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Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon, 305-701, Republic of Korea
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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ABSTRACT |
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9c) supported the classification of strain Kw05T within the genus Flavobacterium. Kw05T therefore represents a novel species, for which the name Flavobacterium granuli sp. nov. is proposed. The type strain is Kw05T (=KCTC 12201T=IAM 15099T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Kw05T is AB180738.
Fatty acid profiles of strain Kw05T and related type strains of Flavobacterium species are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). The formal description of the genus has been emended several times since then. At present, the genus comprises 28 species. Bernardet et al. (1996) proposed an amendment to the description, which states that the genus Flavobacterium represents predominantly gliding, pigmented bacteria that have menaquinone-6 as the primary respiratory quinone. Species of the genus Flavobacterium have DNA G+C contents of 32–37 mol%. Through emendation of classification, several species previously placed in the genus Flavobacterium have been reclassified and placed in new or different genera, including the genera Microbacterium (Takeuchi & Hatano, 1998), Salegentibacter (McCammon & Bowman, 2000) and Planococcus (Nakagawa et al., 1996). Several species previously placed in other genera, including Cytophaga and Flexibacter, have been reclassified and placed in the genus Flavobacterium (Bernardet et al., 1996). Flavobacterium species have been isolated from diverse habitats such as fresh and salt water, soil, sediment, sea ice, diseased fish and microbial mats.
Strain Kw05T was isolated from granules used in the wastewater treatment plant of a beer-brewing factory in Kwang-Ju, Republic of Korea. Anaerobic granules are bacterial aggregates that result from the flocculation of sludge in an upflow anaerobic sludge blanket (UASB) reactor (de Zeeuw & Lettinga, 1980). They are composed of micro-organisms, inorganic nuclei and extracellular polymers (Fukuzaki et al., 1991; Shen et al., 1993). Great attention has been paid to the internal structure and catalytic activities of these granules (MacLeod et al., 1990; Schmidt & Ahring, 1996). In our laboratory, the relationship between the structure and resistance to toxic chemicals of anaerobic granules from a brewery wastewater treatment UASB reactor was studied (Bae & Lee, 1999; Bae et al., 2000). In a series of studies, we attempted to isolate micro-organisms from the anaerobic granules in order to investigate the community structure based on a culture system. Interestingly, the granules contained aerobic bacteria even though they had been kept under anaerobic conditions for 2 years. Strain Kw05T was one of the dominant bacterial isolates that was grown under aerobic conditions.
A polyphasic approach, including phylogenetic analysis based on 16S rRNA gene sequences, chemotaxonomic and phenotypic properties, was conducted to determine the precise taxonomic position of strain Kw05T. The results obtained indicated that it can be assigned as a member of the genus Flavobacterium, but it is clearly distinguished from recognized Flavobacterium species. Here, we propose Kw05T as the type strain of a novel species, Flavobacterium granuli sp. nov.
For isolation of aerobic bacteria, brownish black granules (around 2 mm in diameter) from a brewery wastewater treatment UASB reactor were homogenized using an Ace homogenizer (Nihonseiki Kaisha Ltd). The suspension was spread on R2A agar plates (Difco) after being serially diluted with 50 mM phosphate buffer (pH 7·0). The plates were incubated at 30 °C for 2 weeks. Single colonies on the plates were purified by transferring them onto new plates and incubating again under the same conditions. The purified colonies were tentatively identified based on partial sequences of the 16S rRNA gene. Strain Kw05T was one of the dominant isolates that appeared on the plates following aerobic incubation.
Gram reaction was performed using the non-staining method as described by Buck (1982). Cell morphology was observed under a Nikon light microscope at x1000 magnification, with cells grown for 3 days at 30 °C on R2A agar. Catalase and oxidase tests were performed by the procedures outlined by Cappuccino & Sherman (2002). Substrate utilization as the sole carbon source and physiological characteristics were determined with API 32GN, API 20E and API 20NE galleries according to the manufacturer's instructions (bioMérieux). Congo red absorption, presence of gliding motility (Bernardet et al., 2002), production of flexirubin-type pigments (Reichenbach, 1989), degradation of DNA [using DNA agar (Difco) supplemented with 0·01 % toluidine blue (Merck)], degradation of casein, chitin, starch and L-tyrosine (Atlas, 1993), production of a brown diffusible pigment on L-tyrosine agar and precipitation on egg-yolk agar (Atlas, 1993) were also investigated; reactions were read after 5 days. Hydrolysis of carboxymethyl cellulose was tested as described by Ten et al. (2004). Growth at different temperatures and pH was assessed after 5 days of incubation. Salt tolerance was tested on R2A medium supplemented with 1–10 % (w/v) NaCl after 7 days of incubation. Duplicate antibiotic-sensitivity tests were performed using filter-paper discs containing the following: streptomycin (5, 10 and 15 µg ml–1) (Mast Diagnostics), tetracycline (5, 10 and 15 µg–1), kanamycin (1·0, 1·5 and 2·0 mg ml–1) and ampicillin (20, 25 and 30 µg ml–1) (Sigma). Discs were placed on R2A plates containing cultured Kw05T and were then incubated at 30 °C for 7 days. Growth was tested against various dissolved antibiotics in duplicate at three different concentrations. A discrete Kw05T colony (grown at 30 °C on R2A medium) was then spread onto each plate. (Humphry et al., 2001). Physiological and biochemical characteristics of strain Kw05T and related type strains of Flavobacterium species are summarized in Table 1.
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. 16S rRNA gene sequences of related taxa were obtained from GenBank. Multiple alignments were performed 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). The phylogenetic tree was constructed using a neighbour-joining method (Saitou & Nei, 1987) in the MEGA 2 program (Kumar et al., 2001) with bootstrap values based on 1000 replications (Felsenstein, 1985) (Fig. 1).
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using reversed-phase HPLC. Quinones were extracted from cells grown on R2A broth (Difco) and analysed as described by Komagata & Suzuki (1987) using reversed-phase HPLC. Cellular fatty acids were analysed in organisms grown on trypticase soy agar (TSA; Difco) for 2 days. Cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acids analysed by GC (Hewlett Packard 6890) were identified using the Microbial Identification software package (Sasser, 1990).
Cells of strain Kw05T are aerobic, Gram-negative and rod-shaped. Movement via flagella was not observed. Colonies grown on R2A agar plates (Difco) for 2 days were smooth, circular, non-glossy, yellow in colour and 2–4 mm in diameter. On R2A agar, strain Kw05T was able to grow at 15–30 °C but not at 4 or 45 °C. Physiological characteristics of strain Kw05T are summarized in the species description below and comparison of selective characteristics with related type strains of Flavobacterium species is given in Table 1
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The 16S rRNA gene sequence of strain Kw05T was a continuous stretch of 1442 bp. Sequence similarity calculations based on a neighbour-joining analysis indicated that the closest relatives of strain Kw05T were Flavobacterium limicola (96·6 %), Flavobacterium hibernum (96·3 %), Flavobacterium hydatis (96·1 %) and Flavobacterium xinjiangense (96·1 %). Lower sequence similarities (<97·0 %) were found with all recognized species of the genus Flavobacterium. It has been suggested that in bacterial strains with less than 97 % 16S rRNA gene sequence similarity, levels of DNA–DNA hybridization are less than 70 % (Stackebrandt & Goebel, 1994), which defines a genomic species (Wayne et al., 1987). Thus, based on the 16S rRNA gene sequence analysis, strain Kw05T represents a novel species.
The G+C content of the genomic DNA of strain Kw05T was 36·2 mol% and its major quinone was MK-6. The fatty acid profile of strain Kw05T comprised iso-C15 : 0 (28·2 %), iso-C17 : 0 3-OH (11·2 %), iso-C15 : 0 3-OH (10·3 %), iso-C17 : 19c (7·5 %), C15 : 16c (5·6 %), C15 : 0 (5·0 %), iso-C15 : 1G (4·5 %), unknown 13·566 (4·3 %), C17 : 16c (4·1 %), anteiso-C15 : 0 (3·1 %), iso-C13 : 0 (2·9 %), summed feature 4 (C16 : 17c/iso-C15 : 0-2OH, 2·76 %), summed feature 5 (anteiso-C17 : 1B/I, 2·2 %), iso-C16 : 0 3-OH (1·6 %), C16 : 0 (1·42 %), iso-C14 : 0 (1·0 %), C17 : 18c (1·0 %) and C18 : 0 (1·0 %). No significant differences in the fatty acid profiles for the other Flavobacterium species were found, except that some strains have C16 : 17c as a major component. The fatty acid profiles of strain Kw05T and related type strains of Flavobacterium species are available as supplementary material in IJSEM Online.
The results of our polyphasic analysis support the recognition of a novel species within the genus Flavobacterium, for which the name Flavobacterium granuli sp. nov. is proposed.
Description of Flavobacterium granuli sp. nov.
Flavobacterium granuli (gra.nu'li. L. gen. n. granuli of a small grain, pertaining to a granule, from which the type strain was isolated).
Cells are aerobic, Gram-negative, rod-shaped, non-motile and non-gliding (i.e. non-flagellated), 0·3–0·5 µm wide by 2·0–5·0 µm long. Colonies grown on R2A are circular, convex and yellow-coloured: flexirubin-type pigments are not detected. Congo red is not absorbed. Temperature range for growth is 15–37 °C; no growth occurs at 45 °C. Optimum temperature for growth is 25–30 °C. Growth occurs in the absence of NaCl and in the presence of 1·0 % (w/v) NaCl, but not more than 2 % (w/v) NaCl. Catalase- and oxidase-positive and strictly heteroorganotrophic. Cannot grow anaerobically. H2S is not produced. Nitrate is not reduced to nitrite. No precipitation is produced on egg-yolk agar. Urease, 
-glucosidase and -galactosidase are positive. Acetoin is produced. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, gelatinase and citrate utilization are negative. Does not produce any acid or gas from glucose. Casein, xylan, chitin, DNA, tyrosine, carboxymethyl cellulose, gelatin and starch are not degraded. The following are utilized as sole carbon sources: glucose, mannose, N-acetylglucosamine, maltose, propionate and L-proline. The following are not utilized as sole carbon sources: L-arabinose, mannitol, gluconate, caprate, adipate, malate, citrate, phenylacetate, salicin, D-melibiose, L-fucose, D-sorbitol, valerate, histidine, 2-ketogluconate, 3-hydroxybutyrate, 4-hydroxybutyrate, rhamnose, D-ribose, inositol, D-sucrose, itaconate, suberate, malonate, acetate, lactate, L-alanine, 5-ketogluconate, glycogen, 3-hydroxybenzoate and L-serine. Resistant to 20 µg ampicillin ml–1, 15 µg tetracycline ml–1 and 15 µg streptomycin ml–1, and sensitive to 0·5 mg kanamycin ml–1. Major cellular fatty acids are iso-C15 : 0 (28·2 %), iso-C17 : 0 3-OH (11·2 %), iso-15 : 0 3-OH (10·3 %) and iso-C7 : 19c (7·5 %). The G+C content of the genomic DNA of the type strain is 36·2 mol% (as determined by HPLC).
The type strain, Kw05T (=KCTC 12201T=IAM 15099T), was isolated from granules used in the wastewater treatment plant of a beer-brewing factory in Kwang-Ju, Republic of Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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