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State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Zhong-Guan-Cun, Haidian, Beijing 100080, China
Correspondence
Shuang-Jiang Liu
shuangjiang{at}hotmail.com
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9c (19·0 %). It is proposed that S13T (=CGMCC 1.3801T=JCM 13331T) represents the type strain of a novel species, Flavobacterium saliperosum sp. nov.
Published online ahead of print on 13 October 2005 as DOI 10.1099/ijs.0.64065-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Flavobacterium saliperosum S13T is DQ021903.
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), since when many amendments to its description have been made. Bernardet et al. (1996) defined that Flavobacterium species are Gram-negative rods, are motile by gliding, contain menaquinone-6 (MK-6) as the sole respiratory quinone, and have DNA G+C contents in the range 32–37 mol% (among other properties). The 26 species within the genus Flavobacterium that are recognized at the time of writing appear in Fig. 1 (see later). Flavobacterium species are physiologically diverse: they can be psychrophilic, psychrotolerant or mesophilic, and can be halophilic, halotolerant or sensitive to salts. As a reflection of their physiological diversity, Flavobacterium species have been isolated from various habitats such as freshwater sediments and soil (Bernardet et al., 1996; Tamaki et al., 2003; McCammon & Bowman, 2000), a glacier (Zhu et al., 2003) and Antarctic lakes (Van Trappen et al., 2003, 2004; McCammon et al., 1998; McCammon & Bowman, 2000; Humphry et al., 2001; Yi et al., 2005).
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). Thus, efforts to isolate these CFB-related bacteria were made in our laboratory. We report here the isolation and identification of one of these organisms, designated strain S13T. To isolate Flavobacterium species, a method that combines plate culture with PCR-guided screening was established. First, colonies on agar plates were obtained by plating 10-fold dilutions (10–1–10–5) of lake sediment samples on dilute nutrient medium (DNM; 0·5 g beef extract l–1, 1 g fish peptone l–1, 0·5 g NaCl l–1, 15 g agar l–1) and incubating at 25 °C for 10 days. Second, yellow-pigmented colonies were checked by PCR using a forward primer (designed here according to alignment of 16S rRNA gene sequences of some CFB group members: 5'-ACGGGTGGCGGAACACGTACAG-3') and a reverse primer (1468R, a general primer for bacteria: 5'-CTGGCCACAGACGTGGAG-3'). Eight of 40 colonies gave positive signals during PCR detection. DNA sequences of these eight strains showed that they have almost identical 16S rRNA genes (500 bp at the 5'-end). One representative strain, S13T, was selected for phylogenetic analysis and for biochemical and physiological characterization.
Routine cultivation was conducted at 25 °C with modified M1 medium (ATCC) or Shieh medium (Song et al., 1988). Gram reactions were determined according to the method described by Gerhardt et al. (1994). Cell flagellation and morphology were examined by transmission and scanning electron microscopy. Chitin hydrolysis was tested as described by Hsu & Lockwood (1975). Hydrolysis of CM-cellulose (0·5 %, w/v), gelatin (0·5 %, w/v), casein (50 % skimmed milk, v/v), agar (1·5 %, w/v), alginate (0·5 %, w/v), pectin (0·5 %, w/v), aesculin (0·5 %, w/v), starch (0·5 %, w/v), L-tyrosine (0·5 %, w/v), Tween 80 (1 %, v/v) and egg yolk (5 %, w/v) was tested using the standard mineral base of Stanier et al. (1966). After the mineral base was autoclaved, each compound was added. Growth was examined after incubation at 25 °C for 1, 3, 7 and 14 days. Catalase and oxidase activities, Voges–Proskauer reaction, brown diffusible pigment on L-tyrosine agar and production of H2S were also investigated according to the methods of Dong & Cai (2001). Aerobic and anaerobic production of acids (OF reaction) from carbohydrates was determined in OF basal medium (Hugh & Leifson, 1953). Carbohydrate solution sterilized by filtration was added at final concentration of 1 % (w/v), and acid production was recorded after 7 and 14 days incubation.
Flexirubin-type pigments were detected by using 20 % KOH (Fautz & Reichenbach, 1980) and extracellar glycans were identified with the Congo red absorption test (McCammon & Bowman, 2000). Growth temperature was determined with a TN3F temperature-gradient incubator (Advantec). Growth on seawater agar, nutrient agar and trypticase soy agar was tested at 25 °C after 14 days growth. The presence of gliding motility was determined as described by Zhu et al. (2003). Salt tolerance was tested in M1 medium supplemented with 0–2 % NaCl (spun at 100 r.p.m., 25 °C) incubated for 3 days. Duplicate antibiotic-sensitivity tests were performed using filter-paper discs (ø3·25x0·75 mm) containing each of the following: ceftazidime (30 µg), ceftriaxone (30 µg), tobramycin (10 µg), trimethoprim/sulfamethoxazole (1·25/23·75 µg); penicillin (10 IU), erythromycin (15 µg), tetracycline (30 µg), gentamicin (10 µg), chloramphenicol (30 µg), azithromycin (15 µg), kanamycin (30 µg), streptomycin (10 µg) and rifampicin (5 µg). Discs were placed on M1 medium plates spread with S13T culture and were then incubated at 25 °C for 18–24 h.
Cells of strain S13T were Gram-negative and aerobic, with a size range of 0·35–0·55x1·5–2·5 µm. Flagella were not observed. Colonies were yellow, smooth, circular and 2–4 mm in diameter. Growth was observed over a temperature range of 20–34 °C and a pH range of 6·5–8·5. Gelatin and casein were hydrolysed but starch, CM-cellulose, agar, alginate, aesculin, chitin and pectin were not. A detailed description of assimilation of sugars, sensitivity to antibiotics, etc., is provided in the species description. Growth occurred without adding NaCl to the medium and was severely inhibited in the presence of more than 1 % NaCl; the optimal concentration of NaCl for growth was 0·1 % in medium. Differential phenotypic characteristics of strain S13T and related Flavobacterium species are detailed in Table 1.
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and were analysed by HPLC (Wu et al., 1989), with the MK-6 from Salegentibacter holothuriorum (NBRC 100249T) used as a reference. S. holothuriorum NBRC 100249T was cultivated in liquid Zobell 2216E medium (150 r.p.m., 25 °C) for 36 h. Strain S13T was shown to have MK-6 as the major respiratory quinone. DNA base composition was determined by thermal denaturation (Marmur & Doty, 1962) using DNA from Escherichia coli K-12 as a control. The DNA G+C content of strain S13T was 41 mol%, which is higher than that of other Flavobacterium species (32–37 mol%).
The nearly complete 16S rRNA gene of strain S13T (1400 bp) was amplified and sequenced as described by Zhang et al. (2003). Alignments of 16S rRNA gene sequences were performed with the CLUSTAL_X program, version 1.64b (Thompson et al., 1997). A phylogenetic tree (Fig. 1
) was constructed by the neighbour-joining method (Saitou & Nei, 1987) with Kimura's two-parameter calculation model (Kimura, 1980). 16S rRNA gene sequence analysis indicated that strain S13T was phylogenetically related to members of the genus Flavobacterium, with similarity ranging from 89·1 to 93·8 %. Strain S13T had highest 16S rRNA gene sequence similarity to Flavobacterium columnare (93·8 %) and to Flavobacterium aquatile (93·3 %). The phylogenetic tree indicated that strain S13T clustered with F. columnare, and that this cluster was further grouped with the remaining recognized species of the genus Flavobacterium with strong support (100 %). Based on this phylogenetic analysis and the phenotypic properties, we concluded that strain S13T represents a novel, aquatic species within the genus Flavobacterium. Because it is sensitive to NaCl (1 % NaCl completely halted growth), we propose the name Flavobacterium saliperosum sp. nov.
Description of Flavobacterium saliperosum sp. nov.
Flavobacterium saliperosum (sal.i.per.o'sum. L. n. sal, salis salt; L. adj. perosum detesting, hating greatly; N.L. neut. adj. saliperosum salt-hating).
Gram-negative, aerobic and heterotrophic, with cell size of 0·35–0·55x1·5–2·5 µm. Non-flagellated and non-gliding. Colonies are yellow, smooth and circular with entire margins. Grows at temperatures of 20–34 °C (optimum growth at 28 °C). Grows over pH range 6·5–8·5 (optimum growth at pH 7·5). A concentration of NaCl above 1 % severely inhibits growth (optimum growth is at 0·1 % NaCl in medium). Grows on nutrient agar and trypticase soy agar but not on seawater agar. Voges–Proskauer reaction and oxidase are negative but catalase and lipase are positive. Cells contain flexirubin pigments. Does not reduce nitrate or sulfate. Forms a precipitate on egg-yolk agar and produces brown pigment on tyrosine agar. Hydrolyses L-tyrosine, gelatin and casein, but not agar, alginate, aesculin, starch, CM-cellulose, chitin or pectin. Does not produce acid from the following sugars: lactose, arabinose, rhamnose, raffinose, ribose, galactose, melibiose, D-melezitose, sucrose, xylose, mannose, fucose, fructose, glucose, cellobiose, maltose, salicin, laetrile, mannitol or sorbitol. Filter-paper disc tests indicate resistance to ceftazidime, ceftriaxone, tobramycin and trimethoprim/sulfamethoxazole, but susceptibility to penicillin, erythromycin, tetracycline, gentamicin, chloramphenicol, azithromycin, kanamycin, streptomycin and rifampicin. Cells contain menaquinone-6 (MK-6). Cellular fatty acids are iso-C15 : 0 (28·2 %), iso-C17 : 19c (19·0 %), iso-C17 : 0 3-OH (8·6 %), iso-C16 : 0 (6·9 %), iso-C15 : 1G (6·7 %), iso-C15 : 0 3-OH (5·0 %), C15 : 0 (4·6 %), anteiso-C15 : 0 (3·9 %), C16 : 17c (2·6 %), iso-C16 : 1H (2·3 %), iso-C16 : 0 3-OH (2·0 %), iso-C14 : 0 (1·3 %), C17 : 0 2-OH (1·0 %), iso-C15 : 0 2-OH (0·9 %), iso-C13 : 0 (0·5 %), C15 : 16c (0·42 %) and C17 : 16c (0·3 %). DNA G+C content is 41 mol%.
The type strain, S13T (=CGMCC 1.3801T=JCM 13331T), was isolated from freshwater lake sediment.
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