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1 Biological Resource Center, KRIBB, 52 Oeundong, Yusong, Daejeon 305-333, Korea
2 University of Science and Technology, 52, Eoeun-dong, Yuseong-gu, Daejeon 305-333, Korea
3 Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, People's Republic of China
4 Department of Applied Microbiology, Yeungnam University, 214-1 Dae-dong Gyeongsan-si, Gyeongsangbuk-do 712-749, Korea
5 Environmental Biotechnology National Core Research Center, Kyungsang National University, Jinju, Korea
Correspondence
Jin-Woo Bae
baejw{at}kribb.re.kr
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), which includes the genera Photobacterium (Baumann & Baumann, 1984), Salinivibrio (Mellado et al., 1996) and Grimontia (Thompson et al., 2003) amongst others. Micro-organisms belonging to the genus Vibrio occur frequently in aquatic environments, particularly in marine and estuarine waters, where they are often found associated with various organisms ranging from plankton to fish (Thompson et al., 2004). While some Vibrio species act beneficially in host organisms as probionts (Gomez-Gil et al., 1998, 2000, 2002), others are known to cause disease in fish (Hjeltnes & Roberts, 1993), crustaceans (Lightner, 1993) and molluscs (Austin, 1988). The number of species assigned to the genus Vibrio increased from 20 in 1981 to 63 in 2004 (Thompson et al., 2004); at the time of writing, this genus includes 76 species with validly published names.
To clarify our understanding of the genus Vibrio, we searched for novel strains of this taxon in getbol, the Korean term for tidal flats. The western and south-western coastlines of the Korean peninsula consist primarily of such tidal flats (Kim et al., 2004). They are unique among other marine sediments as they alternately undergo flooding with seawater and exposure to air (Kim et al., 2005). Two novel strains were isolated from a tidal flat area near Dae-Chun, Chung-Nam, Korea (36° 17' 45.2'' N 126° 31' 9.5'' E) by employing the dilution plating technique and culturing the micro-organisms at 25 °C for 3 days on marine agar 2216 (MA; Difco). These strains were designated as MANO22DT and MANO22P. Below, we report the phenotypic, genetic and chemotaxonomic analyses that we performed to elucidate the taxonomic position occupied by these strains. These analyses revealed that both strains are very similar; they belong to the family Vibrionaceae and are most closely related to Vibrio rumoiensis.
In our analyses, V. rumoiensis FERM P-14531T, obtained from Dr I. Yumoto (Yumoto et al., 1999), served as the reference strain. Bacterial cultures of the isolates and the reference strain were stored at –80 °C on marine broth (MB) containing 20 % glycerol and were cultured in MB at 25 °C, with shaking, for morphological and physiological characterization. API 20NE and API ZYM test strips (bioMérieux) and Biolog GN metabolic fingerprinting plates were used to analyse these strains biochemically and physiologically; other biochemical tests were performed using the methods and media described by Gordon et al. (1973). The ability to grow on various carbon sources was tested as described by Gonzalez et al. (1997), and catalase activity was determined by assessing bubble production in a 3 % (v/v) hydrogen peroxide solution. Oxidase activity was determined by using an oxidase reagent (bioMérieux), while sensitivity to O/129 (150 µg per disc) was determined with Oxoid discs. Growth under anaerobic conditions was determined on MA with anaerobic incubation for 7 days in GasPak jars (BBL) containing an N2/CO2/H2 (80 : 10 : 10) atmosphere. Growth in MB at various NaCl concentrations, temperatures and pHs was measured. The Gram reaction was determined by using a Gram-stain kit (Difco) according to the manufacturer's instructions. Cellular morphology and sporulation were determined by using microscopy (E600; Nikon). The cellular motility of young bacterial cultures in MB was observed in fresh wet mounts by using the hanging drop method. For observation using transmission electron microscopy, cells from exponentially grown cultures were negatively stained with 1 % (w/v) phosphotungstic acid. After air drying, the grid was examined using a model H-7600 transmission electron microscope (Hitachi). The strains were grown on MA for 3 days at 25 °C to analyse their fatty acid methyl esters, which were extracted and prepared according to standard protocols provided by the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Chromosomal DNA was extracted and purified as described by Sambrook et al. (1989). The 16S rRNA gene was amplified by using a PCR with two universal primers, as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed according to the methods described by Yoon et al. (2003). DNA–DNA hybridization was performed fluorometrically by using the method of Bae et al. (2005) with Cy5-labelled DNA probes and genome-spotted microarrays. The 16S rRNA gene sequences of the two novel isolates were aligned with 17 reference sequences obtained from the Ribosomal Database Project (Fig. 1) by using the multiple sequence alignment program CLUSTAL_X (version 1.8) (Thompson et al., 1997). The phylogenetic relationships between representatives of the genus Vibrio were determined by using MEGA software (version 2.1). Distance matrices were determined by adopting the assumptions described by Kimura (1980). These matrices were used to elaborate dendrograms by using the neighbour-joining method (Saitou & Nei, 1987). A bootstrap analysis designed to establish the stability of the phylogenetic trees obtained was performed by obtaining a consensus tree based on 1000 randomly generated datasets.
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and are described in the species description below. They were motile and Gram-negative and their cells were rod-shaped (measuring 0.6–0.8 by 2.0–3.0 µm on MA). After 3 days growth on MA, their colonies were pale yellow in colour, 1.5–2.0 mm in diameter, smooth, and circular to slightly irregular in shape. They grew at temperatures ranging from 4 to 45 °C and at pH 4.1–8.8; however, they did not grow at below pH 3.1 or above pH 9.3. They grew in the presence of 1–12 % NaCl, but no growth was observed when NaCl was absent or present at 15 %. They grew under anaerobic conditions, showed catalase, oxidase and urease activities and reduced nitrate to nitrite. They grew on the following carbon sources: glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, malate and citrate. They did not hydrolyse gelatin, urea or aesculin and did not show arginine dihydrolase activity or produce indole. Under anaerobic conditions, they produced acid, but no gas, from D-glucose. They were also susceptible to the vibriostatic agent O/129. The predominant fatty acids of strain MANO22DT were 16 : 1
7c and/or 15 : 0 iso 2-OH, 18 : 17c, 16 : 0 and 14 : 0 3-OH and/or 16 : 1 iso I.
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). DNA–DNA relatedness studies were performed to determine the genomic relationship between MANO22P, MANO22DT and V. rumoiensis FERM P-14531T. The value for DNA–DNA relatedness between MANO22P and MANO22DT was 96 %, but the relatedness values between the novel isolates and V. rumoiensis FERM P-14531T were 7.4 % (MANO22DT) and 3.9 % (MANO22P). Taken together, these observations indicate that strains MANO22DT and MANO22P represent a novel species of the genus Vibrio, for which the name Vibrio litoralis sp. nov. is proposed.
Description of Vibrio litoralis sp. nov.
Vibrio litoralis (li.to.ra'lis. L. masc. adj. litoralis of the shore, a shallow-water dweller).
Cells are Gram-negative, slightly curved rods that are 0.6–0.8 µm wide and 2.0–3.0 µm long. Motile due to the presence of at least one polar flagellum per cell. Colonies on MA are pale yellow, smooth, round or slightly irregular in shape and measure 1.5–2.0 mm in diameter after 3 days culture on MA plates. Bioluminescence is not observed. Growth occurs when 1–12 % NaCl is present, but not when NaCl is absent or at concentrations of 15 %. Growth occurs at 4–45 °C and at pH 4.1–8.8. Optimal conditions are 25–30 °C, pH 6.9 and 3 % NaCl. Susceptible to the vibriostatic agent O/129 and is oxidase- and catalase-positive. Reduces nitrate to nitrite, but not further to N2. Facultatively anaerobic; acid, but no gas, is produced from glucose. The following substrates can be utilized as sole carbon and energy sources: glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, malate and citrate. Gelatin, urea and aesculin are not hydrolysed. API ZYM tests show activities for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-
-glucosaminidase. Arginine dihydrolase, lipase (C14), cystine arylamidase, trypsin, 
-chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, -glucosidase, -mannosidase and -fucosidase activities are not observed. The predominant fatty acids are 16 : 17c and/or 15 : 0 iso 2-OH (38.62 %), 18 : 17c (19.45 %), 16 : 0 (13.49 %), 14 : 0 3-OH and/or 16 : 1 iso I (7.11 %), 12 : 0 (3.65 %) and 18 : 0 iso (3.44 %).
The type strain, MANO22DT (=KCTC 12520T=DSM 17657T), and a reference strain, MANO22P (=KCTC 12519), were isolated from a tidal flat area of Dae-Chun, Chung-Nam, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bae, J. W., Rhee, S. K., Park, J. R., Chung, W. H., Nam, Y. D., Lee, I., Kim, H. & Park, Y. H. (2005). Development and evaluation of genome-probing microarrays for monitoring lactic acid bacteria. Appl Environ Microbiol 71, 8825–8835.
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