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-proteobacterium isolated from soil
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-Ku, Tokyo 113-0032, Japan
Correspondence
Cheng-Hui Xie
aa37116{at}mail.ecc.u-tokyo.ac.jp
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains XD10, XD22 and XD53T are AB110496–AB110498.
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). On the basis of 16S rRNA gene sequence analysis, these yellow-pigmented soil isolates seemed to represent a novel and important group within the 
-Proteobacteria and were closely related to the plasticized acetylated starch-degrading bacterium Fulvimonas soli (Mergaert et al., 2002), the lindane-degrading bacterium Rhodanobacter lindaniclasticus (Nalin et al., 1999) and the acidophilic bacterium Frateuria aurantia (Swings et al., 1984). Upon comparison of the metabolism of the novel bacterium with those of its phylogenetic neighbours, we found that it was not acidophilic like members of the genus Frateuria; however, it was not known if the organism possessed the ability to degrade plasticized acetylated starch or lindane. The aim of this study was to elucidate the phylogenetic position of the isolates, using polyphasic taxonomy (physiology, fatty acid composition, quinone system, DNA G+C content, DNA–DNA relatedness and 16S rRNA gene sequence analysis). On the basis of this substantial evidence, it is proposed that the isolates represent a novel species in a novel genus, Dyella japonica gen. nov., sp. nov. The type strain is XD53T (=IAM 15069T=DSM 16301T=ATCC BAA-939T).
The cells used for tests of growth at various temperatures and pH values were incubated in nutrient broth. Cell shape, size and motility were observed by light microscopy (BX 60 apparatus; Olympus). The presence of flagella was determined by transmission electron microscopy (JEM-1011 apparatus; JEOL) after negative staining with uranyl acetate. Gram staining was performed using the method of Oyaizu-Masuchi & Komagata (1988). API 20NE, API 50 CH and API ZYM tests (all bioMérieux) were used to determine physiological and biochemical characteristics. The API ZYM test results were read after 4 h incubation at 28 °C; all other API tests were read after 48 h. H2S formation was detected with lead acetate paper strips in GPY medium containing (w/v) 5 % D-glucose, 0·5 % peptone, 0·2 % yeast exact, 0·1 % L-cysteine and 0·005 % Na2SO4. The bacteria were incubated at 28 °C on a rotary shaker in AE broth [1·5 % (w/v) glucose, 0·2 % (w/v) yeast extract, 0·3 % (w/v) peptone, 6·5 % (v/v) acetic acid and 2 % (v/v) ethanol] (Entani et al., 1985) for 30 days; AE broth was used to identify the genus Frateuria (Swings et al., 1984). Examination of the respiratory quinone system, DNA G+C content and cellular fatty acid composition, PCR-mediated amplification of 16S rRNA gene sequences and sequencing of the PCR products were carried out as described previously (Xie & Yokota, 2003). DNA was prepared according to the method of Marmur (1961) from cells grown on nutrient broth and DNA G+C contents were determined by using the HPLC method of Mesbah et al. (1989). DNA–DNA hybridizations were carried out with photobiotin-labelled probes in microplate wells as described by Ezaki et al. (1989). The hybridization temperature was set at 51 °C.
The DNA sequences of the three strains were compared with sequences obtained from GenBank (National Centre for Biotechnology Information). The sequences were aligned using the CLUSTAL W software package (Thompson et al., 1994) and evolutionary distances and Knuc values (Kimura, 1980) were generated. Alignment gaps and ambiguous bases were not taken into consideration when 1433 bases of the 16S rRNA gene sequences were compared. Phylogenetic trees were constructed using either the neighbour-joining method (Saitou & Nei, 1987) or the maximum-likelihood method (PHYLIP package; Felsenstein, 1989). The topology of the phylogenetic tree was evaluated by using the bootstrap resampling method of Felsenstein (1985), with 1000 replicates. Similarity values were calculated using PAUP 4.0b1 (Swofford, 1998).
The 16S rRNA gene sequences of the three strains were determined and subjected to comparative analysis. The sequence of strain XD53T showed high similarity (more than 99·0 %) to those of the other two strains. The phylogenetic tree (Fig. 1) shows that these strains are clustered within the family ‘Xanthomonadaceae’ of the -Proteobacteria. The levels of 16S rRNA gene sequence similarity between XD53T and Fulvimonas soli LMG 19981T (Mergaert et al., 2002), Frateuria aurantia LMG 1558T and Rhodanobacter lindaniclasticus RP 5557T (Nalin et al., 1999) were 96·5, 95·9 and 95·0 %, respectively. A similarity value of no more than 97 % between 16S rRNA gene sequences is widely accepted for genus-level differentiation (Gillis et al., 2001). The bootstrap value for the branching of Frateuria aurantia LMG 1558T and the novel strains was less than 70 % (data not shown). The phylogenetic tree calculated by the maximum-likelihood method (data not shown) also supported the contention that these novel isolates represented an independent taxon separated from the genera Fulvimonas, Frateuria and Rhodanobacter.
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). They were found to consist primarily of branched fatty acids, with 17 : 1 iso 
9c (25·6–30·8 %), 15 : 0 iso (20·0–23·6 %) and 17 : 0 iso (19·6–20·0 %) as major constituents. The major hydroxy fatty acids were 11 : 0 iso 3-OH, 13 : 0 iso 3-OH and 17 : 0 iso 3-OH. This cellular fatty acid composition can be differentiated from that of Frateuria aurantia, which contains more than 40·6 % 15 : 0 iso and not more than 3 % 17 : 1 iso 9c, and has 12 : 0 3-OH and 12 : 0 2-OH as the major hydroxy fatty acids (Lisdiyanti et al., 2003). On the other hand, we found that the hydroxy fatty acid profile of Fulvimonas soli contained 12 : 0 iso 3-OH and did not contain 17 : 0 iso 3-OH (Mergaert et al., 2002). Ubiquinone Q-8 was detected as the major quinone system in these isolates; this is similar to the situation in Frateuria aurantia.
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). Motility could be observed in the cells of all strains. All strains could grow slowly at 10 or 37 °C, but could not grow at 4 or 40 °C. The optimum temperature for growth was 25–30 °C. The optimum pH was 6·5–7·2; the strains could not grow at pH 4·6. Biochemical and physiological characteristics of the strains were determined by using the API microtest galleries (bioMérieux): the strains displayed similar characteristics. All were catalase-positive, oxidase-negative, nitrate reduction-positive, nitrite reduction-negative, H2S-negative, produced acid from glucose, fructose and mannose and grew on maltose. In terms of biochemical characteristics, positive test results were obtained for N-acetylglucosamine, alkaline phosphatase, C4 esterase lipase, C8 esterase lipase, C14 lipase, leucine arylamidase, valine arylamidase, acid phosphatase and naphthol phosphohydrolase, weak reactions were obtained for N-acetyl-
-D-glucosamine and negative reactions were obtained for -glucuronidase, urease, gelatin liquefaction, indole production and growth on mannitol, caprate, valerate, D-ribose, D-sucrose, acetate, L-alanine, citrate, histidine, D-ribose, hydroxybenzoate, p-nitrophenyl -D-galactopyranoside, trypsin, 
-galactosidase, aesculin, malate, arginine, -galactosidase, -mannosidase, -fucosidase and D-xylose. These strains can be differentiated from Frateuria aurantia and Fulvimonas soli on the basis of some phenotypic features (Table 2). The novel bacterium could use N-acetyl -D-glucosamine but Frateuria strains could not. Moreover, the novel bacterium did not have the general characteristics of Frateuria (i.e. growth at pH 3·6, production of H2S, ketogenesis from D-mannitol, acid production from almost all carbon sources; Swings et al., 1984). Frateuria strains have been isolated from Lilium auratum and from the fruit of Rubus parvifolius. The novel bacterium could be distinguished easily from members of the genera Fulvimonas and Rhodanobacter by DNA G+C content and by motility, respectively.
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Description of Dyella gen. nov.
Dyella (Dy.el'la. L. dim. ending -ella; N.L. fem. n. Dyella of Dye, in honour of Dr Douglas W. Dye, of New Zealand, who contributed to the taxonomic study of the genus Xanthomonas).
Cells are Gram-negative, catalase-positive and oxidase-negative. Colonies on nutrient agar are yellow. They do not grow in AE broth and do not produce H2S. The G+C content of the DNA is approximately 62–64 mol%. The cellular fatty acid composition consists mainly of branched fatty acids, with 17 : 1 iso 9c, 15 : 0 iso and 17 : 0 iso as the major fatty acids and 11 : 0 iso 3-OH, 13 : 0 iso 3-OH and 17 : 0 iso 3-OH as the major hydroxy fatty acids. Q-8 is the major component of the quinone system. The type species of the genus is Dyella japonica.
Description of Dyella japonica sp. nov.
Dyella japonica (ja.po'ni.ca. N.L. fem. adj. japonica pertaining to Japan, from where the type strain and other strains originated).
Cells are straight rods (0·4x1·2 µm), motile by means of a single polar flagellum. Colonies grow well on nutrient agar. The conditions for growth are 10–37 °C and pH 5·6–8; growth does not occur at pH 4·6. The optimum pH for growth is 6·5–7·2. The optimum temperature for growth is 25–30 °C; growth at 4 and 40 °C is very poor. Cells are nitrate reduction-positive; acids are produced from glucose, fructose and mannose, but not from mannitol. The other characteristics of the species can be found in Table 2
. The G+C content of the DNA of the type strain is 63·5 mol%.
The type strain, XD53T (=IAM 15069T=DSM 16301T=ATCC BAA-939T), and strains XD10 (=IAM 15067) and XD22 (=IAM 15068) were isolated from soil in Tokyo, Japan.
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REFERENCES |
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Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
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