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mura1,2
1 Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
2 The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
Correspondence
Yoko Takahashi
ytakaha{at}lisci.kitasato-u.ac.jp
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Published online ahead of print on 2 September 2005 as DOI 10.1099/ijs.0.63799-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Oryzihumus leptocrescens KV-628T, KV-641 and KV-656 are AB193172, AB193173 and AB193174, respectively.
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; Iwai & Takahashi, 1992; Jiang & Xu, 1996; Nioh et al., 1995; Nonomura & Hayakawa, 1988; Suzuki et al., 1998; Takeuchi & Hatano, 1999). In a previous report, a new isolation method using agar medium containing oxidant scavengers, such as superoxide dismutase (SOD) or SOD plus catalase, was described (Takahashi et al., 2003). Using this method, we succeeded in increasing the number of bacterial strains isolated from soil samples. Among 45 strains isolated from paddy soil in Saitama Prefecture, Japan, using this new isolation method, 20 strains belonged to the class Actinobacteria. Three strains were irregular rods, found to have meso-diaminopimelic acid in the peptidoglycan; in the phylogenetic tree based on 16S rRNA gene sequences, they were loosely associated with Ornithinicoccus hortensis. Ornithinicoccus hortensis cells are cocci containing ornithine in their peptidoglycan.
In this paper, we report on the morphological, physiological and biochemical characteristics, cell composition, DNA–DNA hybridization and 16S rRNA gene sequences of the three isolates in comparison with those of Ornithinicoccus hortensis. On the basis of the characteristics studied, these isolates represent a novel genus and species, Oryzihumus leptocrescens gen. nov., sp. nov.
Strains KV-628T, KV-641 and KV-656 were isolated from soil samples collected from a paddy field in Saitama Prefecture, Japan. Soil samples (2 g) were suspended in 18 ml sterile water and then mixed. Soil particles were allowed to sediment, the liquid phase was diluted 105 times and 100 µl was spread onto the surface of four kinds of GPM agar (1·0 % glucose, 0·5 % peptone, 0·5 % meat extract, 0·3 % NaCl, 1·2 % agar; pH 7·0) plate (Takahashi et al., 2003), as follows: (1) unsupplemented; (2) supplemented with SOD (300 U per plate); (3) supplemented with catalase (2100 U per plate); or supplemented with SOD plus catalase. Each plate was cultured at 27 °C. Biomass for biochemical and chemotaxonomic testing was prepared by culture in Todd–Hewitt broth (Difco) or nutrient broth (Difco) at 27 °C for 3 or 4 days, followed by harvesting by centrifugation.
Morphological observation under a scanning electron microscope (model JSM-5600; JEOL) was performed on cultures grown on GPM medium at 27 °C for 6 days. The ability of the strain to grow on a range of sole carbon sources at 1 % (w/v) was determined in carbon-utilization media (Pridham & Gottlieb, 1948) with yeast nitrogen base agar without amino acids (Nihon Pharmaceutical). NaCl tolerance and pH and temperature ranges for growth were determined on 1/5 nutrient agar. The three isolates were characterized biochemically using API ZYM (bioMérieux) according to the manufacturer's instructions. Susceptibility to antibiotics was tested by culture in YD agar (1·0 % glucose, 1·0 % yeast extract, 1·5 % agar; pH 7·0) medium supplemented with the following antibiotics: erythromycin, aztreonam, rifampicin, novobiocin, streptomycin and chloramphenicol.
Whole-cell hydrolysates were analysed for diaminopimelic acid isomers using TLC (Becker et al., 1965; Hasegawa et al., 1983); the N-acyl types of the muramic acid were determined by using the method of Uchida & Aida (1977). Purified cell wall was obtained by following the method of Kawamoto et al. (1981), and the amino acid composition of the hydrolysed cell wall was determined using an automatic amino acid analyser (model L-8500A; Hitachi). Whole-cell sugars were analysed by using the method of Becker et al. (1965), the presence of mycolic acid was examined by using the TLC method of Tomiyasu (1982), and phospholipids were extracted and identified by following the method of Minnikin et al. (1977). Menaquinones were extracted and purified according to the method of Collins et al. (1977) and were then analysed by HPLC (model 802-SC; Jasco) on a chromatograph equipped with a CAPCELL PAK C18 column (Shiseido) (Tamaoka et al., 1983). The cellular fatty acids were determined. The strains were cultured on 1/5 nutrient agar plates. Methyl esters of cellular fatty acids were prepared by direct transmethylation with methanolic hydrochloride and were analysed by GLC (model GC-17A; Shimazu) with a DB-23 capillary column (0·25 mmx30 m; J&W Scientific) (Suzuki & Komagata, 1983).
PCR amplification for selection of Actinobacteria was performed. DNA was prepared by sonication (Yu et al., 2002). A part of domain III of the 23S rRNA gene was amplified by using the method of Yu et al. (2002).
The DNA G+C content and DNA–DNA relatedness values were determined. DNA was isolated as described by Saito & Miura (1983), with some modifications. The G+C content was estimated by using HPLC (Tamaoka & Komagata, 1984). Levels of DNA–DNA relatedness were determined by the method of Ezaki et al. (1989), using photobiotin and a microplate format.
DNA was prepared by sonication (Yu et al., 2002). The 16S rRNA gene sequence was amplified by PCR (Takahashi et al., 2002) and sequenced with an automatic sequence analyser (ABI PRISM 377A; PE Applied Biosystems) using a PRISM Ready Reaction dye primer cycle sequencing kit (PE Applied Biosystems).
Species related to the novel isolate were determined by performing sequence database searches using BLAST. The sequence data of related species were retrieved from GenBank. Nucleotide substitution rates (Knuc values) were calculated (Kimura & Ohta, 1972) and phylogenetic trees were constructed by using the neighbour-joining method (Saitou & Nei, 1987). The statistical significance of the tree topology was evaluated by bootstrap analysis of sequence data with CLUSTAL W software (Thompson et al., 1994). Sequence similarity values were determined by visual comparison and manual calculation.
Strains KV-628T, KV-641 and KV-656 were isolated from isolation GPM agar medium supplemented with SOD, catalase or SOD plus catalase. PCR amplification was performed to determine if these strains were members of the Actinobacteria. This test was established to distinguish strains of the Actinobacteria from both Gram-negative bacteria and Gram-positive bacteria with low-G+C DNA contents by taking advantage of the specific insertion of a 100 bp sequence in domain III of the 23S rRNA gene of the Actinobacteria. The PCR was designed to amplify a 
380 bp fragment from the Actinobacteria and a 270 bp fragment from other bacteria (Roller et al., 1992; Yu et al., 2002). Amplicons of these strains were all 350 bp in length, and the results showed that all three of the isolated strains were members of the Actinobacteria.
Nearly complete 16S rRNA gene sequences were determined for the three isolated strains. A database search demonstrated that these strains belonged to the suborder Micrococcineae of the family Intrasporangiaceae. It was clear from the phylogenetic tree (Fig. 1) that the three strains formed a monophyletic clade loosely associated with Ornithinicoccus hortensis. The sequence similarity values among KV-628T, KV-641 and KV-656 were above 99·9 %, whereas the sequence similarity values between these three strains and Ornithinicoccus hortensis were below 95·6 %. Sequence similarity values for KV-628T and other Intrasporangiaceae family members were as follows: Terrabacter tumescens, 95·2 %; Knoellia sinensis, 95·2 %; Intrasporangium calvum, 94·8 %; Janibacter limosus, 94·8 %; Tetrasphaera japonica, 94·8 %; Arsenicicoccus bolidensis, 94·8 %; Terracoccus luteus, 94·4 %; and Ornithinimicrobium humiphilum, 93·7 %.
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). The cells of the three strains were Gram-positive, catalase-positive and showed no motility. The strains were able to grow at 15–37 °C and at pH 4–9, but at pH 9 only trace growth was observed. NaCl in 1/5 nutrient agar medium was tolerated up to 4 %, while only trace growth was observed at 5 % NaCl.
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The DNA G+C content of KV-628T, KV-641 and KV-656 was 72–73 mol% (with the HPLC nucleoside method). The cell-wall peptidoglycan of KV-628T, KV-641 and KV-656 contained meso-diminopimelic acid, alanine and glutamic acid at molar ratios of 1·0 : 1·6 : 1·0, 1·0 : 1·7 : 1·0 and 1·0 : 1·7 : 1·0, respectively. The three isolated strains contained a peptidoglycan of type A1
(Schleifer & Kandler, 1972). The predominant menaquinone was MK-8(H4). The acyl type was acetyl. Mycolic acids were not detected. The predominant cellular fatty acids were iso-C14 : 0, iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0 (Table 1).
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for species classification.
At present, the family Intrasporangiaceae contains nine genera: Arsenicicoccus (Collins et al., 2004), Intrasporangium (Kalakoutskii et al., 1967), Janibacter (Martin et al., 1997), Knoellia (Groth et al., 2002), Terrabacter (Collins et al., 1989), Terracoccus (Prauser et al., 1997), Tetrasphaera (Maszenan et al., 2000), Ornithinicoccus (Groth et al., 1999) and Ornithinimicrobium (Groth et al., 2001). Table 2 shows the differential characteristics of the members of the family Intrasporangiaceae. The genera Janibacter, Knoellia and Tetrasphaera also have meso-diaminopimelic acid in their cell walls, but Janibacter, Knoellia (iso-C15 : 0, iso-C17 : 0, iso-C16 : 0 and anteiso-C17 : 0) and Tetrasphaera differ in terms of cellular fatty acids.
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Description of Oryzihumus gen. nov.
Oryzihumus (Ory.zi.hu'mus. L. fem. n. oryza rice; L. masc. n. humus soil; N.L. masc. n. Oryzihumus rice soil).
Gram-positive, catalase-positive, aerobic, non-motile irregular rods. The peptidoglycan is of the A type of direct cross-linkage and contains meso-diaminopimelic acid, alanine and glutamic acid. The acyl type of the glycan chain of peptidoglycan is acetyl. Mycolic acids are absent. The major menaquinone is MK-8(H4). The DNA G+C content is 72–73 mol%. Phylogenetically, this genus is a member of the family Intrasporangiaceae, suborder Micrococcineae. The type species is Oryzihumus leptocrescens.
Description of Oryzihumus leptocrescens sp. nov.
Oryzihumus leptocrescens (lep.to.cre'scens. Gr. adj. leptos thin, fine, delicate, slender; L. part. adj. crescens growing; N.L. part. adj. leptocrescens slender growing).
Cells are irregular rods 0·4–0·9 µm by 0·9–1·9 µm. Colonies are pale yellow. Aerobic to microaerophilic. Growth occurs between pH 4 and 9 and at 15 and 37 °C. In 1/5 nutrient agar medium, NaCl is tolerated up to 5 %. D-Glucose, maltose, sucrose and trehalose are assimilated, but L-rhamnose and D-xylose are not. Susceptible to erythromycin (15 µg ml–1), rifampicin (30 µg ml–1), novobiocin (30 µg ml–1), streptomycin (10 µg ml–1) and chloramphenicol (30 µg ml–1). Not susceptible to aztreonam (100 µg ml–1). Esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, 
-galactosidase, 
-glucosidase and -glucosidase are detected by the API ZYM enzyme assay; alkaline phosphatase, cystine arylamidase, trypsin, chymotrypsin, -glucuronidase, N-acetyl--glucosaminase, -mannosidase and -fucosidase are not detected. A weak reaction for lipase (C14) is detected. Variable reactions for -galactosidase are detected. The DNA G+C content is 72–73 mol%.
Habitat: paddy soil in Japan. The type strain is KV-628T (=NRRL B-24347T=JCM 12835T=NBRC 100762T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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