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1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
2 NeoPharm Co., Ltd, BVC-307, KRIBB, 52 Oeun-dong, Yuseong-gu, Daejeon 305-333, Republic of Korea
3 National University of Uzbekistan, Students town, Tashkent 700-174, Uzbekistan
4 Department of Oriental Medicinal Material and Processing, College of Life Science, Kyung Hee University, 1 Seocheon, Kihung Yongin, Kyunggi 449-701, Republic of Korea
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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ABSTRACT |
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-2 subclass of the Proteobacteria, and was most closely related to Starkeya novella IAM 12100T (98.6 %), Angulomicrobium tetraedale DSM 5895T (98.1 %), Angulomicrobium amanitiforme NCIMB 1785T (98.0 %), Ancylobacter aquaticus ATCC 25396T (96.6 %) and Methylorhabdus multivorans DM13T (95.8 %), all of which belong to the family Hyphomicrobiaceae. The genomic DNA G+C content of strain Jip08T was 69 mol%. Chemotaxonomic data [major ubiquinone, Q-10; major fatty acids, C18 : 1 isomer (summed feature of C18 : 1
7c/9t/12t), C19 : 0 cyclo 8c and C16 : 0], 16S rRNA gene sequence analyses and some morphological and physiological features supported the affiliation of strain Jip08T to the genus Starkeya. However, DNA–DNA hybridization data and some phenotypic properties showed that strain Jip08T could be distinguished from the single known Starkeya species and represented a novel species, for which the name Starkeya koreensis sp. nov. is proposed. The type strain is Jip08T (=KCTC 12212T=NBRC 100963T=IAM 15215T).
A comparison of selected characteristics of strain Jip08T and Starkeya novella IAM 12100T is available as supplementary material in IJSEM Online.
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, who proposed that Thiobacillus novellus should be reclassified as Starkeya novella; the genus Starkeya currently comprises only one species, S. novella IAM 12100T (Kelly et al., 2000). The genus Starkeya belongs to the family Hyphomicrobiaceae of the -2 subclass of the Proteobacteria, which also contains the genera Ancylobacter (Raj, 1983), Angulomicrobium (Vasil'eva et al., 1979), Azorhizobium (Dreyfus et al., 1988) and Methylorhabdus (Doronina et al., 1995). All of these genera form monophylogenetic clades in the phylogenetic tree based on 16S rRNA sequences (Fig. 1).
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). Ever since the genus Thiobacillus was first described by Beijerinck (1904), the ability to grow with a reduced sulfur compound as a source of energy has been considered sufficiently important taxonomically to merit classifying all Gram-negative, sulfur-oxidizing, non-phototrophic rods in this genus. However, as studies using modern taxonomic methods have begun to show that some of the species are only superficially related, virtually every paper describing a novel Thiobacillus species in the last decade has mentioned the need to reorganize the genus. Most of this reorganization has now been done (Kelly & Wood, 2000).
During the course of a study on the culturable aerobic bacterial community in environmental samples (Im et al., 2003) such as rice straw, anaerobic granular sludge (An et al., 2006; Aslam et al., 2005a; Bae et al., 2005; La et al., 2005), aerobic activated sludge (Aslam et al., 2005b; Quan et al., 2006), compost (H.-C. Yang et al., 2006) and soil from a ginseng field (D.-C. Yang et al., 2005, 2006) near Daejeon in Korea, a large number of novel bacterial strains were isolated that belonged to the Proteobacteria, Actinobacteria and Bacteroidetes. The taxonomic status of these bacteria is gradually being explored.
In this study, polyphasic approaches, including phylogenetic analyses based on 16S rRNA gene sequences, chemotaxonomic data and phenotypic properties, were used to determine the precise taxonomic position of strain Jip08T, which was isolated in South Korea from rice straw. The results obtained in this study indicated that strain Jip08T can be designated a member of the genus Starkeya, but it can be clearly distinguished from S. novella. Therefore, we propose Jip08T as the type strain of a novel species, namely Starkeya koreensis sp. nov.
Strain Jip08T was isolated from rice straw at a farm near Daejeon, South Korea: the rice straw was cut into small pieces and thoroughly suspended in 50 mM phosphate buffer (pH 7.0). This suspension was spread on R2A agar (Difco) after serial dilution with 50 mM phosphate buffer (pH 7.0) and then the plates were incubated at 30 °C for 3 weeks. Strain Jip08T was one of the dominant morphotypes appearing on the plates in aerobic conditions; it was routinely cultured on R2A agar at 30 °C and maintained as a glycerol suspension (20 %, w/v) at –70 °C. Strain Jip08T was then deposited in the Korean Collection for Type Cultures as strain KCTC 12212T, and at the National Institute of Technology and Evaluation, Japan, as strain NBRC 100963T.
The Gram reaction was determined using the non-staining method, as described by Buck (1982). Cell morphology was observed under a Nikon light microscope at x1000 and in a transmission electron microscope (CM20; Philips) at x27 500. Catalase activity was determined by assessing bubble production in 3 % (v/v) H2O2, and oxidase activity was determined using 1 % (w/v) tetramethyl-p-phenylenediamine. The utilization of various substrates as sole carbon sources was determined, along with some physiological characteristics, using API 32GN and API 20NE galleries according to the instructions of the manufacturer (bioMérieux). For the degradation of casein, starch and L-tyrosine (Atlas, 1993; Lewin & Lounsbery, 1969), reactions were read after 5 days. The hydrolysis of cellulose and xylan was tested as described by Ten et al. (2004). Growth with formate, methanol, methylamine, succinate and oxalate was tested in minimal medium B (Lingens et al., 1985). Tests for growth on sulfur, tetrathionate and thiosufate were done with sulfur medium, tetrathionate broth, thiocyanate utilization medium and thiosufate-oxidizing medium (Atlas, 1993). Negative and positive controls were obtained by inoculating Flavobacterium granuli and S. novella, respectively, and observing the turbidity after 10 days. Growth at different temperatures and pH values was assessed after 5 days incubation. Salt tolerance was measured in R2A broth medium supplemented with 1–10 % (w/v) NaCl after 7 days incubation. To determine the heat resistance of strain Jip08T, 0.1 ml aliquots of 8- and 16-h-old cultures that had been treated at three different temperatures (80 °C for 20 min, 90 °C for 10 min and 100 °C for 10 min) were inoculated into R2A medium; growth was observed for up to 72 h at 30 °C. Duplicate antibiotic-sensitivity tests were done using filter-paper discs containing the following: streptomycin (5, 10 and 15 µg ml–1) (Mast Diagnostics), tetracycline (5, 10 and 15 µg ml–1), kanamycin (1.0, 1.5 and 2.0 mg ml–1) and ampicillin (30, 50 and 60 µg ml–1) (Sigma). Discs were placed on R2A plates spread with Jip08T culture and were then incubated at 30 °C for 7 days.
The extraction of genomic DNA was performed using a commercial genomic DNA extraction kit (Core Biosystem). The 16S rRNA gene sequence was amplified from the chromosomal DNA by using the universal bacterial primer set (primers 9F and 1512R) and the purified PCR products were sequenced by GenoTech in Daejeon, South Korea (Kim et al., 2005). The full sequence of the 16S rRNA gene was compiled using SeqMan software (DNASTAR). The 16S rRNA gene sequences of related taxa were obtained from the GenBank database. Multiple alignments were obtained using the CLUSTAL_X program (Thompson et al., 1997). Gaps were edited in the BioEdit program (Hall, 1999). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). A phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) and the maximum-parsimony method (Fitch, 1971) with the MEGA3 program (Kumar et al., 2004) and bootstrap values based on 1000 replications (Felsenstein, 1985).
Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v), purified via TLC and subsequently analysed by HPLC, as described previously (Collins & Jones, 1981; Shin et al., 1996). The cellular fatty acids were analysed using cells grown on R2A agar (Difco) for 2 days. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI; Sasser, 1990). The fatty acids were analysed by GC (Hewlett Packard 6890) and identified by using the software package of the Microbial Identification System.
Genomic DNA of strain Jip08T was extracted and purified with the 100/G genomic-tip system (Qiagen) and then degraded enzymically into nucleosides, as described previously (Mesbah et al., 1989). DNA–DNA hybridization was performed fluorometrically, according to the method developed by Ezaki et al. (1989), using photobiotin-labelled DNA probes and microdilution wells. Five replicate hybridizations were conducted for each sample. The highest and lowest values obtained for each sample were excluded, and the remaining three values were used to calculate similarity values. The DNA relatedness values quoted are the means of these three values.
Strain Jip08T was found to comprise Gram-negative, highly curved rods (0.4–0.8x1.2–2.0 µm) (Fig. 2) that occurred singly, in pairs and sometimes in clumps of three to four cells; no filaments were found, even in old cultures. Colonies grown on R2A agar plates for 3 days were smooth, circular, convex and colourless. Spores were never observed and the cells were found to be heat-sensitive. The physiological characteristics of strain Jip08T are summarized in the species description. The distinguishing characteristics of strain Jip08T and related type strains are shown in Table 1.
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). DNA–DNA hybridization experiments were performed between the type strain of S. novella (IAM 12100T) and Jip08T for confirmation of a novel species: the DNA–DNA relatedness value was 15 %, which showed that strain Jip08T belongs to a distinct genomic species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Moreover, the two strains could be easily differentiated using tests for the utilization of substrates as sole carbon sources (see Supplementary Table S1 available in IJSEM Online). Although the genera Angulomicrobium, Ancylobacter and Methylorhabdus show high levels of 16S rRNA gene sequence similarity with respect to strain Jip08T, they are clearly distinguishable in the phylogenetic tree (Fig. 1) and on the basis of phenotypic characteristics (Table 1) (Vasil'eva et al., 1979; Raj, 1983; Doronina et al., 1995; Kelly et al., 2000).
Strain Jip08T had a genomic DNA G+C content of 69 mol% and a ubiquinone of the Q-10 type. The major fatty acids were C18 : 1 isomer (summed feature of C18 : 17c/9t/12t) (66.1 %), C19 : 0 cyclo 8c (23.7 %), C16 : 0 (7.5 %) and C18 : 0 (2.7 %). The major fatty acids of strain Jip08T were similar to those of the type strain of S. novella (Kelly et al., 2000).
A phylogenetic analysis based on 16S rRNA gene sequence similarity, chemotaxonomic characteristics [G+C content of genomic DNA, ubiquinone (Q-10) type, and cellular fatty acid profile] and some ecophysiological and biochemical characteristics (such as the oxidation of thiosulfate and tetrathionate for growth, the optimum temperature and pH for growth, and the absence of gas vacuoles) clearly shows that strain Jip08T belongs to the genus Starkeya. However, strain Jip08T can be distinguished by its low level of DNA–DNA relatedness (15 %) with respect to S. novella IAM 12100T and its cell morphology (Fig. 2). On the basis of the data and observations described above, strain Jip08T should be assigned to the genus Starkeya as the type strain of a novel species, for which the name Starkeya koreensis sp. nov. is proposed.
Description of Starkeya koreensis sp. nov.
Starkeya koreensis (ko.re.en'sis. N.L. fem. adj. koreensis pertaining to Korea, from where the novel micro-organism was isolated).
Cells are strictly aerobic, Gram-negative, non-spore-forming, non-motile, highly curved rods 0.4–0.8 µm wide and 1.2–2.0 µm long. Cells occur singly, in pairs and sometimes in clumps comprising three or four cells, but do not form filaments. Colonies on R2A and complex medium are circular, convex and colourless and are 0.7–1.2 mm in diameter after 3 days. The temperature range for growth is 4–40 °C; no growth occurs at 42 °C. The optimum temperature for growth is 28–30 °C. The pH range for growth is between pH 6.5 and 8.5, with an optimum between pH 7 and 8. Growth occurs in the absence of NaCl and in the presence of 3.0 % (w/v) NaCl, but not with 4 % (w/v) NaCl. Positive for catalase, oxidase, urease, 
-glucosidase and -galactosidase. H2S is not produced. Nitrate is not reduced. Negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and gelatinase. Does not produce any acid or gas from glucose. Casein, xylan, chitin, tyrosine, CM-cellulose, DNA and starch are not degraded. The following compounds are utilized as sole carbon sources: glucose, formate, methanol, oxalate, citrate, L-alanine, L-arabinose, propionate, 3-hydroxybutyrate, acetate, DL-lactate and L-proline. The following compounds are not utilized as sole carbon sources: adipate, methylamine, 4-hydroxybenzoate, valerate and caprate, mannose, N-acetylglucosamine, maltose, propionate, mannitol, gluconate, malate, phenylacetate, salicin, D-melibiose, L-fucose, D-sorbitol, histidine, 2-ketogluconate, 4-hydroxybutyrate, rhamnose, D-ribose, inositol, D-sucrose, itaconate, malonate, lactate, 5-ketogluconate, glycogen and L-serine. As this species can also utilize thiosulfate or tetrathionate as an energy source, it is facultatively chemolithoautotrophic. Sensitive to 20 µg ampicillin ml–1, 5 µg tetracycline ml–1 and 1.0 mg kanamycin ml–1; resistant to 15 µg streptomycin ml–1. The major cellular fatty acids are C18 : 1 isomer (66.1 %), C19 : 0 cyclo 8c (23.7 %), C16 : 0 (7.5 %) and C18 : 0 (2.7 %). The G+C content of the genomic DNA of strain Jip08T is 69 mol% (as determined by HPLC).
The type strain, Jip08T (=KCTC 12212T=NBRC 100963T=IAM 15215T), was isolated from rice straw in Daejeon, South Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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