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1 NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE), 2-5-8 Kazusa-kamatari, Kisarazu-shi, Chiba 292-0818, Japan
2 Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
3 Coastal Branch, Natural History Museum and Institute, 123 Yoshio, Katsuura, Chiba 299-5242, Japan
Correspondence
Yukiyo Fukunaga
fukunaga-yukiyo{at}nite.go.jp
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Published online ahead of print on 13 October 2005 as DOI 10.1099/ijs.0.63879-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Pseudovibrio ascidiaceicola F423T is AB175663.
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). As part of a continuing study to isolate bacteria from various marine invertebrates, two novel isolates from ascidians were tentatively classified by 16S rRNA gene sequence analysis as belonging to the genus Pseudovibrio, a member of the Alphaproteobacteria. Since this genus currently contains only a single species, Pseudovibrio denitrificans isolated from sea water (Shieh et al., 2004), we were interested in characterizing these newly isolated strains. The present paper reports the polyphasic characterization of the two novel strains. Based on the results of phylogenetic and phenotypic analyses, the strains are proposed to represent a novel species, Pseudovibrio ascidiaceicola sp. nov. In 2003 and 2004, several ascidians were collected from a Pacific Ocean beach near the Natural History Museum and Institute at Katsuura, located on the Boso peninsula, Chiba, Japan. The ascidians were washed with sterile artificial sea water (ASW; Naigai Chemical Products) and their tissues were homogenized using a hand-held homogenizer. Subsequently, the ascidian homogenates were serially diluted in sterile ASW and suitable 10-fold dilutions were plated onto marine agar 2216 (Difco) or onto 1/5 strength marine agar. The plates were incubated at 20 °C for 8 days and the colonies that grew were purified on the same medium. Strains F423T and F10102 were isolated in this manner. The sources of these strains were two ascidian species, Polycitor proliferus and Botryllidae sp., respectively.
Colonies of the strains were circular, entire, smooth and brownish-green in colour. Phase-contrast microscopy showed that cells of the strains grown on marine broth 2216 were motile and pleomorphic. In the exponential growth phase, they were predominantly straight or curved rods, but in the late stationary phase, cells became coccus-shaped.
Transmission electron microscopy of F423T and F10102 cells that were negatively stained with phosphotungstic acid revealed that the cells possessed subpolar flagella and intercellular granules and formed star-shaped aggregates. For the preparation of ultrathin sections, cells of F423T were processed by rapid freezing in liquid propane cooled with liquid nitrogen, cryosubstituted with OsO4 in acetone and embedded in epoxy resin. The presence of intercellular granules in ultrathin sections (Fig. 1b) led us to look for the presence of intercellular granules in Pseudovibrio denitrificans JCM 12308T, which is most closely related to strains F423T and F10102 (see below). The cell morphology of P. denitrificans JCM 12308T was very similar to that of strain F423T (data not shown).
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and 16S rRNA genes were amplified according to the protocol of Hiraishi (1992). PCR products were sequenced by using a BigDye Terminator kit (Applied Biosystems) according to the manufacturer's instructions. A model 3100 Genetic Analyzer (Applied Biosystems) was employed for automated sequencing. Multiple alignments and construction of phylogenetic trees by the neighbour-joining method (Saitou & Nei, 1987) were performed by using the CLUSTAL W program (Thompson et al., 1994). Alignment gaps, primer regions for PCR amplification and unidentified base positions were not taken into consideration for the calculations. The robustness of the topology of the phylogenetic trees was evaluated by a bootstrap analysis with 1000 replications. The result of the phylogenetic analysis based on 16S rRNA gene sequences indicated that strains F423T and F10102 were members of the genus Pseudovibrio (Fig. 2) and the 16S rRNA gene sequence similarity between strain F423T and P. denitrificans JCM 12308T was 98·9 %. Strains F423T and F10102 formed a cluster with ‘alpha proteobacterium CRA 5GI’ which was isolated from a marine sponge.
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. DNA–DNA hybridizations were performed using photobiotin-labelled DNA and microplates (Ezaki et al., 1988, 1989). The DNA–DNA hybridization value between strains F423T and F10102 was above 65 %, while that between strain F423T and P. denitrificans JCM 12308T was less than 30 %.
Extraction of cellular fatty acids from cells grown for 48 h at 25 °C on marine agar and the determination of the fatty acid content by gas chromatography were carried out using the Microbial Identification System (MIDI) according to the manufacturer's instructions. The cellular fatty acid profiles of strain F423T, strain F10102 and P. denitrificans JCM 12308T are shown in Table 1. The major fatty acids in strains F423T and F10102 were similar, being 18 : 1
7c (60·2–87·8 %), 19 : 0 cyclo 8c (8·2–30·9 %) and 18 : 0 3-OH (1·6–3·7 %). The dominant fatty acids in P. denitrificans have been reported to be 14 : 0 2-OH (60·4–60·5 mol%), 14 : 0 3-OH (13·0–14·8 mol%) and 16 : 0 (9·4–13·3 mol%) by Shieh et al. (2004). However, in this study, they were 18 : 17c (87·7 %), 16 : 0 (4·0 %) and 18 : 0 3-OH (1·4 %). Thus, in this study, the fatty acid profiles of F423T and F10102 were to some extent similar to that of P. denitrificans JCM 12308T, although the relative proportions of some of the fatty acids were different. For example, 19 : 0 cyclo 8c is a major fatty acid in strains F423T and F10102, but the amount of this fatty acid in P. denitrificans JCM 12308T was less than 1 %.
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) and 0·5 ml 5 M NaCl was added to the CHE medium for the API 50 test. P. denitrificans JCM 12308T was also included for comparison. Table 2 includes a summary of the physiological test results that differentiated strain F423T and P. denitrificans JCM 12308T. Of the carbohydrates on the API 50 CH test strip, strains F423T and F10102 produced acid from D-arabinose, ribose, D-glucose, D-mannose, methyl 
-D-glucoside, maltose, melibiose, sucrose, trehalose, D-turanose, D-fucose and gluconate. Acid production from glycerol, L-arabinose, galactose and L-fucose was weak and differed between strains F423T and F10102. By using the API 20 NE test strip, arginine dihydrolase activity was positive in strain F423T, but was negative in P. denitrificans JCM 12308T. The activities of valine arylamidase, naphthol-AS-BI-phosphohydrolase and N-acetyl-
-glucosaminidase differed between strain F423T and P. denitrificans JCM 12308T.
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The test for anaerobic growth was conducted on either M9 minimal medium plates (Sambrook & Russell, 2001) containing 0·2 % (w/v) glucose and 1·5 % (w/v) NaCl or marine agar at room temperature in a GasPak anaerobic jar (Becton Dickinson). Strains F423T and F10102 grew weakly on M9 or marine agar plates under anaerobic conditions. To check for acid production from glucose under anaerobic conditions, assay tubes were prepared with M9 minimal medium containing 1·0 % (w/v) glucose, 1·5 % (w/v) NaCl, 0·00002 % (w/v) phenol red and 1·5 % (w/v) agar. The tubes were stabbed with strains F423T and F10102, overlaid with sterile mineral oil and incubated at room temperature. The colour of the cultures changed to yellow in 7 days indicating acid production. This result indicated that glucose was potentially fermented by these strains.
For the salt tolerance test, TY medium (2 g tryptone and 1 g yeast extract dissolved in 1 l of water) was supplemented with NaCl at concentrations of 0, 1, 3, 5, 7 or 10 % (w/v) and the cells were grown at 25 °C. Growth was observed in TY supplemented with either 3 or 5 % (w/v) NaCl, with optimal growth at 3 % (w/v) NaCl. To test the pH range for growth, 20 µl cell suspension was transferred to test tubes containing 5 ml filter-sterilized TYSW (2 g tryptone and 1 g yeast extract dissolved in 1 l ASW) adjusted to pH values between 1 and 11 and incubated at 25 °C. Strains F423T and F10102 grew in TYSW with a final pH ranging from 5·0 to 9·0; however P. denitrificans could not grow at pH 6·0 (Shieh et al., 2004). The optimum pH range for growth of the novel isolates was between pH 7·0 and 8·0.
To test the temperature range for growth, marine agar plates were incubated at 4, 10, 15, 20, 25, 30, 35, 37, 40 or 45 °C for 7 days. Strains F423T grew at 10–30 °C, but could not grow at 4 or 35 °C. P. denitrificans could grow at 35 °C (Shieh et al., 2004). Optimum growth occurred between 25 and 30 °C.
On the basis of DNA–DNA hybridization values, fatty acid profiles and some phenotypic characteristics (Tables 1 and 2
), the phylogenetic distinctiveness found in this study is sufficient to categorize strains F423T and F10102 as members of a novel species that is distinct from the previously recognized Pseudovibrio species (Shieh et al., 2004). Strains F423T and F10102 are distinguished from the known species of the genus Pseudovibrio by some phenotypic characteristics, including the activities of arginine dihydrolase, valine arylamidase, naphthol-AS-BI-phosphohydrolase and N-acetyl--glucosaminidase, the utilization of D-galacturonic acid and succinic acid and the temperature and pH ranges (Table 2). Therefore, on the basis of the data presented, strains F423T and F10102 should be placed in the genus Pseudovibrio as representatives of a novel species, for which the name Pseudovibrio ascidiaceicola sp. nov. is proposed.
Description of Pseudovibrio ascidiaceicola sp. nov.
Pseudovibrio ascidiaceicola (as.ci.di'ace.i.co.la. N.L. fem. n. Ascidiacea name of a zoological class; L. suff. cola dweller; N.L. n. ascidiaceicola Ascidiacea dweller).
Cells in exponential to early stationary phase are predominantly straight to curved rods. Late stationary phase cells are predominantly cocci, 0·8–1·3x1·2–8·0 µm, occurring singly or in star-shaped aggregates. Cells are Gram-negative and motile by subpolar flagella. Colonies on marine agar are circular, smooth and brownish-green in colour. Catalase, oxidase, -glucosidase, arginine dihydrolase and urease tests are positive. -Galactosidase activity is weakly positive. Indole is produced from tryptophan. Gelatin and aesculin are hydrolysed. Can grow by fermenting glucose under anaerobic conditions. Nitrate is reduced to nitrogen. Grows on marine agar at temperatures between 10 and 30 °C. Grows in TY containing 3–5 % NaCl and grows in 1/5-strength TYSW at pH 5·0–9·0. The following substrates are utilized for growth: dextrin, Tween 80, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, L-fucose, D-galactose, -D-glucose, myo-inositol, maltose, D-mannose, D-melibiose, D-raffinose, sucrose, D-trehalose, turanose, D-gluconic acid, -hydroxybutyric acid, DL-lactic acid, succinic acid, L-alanine, L-alanylglycine, L-glutamic acid, glycyl-L-aspartic acid, glycyl-L-glutamic acid, hydroxy-L-proline, L-proline, L-serine, inosine, uridine, thymidine, 2-aminoethanol, glycerol and D-glucose 6-phosphate. Activities are detected for alkaline phosphatase, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase and -glucosidase. Weakly positive reaction for esterase C4, ester lipase C8, leucine arylamidase and valine arylamidase activities. The major fatty acid is 18 : 17c. The DNA G+C content is 51·2 mol%.
The type strain, F423T (=NBRC 100514T=IAM 15084T=DSM 16392T=KCTC 12308T), was isolated from a sea squirt, Polycitor proliferus, from the Boso peninsula, Japan. Strain F10102 (=NBRC 10097) is a reference strain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
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