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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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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KV-657T is AB282887.
Scanning electron micrographs of cells of strain KV-657T and HPLC traces from menaquinone analysis of the novel strain and I. calvum NBRC 12989T are available as supplementary material with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Yamamura et al., 2003; Hayakawa et al., 2004). In a previous report, we described a new isolation method using agar medium containing oxidant scavengers, for example superoxide dismutase with or without catalase (Takahashi et al., 2003). Using this method, we have succeeded in isolating representatives of two new genera, Oryzihumus leptocrescens (Kageyama et al., 2005) and Patulibacter minatonensis (Takahashi et al., 2006). Among 45 strains isolated from paddy soil in Saitama Prefecture, Japan, using this new isolation method (Takahashi et al., 2003), 20 strains belonged to the class Actinobacteria, including strain KV-657T. Strain KV-657T forms branching hyphae, has LL-diaminopimelic acid (A2pm) in its peptidoglycan and, on the basis of a phylogenetic tree constructed using 16S rRNA gene sequences, it was thought to be closely associated with Intrasporangium calvum. In this paper, we report on the morphological, physiological and biochemical characteristics of strain KV-657T and DNA–DNA hybridization and 16S rRNA gene sequence analysis in comparison with I. calvum NBRC 12989T. Based on these results, KV-657T represents a novel genus and species.
Strain KV-657T was isolated from soil samples collected from a paddy field in Saitama Prefecture, Japan. Biomass for biochemical and chemotaxonomic studies was prepared by culture in TSB (Difco) at 27 °C for 3 or 4 days, followed by harvesting by centrifugation.
Morphological observations were carried out using a scanning electron microscope (model JSM-5600; JEOL) and cultures grown on GPM agar medium (1 % D-glucose, 0.5 % peptone, 0.5 % meat extract, 0.3 % NaCl and 1.2 % agar; pH 7.0) at 27 °C for 2 days. The ability of the strain to grow on a range of sole carbon sources at 1 % (w/v) was determined using carbon utilization media (Pridham & Gottlieb, 1948) (Nihon Pharmaceutical Co., Ltd). NaCl tolerance (0–7 %) and pH (3–11) and temperature (5–45 °C) ranges for growth were determined on 1/5-strength nutrient agar. The isolate was characterized biochemically using API ZYM in accordance with the manufacturer's instructions (bioMérieux).
Whole-cell hydrolysates were analysed for A2pm isomers using TLC (Becker et al., 1965; Hasegawa et al., 1983). Purified cell walls were obtained by using the method of Kawamoto et al. (1981). One milligram of purified cell wall was hydrolysed at 100 °C with 1 ml 6 M HCl for 16 h. The residue was dissolved in 100 µl water and this solution was used for amino acid analysis. Amino acid composition was determined by HPLC using the Pico Tag method (Waters). Samples were derivatized with phenylisothiocyanate and UV detection (at 254 nm) was used. The presence of mycolic acids was assessed by using the TLC method of Tomiyasu (1982). Menaquinones were extracted and purified by using the method of Collins et al. (1977) and then analysed by HPLC (model 802-SC; Jasco) using a chromatograph equipped with a Capcell Pak C18 column (Shiseido) (Tamaoka et al., 1983). Methyl esters of cellular fatty acids were prepared and analysed by GLC (model HP6890; Hewlett Packard).
DNA for determination of base composition and DNA–DNA relatedness values was isolated as described by Saito & Miura (1963), with some modifications. The DNA base composition was estimated by HPLC (Tamaoka & Komagata, 1984). Levels of DNA–DNA relatedness were determined by using the method of Ezaki et al. (1989), using photobiotin and a microplate format.
DNA for PCR was prepared by sonication (Yu et al., 2002). The 16S rRNA gene was amplified by PCR and sequenced using an automatic sequence analyser (ABI Prism 3130; PE Applied Biosystems) using a dye terminator cycle-sequencing kit (PE Applied Biosystems).
Species related to the new isolate were identified by performing sequence database searches using BLAST. Sequence data for 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 using CLUSTAL W software (Thompson et al., 1994). Sequence similarity values were determined by visual comparison and manual calculation.
A nearly complete 16S rRNA gene sequence was determined for strain KV-657T. A database search demonstrated that the strain belonged to the family Intrasporangiaceae. The phylogenetic tree was calculated using 16S rRNA gene sequence data as follows: I. calvum DSM 43043T (1469 bp), Terracoccus luteus DSM 44263T (1479 bp), Terrabacter terrae PPLBT (1475 bp) and Terrabacter tumescens DSM 20306T (1461 bp). In the 16S rRNA gene phylogenetic tree (Fig. 1), the strain was closely associated with I. calvum DSM 43043T. The sequence similarity between KV-657T and I. calvum DSM 43043T was 97.6 %.
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(Schleifer & Kandler, 1972). The predominant menaquinone was MK-8(H4). Mycolic acids were not detected. The cellular fatty acid components were iso-C14 : 0 (7.3 %), iso-C15 : 0 (39.6 %), anteiso-C15 : 0 (5.8 %), C15 : 0 (4.8 %), iso-C16 : 0 (13.3 %), iso-C17 : 0 (2.0 %), anteiso-C17 : 0 (2.0 %), C17 : 0
8c (6.4 %) and C17 : 0 (5.8 %).
In order to confirm that the strain belonged to a novel species, DNA–DNA hybridization relatedness was determined. The level of DNA–DNA relatedness between the isolated strain and I. calvum NBRC 12989T was determined to be 33–34 %, which was well below the 70 % cut-off point for species classification recommended by Wayne et al. (1987).
The differential characteristics of strain KV-657T and other LL-A2pm-containing members of the family Intrasporangiaceae are listed in Table 1. The major cellular fatty acids of these isolates are a complex mixture of straight-chain saturated, monounsaturated, iso- and anteiso-methyl-branched acids. Members of the genus Arsenicicoccus have a similar complex of major cellular fatty acids, but differ in cell morphology. On the basis of the 16S rRNA gene sequence-based phylogenetic tree, KV-657T is thought to be closely related to the genus Intrasporangium, but can be differentiated by cell morphology and the major menaquinones (Supplementary Fig. S2). Our findings show that KV-657T belongs to new genus in the family Intrasporangiaceae.
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Description of Humihabitans gen. nov.
Humihabitans (Hu.mi.ha'bi.tans. L. masc. n. humus soil; L. part. adj. habitans inhabiting; N.L. part. adj. used as a masc. n. Humihabitans inhabitant of soil).
Gram-positive, catalase-positive, aerobic organisms that form branching hyphae. Fragmentary vegetative mycelium is produced. Peptidoglycan is of the A type of direct cross-linkage and contains LL-A2pm, glycine, alanine and glutamic acid. Mycolic acids are absent. The major menaquinone is MK-8(H4). Phylogenetically, the genus is a member of the family Intrasporangiaceae. The type species is Humihabitans oryzae.
Description of Humihabitans oryzae sp. nov.
Humihabitans oryzae (o.ry'zae. L. gen. n. oryzae of rice, pertaining to the isolation of the type strain from rice paddy soil).
In addition to the characteristics that define the genus, it has the characteristics described below. Cell length is 1.0–2.2 µm with a mean diameter of around 0.5 µm. Colonies are pale yellow. Growth occurs at pH 5–11 and at 8 and 40 °C. In 1/5-strength nutrient agar medium, NaCl is tolerated up to 3 %. D-Fructose, D-glucose, maltose, D-mannitol, D-mannose, sucrose, trehalose and D-xylose are assimilated, but L-arabinose, D-galactose, raffinose and L-rhamnose are not. Esterase (C4), esterase lipase (C8), valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acid phosphatase, 
-galactosidase, 
-galactosidase and -glucosidase are detected by the API ZYM enzyme assay; alkaline phosphatase, -glucuronidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are negative. Weak reactions for lipase (C14), leucine arylamidase, naphthol-AS-BI-phosphohydrolase and -glucosidase are detected in API ZYM tests. The DNA G+C content of the type strain is 70 mol%.
The type strain, KV-657T (=NRRL B-24470T =NBRC 101802T), was isolated from rice paddy soil in Japan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977). Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, 221–230.
Collins, M. D., Routh, J., Saraswathy, A., Lawson, P. A., Schumann, P., Welinder-Olsson, C. & Falsen, E. (2004). Arsenicicoccus bolidensis gen. nov., sp. nov., a novel actinomycete isolated from contaminated lake sediment. Int J Syst Evol Microbiol 54, 605–608.
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.
Groth, I., Schumann, P., Schuetze, B., Augsten, K., Kramer, I. & Stackebrandt, E. (2002). Knoellia sinensis gen. nov., sp. nov. and Knoellia subterranea sp. nov., two novel actinobacteria isolated from a cave. Int J Syst Evol Microbiol 52, 77–84.[Abstract]
Hasegawa, T., Takizawa, M. & Tanida, S. (1983). A rapid analysis for chemical grouping of aerobic actinomycetes. J Gen Appl Microbiol 29, 319–322.[CrossRef]
Hayakawa, M., Yoshida, Y. & Iimura, Y. (2004). Selective isolation of bioactive soil actinomycetes belonging to the Streptomyces violaceusniger phenotypic cluster. J Appl Microbiol 96, 973–981.[CrossRef][Medline]
Kageyama, A., Takahashi, Y., Seki, T., Tomoda, H. & mura, S. (2005). Oryzihumus leptocrescens gen. nov., sp. nov. Int J Syst Evol Microbiol 55, 2555–2559.
Kawamoto, I., Oka, T. & Nara, T. (1981). Cell wall composition of Micromonospora olivoasterospora, Micromonospora sagamiensis, and related organisms. J Bacteriol 146, 527–534.
Kimura, M. & Ohta, T. (1972). On the stochastic model for estimation of mutation distance between homologous proteins. J Mol Evol 2, 87–90.[CrossRef][Medline]
Magarvey, N. A., Keller, J. M., Bernan, V., Dworkin, M. & Sherman, D. H. (2004). Isolation and characterization of novel marine-derived actinomycete taxa rich in bioactive metabolites. Appl Environ Microbiol 70, 7520–7529.
Martin, K., Schumann, P., Rainey, F. A., Schuetze, B. & Groth, I. (1997). Janibacter limosus gen. nov., sp. nov., a new actinomycete with meso-diaminopimelic acid in the cell wall. Int J Syst Bacteriol 47, 529–534.
Pridham, T. G. & Gottlieb, D. (1948). The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J Bacteriol 56, 107–114.
Saito, H. & Miura, K. (1963). Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72, 619–629.[Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.
Schumann, P., Prauser, H., Rainey, F. A., Stackebrandt, E. & Hirsch, P. (1997). Friedmanniella antarctica gen. nov., sp. nov., an LL-diaminopimelic acid-containing actinomycete from Antarctic sandstone. Int J Syst Bacteriol 47, 278–283.
Takahashi, Y., Katoh, S., Shikura, N., Tomoda, H. & Omura, S. (2003). Superoxide dismutase produced by soil bacteria increases bacterial colony growth from soil samples. J Gen Appl Microbiol 49, 263–266.[CrossRef][Medline]
Takahashi, Y., Matsumoto, A., Morisaki, K. & mura, S. (2006). Patulibacter minatonensis gen. nov., sp. nov., a novel actinobacterium isolated using an agar medium supplemented with superoxide dismutase, and proposal of Patulibacteraceae fam. nov. Int J Syst Evol Microbiol 56, 401–406.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.[CrossRef]
Tamaoka, J., Katayama-Fujimura, Y. & Kuraishi, H. (1983). Analysis of bacterial menaquinone mixtures by high performance liquid chromatography. J Appl Bacteriol 54, 31–36.[CrossRef]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Tomiyasu, I. (1982). Mycolic acid composition and thermally adaptative changes in Nocardia asteroides. J Bacteriol 151, 828–837.
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Yamamura, H., Hayakawa, M. & Iimura, Y. (2003). Application of sucrose-gradient centrifugation for selective isolation of Nocardia spp. from soil. J Appl Microbiol 95, 677–685.[CrossRef][Medline]
Yu, L., Takahashi, Y., Matsumoto, A., Seino, A., Iwai, Y. & Omura, S. (2002). Application of PCR for selection of gram-positive bacteria with high DNA G+C content among new isolates. Actinomycetologica 16, 1–5.[CrossRef]
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