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1 Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-0032, Japan
2 Marine Biotechnology Institute Co. Ltd, 3-75-1, Heita, Kamaishi, Iwate 026-0001, Japan
Correspondence
Jaewoo Yoon
aa57058{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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7c and C16 : 15c were the major fatty acids. On the basis of this polyphasic evidence, it was concluded that strain Shu-F-UV2-2T represents a novel species in a new genus of the family ‘Flammeovirgaceae’, for which the name Perexilibacter aurantiacus gen. nov., sp. nov. is proposed. The type strain is Shu-F-UV2-2T (=MBIC06993T=IAM 15413T=KCTC 12867T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Shu-F-UV2-2T is AB276355.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Molecular phylogenetic studies based on 16S rRNA gene sequence analysis have revealed that members of the phylum Bacteroidetes are ubiquitous in aquatic environments (DeLong et al., 1993; Bowman et al., 1997; Glöckner et al., 1999; Cottrell & Kirchman, 2000; O'Sullivan et al., 2002). They play a significant role in the decomposition of organic matter and in carbon cycling within the global ecosystem (Cottrell & Kirchman, 2000; Nedashkovskaya et al., 2003). However, despite the significance of their ecological niche, the majority of members of this phylogenetic group remain unknown and as yet uncultured (O'Sullivan et al., 2004). At the time of writing, genera such as Flammeovirga, Flexithrix, Persicobacter and Thermonema (Lewin, 1970; Hudson et al., 1989; Nakagawa et al., 1997; Takahashi et al., 2006) are incorporated within the family ‘Flammeovirgaceae’.
Strain Shu-F-UV2-2T was isolated from a sandy sediment, collected in August 2003 at a shallow beach 1 m deep on Carp Island, Republic of Palau (7° 5.25' N 134° 16.75' E). Surface sediment plus seawater from the same locality were collected by using a spatula. They were kept in a 15 ml tube for 5 h at 15 °C. For microbiological analyses, 1 g sediment was suspended in 15 ml autoclaved artificial seawater (Lyman & Fleming, 1940). A 50 µl sample of this suspension was applied to marine agar 2216 (Difco) and UV-irradiated for 2 min in a laminar flow cabinet. Dull-orange-coloured colonies appeared after incubation at 25 °C. We investigated the phylogenetic position of strain Shu-F-UV2-2T by using a polyphasic taxonomic approach, including 16S rRNA gene sequence analysis, fatty acid composition analysis, quinone analysis and genotypic and physiological comparisons. Based on these data, it is proposed that the new isolate represents a novel species in a new genus of the family ‘Flammeovirgaceae’ in the phylum Bacteroidetes.
The temperature and pH range for growth were determined by incubating on ten-fold diluted marine agar 2216 (MA). Salt tolerance was tested on R2A agar (Difco) with artificial seawater containing 0–5 % (w/v) NaCl. Gram staining was performed as described by Murray et al. (1994). Cell morphology was observed by using light microscopy (BX60; Olympus) and transmission electron microscopy (TEM). Gliding motility was determined as described by Perry (1973). For the TEM observations, cells were mounted on Formvar-coated copper grids and negatively stained with 1 % (w/v) aqueous uranyl acetate. Grids were observed in a JEOL 1010 transmission electron microscope (JEOL) operated at 100 kV. In the course of TEM, cells of various sizes were observed. Cells were mostly straight and rod-shaped. Cells varied between 0.3 and 0.5 µm in width and 10 and 20 µm in length (Fig. 1). Cells were motile by gliding and no flagella were seen by electron microscopy. Growth under anaerobic conditions was determined after 2 weeks incubation in an AnaeroPack (Mitsubishi Gas Chemical Co., Inc.) on MA. Catalase activity was determined by bubble formation in a 3 % H2O2 solution. Oxidase activity was determined by use of cytochrome oxidase paper (Nissui Pharmaceutical Co., Ltd). API 20E, API 50CH and API ZYM strips (bioMérieux) were used to determine physiological and biochemical characteristics. The API 20E results were read after 48 h incubation at 30 °C, and the API 50CH and API ZYM results after 4 h incubation at 37 °C. Determination of the respiratory quinone system and cellular fatty acid content (MIDI system) were carried out as described by Xie & Yokota (2003). DNA was prepared according to the method of Marmur (1961) from cells grown on MA medium and the DNA base composition was determined by using the HPLC method of Mesbah et al. (1989). An approximately 1500 bp fragment of the 16S rRNA gene was amplified from the extracted DNA by using bacterial universal primers specific to the 16S rRNA gene: 27F and 1492R (Weisburg et al., 1991). To ascertain the phylogenetic position of the novel isolate, the 16S rRNA gene sequence of strain Shu-F-UV2-2T was compared with sequences obtained from GenBank (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov). Multiple alignments of the sequences were performed by using CLUSTAL_X (version 1.83) (Thompson et al., 1997). Alignment gaps and ambiguous bases were not taken into consideration when the 1383 bases of 16S rRNA gene nucleotides were compared. Phylogenetic relationships were analysed by using the same software. Distances were calculated by using the Kimura two-parameter model (Kimura, 1980). Clustering with the neighbour-joining method (Saitou & Nei, 1987) was determined by using bootstrap values based on 1000 replications (Felsenstein, 1985). Similarity values were calculated via MEGA3 (Kumar et al., 2004).
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), followed by Flammeovirga arenaria NBRC 15982T (84.9 %; Takahashi et al., 2006) and Flammeovirga aprica NBRC 15941T (84.7 %; Takahashi et al., 2006). All other recognized species of the family ‘Flammeovirgaceae’ were more distantly related, showing 16S rRNA gene sequence similarity to strain Shu-F-UV2-2T of less than 84 %. Thus, on the basis of the phylogenetic data presented, strain Shu-F-UV2-2T should be identified as representing a novel species in a new genus of the family ‘Flammeovirgaceae’ within the phylum Bacteroidetes (Fig. 2).
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6 (arachidonic acid) (Table 1).
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). Gliding motility was observed via phase-contrast microscopy.
The physiological and biochemical features further supported the phylogenetic results. The major quinone system was menaquinone MK-7. The G+C content of the DNA of strain Shu-F-UV2-2T was 43.0 mol%, a value different from those for members of the genus Flammeovirga (Table 2).
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). Based on the results of phylogenetic analysis and its physiological and biochemical properties, strain Shu-F-UV2-2T is considered to represent a novel species in a new genus of the family ‘Flammeovirgaceae’, phylum Bacteroidetes. We propose the name Perexilibacter aurantiacus gen. nov., sp. nov. for this organism.
Description of Perexilibacter gen. nov.
Perexilibacter [Pe.rex.i.li.bac'ter. L. adj. perexilis very slender; N.L. masc. n. bacter (equivalent of Gr. neut. n. baktron) rod or staff; N.L. masc. n. Perexilibacter very slender rod, referring to its cell shape].
Cells are straight and rod-shaped, Gram-negative and strictly aerobic. Motile by gliding. Do not form endospores. Catalase- and oxidase-positive. Nitrate and nitrite reduction are negative. The major respiratory menaquinone is MK-7. The G+C content of the genomic DNA is 43.0 mol%. Predominant cellular fatty acids are iso-C15 : 0, C16 : 17c and C16 : 15c. The genus Perexilibacter is a member of the family ‘Flammeovirgaceae’, phylum Bacteroidetes. The type species is Perexilibacter aurantiacus.
Description of Perexilibacter aurantiacus sp. nov.
Perexilibacter aurantiacus (au.ran'ti.a.cus. N.L. masc. adj. aurantiacus orange-coloured).
Main characteristics are as given for the genus. In addition, cells are 0.3–0.5 µm in width and 10–20 µm in length. Swarming growth is not observed. Colonies grown on ten-fold diluted marine agar are circular, convex and dull orange-pigmented. The optimum temperature range for growth is 30–37 °C; no growth occurs at 4 or 45 °C. The pH range for growth is 5–10, with an optimum around neutral pH. Seawater is required for growth. NaCl alone does not support growth. Growth occurs in artificial seawater medium containing 0–3.5 % NaCl. Gelatin is hydrolysed but agar and starch are not. Negative for the Voges–Proskauer reaction, ONPG, citrate utilization and production of arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, hydrogen sulfide and indole. Acid is produced from aesculin ferric citrate and 5-keto-gluconate, but not from D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, methyl 
-D-xylopyranoside, galactose, glucose, fructose, mannose, rhamnose, methyl 
-D-mannnopyranoside, methyl -D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, melezitose, inulin, raffinose, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, glycerol, erythritol, adonitol, sorbose, dulcitol, inositol, mannitol, sorbitol, starch, glycogen, xylitol, D-arabitol, L-arabitol, gluconate or 2-keto-gluconate. Positive for alkaline phosphatase, leucine arylamidase, valine arylamidase, trypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for esterase (C4), esterase lipase (C8), lipase (C4), cystine arylamidase, chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. Major fatty acid components (>2.0 % of the total) are iso-C15 : 0 (50.6 %), C16 : 17c (16.7 %), C16 : 15c (4.9 %), C16 : 0 (3.8 %), iso-C15 : 1 G (2.9 %), iso-C17 : 0 3-OH (2.4 %), iso-C15 : 0 3-OH (2.2 %) and C16 : 0 3-OH (2.2 %).
The type strain, Shu-F-UV2-2T (=MBIC06993T=IAM 15413T=KCTC 12867T), was isolated from sediment from Carp Island, Republic of Palau.
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ACKNOWLEDGEMENTS |
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