol% (Table 1), which are in the range characteristic of recognized Paenibacillus species (Shida et al., 1997).
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). The five novel strains showed high levels of nifH gene sequence similarity with P. azotofixans ATCC 35681T (97.20–98.15 %), P. polymyxa (93.18–95.45 %) and P. odorifer (88.79 %). Levels of nifH gene sequence similarity among the five novel strains were 97.22–100 %.
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). As shown in Table 2, all of the novel strains exhibited significant nitrogenase activity.
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The cellular fatty acid profile of one of the novel isolates, JH29T, was determined together with those of P. azotofixans ATCC 35681T, P. stellifer DSM 4472T, P. polymyxa DSM 36T and P. macerans ATCC 8244T (Table 3). Anteiso-branched C15 : 0, the major fatty acid characteristic for the genus Paenibacillus, was also the major fatty acid component (32.19 %) of strain JH29T.
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Description of Paenibacillus zanthoxyli sp. nov.
Paenibacillus zanthoxyli (zan'th.ox.y.li. N.L. gen. n. zanthoxyli of Zanthoxylum, referring to the plant Zanthoxylum simulans, the source of the rhizosphere soil from which the type strain was isolated).
Gram-positive, facultatively anaerobic, motile, straight rods (0.35–0.4x4.0–4.8 µm). Oval or ellipsoidal endospore located subterminally or centrally in swollen sporangia. Colonies on nutrient agar are circular, convex and glossy with entire margins. Growth occurs at pH 4.2–10.0 and at 4–37 °C. Growth occurs in 3 % (w/v) NaCl, but not in 0.001 % (w/v) lysozyme. Catalase activity is positive and oxidase activity is negative. Voges–Proskauer and methyl-red reactions are positive. Nitrogen fixation is detected by the presence of the nifH gene and acetylene reduction in all strains. Acid and gas are not produced from glucose, sucrose, lactose, fructose, glycerol, xylose, maltose, D-sorbitol, sodium succinate, sodium citrate, glycine or L-aspartate. Gelatin, casein and starch are not hydrolysed. The major cellular fatty acids of the type strain, JH29T, are anteiso-C15 : 0, iso-C16 : 0 and C16 : 0. The DNA G+C content of the type strain is 53.2 mol%.
The type strain, JH29T (=CCBAU 10243T=DSM 18202T), was isolated from the rhizosphere soil of Zanthoxylum simulans planted in Beijing, China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Ash, C., Priest, F. G. & Collins, M. D. (1993). Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test. Proposal for the creation of a new genus Paenibacillus. Antonie van Leeuwenhoek 64, 253–260.[CrossRef][Medline]
Berge, O., Guinebretière, M. H., Achouak, W., Normand, P. & Heulin, T. (2002). Paenibacillus graminis sp. nov. and Paenibacillus odorifer sp. nov., isolated from plant roots, soil and food. Int J Syst Evol Microbiol 52, 607–616.[Abstract]
Claus, D. & Berkeley, R. C. W. (1986). Genus Bacillus Cohn 1872, 174AL. In Bergey's Manual of Systematic Bacteriology, vol. 2, pp. 1105–1139. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. London: Cambridge University Press.
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
Ding, Y., Wang, J., Liu, Y. & Chen, S. (2005). Isolation and identification of nitrogen-fixing bacilli from plant rhizospheres in Beijing region. J Appl Microbiol 99, 1271–1281.[CrossRef][Medline]
Elo, S., Suominen, I., Kämpfer, P., Juhanoja, J., Salkinoja-Salonen, M. & Haahtela, K. (2001). Paenibacillus borealis sp. nov., a nitrogen-fixing species isolated from spruce forest humus in Finland. Int J Syst Evol Microbiol 51, 535–545.[Abstract]
Grau, F. H. & Wilson, P. W. (1962). Physiology of nitrogen-fixation by Bacillus polymyxa. J Bacteriol 83, 490–496.
Komagata, K. & Suzuki, K. (1987). Lipid and cell wall analysis in bacterial systematics. Methods Microbiol 19, 161–207.
Priest, F. G., Goodfellow, M. & Todd, C. (1981). The genus Bacillus: a numerical analysis. In The Aerobic Endospore-forming Bacteria Classification and Identification, pp. 91–103. Edited by R. C. W. Berkeley & M. Goodfellow. London: Academic Press.
Rhodes-Roberts, M. (1981). The taxonomy of some nitrogen-fixing Bacillus species with special reference to nitrogen fixation. In The Aerobic Endospore-Forming Bacteria. Classification and Identification, pp. 315–335. Edited by R. C. W. Berkeley & M. Goodfellow. London: Academic Press.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Seldin, L., van Elsas, J. D. & Penido, E. G. C. (1984). Bacillus azotofixans sp. nov., a nitrogen-fixing species from Brazilian soils and grass roots. Int J Syst Bacteriol 34, 451–456.
Shida, O., Takagi, H., Kadowaki, K., Nakamura, L. K. & 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.
Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16, 313–340.[Medline]
Slepecky, E. B. & Hemphill, E. (1991). The genus Bacillus nonmedical. In The Prokaryotes, vol. 2, pp. 1663–1696. Edited by A. Balows, H. G. Truper, M. Dworkin, W. Harder & K.-H. Schleifer. New York: Springer.
Suominen, I., Spröer, C., Kämpfer, P., Rainey, F. A., Lounatmaa, K. & Salkinoja-Salonen, M. S. (2003). Paenibacillus stellifer sp. nov., a cyclodextrin-producing species isolated from paperboard. Int J Syst Evol Microbiol 53, 1369–1374.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Van der Peer, Y. & de Wachter, R. (1994). TREECON for Windows: a software package for construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570.
von der Weid, I., Duarte, G. F., van Elsas, J. D. & Seldin, L. (2002). Paenibacillus brasilensis sp. nov., a novel nitrogen-fixing species isolated from the maize rhizosphere in Brazil. Int J Syst Evol Microbiol 52, 2147–2153.[Abstract]
Witz, D. F., Detroy, R. W. & Wilson, P. W. (1967). Nitrogen fixation by growing cells and cell-free extracts of the Bacillaceae. Arch Mikrobiol 55, 369–381.[CrossRef][Medline]
Yanagi, M. & Yamasato, K. (1993). Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer. FEMS Microbiol Lett 107, 115–120.[CrossRef][Medline]
Yoon, J. H., Oh, H. M., Yoon, B. D., Kang, K. H. & Park, Y. H. (2003). Paenibacillus kribbensis sp. nov. and Paenibacillus terrae sp. nov., bioflocculants for efficient harvesting of algal cells. Int J Syst Evol Microbiol 53, 295–301.
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