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1 Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences, Prospekt 100 let Vladivostoku 159, 690022 Vladivostok, Russia
2 Korean Collection for Type Cultures, Genetic Resources Center, Korea Institute of Bioscience and Biotechnology, Yusong, Daejon 305-333, Republic of Korea
3 Institute of Microbiology of the Russian Academy of Sciences, Prospekt 60 let October 7/2, 117811 Moscow, Russia
Correspondence
Olga I. Nedashkovskaya
olganedashkovska{at}piboc.dvo.ru
or
olganedashkovska{at}yahoo.com
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ABSTRACT |
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) and Arenibacter troitsensis (Nedashkovskaya et al., 2003b), accommodates Gram-negative, strictly aerobic, heterotrophic, dark orange-pigmented, non-motile, marine bacteria that belong to the family Flavobacteriaceae. Strains of this genus were isolated from bottom-sediment samples, the brown alga Chorda filum and the holothurian Apostichopus japonicus. The genus Arenibacter forms a phylogenetic cluster with the recently described genera Muricauda (Bruns et al., 2001) and Zobellia (Barbeyron et al., 2001). In June 2000, we isolated an unknown bacterium (strain KMM 3941T) from a green alga, Ulva fenestrata, collected in Troitsa Bay, Gulf of Peter the Great, Sea of Japan. Polyphasic taxonomic study of the phenotypic, chemotaxonomic and genotypic characteristics and phylogenetic position of strain KMM 3941T cultured on marine agar 2216 (Difco), obtained and presented in this work, indicates that the algal isolate represents a novel species of the genus Arenibacter, for which the name Arenibacter certesii sp. nov. is proposed.
Genomic DNA extraction, PCRs and sequencing of the 16S rRNA gene were performed by using previously described procedures (Kim et al., 1998). The sequence data obtained were aligned with those of representative members of selected genera belonging to the family Flavobacteriaceae by using PHYDIT version 3.2 (http://plaza.snu.ac.kr/
jchun/phydit/). Phylogenetic trees were inferred by using suitable programs of the PHYLIP package (Felsenstein, 1993). Phylogenetic distances were calculated from the models of Jukes & Cantor (1969) and trees were constructed on the basis of the neighbour-joining (Saitou & Nei, 1987), least-squares (Fitch & Margoliash, 1967) and maximum-likelihood (Felsenstein, 1993) algorithms. Bootstrap analysis was performed with 1000 resampled datasets, using the SEQBOOT and CONSENSE programs of the PHYLIP package.
To establish the precise taxonomic position of strain KMM 3941T, 1438 nt of 16S rRNA gene sequence was determined; 1369 bp of the determined sequence was used for comparative analysis with the published sequences of related representatives of the phylum Cytophaga–Flavobacterium–Bacteroides. Analysis revealed that strain KMM 3941T is phylogenetically a member of the family Flavobacteriaceae and forms a distinct subline within the genus Arenibacter (Fig. 1). Levels of 16S rRNA gene sequence similarity between strains KMM 3941T and A. latericius KMM 426T and A. troitsensis were 98·7 and 94·8 %, respectively.
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and the DNA G+C content was determined by using the thermal denaturation method (Marmur & Doty, 1962). The DNA G+C content of strain KMM 3941T was 37·7 mol%. DNA–DNA hybridization was performed spectrophotometrically and initial renaturation rates were recorded as described by De Ley et al. (1970). The level of DNA–DNA binding between strain KMM 3941T and A. latericius KMM 426T was 54 %, and that between strain KMM 3941T and A. troitsensis KMM 3674T was 43 %. To determine whole-cell fatty acid profiles, the strains studied were grown at 28 °C for 48 h on marine agar 2216 (Difco). Analysis of fatty acid methyl esters was carried out according to the standard protocol of the Microbial Identification system (Microbial ID). The predominant cellular fatty acids of KMM 3941T were straight-chain unsaturated, branched-chain unsaturated and saturated, namely the fatty acids i15 : 0 (11·7 %), a15 : 0 (8·6 %), i15 : 1 (14·3 %), 15 : 0 (15·3 %) and i17 : 0 3-OH (6·1 %).
The medium of Hugh & Leifson (1953), modified for marine bacteria (Lemos et al., 1985), was used to determine whether glucose was utilized oxidatively or fermentatively. Degradation of agar, starch, casein, gelatin, cellulose (filter paper and CM-cellulose), chitin, DNA, urea and alginic acids, flexirubin production, growth at different pH values, production of acid from carbohydrates and susceptibility to antibiotics were tested as described previously (Nedashkovskaya et al., 2003a). Gram-staining reaction, hydrolysis of Tweens 20, 40 and 80, nitrate reduction, production of hydrogen sulphide and indole and 
-galactosidase, oxidase, catalase and alkaline phosphatase activities were tested according to the methods of Gerhardt et al. (1994). To examine carbon-source utilization, a medium that contained 0·2 g NaNO3, 0·2 g NH4Cl, 0·05 g yeast extract (Difco) and 0·4 % (w/v) carbon source in 1000 ml artificial sea water was used. Carbon sources tested were arabinose, glucose, lactose, mannose, sucrose, inositol, sorbitol, mannitol, fumarate, citrate and malonate. Spreading growth was observed by cultivation on medium B, which contained (l–1): 1 g Bacto peptone (Difco), 1 g yeast extract (Difco), 15 g agar and half-strength natural sea water under high-moisture conditions. Gliding motility was determined as described by Bowman (2000).
Physiological, morphological and biochemical characteristics of the strains studied are listed in the species description and in Table 1. Similarities in phenotypic characteristics support the inclusion of strain KMM 3941T in the genus Arenibacter. However, strain KMM 3941T differed from strains of A. latericius by growing at 10 % NaCl, having a maximum growth temperature of 38 °C, being unable to oxidize glycerol and being resistant to carbenicillin. The absence of gelatin and Tween 40 hydrolysis, absence of H2S production, presence of urease activity, ability to oxidize galactose, glucose, lactose, melibiose and N-acetylglucosamine, susceptibility to ampicillin and lower DNA G+C content (37·7 mol%) are characteristics that clearly distinguish strain KMM 3941T from A. troitsensis (Table 1).
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), together with molecular differences, allow the differentiation of strain KMM 3941T from A. latericius and A. troitsensis. Thus, we propose that strain KMM 3941T should be placed in the genus Arenibacter as Arenibacter certesii sp. nov.
Description of Arenibacter certesii sp. nov.
Arenibacter certesii (cer.te'si.i. N.L. gen. n. certesii of Certes, to honour A. Certes for his contributions to the development of marine microbiology).
Cells are Gram-negative, strictly aerobic with respiratory metabolism, chemo-organotrophic, non-motile, asporogenic rods, 0·4–0·7 µm wide and 3–5 µm long. Oxidase-, catalase-, -galactosidase-, urease- and alkaline phosphatase-positive. Colonies are circular, low convex, shiny with entire edges and 1–3 mm in diameter on marine agar 2216. Dark orange, non-diffusible pigments are produced. No growth is observed without Na+. There is growth at 1–10 % NaCl. Flexirubin pigments are absent. Growth occurs at 4–38 °C. Agar, casein, gelatin, starch, alginic acids, cellulose (CM-cellulose and filter paper), chitin, DNA and Tweens 20, 40 and 80 are not hydrolysed. Acid is formed from cellobiose, fucose, galactose, glucose, lactose, maltose, melibiose, sucrose, raffinose and N-acetylglucosamine, but not from arabinose, rhamnose, xylose, adonitol, dulcitol, glycerol, inositol or mannitol. Arabinose, glucose, lactose, mannose and sucrose are utilized as sole sources of carbon and energy. Utilization of inositol, mannitol, sorbitol, citrate, fumarate and malonate was not observed. Positive for nitrate reduction. H2S, indole and acetoin (Voges–Proskauer reaction) are not produced. Susceptible to ampicillin, oleandomycin and lincomycin. Resistant to carbenicillin, kanamycin, benzylpenicillin, neomycin, streptomycin, gentamicin, polymyxin B and tetracycline. Predominant cellular fatty acids are straight-chain unsaturated, branched-chain unsaturated and saturated, namely i15 : 0 (11·7 %), a15 : 0 (8·6 %), i15 : 1 (14·3 %), 15 : 0 (15·3 %) and i17 : 0 3-OH (6·1 %). Major isoprenoid quinone is MK-6. DNA G+C content is 37·7 mol%.
The type strain is KMM 3941T (=KCTC 12113T=CCUG 48006T). Isolated from a bottom-sediment sample.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Bowman, J. P. (2000). Description of Cellulophaga algicola sp. nov., isolated from the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol 50, 1861–1868.
Bruns, A., Rohde, M. & Berthe-Corti, L. (2001). Muricauda ruestringensis gen. nov., sp. nov., a facultatively anaerobic, appendaged bacterium from German North Sea intertidal sediment. Int J Syst Evol Microbiol 51, 1997–2006.[Abstract]
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
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Fitch, W. M. & Margoliash, E. (1967). Construction of phylogenetic trees. Science 155, 279–284.
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.
Hugh, R. & Leifson, E. (1953). The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram-negative bacteria. J Bacteriol 66, 24–26.
Ivanova, E. P., Nedashkovskaya, O. I., Chun, J. & 7 other authors (2001). Arenibacter gen. nov., new genus of the family Flavobacteriaceae and description of a new species, Arenibacter latericius sp. nov. Int J Syst Evol Microbiol 51, 1987–1995.[Abstract]
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
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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 phylum Cytophaga–Flavobacterium–Bacteroides. Int J Syst Evol Microbiol 53, 81–85.
Nedashkovskaya, O. I., Suzuki, M., Vysotskii, M. V. & Mikhailov, V. V. (2003b). Arenibacter troitsensis sp. nov., isolated from marine bottom sediment. Int J Syst Evol Microbiol 53, 1287–1290.
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