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1 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-Ku, Tokyo 113-0032, Japan
2 Marine Biotechnology Institute Co. Ltd, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan
3 Microbiological and Analytical Group, Food Research Laboratories, Mitsui Norin Co. Ltd, 223-1 Miyahara, Fujieda, Shizuoka 426-0133, Japan
Correspondence
Sun-Young An
an12su{at}hotmail.com
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ABSTRACT |
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. At present, the genus Sporosarcina contains six recognized species (Claus & Fahmy, 1986; Larkin & Stokes, 1967; Miquel, 1889; Nakamura, 1984; Reddy et al., 2003; Yoon et al., 2001), with Sporosarcina ureae as the type species. Members of the genus Sporosarcina are Gram-positive or Gram-variable, endospore-forming, rod-shaped or spherical bacteria. They have L-lysine as the diagnostic amino acid at position 3 of the peptide subunit of the peptidoglycan and MK-7 as the predominant menaquinone. In this study, we report two strains, HG645T and HG711, respectively isolated from surface water of Lake Saroma (Hokkaido, Japan) and from sediment of Nagasuka fishery harbour (Miyagi, Japan). On the basis of phenotypic characteristics, chemotaxonomic data and phylogenetic analysis of the 16S rRNA gene sequence, these isolates represent a novel species of Sporosarcina.
Strain HG645T was isolated by incubation for 1 month at 25 °C on JCM57 medium (JCM online catalogue; http://www.jcm.riken.go.jp/). Strain HG711 was isolated by incubation for 1 month at 25 °C on 1/10-diluted marine agar 2216 (BD). Isolates HG645T and HG711 were routinely cultivated on trypticase soy agar (TSA; BD BBL) containing 50 % Herbst's artificial seawater at 25 °C. Herbst's artificial seawater contains the following (per l distilled water): NaCl, 30 g; KCl, 0.7 g; MgSO4 . 7H2O, 5.3 g; CaSO4 . 2H2O, 1.3 g; and MgCl2 . 6H2O, 10.8 g. Cell morphology was observed by scanning electron microscopy (JSM-6700F apparatus; JEOL). Cell motility was examined by the hanging drop method using phase-contrast microscopy (BX60 microscope; Olympus). The temperature for growth was measured over the range 5 to 50 °C). The pH range for growth was assessed at pH 5.0–10.0 at intervals of 0.5 pH units. Growth under anaerobic conditions was determined after 2 weeks of incubation in an AnaeroPack (Mitsubishi Gas Chemical Co., Inc.). Catalase activity was tested by adding a drop of 3 % H2O2 to a single colony and was recorded as positive when development of bubbles was observed. Oxidase activity was determined by using cytochrome oxidase paper (Nissui Pharmaceutical Co., Inc.). Hydrolysis of casein and starch was tested according to the method of Smibert & Krieg (1994). API 20E and API 50CH microtest galleries (bioMérieux) were used to determine physiological and biochemical characteristics according to the manufacturer's instructions. The API strips were incubated for 2 days at 30 °C. The isolates were Gram-positive, endospore-forming, strictly aerobic, motile and rod-shaped. Morphological and physiological characteristics of the isolates are given in the species description. The isolates showed differences from other species of Sporosarcina in some physiological properties (Table 1).
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, 2002), the strains were grouped into the Sporosarcina cluster. However, they were found to be distinct from previously described species of the genus. 16S rRNA gene sequences were determined using a 16S rRNA Gene kit following the protocols of the manufacturer (Applied Biosystems). The 16S rRNA gene sequences of the strains HG645T and HG711 were used for a BLAST search via NCBI (National Centre for Biotechnology Information). The sequences obtained 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 1303 bases of the 16S rRNA gene nucleotide were compared. The phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987), which was obtained with MEGA3. The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985) with 1000 replicates. Similarity values were calculated using MEGA3 (Kumar et al., 2004).
Almost-complete 16S rRNA gene sequences of the strains HG645T and HG711 shared 100 % similarity. The isolates showed the highest degree of 16S rRNA gene sequence similarity with the type strain of Sporosarcina aquimarina (97.3 %), followed by the type strains of Sporosarcina globispora (96.9 %), Sporosarcina psychrophila (96.8 %), S. ureae (96.8 %), Sporosarcina pasteurii (96.0 %) and Sporosarcina macmurdoensis (95.9 %). The phylogenetic tree indicated that strains HG645T and HG711 were closely related to the genus Sporosarcina (Fig. 1).
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. DNA–DNA hybridization was performed by the photobiotin-labelling method of Ezaki et al. (1989) using a multi-well plate reader (CytoFluoR; PerSeptive Biosystems). The values of DNA–DNA hybridization between strains HG645T and HG711 were 79–86 %. The two strains should therefore be considered to be members of a single species (Stackebrandt et al., 2002). The level of DNA–DNA relatedness between strain HG645T and S. aquimarina JCM 10887T was less than 10 %, i.e. much less than the threshold value of 70 % suggested for separate species delineation by Wayne et al. (1987). The G+C content of the total DNA was measured by HPLC according to the method described by Mesbah et al. (1989). The DNA G+C contents of HG645T and HG711 were 46.0 and 45.2 mol%, which is slightly higher than those reported for other species of genus Sporosarcina (Table 1
). Cellular fatty acids of strains HG645T and HG711 were prepared from cell biomass grown on TSA medium for 48 h at 27 °C and then separated and identified with the Microbial Identification System (MIDI Inc.). The fatty acids (>1 % of the total fatty acids in at least one strain) of strains HG645T and HG711 were iso-C15 : 0 (49.5/45.6 %, respectively), anteiso-C15 : 0 (33.3/35.2 %), iso-C14 : 0 (5.6/5.3 %), iso-C16 : 0 (4.2/2.1 %), anteiso-C17 : 0 (2.4/3.01 %), C16 : 0 (1.4/0 %), C16 : 0
7c alcohol (0.4/2.0 %) and summed feature 4 (0/1.5 %). Summed feature 4 comprises iso-C15 : 0 2-OH and/or C16 : 17t. Analysis of cell-wall peptidoglycan of strains HG645T and HG711 was carried out by the methods of Schleifer & Kandler (1972); strains HG645T and HG711 possessed a cell-wall type of Lys–Glu. Respiratory quinone analyses of strains HG645T and HG711 were performed by the method of Collins & Jones (1981); the major isoprenoid quinone was MK-7. Polar lipids were analysed according to the method of Tindall (1990a, b); the predominant polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. On the basis of phenotypic, chemotaxonomic and phylogenetic characteristics, we conclude that strains HG645T and HG711 belong to a novel species of genus Sporosarcina, for which the name Sporosarcina saromensis sp. nov. is proposed.
Description of Sporosarcina saromensis sp. nov.
Sporosarcina saromensis (sa.ro.men'sis. N.L. fem. adj. saromensis pertaining to Lake Saroma, where the type strain was collected).
Cells are Gram-positive, strictly aerobic rods (0.8–1.0x2.0–3.2 µm), motile by means of lateral flagella. Spherical endospores are formed in a terminal position. Colonies grown on TSA medium containing 50 % Herbst's artificial seawater are circular, convex and beige. Optimal temperature for growth is 27 °C; growth occurs at 5–40 °C but not at 45 °C. Optimal pH for growth is 6.5; growth occurs at pH 5.5–9.0. NaCl is not required for growth but can be tolerated up to 9 % (w/v). Tests for catalase and oxidase activities are positive. Starch is hydrolysed but casein is not. H2S, indole and acetoin are not produced. Nitrate is not reduced. Tests for urease, gelatinase and 
-galactosidase activities are positive. Tests for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and citrate utilization are negative. Acid is not produced from carbohydrates in the API 50CH gallery. The cell wall contains peptidoglycans of the Lys–Glu type. The major isoprenoid quinone system is MK-7. The major cellular fatty acids are iso-C15 : 0 and anteiso-C15 : 0. Predominant polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The genomic DNA G+C content of the type strain is 46.0 mol%.
The type strain, HG645T (=MBIC08270T =IAM 15429T =KCTC 13119T), was isolated from surface water in Lake Saroma (Hokkaido, Japan). Strain HG711, isolated from sediment in Nagasuka fishery harbour (Miyagi, Japan), is a reference strain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Collins, M. D. & Jones, D. (1981). Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implications. Microbiol Rev 45, 316–354.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in micro-dilution 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.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Goto, K., Omura, T., Hara, Y. & Sadaie, Y. (2000). Application of the partial 16S rDNA sequence as an index for rapid identification of species in the genus Bacillus. J Gen Appl Microbiol 46, 1–8.[CrossRef][Medline]
Goto, K., Mochida, K. M., Asahara, M., Suzuki, M. & Yokota, A. (2002). Application of the hypervariable region of the 16S rDNA sequence as an index for the rapid identification of species in the genus Alicyclobacillus. J Gen Appl Microbiol 48, 243–250.[CrossRef][Medline]
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Kluyver, A. J. & van Niel, C. B. (1936). Prospects for a natural classification of bacteria. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Abt II 94, 369–403.
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
Larkin, J. M. & Stokes, J. L. (1967). Taxonomy of psychrophilic strains of Bacillus. J Bacteriol 94, 889–895.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Miquel, P. (1889). Étude sur la fermantation ammoniacale et sur les ferments de l'urée. Ann Microgr 1, 506–519 (in French)
Nakamura, L. K. (1984). Bacillus psychrophilus sp. nov., nom. rev. Int J Syst Bacteriol 34, 121–123.
Reddy, G. S. N., Matsumoto, G. I. & Shivaji, S. (2003). Sporosarcina macmurdoensis sp. nov., from a cyanobacterial mat sample from a pond in the McMurdo Dry Valleys, Antarctica. Int J Syst Evol Microbiol 53, 1363–1367.
Rüger, H.-J. (1983). Differentiation of Bacillus globisporus, Bacillus marinus comb. nov., Bacillus aminovorans, and Bacillus insolitus. Int J Syst Bacteriol 33, 157–161.
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.
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Manual of Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt. Washington, DC: American Society for Microbiology.
Stackebrandt, E., Frederiksen, W., Garrity, G. M., Grimont, P. A. D., Kämpfer, P., Maiden, M. C. J., Nesme, X., Rosselló-Mora, R., Swings, J. & other authors (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043–1047.[Abstract]
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.
Tindall, B. J. (1990a). A comparative study of the lipid composition of Halobacterium saccharovorum from various sources. Syst Appl Microbiol 13, 128–130.
Tindall, B. J. (1990b). Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol Lett 66, 199–202.[CrossRef]
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 systematic. Int J Syst Bacteriol 37, 463–464.
Yoon, J.-H., Lee, K.-C., Weiss, N., Kho, Y. H., Kang, K. H. & Park, Y.-H. (2001). Sporosarcina aquimarina sp. nov., a bacterium isolated from seawater in Korea, and transfer of Bacillus globisporus (Larkin and Stokes 1967), Bacillus psychrophilus (Nakamura 1984) and Bacillus pasteurii (Chester 1898) to the genus Sporosarcina as Sporosarcina globispora comb. nov., Sporosarcina psychrophila comb. nov. and Sporosarcina pasteurii comb. nov., and emended description of the genus Sporosarcina. Int J Syst Evol Microbiol 51, 1079–1086.[Abstract]
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