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1 Thailand Institute of Scientific and Technological Research (TISTR), 35 Moo 3, Technopolis, Khlong 5, Khlong Luang, Pathum Thani 12120, Thailand
2 Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai 90112, Thailand
3 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyu-ku, Tokyo 113-0032, Japan
Correspondence
Pornpoj Srisukchayakul
pornpoj{at}tistr.or.th
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5c (cis-5-hexadecenoic acid) and 15 : 0 (pentadecanoic acid). The major hydroxyl fatty acids were 3-OH 17 : 0 (3-hydroxyheptadecanoic acid), 3-OH 15 : 0 (3-hydroxypentadecanoic acid) and 3-OH 16 : 0 (3-hydroxyhexadecanoic acid). On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic data, these marine bacteria are considered to represent a novel species of a new genus, for which the name Rapidithrix thailandica gen. nov., sp. nov. is proposed. The type strain of Rapidithrix thailandica is TISTR 1750T (=IAM 15448T).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains TISTR 1736, TISTR 1741 and TISTR 1750T are AB284517, AB265183 and AB265192, respectively.
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). The taxonomic position of the CFB complex remains unclear (Shah & Collins, 1988; Bernardet et al., 1996; Suzuki et al., 2001; Nakagawa et al., 2002). However, this complex has recently been classified as the phylum Bacteroidetes (Ludwig & Klenk, 2001). Reclassifications and emended descriptions have since been applied to bacteria belonging to this phylum. New genera and novel species of bacteria from marine coastal ecosystems have been found and described as a result of polyphasic taxonomic studies.
The family ‘Flammeovirgaceae’, phylum Bacteroidetes, consists of five genera: Flammeovirga, Flexithrix, Persicobacter, Perexilibacter and Thermonema. The genus Flammeovirga now has four species, Flammeovirga kamogawensis (Hosoya & Yokota, 2007) and Flammeovirga aprica, Flammeovirga arenaria and Flammeovirga yaeyamensis (Nakagawa et al., 1997), the genus Flexithrix comprises one species, Flexithrix dorotheae (Lewin, 1970), the genus Persicobacter comprises one species, Persicobacter diffluens (Nakagawa et al., 1997), and the genus Thermonema comprises two species, Thermonema lapsum and Thermonema rossianum (Hudson et al., 1989).
The aim of the present study was to establish the taxonomic position of gliding bacteria recovered from the Andaman Sea along the southern coastline of Thailand.
Strains TISTR 1736, TISTR 1741 and TISTR 1750T were isolated from decayed wood from Phuket Province, seashell material from Trang Province and marine algae from Krabi Province, respectively. The medium used for isolation was SAP2 agar (Hosoya et al., 2006). Purification of the isolates was performed by using the Skerman micromanipulation technique as described in Sly & Arunpairojana (1987). These isolates were cultured and maintained at 25 °C on SAP2 agar.
For determination of phylogenetic relationships, the 16S rRNA gene from the novel strains was amplified via PCR by using 16S rRNA gene universal primers. The PCR products were purified by using a GFX PCR DNA and gel band purification kit (Amersham). Sequencing reactions were performed with the ABI PRISM BigDye Terminator cycle sequencing ready kit (Applied Biosystems). Sequences were edited and assembled in the BioEdit program (Hall, 2004) and were compared with sequences by using the BLAST program within the GenBank database (Altschul et al., 1990). Alignments of the 16S rRNA gene sequences of the novel isolates and phylogenetically related genera were carried out by using the CLUSTAL_X program (version 1.83; Thompson et al., 1997). Nucleotide substitution rates (Knuc values; Kimura, 1980) were determined and a distance matrix tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987). Alignment gaps and unidentified base positions were not taken into consideration in the calculations. The topology of the phylogenetic tree was evaluated by performing a bootstrap analysis with 1000 trials.
Phylogenetic analysis of the 16S rRNA gene sequence data revealed that the novel isolates formed a distinct lineage within the phylum Bacteroidetes and belonged to the family ‘Flammeovirgaceae’ (Fig. 1). The 16S rRNA gene sequence of strain TISTR 1750T was identical to that of strains TISTR 1736 and TISTR 1741. For analysis of DNA–DNA relatedness, DNA–DNA hybridization experiments were carried out at 40 °C and levels were measured fluorometrically by using the method of Ezaki et al. (1989). High levels of DNA–DNA relatedness (87–100 %) were found between these three strains. A BLAST search with the 16S rRNA gene sequence of strain TISTR 1750T in the GenBank database and subsequent phylogenetic analysis based on 16S rRNA gene sequences showed that strain TISTR 1750T was most closely related to Flexithrix dorotheae IFO 15987T (91 % similarity). Strain TISTR 1750T showed gene sequence similarity levels of 90–91 % to Persicobacter diffluens NBRC 15940T, Flammeovirga arenaria NBRC 15982T and Reichenbachiella agariperforans KMM 3525T.
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. The DNA G+C contents of the novel strains were in the range 40–43 mol%.
Respiratory quinones were identified according to the method of Uchino et al. (1998). Quinone fractions were extracted with chloroform/methanol (2 : 1, v/v) from lyophilized cells. The fractions were then separated with TLC developed with hexane/diethyl ether (85 : 15, v/v). Quinones were detected under UV light at 275 nm. The quinone spots were scraped off and quinones were extracted with acetone. After concentration, the quinone samples were analysed by reversed-phase HPLC (Komagata & Suzuki, 1987). The major respiratory quinone of the novel strains was MK-7.
Cellular fatty acids of the novel strains were analysed as methyl esters by GC according to the instructions of the Microbial Identification System (MIDI, Inc.). Fatty acid methyl esters were prepared from bacterial cell mass which was separated from trypticase soy agar medium supplemented with artificial seawater and incubated at 25 °C for 3 days. The major cellular fatty acids were 16 : 15c (cis-5-hexadecenoic acid) and 15 : 0 (pentadecanoic acid). Additionally, the major hydroxyl fatty acids were 3-OH 17 : 0 (3-hydroxyheptadecanoic acid), 3-OH 15 : 0 (3-hydroxypentadecanoic acid) and 3-OH 16 : 0 (3-hydroxyhexadecanoic acid) (Table 1).
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), Tweens 20 and 80, and L-tyrosine (Barrow & Feltham, 1993) were determined by using media in which 0.5x artificial seawater had been added. Acid production from carbohydrates was studied by using the commercial API 50 CH and API 20 E systems (bioMérieux). Detection of bacterial enzymes was performed by using the commercial API ZYM system (bioMérieux) according to the manufacturer's instructions.
All of the novel strains were Gram-negative, aerobic, non-sporulating, non-fruiting, gliding bacteria. The isolates formed light olive grey colonies. Cells were flexible filaments of 0.7x20–100 µm (Fig. 2). The swarm colony spread through the culture plate as a thin film on the agar surface. The typical swarm edge expanded like a sea wave. The phenotypic characteristics of the novel strains are given in the species description below and in Table 2. The novel strains showed many similar characteristics to those of the genera Flexithrix and Persicobacter, but there were some differences in cell morphology, colony colour, optimum temperature and pH for growth, oxidase and catalase activity, cystine arylamidase activity, trypsin activity, acid phosphatase activity and DNA G+C content.
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Description of Rapidithrix gen. nov.
Rapidithrix (Ra.pid.i.thrix. L. adj. rapidus rapid; Gr. n. thrix hair; N.L. fem. n. Rapidithrix rapidly moving hair).
Cells are unbranched, flexible filaments, 0.7x20–100 µm. Motile by gliding. Gram-negative. Resting stages are not known. Chemo-organotrophs. Strict aerobes. Require seawater for growth. The major respiratory quinone is MK-7. The major cellular fatty acids are 16 : 1 and 15 : 0. This genus belongs to the family ‘Flammeovirgaceae’, phylum Bacteroidetes. The type species is Rapidithrix thailandica.
Description of Rapidithrix thailandica sp. nov.
Rapidithrix thailandica (thai.lan.dic.a. N.L. fem. adj. thailandica pertaining to Thailand, from where the organisms were isolated).
The main characteristics are as those given in the genus description. In addition, the optimal growth temperature is 25–30 °C. The pH range for growth is 5–10. Colonies are light olive grey. Peptone and ammonium are useful nitrogen sources for growth. Positive in tests for oxidase, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, N-acetyl-
-glucosaminidase and 
-fucosidase. Negative in tests for catalase, lipase (C14), -chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase and -mannosidase. Decomposes starch, gelatin, L-tyrosine, and Tweens 20 and 80. Acid is produced from glycerol, D-arabinose, D-xylose, galactose, glucose, mannose, amygdalin, aesculin, cellobiose, maltose, lactose, melibiose, trehalose, gentiobiose and L-fucose, but not from erythritol, L-arabinose, ribose, L-xylose, adonitol, fructose, sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, arbutin, inulin, melezitose, raffinose, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, D-arabitol, L-arabitol or gluconate. The DNA G+C content is in the range 40–43 mol%. The DNA G+C content of the type strain is 40 mol%.
The type strain, TISTR 1750T (=IAM 15448T), was isolated from marine algae from the Andaman Sea, Krabi Province, Thailand. Strains TISTR 1736 (isolated from decayed wood from the Andaman Sea, Phuket Province, Thailand) and TISTR 1741 (isolated from seashell material from the Andaman Sea, Trang Province, Thailand) are reference strains.
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