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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
aa37116{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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7c as the major fatty acid and 14 : 0 3-OH and 16 : 0 3-OH as the major hydroxy fatty acids) were also similar to those of the genus Azospirillum. Based on its physiological characteristics, strain COC8T can clearly be differentiated from recognized species of Azospirillum. The name Azospirillum oryzae sp. nov. is proposed to accommodate this bacterial strain; the type strain is COC8T (=IAM 15130T=CCTCC AB204051T).
-hydroxybutyratePublished online ahead of print on 11 February 2005 as DOI 10.1099/ijs.0.63503-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and nifH gene sequences of strain COC8T are AB185396 and AB185395, respectively.
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MAIN TEXT |
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are associated with grasses, cereals and crops. The genus currently comprises seven species; the type species is Azospirillum lipoferum. Cells are nitrogen-fixing, curved rods that are motile with a single polar flagellum; they have been found previously in association with the rice plant (Khammas et al., 1989; Ladha et al., 1987). Strain COC8T was isolated from the paddy soil of a rice plant in 1982, and was reported to be similar to A. lipoferum based on its phenotypic characteristics (Oyaizu-Masuchi & Komagata, 1988). However, the strain has remained unidentified at the species level. The purpose of this study was to clarify the taxonomic position of this strain based on phylogenetic analysis of its 16S rRNA gene sequence together with those of other representatives of the genus Azospirillum.
All strains investigated were incubated on M medium (5·0 g sodium malate, 0·02 g CaCl2.2H2O, 0·2 g MgSO4.7H2O, 0·1 g K2HPO4, 0·4 g KH2PO4, 0·1 g NaCl, 10 mg FeCl3, 2 mg Na2MoO4.2H2O, 0·1 g yeast extract, 2 µg biotin, 1·0 l distilled water, pH 6·8) and NFG medium (10·0 g glucose, 20 mg CaCl2.2H2O, 0·2 g MgSO4.7H2O, 1·0 g K2HPO4, 5 g CaCO3, 50 mg FeSO4.7H2O, 1 mg Na2MoO4.2H2O, 1·0 l distilled water, pH 7·3). Methods used for phenotypic characterization (determination of the DNA base composition and quinone system and acetylene reduction assay) were as described by Oyaizu-Masuchi & Komagata (1988). Cellular fatty acid methyl esters were prepared, separated and identified using the Microbial Identification system as described by Xie & Yokota (2003). The fatty acid composition could not be clearly identified by the MIDI system (Microbial ID, Inc.). For example, summed features 2 and 3 were further analysed as follows: fatty acid samples and standard non-polar fatty acids and hydroxy fatty acids used for comparison were developed on a TLC plate (silica-gel F254; Merck) with hexane/ethyl ether (1 : 1), sprayed with a 0·02 % dichlorofluorescein ethanol solution and dried and detected under UV light. The separated spots of non-polar fatty acids and hydroxy fatty acids were scraped from the plates, transferred to tubes and extracted with ethyl ether. These extracts were then concentrated under a N2 gas stream and dissolved in hexane/methyl tert-butyl ether (1 : 1). The separated and purified non-polar fatty acids and hydroxy fatty acids were then again identified by using the MIDI system. Summed features 2 and 3 were identified to be 14 : 0 3-OH and 16 : 17c, respectively. PCR-mediated amplification of the 16S rRNA gene and sequencing of the PCR products were carried out as described by Xie & Yokota (2003). A 420-base fragment of the nifH gene (encoding dinitrogenase reductase) was amplified from the extracted DNA using the forward primer IGK (5'-TACGGYAARGGBGGYATCGG) and the backward primer AQE (5'-GACGATGATYTCCTG) (Y=C/T; S=G/C; R=A/G; B=C/G/T) (Xie & Yokota, 2004). The DNA sequences were compared with sequences obtained from DDBJ/GenBank and aligned with the CLUSTAL W software package (Thompson et al., 1994); evolutionary distances and the Knuc value (Kimura, 1980) were then calculated. Alignment gaps and ambiguous bases were excluded from the calculations. A phylogenetic tree based on comparison of 1108 bases was constructed using the neighbour-joining method (Saitou & Nei, 1987). The topology of this phylogenetic tree was evaluated by using the bootstrap resampling method of Felsenstein (1985) with 1000 replicates, and similarity values were calculated using PAUP 4.0b1 (Swofford, 1998). Using similar methodology, 408-base partial nifH sequences were also aligned and a phylogenetic tree was constructed.
16S rRNA gene sequence analyses indicated that the closest relatives to strain COC8T were A. lipoferum and Azospirillum largimobile (96·0 % similarity); values of not more than 95·0 % were obtained against other recognized species of Azospirillum. When alignment gaps and ambiguous bases in the 16S rRNA gene sequences were excluded from the calculations, similarity values for strain COC8T against A. lipoferum and A. largimobile were 96·3 and 96·8 %, respectively. A. lipoferum was the first species assigned to Azospirillum (Tarrand et al., 1978), and A. largimobile was subsequently reclassified from the genus Conglomeromonas (Skerman et al., 1983). Unfortunately, the type strain ACM 2041T of this species is no longer extant, either with the authors who originally described it (Ben Dekhil et al. 1997) or with the ACM Bacteria Collection, where the strain was deposited. However, based on the fact that the 16S rRNA gene sequences differed by more than 3 %, which is a level sufficient to allow the proposal of a new species (Stackebrandt & Goebel, 1994), strain COC8T was considered to represent a novel species of the genus Azospirillum. The results of the neighbour-joining analysis of the 16S rRNA gene sequence are shown in Fig. 1.
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). Moreover, a partial fragment of the nifH gene was amplified in this study, suggesting that strain COC8T is a nitrogen-fixing bacterium. The expected 420-bp nifH sequence of strain COC8T was used as a database search, and highest sequence similarities (91 %) were shown by A. lipoferum and Azospirillum brasilense; however, similarities with other diazotrophic bacteria were not more than 89 %. The results of the neighbour-joining analysis of the nifH sequence (Fig. 2) revealed that strain COC8T was closely related to Azospirillum species (69 % bootstrap support).
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-hydroxybutyrate (PHB) granules were observed in the cells. These properties matched the general characteristics of the genus Azospirillum. The physiological properties of strain COC8T were similar to those of its nearest relatives A. largimobile and A. lipoferum: acid production from glucose aerobically and anaerobically; growth with glucose, D-galactose, D-fructose, D-ribose and L-arabinose as the sole carbon source; positive for catalase, oxidase, urease, DNase and nitrate reduction; aesculin hydrolysis; and no growth with lactose or D-cellobiose, or on 3 % NaCl. However, they could be differentiated from each other based on several physiological properties (Table 1). Cells of strain COC8T have only one polar flagellum whereas those of A. largimobile have 1–10 distinctive lateral flagella (Ben Dekhil et al., 1997). On media containing sodium ions, multicellular conglomerates of A. largimobile develop from a single cell and become immobile (Ben Dekhil et al., 1997); strain COC8T shows no such characteristic. Strain COC8T can utilize hydrogen for growth (Oyaizu-Masuchi & Komagata, 1988).
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). As with A. lipoferum and A. brasilense, strain COC8T had a ubiquinone (Q-10) quinone system. The fatty acid profile of strain COC8T included 18 : 17c (55·3 %), 16 : 17c (16·1 %), 16 : 0 (6·2 %), 19 : 0 cyclo 8c (3·8 %), 13 : 1 at 12–13 (3·3 %), unknown ECL 10·922 (3·0 %) and 14 : 0 (0·9 %); hydroxy fatty acids were 14 : 0 3-OH (6·7 %) and 16 : 0 3-OH (4·8 %). This Sherlock MIS result differs slightly from that identified by using GLC (Oyaizu-Masuchi & Komagata, 1988). However, the two methods are in broad agreement that the genus Azospirillum has a large amount of 18 : 17c (55·3 %), contains 16 : 17c, 16 : 0 as a major component, and that the major hydroxy fatty acids are 14 : 0 3-OH and 16 : 0 3-OH. The results obtained from the chemotaxonomic analyses were consistent with phylogenetic analysis, indicating that strain COC8T belongs to the genus Azospirillum and can be differentiated from all recognized species. The name Azospirillum oryzae sp. nov. is therefore proposed for strain COC8T.
Description of Azospirillum oryzae sp. nov.
Azospirillum oryzae (o.ry'zae. L. gen. n. oryzae of rice, from where the type strain was isolated).
Cells are spiral or vibrioid, 1·0x1·5–5·0 µm in size and motile via a single polar flagellum. PHB granules are present in the cells. Nitrogen can be fixed, and the cells can grow on nitrogen-free medium or nutrient medium. Temperature range for growth is 4–37 °C (optimum 30 °C). Optimum pH for growth is between 6·0 and 7·0. Cells do not tolerate 3 % NaCl. Acids are produced from L-arabinose, D-xylose, D-ribose, D-glucose, D-fructose, D-galactose and L-rhamnose but not from sorbitol, lactose, maltose, mannitol, inositol, cellobiose or sucrose. Positive for nitrate reduction but negative for nitrite reduction. Positive reactions for catalase, oxidase, urease, phosphatase, DNase, gelatin liquefaction, aesculin hydrolysis and growth on citrate as a carbon source. Negative reactions for the Voges–Proskauer test and indole production. Malonate and phenylalanine are not assimilated. Biotin is required for growth. Hydrogen is utilized. Major cellular fatty acids are 18 : 17c, 16 : 17c and 16 : 0 and major hydroxy fatty acids are 14 : 0 3-OH and 16 : 0 3-OH. The G+C content of the DNA is 66·8 mol%. The predominant quinone system is ubiquinone (Q-10).
The type strain, COC8T (=IAM 15130T=CCTCC AB204051T), was isolated in 1982 from the roots of Oryza sativa.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Eckert, B., Weber, O. B., Kirchhof, G., Halbritter, A., Stoffels, M. & Hartmann, A. (2001). Azospirillum doebereinerae sp. nov., a nitrogen-fixing bacterium associated with the C4-grass Miscanthus. Int J Syst Evol Microbiol 51, 17–26.[Abstract]
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Khammas, K. M., Ageron, E., Grimont, P. A. D. & Kaiser, P. (1989). Azospirillum irakense sp. nov., a nitrogen-fixing bacterium associated with rice roots and rhizosphere soil. Res Microbiol 140, 679–693.[Medline]
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Oyaizu-Masuchi, Y. & Komagata, K. (1988). Isolation of free-living nitrogen-fixing bacteria from the rhizosphere of rice. J Gen Appl Microbiol 34, 127–164.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Skerman, V. B. D., Sly, L. I. & Williamson, M. (1983). Conglomeromonas largomobilis gen. nov., sp. nov., a sodium-sensitive, mixed-flagellated organism from freshwater. Int J Syst Bacteriol 33, 300–308.
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.
Tarrand, J. J., Krieg, N. R. & Dobereiner, J. (1978). A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov., and two species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Can J Microbiol 24, 967–980.[Medline]
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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. (2004). Phylogenetic analyses of the nitrogen-fixing genus Derxia. J Gen Appl Microbiol 50, 129–135.
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