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1 Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Sendai, Miyagi 980-8579, Japan
2 Department of Biochemistry, School of Medicine, Kanazawa Medical University, Uchinada 1-1, Ishikawa 920-0293, Japan
3 NCIMB Japan, ShimizuOrido 3-20-1, Shizuoka, Shizuoka, 424-8610, Japan
Correspondence
Toru Nakayama
nakayama{at}seika.che.tohoku.ac.jp
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ABSTRACT |
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The cellular fatty acid compositions of strain TNJL143-2T and related type strains are available in a supplementary table in IJSEM Online.
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, b), are widespread in nature, and some species of this genus are of significance in biotechnology, e.g. the amino acid industry (Nakayama & Soda, 1998). During the course of our studies to screen micro-organisms that were able to grow on N-acetyl-D-phenylalanine as the sole nitrogen and carbon source, we found that a yellow-pigmented bacterium, strain TNJL143-2T, isolated from soil at Natori, Miyagi, Japan, produced a novel N-acyl-D-amino acid amidohydrolase (D-aminoacylase). As a result, extensive physiological, chemotaxonomic and phylogenetic analyses were carried out. On the basis of these results, we propose that strain TNJL143-2T represents a novel species of the genus Microbacterium, which we have named Microbacterium natoriense sp. nov.
Strain TNJL143-2T was isolated from soil, using an agar medium (at pH 7·0) containing 0·1 % (w/v) N-acetyl-D-phenylalanine, 0·01 % (w/v) yeast extract, 0·5 % (w/v) NaCl, 0·1 % (w/v) KH2PO4, 0·0005 % (w/v) MgSO4.7H2O and 1·5 % (w/v) agar at 30 °C. The type strains Microbacterium aerolatum JCM 12137T (=DSM 14217T) (Zlamala et al., 2002), Microbacterium foliorum JCM 11569T (=DSM 12966T) (Behrendt et al., 2001) and Microbacterium phyllosphaerae JCM 11571T (=DSM 13468T) (Behrendt et al., 2001) were obtained from the Japan Collection of Microorganisms, Wako, Saitama, Japan. Cells of strains TNJL143-2T, JCM 12137T, JCM 11569T and JCM 1571T were stored at –80 °C in Luria–Bertani (LB) broth at pH 6·8 (Sambrook et al., 1989) supplemented with 15 % (w/v) glycerol.
General laboratory cultivation was performed using LB broth (5 ml) at 30 °C. Bacterial growth was monitored for up to 7 days after inoculation by measuring the turbidity, at 600 nm, of cultures in 25 ml test tubes, which were capped with air-permeable silicone plugs and incubated in a water-bath incubator. For measurement of turbidity, an uninoculated control was used as a blank. Cell morphology was observed by light microscopy after 24 h cell growth. Sporulation was observed by phase-contrast microscopy after the cells had reached stationary phase. Gram staining was carried out using exponentially growing cells, according to Hucker's modification (Cowan & Steel, 1965), with reagents (Favour G) produced by Nissui. Flagellation was examined using Leifson's method (Cowan & Steel, 1965). The temperature range for growth of the organism was examined at 4 °C and at 10–50 °C in 5 °C steps. To determine the pH range for growth, the organisms were grown in LB medium supplemented with 0·1 % (w/v) potassium phosphate at 30 °C, as described above, except that the pH of the medium was adjusted to different values with 1·0 M H2SO4 or 1·0 M NaOH. Anaerobic growth was tested by incubation at 30 °C in 10 ml rubber-sealed screw-cap tubes containing LB medium (9 ml) that was covered with liquid paraffin.
Biochemical examinations (nitrate reduction, pyrazinamidase, pyrrolidonyl arylamidase, alkaline phosphatase, 
-glucuronidase, -galactosidase, 
-glucosidase, N-acetyl--glucosaminidase, -glucosidase, urease and gelatinase) were examined using API Coryne test strips (bioMérieux). Cells were resuspended in API GP suspension medium (bioMérieux) at a cell density corresponding to tube no. 6 of the MacFarland series of standard opacities (Cowan & Steel, 1965). The cell suspensions (150 µl) were added to API Coryne test-strip wells as recommended by the manufacturer, then incubated at 30 °C. Acid production from carbon sources was examined under aerobic conditions by using API 50 CH test strips (bioMérieux). Cells were resuspended in API 50 CHB/CHE suspension medium (bioMérieux) at a cell density corresponding to tube no. 2 of the MacFarland series. The cell suspensions (150 µl) were added to API 50 CH test-strip wells as recommended by the manufacturer, then incubated at 30 °C. Acidification was observed daily for 7 consecutive days; if the medium turned yellow, the cells were regarded as positive with respect to acid production. Strain TNJL143-2T was tested (as described previously; Barrow & Feltham, 1993) for catalase activity, oxidase activity and the hydrolysis of casein, and was subjected to an oxidation/fermentation test. The cellular menaquinones, cellular fatty acids and cell-wall diamino acids of TNJL143-2T were analysed as described previously (Komagata & Suzuki, 1987).
Isolation and purification of chromosomal DNA and estimation of DNA base composition (by HPLC) were performed according to Tamaoka & Komagata (1984). The 16S rRNA gene was amplified by a PCR (Weisburg et al., 1991) with the universal bacterial primers 10F (5'-AGTTTGSTCCTGGCTC-3') and 1500R (5'-GGCTACCTTGTTACGA-3'). PCR products were purified on Microspin S-400 HR columns (Amersham Biosciences) and sequenced using a CEQ2000XL DNA system (Beckman Coulter). Previously published 16S rRNA gene sequences were obtained from the GenBank/DDBJ/EMBL databases. Multiple alignment of the sequences, calculation of nucleotide substitution rates (Knuc values; Kimura, 1980), construction of a neighbour-joining phylogenetic tree (Saitou & Nei, 1987) and bootstrap analysis with 1000 replicates for the evaluation of phylogenetic tree topology (Felsenstein, 1985) were carried out using the CLUSTAL X (Thompson et al., 1997) and MEGA (Kumar et al., 1993) software programs. DNA reassociation values were determined as described by Ezaki et al. (1989). In a typical experiment, probes for DNA hybridization were prepared from cells of M. aerolatum JCM 12137T, M. foliorum JCM 11569T and M. phyllosphaerae JCM 11571T. Probe DNAs were biotinylated with photobiotin and hybridized with single-stranded unlabelled chromosomal DNA fragments of strain TNJL143-2T. Hybridized DNA fragments were visualized using alkaline-phosphatase-based fluorometric methods (Ezaki et al., 1989). Means from three independent determinations of DNA–DNA reassociation values were determined.
Cells of strain TNJL143-2T were rod-shaped (0·5–0·6x1·5 µm) and stained Gram-positive. Neither spore formation nor motility was observed and anaerobic growth did not occur. TNJL143-2T cells tested positive in a catalase test but gave a negative result in an oxidase test (Collins & Keddie, 1986). Colonies on LB agar were slightly yellow, shiny, moist, circular, slightly convex and 1 mm in diameter after 30 h growth at 30 °C.
A 1429 nt stretch of the 16S rRNA gene of strain TNJL143-2T was sequenced and compared with available 16S rRNA gene sequences to construct a phylogenetic tree (Fig. 1). It showed the highest similarities to the 16S rRNA gene sequences of M. phyllosphaerae DSM 13468T (98·7 %), M. foliorum DSM 12966T (98·6 %), Microbacterium keratanolyticum DSM 8606T (98·1 %) (Yokota et al., 1993; Takeuchi & Hatano, 1998a) and M. aerolatum DSM 14217T (97·6 %). Phylogenetic analysis showed that TNJL143-2T fell within the radiation of the genus Microbacterium and was most closely related to M. aerolatum DSM 14217T. The G+C contents of the genomic DNAs of strain TNJL143-2T, M. aerolatum JCM 12137T, M. foliorum JCM 11569T and M. phyllosphaerae JCM 11571T were determined (by HPLC) as 69·1, 68·1, 68·5 and 68·7 mol%, respectively. DNA–DNA reassociation studies on strain TNJL143-2T were performed using M. aerolatum JCM 12137T, M. foliorum JCM 11569T and M. phyllosphaerae JCM 11571T. The DNA–DNA reassociation values for strain TNJL143-2T with these type strains were 12, 11 and 12 %, respectively. In comparison, the DNA–DNA reassociation value between M. foliorum and M. phyllosphaerae, determined by the method described by Ezaki et al. (1989), was 11·5 %, which is even lower than the value (41·0 %) previously obtained by the spectrophotometric method (Behrendt et al., 2001; Martin et al., 1997). The values for strain TNJL143-2T with respect to type strains are significantly lower than the threshold value (70 %) recommended for the delineation of a novel species (Wayne et al., 1987). Thus, strain TNJL143-2T can be distinguished from representatives of all previously described species of Microbacterium.
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), whereas M. foliorum DSM 12966T and M. phyllosphaerae DSM13468SUP>T contain MK-10, MK-11 and MK-12.
Strain TNJL143-2T was able to grow in LB medium at pH 5–9, with a broad optimum at around pH 5–7 (at 30 °C). Optimum growth was observed at 30 °C in LB medium; no growth occurred below 4 °C or above 45 °C. The organism was able to grow in the presence of 6 % (w/v) NaCl in LB medium; however, no growth was observed at NaCl concentrations at or above 7 % (w/v). Strain TNJL143-2T gave a negative result in an oxidation/fermentation test. Several other biochemical and physiological characteristics of TNJL143-2T were also compared with those of M. aerolatum JCM 12137T, M. foliorum JCM 11569T and M. phyllosphaerae JCM 11571T (Tables 1 and 2). The results obtained with these three related species in the present study were generally in good agreement with those reported in the literature; exceptions are noted in the footnotes of Tables 1 and 2. TNJL143-2T was positive for catalase, pyrazinamidase, alkaline phosphatase, N-acetyl--glucosaminidase and gelatinase, but was negative for nitrate reduction (Table 1). Acid production under aerobic conditions was observed with N-acetylglucosamine, inulin and glycogen, but not with L-rhamnose or D-mannitol (Table 2). These characteristics distinguish strain TNJL143-2T from the phylogenetically related species of Microbacterium.
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Rod-shaped, Gram-positive, non-spore-forming, strictly aerobic organism. The rods measure 0·5–0·6x1·5 µm. No motility is observed. The temperature range for growth is 10–40 °C (optimum 30 °C). The pH range for growth is 5–9 (optimum pH 5–7). The organism grows in the presence of 6 % (w/v) NaCl in LB medium. Positive results are obtained for catalase, pyrazinamidase, alkaline phosphatase, -galactosidase, -glucosidase, N-acetyl--glucosaminidase, -glucosidase and gelatinase, but test results for oxidation/fermentation, oxidase, casein hydrolysis, nitrate reduction, pyrrolidonyl arylamidase, -glucuronidase and urease are negative. Test results for acid production from the following carbohydrates are positive: glycerol, L-arabinose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, methyl -D-glucopyranoside, N-acetyl-D-glucosamine, amygdalin, arbutin, aesculin, salicin, D-cellobiose, D-maltose, D-melibiose, sucrose, trehalose, inulin, D-melezitose, D-raffinose, starch, glycogen and D-turanose. Acid production is not observed from the following: erythritol, D-arabinose, D-ribose, L-xylose, D-adonitol, methyl -D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, lactose, xylitol, gentiobiose, D-tagatose, D-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate or potassium 5-ketogluconate. The cell-wall diamino acid is ornithine. The major fatty acids are C15 : 0 anteiso (45·0 mol%), C15 : 0 iso (17·0 mol%) and C17 : 0 anteiso (16·9 mol%). The major menaquinones are MK-9, MK-10, MK-11 and MK-12. The G+C content of the genomic DNA of the type strain is 69·1 mol%.
The type strain, strain TNJL143-2T (=JCM 12611T =ATCC BAA-1032T), was isolated from a soil sample obtained at Natori, Miyagi, Japan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Behrendt, U., Ulrich, A. & Schumann, P. (2001). Description of Microbacterium foliorum sp. nov. and Microbacterium phyllosphaerae sp. nov., isolated from the phyllosphere of grasses and the surface litter after mulching the sward, and reclassification of Aureobacterium resistens (Funke et al. 1998) as Microbacterium resistens comb. nov. Int J Syst Evol Microbiol 51, 1267–1276.[Abstract]
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Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. Cambridge: Cambridge University Press.
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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Martin, K., Schumann, P., Rainey, F. A., Schuetze, B. & Groth, I. (1997). Janibacter limosus gen. nov., a new actinomycete with meso-diaminopimelic acid in the cell wall. Int J Syst Bacteriol 47, 529–534.
Nakayama, T. & Soda, K. (1998). Production of amino acids. In Nutritional Requirements of Commercially Important Microorganisms, pp. 202–235. Edited by T. Nagadawithana & G. Reed. Milwaukee, WI: Esteekay Associates.
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Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Takeuchi, M. & Hatano, K. (1998a). Union of the genera Microbacterium Orla-Jensen and Aureobacterium Collins et al. in a redefined genus Microbacterium. Int J Syst Bacteriol 48, 739–747.
Takeuchi, M. & Hatano, K. (1998b). Proposal of six new species in the genus Microbacterium and transfer of Flavobacterium marinotypicum Zobell and Upham to the genus Microbacterium as Microbacterium maritypicum comb. nov. Int J Syst Bacteriol 48, 973–982.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 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.
Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697–703.
Yokota, A., Takeuchi, M. T. S. & Weiss, N. (1993). Proposal of six new species in the genus Aureobacterium and transfer of Flavobacterium esteraromaticum Omelianski to the genus Aureobacterium as Aureobacterium esteraromaticum comb. nov. Int J Syst Bacteriol 43, 555–564.
Zlamala, C., Schumann, P., Kämpfer, P., Valens, M., Rosselló-Mora, R., Lubitz, W. & Busse, H. J. (2002). Microbacterium aerolatum sp. nov., isolated from the air in the ‘Virgilkapelle’ in Vienna. Int J Syst Evol Microbiol 52, 1229–1234.[Abstract]
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