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1 Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural Development Administration (RDA), Suwon 441-707, Republic of Korea
2 Korean Agricultural Culture Collection (KACC), Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration (RDA), Suwon 441-707, Republic of Korea
3 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7b, 38124 Braunschweig, Germany
4 Department of Environmental Sciences, University of California, Riverside, CA 92521-0424, USA
Correspondence
Soon-Wo Kwon
swkwon{at}rda.go.kr
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ABSTRACT |
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D-Asp and the main cellular fatty acid was iso-C16 : 0. The DNA G+C contents of strains HC145T and HC148T were 42.4 and 38.5 mol%, respectively. On the basis of the data from this polyphasic study, strains HC145T and HC148T represent members of the genus Ureibacillus, for which the names Ureibacillus composti sp. nov. and Ureibacillus thermophilus sp. nov., respectively, are proposed. The type strain of U. composti is HC145T (=KACC 11361T =DSM 17951T) and the type strain of U. thermophilus is HC148T (=KACC 11362T =DSM 17952T).
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), soil (Ureibacillus terrenus; Fortina et al., 2001) and composts (Ureibacillus suwonensis; Kim et al., 2006). The temperature range for growth for members of this genus is 35–65 °C, the optimum being 50–60 °C (Kim et al., 2006). Composting is a self-heating, aerobic, solid-phase, biodegradative process occurring in organic waste materials (de Bertoldi et al., 1983; Finstein & Morris, 1975). During the thermogenic phase of the composting process, the temperature usually rises to 65–80 °C for a certain period of time. Thus, hot compost is considered to represent a favourable habitat for thermophilic strains. During the characterization of thermophilic bacteria isolated from livestock-manure composts, strains HC145T and HC148T were recovered on trypticase soy agar at pH 7.0 and incubated at 55 °C. Strains HC145T and HC148T formed round, brown, convex colonies on both trypticase soy agar and nutrient agar (Oxoid). Gram staining was performed using a Difco Gram-stain kit according to the manufacturer's recommended protocol. A KOH test and an L-alanine aminopeptidase assay were also performed (Gregersen, 1978). Cell morphology was observed under a phase-contrast microscope after 2 days incubation on CASO agar (DSMZ medium no. 220; http://www.dsmz.de/media/media.htm) supplemented with MnSO4 at 10–100 mg l–1. The Voges–Proskauer test and tests for the following physiological traits were carried out according to Gordon et al. (1973) and Claus & Berkeley (1986): catalase, anaerobic growth, temperature range for growth (30–70 °C, in increments of 5 °C), growth in the presence of NaCl (0, 2, 5, 7 and 10 %, w/v), growth at pH 4–10, acids from carbohydrates (D-glucose, L-arabinose, D-xylose and D-mannitol), formation of gas from glucose, hydrolysis of starch, nitrate reduction, production of indole, deamination of phenylalanine, decomposition of casein and tyrosine and liquefaction of gelatin. The oxidase test and the determination of aesculin hydrolysis were conducted according to Smibert & Krieg (1994). Motility tests were performed on 1/10-strength CESP agar (1.5 g Casitone, 0.5 g yeast extract, 0.3 g soytone, 0.2 g peptone, 0.015 g MgSO4, 0.007 g FeCl2 and 0.002 g MnCl2, made up to 1 l with distilled water; pH 7.2) (Fortina et al., 2001). Strain HC148T did not form spores on CASO agar medium supplemented with MnSO4 at 10–100 mg l–1, unlike other strains within the genus Ureibacillus. The physiological and biochemical properties of strains HC145T and HC148T and the three existing species within the genus Ureibacillus are shown in Table 1.
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). The sequences were aligned together with those of the type strains of the genus Ureibacillus by using the CLUSTAL W program (Thompson et al., 1994). The evolutionary tree for the dataset was inferred with the neighbour-joining and maximum-parsimony methods, using MEGA 3.1 (Kumar et al., 2004). The stability of relationships was assessed by performing bootstrap analyses based on 1000 resamplings. Phylogenetic analyses based on 16S rRNA gene sequences confirmed that strains HC145T and HC148T are members of the genus Ureibacillus (Fig. 1). The level of 16S rRNA gene sequence similarity between strains HC145T and HC148T was 98.4 % and the levels of similarity between strains HC145T and HC148T and the type strains of other Ureibacillus species were in the ranges 97.8–98.1 % and 97.4–98.7 %, respectively. Strains HC145T and HC148T and the type strains of the three existing species within the genus Ureibacillus were clustered into one clade with a high level of bootstrap support (100 %). This was also supported by the tree constructed using the maximum-parsimony method.
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. Probe labelling was conducted using the non-radioactive DIG-High Prime system (Roche) and DNA–DNA relatedness was quantified using a densitometer (Bio-Rad). The DNA relatedness between strains HC145T and HC148T was 42 %. Strains HC145T and HC148T exhibited only 42–51 and 38–45 % relatedness, respectively, to the type strains of the three recognized species.
DNA G+C contents were determined by means of HPLC analysis of deoxyribonucleosides, as described by Mesbah et al. (1989), using a reversed-phase column (Supelcosil LC-18 S; Supelco). The preparation of cell walls and the determination of peptidoglycan structure was carried out at the DSMZ as described by Schleifer (1985) and Schleifer & Kandler (1972) except that TLC on cellulose was used instead of paper chromatography. Polar lipid analyses were carried out by the Identification Service of the DSMZ (Tindall, 1990). After growth of the cells on R2A for 2 days at 50 °C, fatty acid methyl esters were extracted and prepared using the standard protocol of the Microbial Identification system (MIDI; Microbial ID). The DNA G+C content of strain HC148T was 38.5 mol%, which lies within the range observed for members of the genus Ureibacillus, while the content of HC145T was 42.4 mol%, which is a little higher than those reported previously for Ureibacillus type strains (35.7–41.5 mol%). Fatty acid profiles for members of the genus Ureibacillus (Table 1
) revealed that all of the strains contained iso-C16 : 0 as the predominant fatty acid (60.0–77.3 %). The minor fatty acid compositions among the strains were found to be very variable. Strain HC148T was unique in containing summed feature 4 (iso-C17 : 1 I and/or anteiso-C17 : 1 B; 2.6 %). The peptidoglycan cross-linkage in the two novel strains was of the L-LysD-Asp type (variation A4
). The polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phospholipids and glycolipids of unknown composition. These results were in accordance with those given in the description of the genus Ureibacillus (Fortina et al., 2001).
Phenotypic features that serve to distinguish strains HC145T and HC148T from recognized Ureibacillus species are shown in Table 1. Strains HC145T and HC148T could be differentiated from each other on the basis of the following features: temperature range for growth, tyrosine hydrolysis and DNA G+C content. The two novel strains can be distinguished from recognized Ureibacillus strains on the basis of several phenotypic properties as well as some chemotaxonomic properties such as fatty acid composition and menaquinone content (Table 1). Furthermore, the DNA–DNA reassociation values (below 70 %) and the 16S rRNA gene sequence analysis confirmed the distinct positions of strains HC145T and HC148T within the genus Ureibacillus.
On the basis of the data from this polyphasic taxonomic study, strains HC145T and HC148T represent two novel species of the genus Ureibacillus, for which we propose the names Ureibacillus composti sp. nov. and Ureibacillus thermophilus sp. nov., respectively.
Description of Ureibacillus composti sp. nov.
Ureibacillus composti (com.pos'ti. N.L. gen. n. composti of compost).
Cells are Gram-negative, motile, rod-shaped bacteria, 0.7–0.9 µm wide and 2.5–4.0 µm long. They bear spherical endospores that lie in subterminal or terminal positions. Round, light-brown, convex colonies are formed. Grows at 37–60 °C and pH 6–8. Tolerates up to 5 % NaCl (w/v). Aesculin is hydrolysed. Casein, gelatin, tyrosine, starch and urea are not hydrolysed. Positive for catalase, oxidase and deamination of phenylalanine. Negative for anaerobic growth, glucose fermentation, indole formation, nitrate reduction, acid production from D-glucose, L-arabinose, D-xylose and D-mannitol and in the Voges–Proskauer test. Peptidoglycan cross-linkage is of the L-LysD-Asp type (variation A4). The major cellular fatty acid is iso-C16 : 0. The major quinones are MK-7, MK-8 and MK-9. Polar lipids consist of phosphatidylglycerol, diphosphatidylglycerol, phospholipids and glycolipids of unknown composition. The DNA G+C content of the type strain is 42.4 mol%.
The type strain, HC145T (=KACC 11361T=DSM 17951T), was isolated from livestock-manure compost from Ichon, Korea.
Description of Ureibacillus thermophilus sp. nov.
Ureibacillus thermophilus (ther.mo'phi.lus. Gr. adj. thermos hot; Gr. adj. philos loving; N.L. masc. adj. thermophilus heat-loving).
Cells are Gram-negative, motile, rod-shaped bacteria, 0.8–1.2 µm wide and 2.5–3.5 µm long. Round, light-brown, convex colonies are formed. Grows at 30–65 °C and pH 6–8. Tolerates up to 5 % NaCl (w/v). Aesculin and tyrosine are hydrolysed. Casein, gelatin, starch and urea are not hydrolysed. Positive for catalase, oxidase and deamination of phenylalanine. Negative for anaerobic growth, glucose fermentation, indole formation, nitrate reduction, acid production from D-glucose, L-arabinose, D-xylose and D-mannitol and in the Voges–Proskauer test. Peptidoglycan cross-linkage is of the L-LysD-Asp type (variation A4). The major cellular fatty acid is iso-C16 : 0. The major quinones are MK-8, MK-9 and MK-7. Polar lipids consist of phosphatidylglycerol, diphosphatidylglycerol, phospholipids and glycolipids of unknown composition. The DNA G+C content of the type strain is 38.5 mol%.
The type strain, HC148T (KACC 11362T=DSM 17952T), was isolated from livestock-manure compost from Ichon, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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