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School of Earth and Environmental Sciences and Research Institute of Oceanography, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
Correspondence
Byung C. Cho
bccho{at}snu.ac.kr
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CL-TF09T is AY962293.
The complete fatty acid profile of strain CL-TF09T is given in Supplementary Table S1 available in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Since the proposal of the family Flavobacteriaceae by Jooste (1985) and subsequent validation and emended description (Reichenbach, 1992; Bernardet et al., 1996, 2002), more than 40 genera have been assigned to the family Flavobacteriaceae, about half of which have been described since 2004. The family Flavobacteriaceae includes species from various habitats, including freshwater, brackish and marine waters, soil and epibenthic fauna, and members of the family are known to be proficient in degrading various biopolymers such as cellulose, chitin and pectin (Kirchman, 2002). Recently, phylogenetic analyses have revealed that many marine species in the family clustered together into a well-defined ‘marine clade’ (Bowman & Nichols, 2005). These marine flavobacteria are known to be important members of the bacterial community in many aquatic environments and have a specialist role in using high-molecular-mass dissolved organic matter (Kirchman, 2002). In this study, a bacterium, designated strain CL-TF09T, affiliated with the ‘marine clade’ of the family Flavobacteriaceae, was isolated from tidal flat sediment in Ganghwa, Korea. Sediment slurry was spread on a marine agar 2216 (MA; Difco) plate, which was then incubated at 30 °C for 1 week. Strain CL-TF09T was isolated and subsequently purified on MA at 30 °C four times. The strain was maintained both on MA at 4 °C and in marine broth 2216 (MB; Difco) supplemented with 30 % (v/v) glycerol at –80 °C.
The 16S rRNA gene was amplified from a single colony by PCR with Taq DNA polymerase (Bioneer) and primers 27F and 1492R (Lane, 1991). The PCR product was purified using the AccuPrep PCR purification kit (Bioneer) and cloned using the pCR2.1 TOPO TA cloning kit (Invitrogen). Sequencing of the 16S rRNA gene was performed with an Applied Biosystems automatic sequencer (ABI3730XL) at Macrogen, Seoul, Korea. The almost complete 16S rRNA gene sequence of strain CL-TF09T (1444 bp) was obtained. The sequence was compared with those available in the GenBank using BLAST-N searches (Altschul et al., 1990). The 16S rRNA gene sequence of strain CL-TF09T was manually aligned with those of the type strains of species belonging to genera phylogenetically related to CL-TF09T and of the type species of other genera in the family Flavobacteriaceae obtained from GenBank and the Ribosomal Database Project (Cole et al., 2003) databases using known 16S rRNA gene secondary structure information. Phylogenetic trees were obtained by use of the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods. An evolutionary distance matrix for the neighbour-joining method was generated according to the model of Jukes & Cantor (1969). The robustness of tree topologies was assessed by bootstrap analyses based on 1000 replications for the neighbour-joining and maximum-parsimony methods and 100 replications for the maximum-likelihood method. Alignment analysis was carried out using the jPHYDIT program (version 1.0, available at http://chunlab.snu.ac.kr/jphydit/), and phylogenetic analyses were carried out using MEGA 3 (Kumar et al., 2004) and PAUP* 4.0 (Swofford, 1998). Likelihood parameters were estimated by the hierarchical ratio tests in MODELTEST version 3.04 (Posada & Crandall, 1998). Sequence similarity indicated that the closest relatives of strain CL-TF09T belonged to the genera Tenacibaculum (90·6–91·8 %) and Polaribacter (91·0–91·5 %). Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain CL-TF09T formed a very robust clade with Tenacibaculum and Polaribacter species, but could not be linked to any of the known genera in the family Flavobacteriaceae (Fig. 1). Thus, strain CL-TF09T was recognized as representing a new genus. The DNA G+C content was determined by HPLC analysis of deoxyribonucleosides as described by Mesbah et al. (1989) after DNA purification following the method of Marmur (1961). The DNA G+C content was 33·9 mol%.
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. Cell morphology was examined by phase-contrast microscopy and transmission electron microscopy (JEOL EX2) with cells grown at 30 °C in MB. Gliding motility was observed by the hanging drop method (Suzuki et al., 2001). Anaerobic growth was checked on MA using the GasPak anaerobic system (BBL). Catalase and oxidase activities were determined according to the protocols described by Smibert & Krieg (1994), and gelatinase, amylase, DNase, nitrate reductase activities and degradation of Tween 80 were examined as described by Hansen & Sørheim (1991). Cells of strain CL-TF09T were rods 0·3–0·8 µm in width and 1·0–5·7 µm in length when in the exponential growth phase, but irregular rods or spherical cells were observed in older cultures (Fig. 2). Cells were non-motile (Table 1). Colonies on MA were circular, smooth, entire, convex, shining and yellow. After 1 week incubation, colonies were about 2 mm in diameter.
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-galactosidase, acid production from glucose, and hydrolysis of aesculin were tested using the API 20NE kit (bioMérieux) according to the manufacturer's instructions, except that cell suspension was prepared using artificial sea water [(ASW) 24 g NaCl, 5·1 g MgCl2, 4 g Na2SO4, 1·1 g CaCl2, 0·7 g KCl, 0·2 g NaHCO3, 0·1 g KBr, 0·027 g H3BO3, 0·024 g SrCl2, 0·003 g NaF, per litre distilled water; Lyman & Fleming, 1940] as a suspension medium. Carbon utilization was tested on basal agar medium supplemented with yeast extract (23·6 g NaCl, 0·64 g KCl, 4·53 g MgCl2.6H2O, 5·94 g MgSO4.7H2O, 1·3 g CaCl2.2H2O, 0·2 g NaNO3, 0·2 g NH4Cl, 15 g Bacto agar, 0·05 g yeast extract, per litre distilled water; Bruns et al., 2001) containing 0·2 % of the carbon source. Incubation was prolonged for 1 month and growth was scored as positive when visible colonies were observed. Growth of CL-TF09T was observed at temperatures of 5–30 °C, with optimum growth at 25–30 °C. Growth occurred from pH 7 to 8. The strain could grow at sea salt concentrations from 1 to 5 % but could not grow on agar plates with 3 % NaCl as the sole salt. Strain CL-TF09T could degrade starch, gelatin and aesculin (Table 1
). However, it was not able to reduce nitrate to nitrite. CL-TF09T was negative for oxidase and weakly positive for -galactosidase.
Isoprenoid quinones were isolated according to the method of Minnikin et al. (1984) and analysed by HPLC as described by Collins (1985). The major isoprenoid quinone in CL-TF09T was menaquinone-6 (MK-6). Pigments were extracted using 90 % acetone from cells cultured in the dark, and then examined by spectroscopy. Flexirubin-type pigments were detected by using a colour shift test using a 20 % (w/v) KOH solution (Reichenbach, 1992). The spectrum of CL-TF09T showed two peaks at 450 and 475 nm that are typical for carotenoids. The strain did not contain flexirubin-type pigments. Fatty acid methyl esters in whole cells were analysed by GC according to the instructions of the Microbial Identification System (MIDI) at the Korean Culture Center of Microorganisms in Korea. The fatty acid profile for strain CL-TF09T was dominated by iso-C15 : 0 3-OH (17·4 %), iso-C15 : 0 (16·7 %), anteiso-C15 : 0 (15·1 %) and iso-C16 : 0 3-OH (13·4 %) (Table 1; the complete fatty acid profile of CL-TF09T is given in Supplementary Table S1 available in IJSEM Online).
In terms of its phenotypic features, strain CL-TF09T could be differentiated from members of the closely related genera Tenacibaculum and Polaribacter based on colony morphology, pH and temperature range for growth, and biochemical characteristics (Table 1). In particular, hydrolysis of aesculin, a negative response for oxidase, no growth at pH 6, and utilization of pyruvic acid and succinate distinguish strain CL-TF09T from members of the genus Tenacibuculum; and growth at 25 °C and utilization of citrate, L-leucine, tartrate, pyruvic acid and succinate distinguish it from members of the genus Polaribacter (Table 1). In addition, the fatty acid profile of strain CL-TF09T clearly distinguishes it from members of the genera Tenacibaculum and Polaribacter (Table 1). In this regard, the major distinctive difference is a large fraction of anteiso-C15 : 0 in strain CL-TF09T. In addition, they were clearly differentiated by the proportions of several fatty acids, including iso-C16 : 0 3-OH, iso-C15 : 1-G and summed feature 3 (C16 : 1
7c/iso-C15 : 0 2-OH).
In conclusion, phylogenetic analyses based on 16S rRNA gene sequences, fatty acid profiles and phenotypic features suggest that strain CL-TF09T should be classified as the type strain of a novel genus and species, for which the name Lutibacter litoralis gen. nov., sp. nov. is proposed.
Description of Lutibacter gen. nov.
Lutibacter (Lu.ti.bac'ter. L. n. lutum mud; N.L. masc. n. bacter rod; N.L. masc. n. Lutibacter rod from mud).
Cells are Gram-negative and rod-shaped. Growth is heterotrophic and aerobic. Catalase-positive and oxidase-negative. The predominant menaquinone is MK-6. Dominant fatty acids are iso-C15 : 0 3-OH, iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0 3-OH. Cells contain carotenoids but no flexirubin-type pigments. The genus is a member of the family Flavobacteriaceae. The type species is Lutibacter litoralis.
Description of Lutibacter litoralis sp. nov.
Lutibacter litoralis (li.to.ra'lis. L. masc. adj. litoralis of the shore).
Exhibits the following properties in addition to those given in the genus description. Cells are approximately 0·3–0·8 µm wide and 1·0–5·7 µm long. Spherical cells appear in ageing culture. Cells are non-motile. On MA medium, colonies are circular, entire, convex, shining, opaque and yellow. Absorption spectral peaks of the pigments are observed at 450 and 475 nm. Growth occurs at 5–30 °C (optimum 25–30 °C) and at pH values of between 7 and 8. Growth occurs at sea salt concentrations of 1–5 % (w/v). Catalase, amylase, gelatinase and DNase activities are positive. Cytochrome oxidase, nitrate reductase and Tween 80 hydrolysis activities are negative. According to API 20NE tests, aesculin hydrolysis and gelatinase activities are positive, whereas nitrate reductase, indole production, acid production from glucose, arginine dihydrolase and urease are negative. Growth occurs on acetone, citrate, D-fructose, D-raffinose, D-salicin, D-sorbitol, glycine, glycogen, myo-inositol, L-arginine, L-lysine, L-ornithine, pyruvic acid, succinate, tartrate, urea, Casamino acids, L-leucine, peptone, tryptone and yeast extract. No growth occurs on acetate, acetamide, 
-ketobutyric acid, benzoate, DL-cysteine, D-cellobiose, D-galactose, D-glucose, D-mannitol, D-mannose, D-ribose, D-trehalose, D-xylose, ethanol, formic acid, glycerol, inulin, 2-propanol, lactose, L-arabinose, L-ascorbate, L-asparagine, L-rhamnose, maleic acid, N-acetylglucosamine, polyethylene glycol, salicylate, sucrose, thiamine, DL-aspartate, L-proline or L-glutamate. The DNA G+C content is 33·9 mol%.
The type strain, CL-TF09T (=KCCM 42118T=JCM 13034T), was isolated from a tidal flat sediment in Ganghwa, Korea.
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ACKNOWLEDGEMENTS |
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