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1 Korean Agricultural Culture Collection, Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration (RDA), Suwon 441-707, South Korea
2 Applied Microbiology Division, National Institute of Agricultural Science and Technology, RDA, Suwon 441-707, South Korea
3 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7b, D-38124 Braunschweig, Germany
Correspondence
Byung-Yong Kim
kimby{at}rda.go.kr
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type (L-Lys–D-Glu) and the major menaquinone was MK-7. Diaminopimelic acid was absent from both strains. The genomic DNA G+C contents of strains F73T and I80T were 46.5 and 44.5 mol%, respectively. On the basis of the phylogenetic analysis and physiological and chemotaxonomic data, the isolates represent two novel species of the genus Sporosarcina, for which the names Sporosarcina koreensis sp. nov. (type strain F73T =KACC 11299T =DSM 16921T) and Sporosarcina soli sp. nov. (type strain I80T =KACC 11300T =DSM 16920T) are proposed.
Two-dimensional thin-layer chromatograms of the polar lipids of strains F73T and I80T are available as supplementary material with the online version of this paper.
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for strains that have spherical or oval-shaped cells. The first species of this genus to be described was Sporosarcina ureae, the type species. The second species, Sporosarcina halophila, was described by Claus et al. (1983) but was reclassified within the newly described genus Halobacillus as Halobacillus halophilus (Spring et al., 1996). The species Sporosarcina globispora, Sporosarcina psychrophila and Sporosarcina pasteurii were created by reclassification of Bacillus globisporus, Bacillus psychrophilus and Bacillus pasteurii, respectively, and Sporosarcina aquimarina, isolated from seawater, was also added to the genus (Yoon et al., 2001). More recently, Sporosarcina macmurdoensis, a psychrophilic bacterium isolated from a pond in Antarctica, was described by Reddy et al. (2003). In this study, we describe two spore-forming bacteria, F73T and I80T, that were isolated from soil in Suwon, Korea, and subjected to detailed phylogenetic, phenotypic and chemotaxonomic analyses.
The two strains were isolated from soil samples by using a dilution-plating technique on trypticase soy agar (TSA; BBL) and cultured at 30 °C for 2 days. Yellowish colonies were produced on TSA and on nutrient agar (Difco). Gram staining was performed using a Gram-stain kit (Difco) according to the manufacturer's protocol. After incubation for 2 days on TSA, cells were fixed using a glass slide coated with 0.8 % agar (to keep the cells in focus) and observed under a phase-contrast microscope (Zeiss). The following physiological tests were carried out according to Gordon et al. (1973) and Claus & Berkeley (1986): catalase and oxidase reactions, Voges–Proskauer test, growth temperature determinations (5–45 °C, using increments of 5 °C), determinations of growth in the presence of NaCl (0, 5, 7, 10 and 15 %) and at pH 5.7, formation of acid from carbohydrates (D-glucose, L-arabinose, D-xylose and D-mannitol), hydrolysis of starch, casein, gelatin and Tween 80, utilization of citrate and propionate, reduction of nitrate, production of indole, deamination of phenylalanine and the urease test. Motility tests were performed on motility medium (0.1 % yeast extract, 0.01 % K2HPO4 and 0.2 % agar). Growth under anaerobic conditions was tested by incubation in an anaerobic chamber (BBL) for 2 weeks.
The cells of strains F73T and I80T were Gram-positive and rod-shaped, occurring singly or in filamentous chains. Single cells were 0.5–0.7 (F73T) or 0.7–1.0 (I80T) µm in diameter and 2.5–3.0 (F73T) or 2.0–3.0 (I80T) µm in length. They did not grow anaerobically. In both cases, spores were spherical or oval. However, the cellular positions of the sporangia differed: like spores in described Sporosarcina species, those of F73T were positioned terminally, whereas the spores of I80T were positioned centrally (Table 1). Cells of strain F73T were motile, but those of strain I80T were non-motile on the medium used in this study. The growth temperature range for strain F73T was 15–40 °C, while that for strain I80T was 15–37 °C. Isolate F73T grew at pH 6.0–9.0 (optimum, pH 7.0), whereas isolate I80T grew at pH 7.0–9.0 (optimum, pH 8.0). Strain F73T grew in the presence of 7 % NaCl whereas I80T grew in the presence of up to 5 % NaCl. These data indicate that the two isolates are very different physiologically. The two strains were positive for catalase, urease and oxidase and negative for phenylalanine deamination, the formation of indole and dihydroxyacetone, in the Voges–Proskauer test, for acid production from D-glucose, L-arabinose, D-xylose and D-mannitol, and for hydrolysis of starch, casein and Tween 80. The detailed results of the physiological characterizations are given in the species descriptions; features that serve to distinguish between the two strains and related Sporosarcina species are listed in Table 1.
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). The 16S rRNA gene sequences were analysed using an Applied Biosystems DNA sequencer (ABI 3100). Phylogenetic analyses were performed by using MEGA version 3.1 (Kumar et al., 2004) after multiple alignment of the data by CLUSTAL W (Thompson et al., 1994). Distances were obtained using Kimura's two-parameter model (Kimura, 1980) and clustering was performed using the neighbour-joining algorithm (Saitou & Nei, 1987). The stability of relationships was evaluated by performing a bootstrap analysis based on 1000 resamplings. The phylogenetic tree indicated that our isolates belong to the genus Sporosarcina (at a high bootstrap value) (Fig. 1). Strain F73T showed the highest sequence similarity with strain I80T (98.9 %), followed by the type strains of S. globispora (97.3 %), S. aquimarina (97.2 %) and S. psychrophila (97.2 %). Strain I80T showed the closest relationships with strain F73T (98.9 %) and the type strains of S. globispora (97.2 %), S. aquimarina (97.2 %) and S. psychrophila (96.9 %). Lower levels of sequence similarity (<96.0 %) were found with species from other genera. Furthermore, five of the Sporosarcina type strains plus F73T and I80T formed a compact cluster with 97 % bootstrap support. Thus, it is clear that these two isolates belong to the genus Sporosarcina.
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). Probe labelling was conducted by using the non-radioactive DIG-High Prime system (Roche), and hybridized DNA was visualized using the DIG luminescent detection kit (Roche). DNA–DNA relatedness was quantified by using a densitometer (Bio-Rad). The levels of DNA–DNA hybridization between the isolates and related type strains of Sporosarcina species were less than the 70 % threshold value suggested by Wayne et al. (1987) for separate species delineation. Isolate F73T showed low values for DNA relatedness with respect to isolate I80T (28.0 %), S. globispora DSM 4T (20.0 %), S. aquimarina KCCM 41039T (15.2 %) and S. psychrophila DSM 3T (18.5 %). Likewise, isolate I80T showed low values for DNA relatedness with respect to S. globispora DSM 4T (19.3 %), S. aquimarina KCCM 41039T (15.2 %) and S. psychrophila DSM 3T (10.0 %).
Analysis of the peptidoglycan structure was carried out as described previously (Groth et al., 1996; Schleifer & Kandler, 1972). Respiratory lipoquinones and polar lipids were extracted from freeze-dried cell material (100 mg) by using a two-stage method (Tindall, 1990a, b). Biomass for most of the chemotaxonomic studies was prepared following growth of the isolates and standard strains in shake flasks of trypticase soy broth for 3 days at 30 °C; after a purity check, biomass was harvested by centrifugation, washed twice in distilled water and freeze-dried. Whole-cell fatty acid profiles were determined using the Microbial Identification system (MIDI; Microbial ID), from biomass grown on TSA for 2 days at 30 °C. The G+C content of the DNA was determined by HPLC (Mesbah et al., 1989).
The peptidoglycan of the two isolates contained lysine, glutamic acid, ornithine and alanine [i.e. a variant of the A4 type, with L-Lys–D-Glu (A11.33; http://www.dsmz.de/species/murein.htm), as described by Schleifer & Kandler (1972)]. Neither strain contained diaminopimelic acid as the diagnostic amino acid in the cell-wall peptidoglycan. The predominant isoprenoid quinone was an unsaturated menaquinone with seven isoprene units (MK-7). The polar lipids of strain F73T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminolipid and two unidentified aminophospholipids. Meanwhile, the polar lipids of strain I80T were diphosphatidylglycerol, phosphatidylglycerol and an unidentified phospholipid (see Supplementary Fig. S1 available in IJSEM Online). The major fatty acids of the two isolates were anteiso-C15 : 0, iso-C15 : 0 and iso-C14 : 0, being similar to those present in species of the genus Sporosarcina. However, the proportion of iso-C15 : 0 was much higher in the isolates, differentiating them from recognized species in the genus Sporosarcina (Table 2). The G+C contents of the DNAs of strains F73T and I80T were 44.5 and 46.5 mol%, which are slightly higher than those of Sporosarcina species (Table 1
). These chemotaxonomic characteristics, together with the morphological features, also support the affiliation of the isolates to the genus Sporosarcina.
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Description of Sporosarcina koreensis sp. nov.
Sporosarcina koreensis (ko.re.en'sis. N.L. fem. adj. koreensis referring to Korea, where the isolates were collected).
Cells are Gram-positive, aerobic, motile, spore-forming rods (0.5–0.7x2.5–3.0 µm) that occur singly or in short chains. Endospores are mainly oval and positioned terminally in swollen sporangia. Light-orange-coloured colonies are formed after incubation for 2 days on nutrient agar or TSA at 30 °C. Growth occurs at temperatures ranging from 15 to 40 °C, with optimum growth occurring at 30 °C. Growth does not occur in the presence of >7 % NaCl. The pH range for growth is 6.0–9.0, the optimum being pH 7.0; growth is not observed at pH 5.7 or 10.0. Positive for catalase, oxidase, urease and gelatinase. Negative for anaerobic growth, formation of indole and dihydroxyacetone, in the Voges–Proskauer test, for phenylalanine deamination, nitrate reduction, acid production from D-glucose, L-arabinose, D-xylose and D-mannitol and for hydrolysis of starch, casein and Tween 80. Does not utilize citrate or propionate. The peptidoglycan is of the L-Lys–D-Glu type (variation A4). The major cellular fatty acids are iso-C15 : 0 and anteiso-C15 : 0. The major menaquinone is MK-7. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid and two unidentified aminophospholipids. The G+C content of the DNA is 46.5 mol%.
The type strain, F73T (=KACC 11299T =DSM 16921T), was isolated from upland soil in Suwon, Korea.
Description of Sporosarcina soli sp. nov.
Sporosarcina soli (so'li. L. neut. gen. n. soli of soil, the source of the organism).
Cells are Gram-positive, aerobic, non-motile, spore-forming rods (0.7–1.0x2.0–3.0 µm) that occur singly or in pairs and occasionally in short chains. Endospores are round and are positioned centrally in non-swollen sporangia. Light-orange-coloured colonies are formed after incubation for 2 days on nutrient agar or TSA at 30 °C. Growth occurs at temperatures ranging from 15 to 37 °C, with optimum growth occurring at 30 °C. Growth does not occur in the presence of >5 % NaCl. The pH range for growth is 7.0–9.0, the optimum being pH 8.0; growth is not observed at pH 5.7 or 10.0. Positive for catalase, oxidase, urease, phenylalanine deamination and nitrate reduction. Negative for anaerobic growth, formation of indole and dihydroxyacetone, in the Voges–Proskauer test, for acid production from D-glucose, L-arabinose, D-xylose and D-mannitol and for hydrolysis of starch, casein, gelatin and Tween 80. Does not utilize citrate or propionate. The peptidoglycan is of the L-Lys–D-Glu type (variation A4). The major cellular fatty acids are anteiso-C15 : 0 and iso-C15 : 0. The major menaquinone is MK-7. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol and an unidentified phospholipid. The G+C content of the DNA is 44.5 mol%.
The type strain, I80T (=KACC 11300T =DSM 16920T), was isolated from upland soil in Suwon, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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