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1 Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences, Pr. 100 Let Vladivostoku 159, 690022, Vladivostok, Russia
2 BCCM/LMG Bacteria Collection, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium
3 Laboratory of Microbiology, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium
Correspondence
Olga I. Nedashkovskaya
olganedashkovska{at}yahoo.com
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ABSTRACT |
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), accommodates heterotrophic, Gram-negative, aerobic, dark-yellow or brownish-coloured, gliding flavobacteria producing flexirubin-type pigments that have been isolated from seawater samples (Nedashkovskaya et al., 2005). Sufficient phylogenetic divergence, as well as a number of phenotypic differences and distinctive fatty acid contents, allowed the separation of the representatives of the single species of the genus, Aquimarina muelleri, from their nearest neighbour, [Cytophaga] latercula. The latter species was reclassified as Stanierella latercula in the same study. In the course of a study of cultured bacteria isolated from the sea urchin Strongylocentrotus intermedius, we found a novel reddish-coloured bacterium, strain KMM 6258T. Phylogenetic analysis of the 16S rRNA gene sequence of strain KMM 6258T revealed that the closest relatives of the novel isolate were members of the genera Aquimarina, Stanierella and Gaetbulimicrobium. The novel strain occupied an intermediate position between the three genera. Based on the results of phylogenetic, phenotypic and fatty acid analyses, we propose that strain KMM 6258T be placed in the genus Aquimarina as a separate species and that the nearest neighbours, Stanierella latercula and Gaetbulimicrobium brevivitae, are reclassified as members of the genus Aquimarina.
Strain KMM 6258T was isolated from the sea urchin Strongylocentrotus intermedius collected in Troitsa Bay, Gulf of Peter the Great, Sea of Japan. To isolate the strain, 0.1 ml tissue homogenate was transferred onto plates of marine agar 2216 (Difco). After primary isolation and purification, the novel strain was cultivated at 28 °C on the same medium and stored at –80 °C in marine broth 2216 (Difco) supplemented with 20 % (v/v) glycerol.
The almost-complete 16S rRNA gene sequence of strain KMM 6258T was determined following a previously described procedure (Vancanneyt et al., 2004). This sequence (a continuous stretch of 1476 bp) was aligned with sequences retrieved from EMBL and a phylogenetic tree was constructed by the neighbour-joining method using the BioNumerics software package, version 4.0 (Applied Maths). Unknown bases were discarded for analyses. Bootstrapping analysis was undertaken to test the statistical reliability of the topology of the neighbour-joining tree using 500 bootstrap resamplings of the data (Fig. 1). The tree topology obtained with the neighbour-joining method was evaluated and confirmed by maximum-parsimony analysis using BioNumerics (data not shown). Strain KMM 6258T showed 16S rRNA gene sequence similarities of 99.7, 96.4, 96.3 and 96.2 % with its nearest neighbours, [Flexibacter] tractuosus IFO 15980, S. latercula ATCC 23177T, A. muelleri KMM 6020T and G. brevivitae SMK-19T, respectively (Fig. 1). These observations support the reclassification of S. latercula and G. brevivitae in the genus Aquimarina.
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and G+C content was determined by using the thermal denaturation method (Marmur & Doty, 1962). The G+C content of the DNA of strain KMM 6258T was 37.1 mol%.
Analysis of fatty acid methyl esters of strain KMM 6258T and of the type strains of A. muelleri and S. latercula grown on marine agar at 28 °C for 24 h was carried out according to the standard protocol of the Sherlock Microbial Identification System (Microbial ID). The major cellular fatty acids for strain KMM 6258T were: iso-C17 : 0 3-OH (32.6 %), iso-C15 : 0 (25.9 %), iso-C17 : 1
9c (12.7 %), iso-C15 : 1 (7.8 %), iso-C15 : 0 3-OH (7.0 %) and summed feature 3 (5.0 %), comprising C16 : 17 and/or iso-C15 : 0 2-OH (Table 1).
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, b). In order to determine susceptibility to antibiotics, the novel strain was grown on marine agar at 28 °C for 48 h. In addition to the antibiotics tested in previous studies, the susceptibility of the strain to chloramphenicol (30 µg), doxycycline (10 µg) and erythromycin (15 µg) was also determined.
The physiological, biochemical and morphological characteristics of strain KMM 6258T are given in the species description and in Table 2
. Phenotypic examination revealed many common traits between the novel strain and its closest relatives, A. muelleri, S. latercula and G. brevivitae. However, strain KMM 6258T could be clearly differentiated from these species by its ability to utilize carbohydrates and by a higher DNA G+C content (Table 2). Strain KMM 6258T could also be differentiated from A. muelleri by the absence of chitinase activity, the presence of 
-galactosidase activity, hydrogen sulfide production and susceptibility to benzylpenicillin. Some features of strain KMM 6258T, including catalase and amylase activities, the ability to grow in the presence of 10 % NaCl and the absence of agar and chitin hydrolysis, may be helpful for separating the novel strain from S. latercula. A combination of phenotypic properties, such as the ability to produce flexirubin-type pigments and hydrogen sulfide, to utilize L-arabinose, D-mannose and sucrose, a lower temperature range for growth and the inability to form acid from carbohydrates, distinguish strain KMM 6258T from G. brevivitae (Table 2).
Consequently, on the basis of the combination of significant molecular, genotypic and phenotypic similarities between strain KMM 6258T and A. muelleri, we suggest that strain KMM 6258T represents a novel species in the genus Aquimarina, for which the name Aquimarina intermedia sp. nov. is proposed.
Furthermore, the high level of 16S rRNA gene sequence similarity of S. latercula and G. brevivitae with members of the genus Aquimarina (96.2–96.4 %) supports the placement of these two species in the genus Aquimarina (Stackebrandt & Goebel, 1994). Taken together with the phylogenetic data, the similarity in fatty acid content of all the strains tested (Table 1) supports the transfer of Stanierella latercula and Gaetbulimicrobium brevivitae to the genus Aquimarina as Aquimarina latercula comb. nov. and Aquimarina brevivitae comb. nov., respectively. Since gliding motility and catalase production, both characteristics of other members of the genus Aquimarina, were not observed among cells of S. latercula in this and previous studies (Lewin, 1969; Reichenbach, 1989; Nedashkovskaya et al., 2005), an emended description of the genus Aquimarina is proposed.
Emended description of the genus Aquimarina
Aquimarina (A.qui.ma.ri'na. L. fem. n. aqua water; L. fem. adj. marina marine; N.L. fem. n. aquimarina an organism of seawater).
Rod-shaped and strictly aerobic cells. Gram-negative. Do not form endospores. Produce non-diffusible carotenoid and/or flexirubin-type pigments. Cells of some species can move by means of gliding. Chemoorganotroph. Cytochrome oxidase- and alkaline phosphatase-positive. The predominant cellular fatty acids are branched-chain saturated and unsaturated and straight-chain unsaturated fatty acids iso-C15 : 0, iso-C15 : 1, iso-C15 : 0 3-OH, iso-C17 : 19c, iso-C17 : 0 3-OH and summed feature 3 (comprising C16 : 17 and/or iso-C15 : 0 2-OH). The main respiratory quinone is MK-6. 16S rRNA gene sequence analysis indicates that the genus Aquimarina is a member of the family Flavobacteriaceae, phylum Bacteroidetes. The type species is Aquimarina muelleri.
Description of Aquimarina intermedia sp. nov.
Aquimarina intermedia (in.ter.me'di.a. L. fem. adj. intermedia intermediate, referring to the level of 16S rRNA gene sequence similarity with its closest relatives).
The main characteristics are the same as those given for the genus. In addition, cells range from 0.4 to 0.5 µm in width and from 2.1 to 3.2 µm in length and are motile by means of gliding. On marine agar, colonies are 2–3 mm in diameter, circular, shiny with entire edges and reddish-pigmented. Growth is observed at 4–36 °C. Optimal temperature for growth is 25–28 °C. Growth occurs in 1–10 % NaCl, with an optimum of 2–5 % NaCl. -Galactosidase activity is present. Flexirubin-type pigments are produced. Decomposes casein, gelatin, starch, DNA and Tweens 20, 40 and 80. Does not degrade agar, cellulose (carboxymethylcellulose and filter paper) or chitin. Does not form acid from L-arabinose, D-cellobiose, L-fucose, D-galactose, D-glucose, D-lactose, D-maltose, D-melibiose, L-raffinose, L-rhamnose, L-sorbose, sucrose, DL-xylose, N-acetylglucosamine, citrate, adonitol, dulcitol, glycerol, inositol or mannitol. Utilizes L-arabinose, D-glucose, D-mannose and sucrose, but not D-lactose, mannitol, inositol, sorbitol, malonate or citrate. Nitrate is not reduced. Hydrogen sulfide is produced. The results for indole and acetoin (Voges–Proskauer reaction) production are negative. Susceptible to ampicillin, benzylpenicillin, carbenicillin, chloramphenicol, doxycycline, erythromycin, lincomycin, oleandomycin and tetracycline. Resistant to gentamicin, kanamycin, neomycin, polymyxin B and streptomycin. The dominant fatty acids are iso-C17 : 0 3-OH (32.6 %), iso-C15 : 0 (25.9 %), iso-C17 : 19c (12.7 %), iso-C15 : 1 (7.8 %), iso-C15 : 0 3-OH (7.0 %) and summed feature 3 (5.0 %), comprising C16 : 17 and/or iso-C15 : 0 2-OH. The G+C content of the DNA of the type strain is 37.1 mol%.
The type strain, strain KMM 6258T (=DSM 17527T=JCM 13506T=LMG 23204T), was isolated from the sea urchin Strongylocentrotus intermedius collected in Troitsa Bay, Gulf of Peter the Great, Sea of Japan.
Description of Aquimarina latercula (Lewin 1969) comb. nov.
Aquimarina latercula (la.ter'cu.la. L. masc. dim. n. laterculus a small brick; N.L. fem. adj. latercula brick-like, brick-red colour).
Basonym: Cytophaga latercula Lewin 1969 (Approved Lists 1980).
Synonym: Stanierella latercula (Lewin 1969) Nedashkovskaya et al. 2005.
The description is as given for the genus and by Nedashkovskaya et al. (2005), with the addition that the strain produces flexirubin-type pigments. Gliding motility and catalase activity are not observed. Does not utilize L-arabinose, D-glucose, D-lactose, D-mannose, sucrose, inositol, mannitol, sorbitol, malonate or citrate. The predominant fatty acids are iso-C17 : 0 3-OH (30.5 %), iso-C15 : 0 (18.4 %), iso-C15 : 1 (7.3 %), iso-C15 : 0 3-OH (7.0 %), summed feature 3 (6.4 %), comprising C16 : 17 and/or iso-C15 : 0 2-OH, C15 : 0 (5.3 %) and iso-C17 : 19c (4.7 %), and The G+C content of the DNA is 34 mol%.
The type strain, LMG 1343T (=ATCC 23177T=NCIMB 1399T=CIP 104806T), was isolated from the outflow of a marine aquarium in La Jolla, California, USA.
Description of Aquimarina brevivitae (Yoon et al. 2006) comb. nov.
Aquimarina brevivitae (bre.vi.vi.ta'e. L. adj. brevis short; L. gen. n. vitae of life; N.L. gen. n. brevivitae of short life, referring to the short-lived cultures of the type strain).
Basonym: Gaetbulimicrobium brevivitae Yoon et al. 2006.
The description is as given for Gaetbulimicrobium brevivitae by Yoon et al. (2006).
The type strain is SMK-19T (=DSM 17196T=KCTC 12390T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Lewin, R. A. (1969). A classification of flexibacteria. J Gen Microbiol 58, 189–206.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109–118.[Medline]
Nedashkovskaya, O. I., Suzuki, M., Vysotskii, M. V. & Mikhailov, V. V. (2003a). Reichenbachia agariperforans gen. nov., sp. nov., a novel marine bacterium in the Cytophaga-Flavobacterium-Bacteroides. Int J Syst Evol Microbiol 53, 81–85.
Nedashkovskaya, O. I., Kim, S. B., Han, S. K. & 7 other authors (2003b). Mesonia algae gen. nov., sp. nov., a novel marine bacterium of the family Flavobacteriaceae isolated from the green alga Acrosiphonia sonderi (Kütz) Kornm. Int J Syst Evol Microbiol 53, 1967–1971.
Nedashkovskaya, O. I., Kim, S. B., Lysenko, A. M., Frolova, G. M., Mikhailov, V. V., Lee, K. H. & Bae, K. S. (2005). Description of Aquimarina muelleri gen. nov., sp. nov., and proposal of the reclassification of [Cytophaga] latercula Lewin 1969 as Stanierella latercula gen. nov., comb. nov. Int J Syst Evol Microbiol 55, 225–229.
Reichenbach, H. (1989). Genus I. Cytophaga Winogradsky 1929, 577,AL emend. In Bergey's Manual of Systematic Bacteriology, vol. 3, pp. 2015–2050. Edited by J. T. Staley, M. P. Bryant, N. Pfennig & J. C. Holt. Baltimore: Williams & Wilkins.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Vancanneyt, M., Mengaud, J., Cleenwerck, I. & 7 other authors (2004). Reclassification of Lactobacillus kefirgranum Takizawa et al. 1994 as Lactobacillus kefiranofaciens subsp. kefirgranum subsp. nov. and emended description of L. kefiranofaciens Fujisawa et al. 1988. Int J Syst Evol Microbiol 54, 551–556.
Yoon, J.-H., Kang, S.-J., Jung, S.-Y., Oh, H. W. & Oh, T.-K. (2006). Gaetbulimicrobium brevivitae gen. nov., sp. nov., a novel member of the family Flavobacteriaceae isolated from tidal flat of the Yellow Sea in Korea. Int J Syst Evol Microbiol 56, 115–119.
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