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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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A neighbour-joining tree based on nifH gene sequences showing the relationships between strain Y39T and other nitrogen-fixing bacteria is available as supplementary material in IJSEM Online.
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reported the isolation of a number of free-living, nitrogen-fixing bacteria from the rhizosphere or roots of rice plants. These isolates were classified into ten groups on the basis of phenotypic and chemotaxonomic characteristics. Lacking phylogenetic studies, these isolates have remained unidentified. Here, we report on the classification of three strains, Y39T (NBRC 15497T), Y22 (NBRC 15496) and OSG47 (NBRC 15495), which belonged to group 2 in the original scheme. Takeuchi et al. (2001) classified these strains as representing Sphingomonas sensu stricto, based on 16S rRNA gene sequence analysis, and polyamines, fatty acids and some physiological traits. The strains were found to contain ubiquinone Q-10, homospermidine as the sole polyamine, a unique sphingoglycolipid and 2-hydroxy fatty acids, which are common chemotaxonomic features of the genus Sphingomonas (Kämpfer et al., 1997; Takeuchi et al., 2001). Based on the results of Oyaizu-Masuchi & Komagata (1988) and Takeuchi et al. (2001), together with newly obtained data in this study, we propose that the nitrogen-fixing strains Y39T, Y22 and OSG47 represent a single novel species of the genus Sphingomonas.
Strains were grown at 25 °C in nitrogen-free medium (5·0 g glucose, 5·0 g lactose, 0·1 g CaCl2.2H2O, 0·1 g MgSO4.7H2O, 0·9 g K2HPO4, 0·1 g KH2PO4, 5 g CaCO3, 10 mg FeSO4.7H2O, 5 mg Na2MoO4.2H2O, 1 l distilled water, pH 7·3), TY medium (5·0 g tryptone, 0·75 g yeast extract, 0·454 g KH2PO4, 2·388 g Na2HPO4.2H2O, 1·0 g CaCl2, 1 l distilled water, pH 7·0), YMA medium (Jordan, 1984) or nutrient broth. Sphingomonas trueperi NBRC 100456T and Sphingomonas pituitosa CIP 106154T were grown in nutrient broth. Tolerance of salinity was determined by using YMA medium supplemented with 0–4·0 % NaCl (w/v). The isolated strains and S. trueperi NBRC 100456T and S. pituitosa CIP 106154T were grown on TSA medium (trypticase soy agar; Becton Dickinson) for fatty acid extraction. Cellular fatty acid methyl esters were prepared, separated and identified by using the Microbial Identification system, as described by Xie & Yokota (2003). Data from acetylene reduction assays, quinone analyses and macromolecular hydrolysis were taken from Oyaizu-Masuchi & Komagata (1988). Polyamine composition was taken from Takeuchi et al. (2001).
DNA–DNA hybridization was performed by using the photobiotin-labelling method of Ezaki et al. (1989), using a multi-well plate reader (CytoFluoR; Perseptive Biosystems). The hybridization temperature was 52 °C and reciprocal experiments were performed as follows. DNA of strain Y39T was used as a probe for hybridization of itself, and for DNA of strains OSG47 and Y22, and S. trueperi NBRC 100456T, S. pituitosa CIP 106154T and a negative control (Bacillus subtilis IAM 12118T). DNA of S. trueperi NBRC 100456T was also used as a probe. PCR of 16S rRNA gene sequences and sequencing of the products were carried out as described previously (Xie & Yokota, 2003). A 750 bp fragment of the nifH gene sequence (encoding the iron protein of nitrogenase, a key enzyme in nitrogen fixation) was amplified from the extracted DNA using the forward primer IGK (5'-TACGGYAARGGBGGYATCGG-3'; Poly et al., 2001) and the reverse primer R750 (5'-TCCATBGTGATGGGDCGGGATG-3'; this study) (Y=C/T; S=G/C; R=A/G; B=C/G/T). The sequences were compared with sequences obtained from GenBank and aligned using 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 1352 bases was constructed using the neighbour-joining method (Saitou & Nei, 1987). The topology of this tree was evaluated by using the bootstrap resampling method of Felsenstein (1985) with 1000 replicates, whereas similarity values were calculated using PAUP 4.0b1 (Swofford, 1998). Using the same methods, 415 bases of nifH sequences were also aligned and a phylogenetic tree was constructed.
Cells of strains Y39T, Y22 and OSG47 are Gram-negative, straight rods and are motile by means of peritrichous flagella. Colonies are yellow–orange on agar medium and the spectral characteristics of the yellow pigment extracted in acetone had two peaks at 452 and 480 nm, corresponding to those of S. pituitosa DSM 13101T (Denner et al., 2001). The three isolates had only 2-hydroxy fatty acids and no 3-hydroxy fatty acids, consistent with the generic characteristics of Sphingomonas (Takeuchi et al., 2001). The sole cellular polyamine was homospermidine; putrescine, spermidine and agmatine were not present, which differentiates Sphingomonas from other genera (Takeuchi et al., 2001; Kämpfer et al., 1997). Similar to all species of the genus Sphingomonas, the three isolates contained ubiquinone Q-10. On the basis of chemotaxonomic characteristics, we consider the three strains studied to be members of the genus Sphingomonas.
Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains OSG47, Y39T and Y22 are affiliated with the genus Sphingomonas and are most closely related to S. trueperi NBRC 100456T (99·5 % similarity) and S. pituitosa DSM 13101T (99·0 % similarity) (Fig. 1). This subcluster, with a bootstrap confidence level of 100 %, is phylogenetically distant from other species of Sphingomonas, with not more than 96 % similarity, a level that clearly delineates different species. That the three isolates and S. trueperi NBRC 100456T and S. pituitosa CIP 106154T were found to have a close phylogenetic relationship does not imply that they should be considered as representing a single species (Fox et al., 1992). Levels of DNA–DNA relatedness of strain Y39T with the other isolates and Sphingomonas species were 78·9 % (OSG47), 80·6 % (Y22), 25·3 % (S. trueperi NBRC 100456T) and 15·9 % (S. pituitosa CIP 106154T). Levels of DNA–DNA relatedness of S. trueperi NBRC 100456T with the three isolated strains were 29·7 % (Y39T), 28·6 % (OSG47) and 27·7 % (Y22), and 16·5 % with S. pituitosa CIP 106154T. These results suggest that strains OSG47, Y39T and Y22 represent a single species and are distinct from S. trueperi NBRC 100456Tand S. pituitosa CIP 106154T at the genomic level. Moreover, the three isolates could be distinguished from their close relatives by some phenotypic characteristics. When the fatty acid compositions of the strains were compared (Table 1), we found that the fatty acids of S. trueperi NBRC 100456T were quite different from those of the isolated strains and S. pituitosa CIP 106154T, with the former lacking 2-OH 15 : 0, 11-methyl 18 : 1
7c and 19 : 0 cyclo 8c. On the other hand, the three isolates did not contain 16 : 15c, in contrast to the two other strains. Moreover, the three strains could be easily distinguished from their closest phylogenetic neighbours, S. trueperi NBRC 100456T and S. pituitosa CIP 106154T (Table 2). S. trueperi NBRC 100456T, formerly known as ‘Pseudomonas azotocolligans’ Anderson 1955, has been proposed to be a diazotropic bacterium. However, Hill & Postgate (1969) demonstrated that this organism could not fix nitrogen, and it was then reclassified as S. trueperi by Kämpfer et al. (1997).
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, 2005a, b; Moulin et al., 2001; Rosado et al., 1998; Young, 1992). Therefore, the nifH gene appears to fulfil the criteria for a molecule suitable for molecular phylogeny and has been successfully used as a phylogenetic marker (Ueda et al., 1995). Part of the nifH gene of representative strain Y39T was sequenced and a phylogenetic tree was constructed (Supplementary Fig. S1 in IJSEM Online). The highest similarities (98 %) were found with the uncultured diazotrophic bacteria (NIW samples; e.g. GenBank accession nos AF389758 and AF389759) associated with dead aboveground biomass of Spartina alterniflora (Lovell et al., 2001), whereas there was very low similarity (less than 89 %) with other species with validly published names. The nifH phylogeny of the nitrogen-fixing proteobacteria consists of two distinctive monophyletic clades (Alphaproteobacteria and Gammaproteobacteria) and one mixed group (Alphaproteobacteria and Betaproteobacteria). Strain Y39T is closely related to the rhizobial genus Bradyrhizobium within the mixed group, which is distant from other common alphaproteobacteria. The diazotrophic bacteria in the mixed group are not congruent with the phylogeny of the organisms as deduced from 16S rRNA gene sequence analysis, suggesting that the nitrogen fixation of the three isolated strains occurs by lateral transfer. The possibility of lateral transfer of the nifH gene has been reported previously (Xiong et al., 2000; Cantera et al., 2004; Raymond et al., 2004). To our knowledge, the three isolates represent the first species of diazotrophic bacteria to belong to the genus Sphingomonas. Therefore, the name Sphingomonas azotifigens sp. nov. is proposed to accommodate the strains described here.
Description of Sphingomonas azotifigens sp. nov.
Sphingomonas azotifigens [a.zo.ti.fi'gens. French. n. azote (from Gr. pref. a- and Gr. n. zoê) nitrogen; N.L. n. azotum -i nitrogen; L. part. figens (from L. v. figo) fixing; N.L. part. adj. azotifigens nitrogen fixing].
The description is based on Oyaizu-Masuchi & Komagata (1988), Takeuchi et al. (2001) and this study. Cells are Gram-negative, aerobic, straight rods, 0·5–1·0x1·0–3·0 µm in size and motile by means of peritrichous flagella. Nitrogen-fixing. Colonies are circular, smooth, convex, opaque and yellow–orange on agar medium. The visible absorption spectrum of the acetone extract of the yellow pigment has two peaks at 452 and 480 nm. Cells contain poly-
-hydroxybutyrate granules. Optimum temperature for growth is 25–37 °C; growth is inhibited at 42 °C and in 2·5 % NaCl. Starch, aesculin and Tween 80 are hydrolysed but not chitin. Catalase, oxidase, -galactosidase, phosphatase and DNase are present but not indole or arginine dihydrolase. Does not produce H2S or reduce nitrate. Acid is produced from L-arabinose, D-xylose, fructose, galactose, sucrose, maltose, lactose, trehalose, melibiose, cellobiose and D-mannose but not from D-arabinose, melezitose, adonitol, dulcitol, sorbitol, mannitol, inositol, ribose, inulin or ethanol. Assimilates acetate, L-asparate, succinate, malate, pyruvate, arabinose, xylose, glucose, fructose, galactose, trehalose, sucrose, lactose, maltose, cellobiose, raffinose, suberate, -hydroxybutyrate, glutamate and starch, but not citrate, malonate, meso-tartrate, mandelate, benzoate, m-hydroxybenzoate, -aminobutyrate, proline, glycerol, gluconate, 2-ketogluconate, betaine, itaconate, adipate, mucate, L-alanine, methanol or ethanol. Major fatty acids are 18 : 17c, 16 : 0 and 11-methyl 18 : 17c; there is a large amount of the hydroxy fatty acid 2-OH 14 : 0 and a minor amount of 2-OH 15 : 0, but cells do not contain 3-hydroxy fatty acid. Sphingolipids are present. Ubiquinone Q-10 is the major quinone. The G+C content of the DNA is 66·0–68·0 mol%.
The type strain is Y39T (=NBRC 15497T=IAM 15283T=CCTCC AB205007T). Strains Y39T, OSG47 (=NBRC 15495) and Y22 (=NBRC 15496) were isolated from paddy soil and the roots of Oryza sativa in Japan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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