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Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE), 2-5-8 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan
Correspondence
Shams Tabrez Khan
shams-tabrez-khan{at}nite.go.jp
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7c and/or iso-15 : 0 2-OH). The DNA G+C content was 37 mol%. The phylogenetic distance to the type strains of all recognized species in the family Flavobacteriaceae and the phenotypic properties of strain Mok-17T supported its classification as representing a novel species in a new genus, for which the name Galbibacter mesophilus gen. nov., sp. nov. is proposed. The type strain is Mok-17T (=NBRC 101624T=CIP 109219T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Mok-17T is AB255367.
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; Brown & Bowman, 2001) where they perform different functions of ecological importance. Their possible roles in the carbon cycle (Abell & Bowman, 2005), degradation of dissolved and particulate organic matter (Cottrell & Kirchman, 2000; Davey et al., 2001), fish pathology (Bernardet, 1998) and algicidal activity (Maeda et al., 1998) have been discussed. Comprising more than 50 genera at the time of writing, the family Flavobacteriaceae is one of the major branches of the phylum Bacteroidetes. Recently, new members of the family have been isolated from various marine environments (Nedashkovskaya et al., 2003, 2004, 2005). Our group has also characterized several novel species in the family, mostly from sandy sediments collected from the Pacific coasts of Japan (Khan et al., 2006a, b, c, 2007). Here we describe the isolation of a new strain, designated Mok-17T, from a marine sediment sample collected in Okinawa Island, Japan. The sample was diluted in artificial seawater (ASW; Naigai Chemicals) and aliquots (0.1 ml) of serial dilutions were plated on marine agar 2216 (MA; Difco). Strain Mok-17T was one of the yellow colonies that grew on the plates. Unless mentioned otherwise, plates or slants of half-strength MA (HSMA) diluted with ASW were used for routine cultivation at 30 °C. For long-term preservation at –80 °C, bacterial cells were suspended in ASW with 20 % (v/v) glycerol.
Template DNA for 16S rRNA gene amplification was prepared by using Prepman Ultra (Applied Biosystems). The pair of universal primers 27f and 1492r was used to amplify the portion of the 16S rRNA gene corresponding to positions 8–1492 in the Escherichia coli 16S rRNA gene sequence (Brosius et al., 1978). The amplified fragment was sequenced directly by using a BigDye Terminator v3.1 cycle sequencing kit and an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The ATGC program (Genetyx) was used for sequence editing and assembly. The assembled sequence was then compared with the 16S rRNA gene sequences in the DNA Database of Japan (DDBJ) using BLAST searches (Altschul et al., 1990) and was aligned with related sequences by using CLUSTAL X (Thompson et al., 1997). Phylogenetic trees were inferred by using the neighbour-joining (NJ; Saitou & Nei, 1987), maximum-parsimony (MP; Swofford, 2000) and maximum-likelihood (ML; Adachi & Hasegawa, 1996) algorithms. The topology of the trees was evaluated by bootstrap resampling analysis (Felsenstein, 1985) of 1000 replicates for NJ and 100 replicates for MP and ML. The BLAST search result clearly indicated that strain Mok-17T represented a member of the family Flavobacteriaceae. It shared highest 16S rRNA gene sequence similarity (92 %) with an unclassified bacterium, ‘Flavobacterium’ sp. (DDBJ accession no. AJ244702). Strain Mok-17T showed highest 16S rRNA gene sequence similarity to recognized species of 91 % with both Leeuwenhoekiella marinoflava (DDBJ accession no. AY167315) and Robiginitalea biformata (DDBJ accession no. AY424900). Pairwise 16S rRNA gene sequence comparison of strain Mok-17T with other recognized members of the family Flavobacteriaceae revealed similarities of only 80–91 %. In an NJ tree based on 16S rRNA gene sequences, strain Mok-17T formed a novel lineage in the family Flavobacteriaceae, although the branching order of the lineage was not strongly supported by bootstrap analysis (Fig. 1). Similar results were obtained with MP and ML analyses (data not shown).
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). Cell morphology was observed under an Olympus light microscope (CX41LF) without staining and after Gram-staining performed as described by Cowan & Steel (1993). Gliding motility was tested by observing the spread of colony edges on MA plates (Perry, 1973) and by microscopic observation of hanging drops of a marine broth 2216 (MB; Difco) culture (Bernardet et al., 2002) under a 1000x oil-immersion objective (Olympus CX41LF). The absorption spectrum (260–700 nm) of an acetone extract of the cells was obtained with a Shimadzu UV–visible spectrophotometer (UV-1650 PC) to assess the presence of carotenoid pigment(s). The bathochromic shift test with 20 % KOH (w/v) was performed for the detection of flexirubin-type pigments (Fautz & Reichenbach, 1980). Growth at different temperatures (4, 10, 20, 30, 35, 37, 40, 42 and 45 °C) was assessed on HSMA plates. Salt tolerance was tested in MB with final NaCl concentrations adjusted to 3, 5, 6, 7, 8, 9, 10 and 15 % (w/v). Growth was also tested in one-fifth-strength LB [2 g Bacto tryptone (Difco) and 1 g Bacto yeast extract (Difco) dissolved in a final volume of 1000 ml] containing 0, 30, 50 and 70 % (v/v) ASW. Cells from a 2-day-old HSMA culture were spotted on a glass slide and were flooded with 3 % hydrogen peroxide (v/v) to test for the presence of catalase. The presence of oxidase was tested by spotting a cell suspension (in sterile water) on a cytochrome oxidase strip (Nissui Pharmaceuticals). MB solidified with 1.5 % carrageenan (Type I; Sigma) or agar was used to test the ability of the strain to degrade these molecules. Degradation of cellulose was tested by immersing cellulose strips (Whatman No. 1 paper) in a culture of strain Mok-17T in one-fifth-strength LBM (one-fifth-strength LB prepared with ASW) at 30 °C for 1 month. Degradation of carboxymethylcellulose (CM-cellulose) was tested by observing the liquefaction of one-fifth-strength LBM solidified with 3 % (w/v) CM-cellulose (High Viscosity; Sigma), while the methods described by Cowan & Steel (1993) were used to test for the degradation of starch, gelatin, chitin, casein and DNA. The recommendations of the manufacturer were followed for the API 20NE tests (bioMérieux) except that inocula were prepared by suspending cells in sterile ASW. The GN2 MicroPlate system (Biolog) was used to test for the utilization of 95 different carbon sources. Inocula were prepared as suggested by Rüger & Krambeck (1994) and the microplate was incubated at 30 °C for 26–30 h after inoculation.
The Sherlock Microbial Identification system (MIDI) was used for the analysis of fatty acid methyl esters of strain Mok-17T cultivated on MA for 82 h at 20 °C. The predominant cellular fatty acids were iso-17 : 0 3-OH, iso-15 : 0, iso-15 : 1 and summed feature 3 (16 : 17c and/or iso-15 : 0 2-OH). The detailed fatty acid profiles of strain Mok-17T and related organisms are given in Table 1. Isoprenoid quinones were extracted and analysed according to the protocol of Nakagawa & Yamasato (1993).
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. The HPLC method of Mesbah et al. (1989) was used to determine the G+C content of the chromosomal DNA.
Table 2 lists the phenotypic characteristics that can be used to differentiate strain Mok-17T from the three recognized Leeuwenhoekiella species and R. biformata. Other phenotypic characteristics of strain Mok-17T are given in the genus and species descriptions. Based on the large phylogenetic distances and differential phenotypic characteristics, it is proposed that strain Mok-17T be classified as representing a novel species of a new genus in the family Flavobacteriaceae, for which the name Galbibacter mesophilus gen. nov., sp. nov. is proposed.
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Cells are Gram-negative rods. Catalase- and oxidase-positive. The main respiratory quinone is menaquinone-6. Major fatty acids are iso-17 : 0 3-OH, iso-15 : 0, iso-15 : 1 and summed feature 3 (16 : 17c and/or iso-15 : 0 2-OH). Member of the family Flavobacteriaceae. The type species is Galbibacter mesophilus.
Description of Galbibacter mesophilus sp. nov.
Galbibacter mesophilus [me.so.phi'lus. Gr. adj. mesos middle; Gr. adj. philos loving; N.L. masc. adj. mesophilus middle (temperature)-loving, i.e. mesophilic].
The description is as for the genus with the following additional properties. Cells are 0.5–0.7 µm in width and 1.0–3.0 µm in length. Colonies on MA are yellow with irregular margins after 3 days incubation at 30 °C. The edge of some colonies may show swarming growth; however, gliding motility is not observed in hanging-drop preparations. Carotenoid-type pigments are produced but flexirubin-type pigments are not. Growth occurs at temperatures between 10 and 42 °C, with optimal growth at 25–30 °C. No growth is observed at 4 or 45 °C. Growth occurs in the presence of 30–70 % (v/v) ASW but not in the absence of seawater. Growth occurs in the presence of 3–7 % NaCl (w/v) and optimally with 3–5 % NaCl (w/v). Positive for the degradation of starch, aesculin and gelatin, reduction of nitrate to nitrite, and production of 
-galactosidase. Negative for arginine dihydrolase activity and for the degradation of agar, carrageenan, urea, arginine, DNA, chitin, cellulose and CM-cellulose. Weakly positive for the production of indole from tryptophan and for casein degradation. Acid is not produced from glucose. The following compounds are utilized: 
-cyclodextrin, dextrin, cellobiose, D-fructose, D-galactose, gentiobiose, -D-glucose, -D-lactose, lactulose, maltose, D-mannose, D-melibiose, methyl -D-glucoside, D-raffinose, sucrose, D-trehalose, turanose, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-glutamic acid, L-proline, L-serine and L-threonine. The detailed fatty acid composition is given in Table 1
. The DNA G+C content of the type strain is 37 mol%.
The type strain, Mok-17T (=NBRC 101624T=CIP 109219T), was isolated from a marine sediment sample collected in Okinawa Island, Japan.
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ACKNOWLEDGEMENTS |
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