International Journal of Systematic and Evolutionary (2005), 55, 1033–1038 DOI 10.1099/ijs.0.63410-0

Reclassification of [Cytophaga] marinoflava Reichenbach 1989 as Leeuwenhoekiella marinoflava gen. nov., comb. nov. and description of Leeuwenhoekiella aequorea sp. nov.

Olga I. Nedashkovskaya,1 Marc Vancanneyt,2 P. Dawyndt,3 Katrien Engelbeen,2 Katrien Vandemeulebroecke,2 Ilse Cleenwerck,2 Bart Hoste,2 Joris Mergaert,3 Tjhing-Lok Tan,4 Galina M. Frolova,1 Valery V. Mikhailov1 and Jean Swings2,3

Correspondence 1Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy Olga I. Nedashkovskaya of Sciences, Pr. 100 Let Vladivostoku 159, 690022, Vladivostok, Russia [email protected] 2,3BCCM/LMG Collection2 and Laboratory of Microbiology3, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium 4Alfred-Wegener-Institu¨tfu¨r Polar- und Meeresforschung, Am Handelshfen 12, D-27570 Bremerhaven, Germany

Five heterotrophic, aerobic, halotolerant and pigmented bacterial strains with gliding motility were isolated from Antarctic sea water; one other isolate was collected from the sea urchin Strongylocentrotus intermedius in the Gulf of Peter the Great in the Sea of Japan. 16S rRNA gene sequence analysis indicated that the strains are members of the family , the nearest neighbour (with 97?1 % sequence similarity) being the misclassified [Cytophaga] marinoflava. DNA–DNA hybridization experiments and chemotaxonomic and phenotypic analyses demonstrated that the six novel isolates represent a single species distinct from [C.] marinoflava. On the basis of its separate phylogenetic lineage (the nearest neighbours show 92 % sequence similarity), [C.] marinoflava is reclassified as Leeuwenhoekiella marinoflava gen. nov., comb. nov. A second species of this new genus, Leeuwenhoekiella aequorea sp. nov., is proposed for the six novel isolates, with strain LMG 22550T (=CCUG 50091T) as the type strain.

Many novel taxa that inhabit shallow-water environments (Dobson et al., 1993; McCammon & Bowman, 2000; have been described within the family Flavobacteriaceae in Bowman et al., 1998). recent years (Bernardet et al., 2002). Most coastal-water flavobacteria, e.g. members of the genera Arenibacter, The genus Cytophaga was established by Winogradsky Cellulophaga, Gelidibacter, Mesonia, Muricauda, Polaribacter, (1929) and emended by Reichenbach (1989). Later, Psychroserpens, Tenacibaculum, Ulvibacter, Vitellibacter and Nakagawa & Yamasato (1996) proposed the restriction of Zobellia, require NaCl or sea water for growth and are this genus on the basis of 16S rRNA gene sequence described as weakly or moderately halophilic (Kushner, phylogenetic analysis, and emended the genus description. 1978; Reichenbach, 1989; Bowman et al., 1997; Gosink et al., Currently, the genus Cytophaga sensu stricto (aerobic, 1998; Johansen et al., 1999; Barbeyron et al., 2001; Bruns gliding, pigmented, cellulose-degrading bacteria) comprises two species: Cytophaga aurantiaca and Cytophaga hutchin- et al., 2001; Ivanova et al., 2001; Suzuki et al., 2001; sonii. Some marine bacteria previously included in the Nedashkovskaya et al., 2003b, d, 2004a). The halotolerant genus Cytophaga have been reclassified (Nakagawa & taxa of the family Flavobacteriaceae, e.g. Yamasato, 1996; Nakagawa et al., 1997; Johansen et al., salegens and Psychroflexus gondwanensis, can grow without + 1999; Suzuki et al., 2001; Barbeyron et al., 2001; Na ions or sea water and can tolerate high salinity levels Nedashkovskaya et al., 2005). At present, two misnamed species of the genus Cytophaga that were isolated from Published online ahead of print on 25 November 2004 as DOI marine environments, [Cytophaga] fermentans and [Cyto- 10.1099/ijs.0.63410-0. phaga] marinoflava, remain to be reclassified. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Leeuwenhoekiella aequorea strains LMG 22550T In this work, we report the isolation and identification and KMM 6066 are AJ278780 and AJ780980, respectively. of six novel halotolerant, Gram-negative, aerobic, gliding,

Downloaded from www.microbiologyresearch.org by 63410 G 2005 IUMS Printed in Great Britain 1033 IP: 193.191.134.1 On: Mon, 24 Oct 2016 11:11:10 O. I. Nedashkovskaya and others yellow-pigmented marine bacteria. On the basis of the DNA G+C contents were determined for strains LMG results of genotypic, chemotaxonomic and phenotypic 22550T to LMG 22555 and for [C.] marinoflava LMG 1345T. analyses, it is clear that the isolates represent a novel species, Strains were cultivated on marine agar 2216 for 24 h at with [C.] marinoflava as the nearest neighbour. Both taxa are 37 uC. DNA was extracted from 0?75–1?25 g (wet wt) cells, here classified in a single novel genus, as Leeuwenhoekiella using the DNA extraction protocol of Wilson (1987) as marinoflava gen. nov., comb. nov. and Leeuwenhoekiella modified by Cleenwerck et al. (2002). Cells were lysed in a aequorea sp. nov. Tris/EDTA buffer (10 mM Tris/HCl with up to 200 mM ? T = T = EDTA, pH 8 0) containing RNase A (Sigma), SDS (Serva) Strains LMG 22550 ( ANT 14 ), LMG 22551 ( ANT and proteinase K (Merck) to final concentrations of 18d/2), LMG 22552 (=ANT 26b), LMG 22553 (=ANT 35/ 21 21 400 mgml , 2 % (w/v) and 200 mgml , respectively. 2) and LMG 22554 (=ANT 54b/2) were isolated previously NaCl (5 M stock solution) and CTAB/NaCl solution (10 %, from Antarctic sea-water samples at stations above w/v, hexadecyltrimethylammonium bromide in 0?7M Gunnerus Ridge and Astrid Ridge (Tan et al., 1999), using NaCl) were added to final concentrations of 1 M and enrichment in dialysis chambers (Tan, 1997). Strain KMM 13?3 % (v/v), respectively. For determination of G+C 6066 (=LMG 22555) was isolated from the sea urchin content, DNA was enzymically degraded into nucleosides Strongylocentrotus intermedius in Troitsa Bay, Gulf of Peter the Great, Sea of Japan. For the isolation of the latter as described by Mesbah et al. (1989). The nucleoside mix- strain, 0?1 ml tissue homogenate was transferred onto plates ture obtained was then separated by HPLC using a Waters of marine agar 2216 (Difco). After primary isolation and Symmetry Shield C8 column maintained at a temperature u purification, strains were cultivated at 28 uC on the same of 37 C. The solvent was 0?02 M NH4H2PO4 (pH 4?0) with medium and stored at 280 uC in marine broth 2216 (Difco) 1?5 % acetonitrile. Non-methylated phage l DNA (Sigma) supplemented with 20 % (v/v) glycerol. was used as the calibration reference. The DNA G+C contents were 35–36 mol% for the novel An almost-complete 16S rRNA gene sequence (1475 nt) T of one representative of the Antarctic isolates, strain isolates (LMG 22550 to LMG 22555) and 38 mol% for [ ] T LMG 22550T, was determined previously in a study on the type strain of C. marinoflava (LMG 1345 ). DNA– the diversity of facultative oligotrophic bacteria from polar DNA hybridizations were performed between strains LMG T [ ] T seas (Mergaert et al., 2001). The sequence of strain KMM 22550 to LMG 22555 and C. marinoflava LMG 1345 6066 (1474 nt) was determined in the present study by with DNA prepared as described above. The microplate following a procedure described previously (Vancanneyt method was used as described by Ezaki et al. (1989) and et al., 2004) and showed a similarity of 99?8 % with respect Goris et al. (1998), using an HTS7000 Bio Assay Reader to LMG 22550T. The nearest phylogenetic neighbour of (Perkin Elmer) for the fluorescence measurements. both strains was [C.] marinoflava ATCC 19326T (=LMG Biotinylated DNA was hybridized with single-stranded 1345T): the 16S rRNA gene sequence similarity was 97?1%. unlabelled DNA, non-covalently bound to microplate The three strains formed a distinct lineage within the wells. Hybridizations were performed at 36 uC in a hybri- family Flavobacteriaceae, showing sequence similarity levels dization mixture [26 SSC, 56 Denhardt’s solution, 2?5% below 92?2 % with the genera Vitellibacter, Aequorivita, dextran sulphate, 50 % formamide, 100 mg denatured Arenibacter, Muricauda, Zobellia and Maribacter (Fig. 1). low-molecular-mass salmon DNA ml21, 1250 ng These observations allow reclassification of members of biotinylated probe DNA ml21]. Hybridization levels of the [C.] marinoflava branch within a novel genus. 79–100 % were found between strains LMG 22550T to LMG

Fig. 1. Phylogenetic tree based on the 16S rRNA gene sequences of strains ATCC 19326T and LMG 22550T and representa- tive members of related genera in the family Flavobacteriaceae. The tree was generated by using the neighbour-joining method (Saitou & Nei, 1987). The numbers at nodes indicate bootstrap values (%). Bar, 0?1 sub- stitutions per nucleotide position.

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22555, which indicates that the strains constitute a single Phenotypic analysis was performed by using previously species. The latter strains had binding values of 9–14 % described methods (Nedashkovskaya et al., 2003a, b). The with [C.] marinoflava LMG 1345T. These data indicate that physiological, biochemical and morphological characteris- the novel isolates constitute a single species distinct from tics of the strains studied are listed in the species descrip- the latter misclassified species (Wayne et al., 1987). tion and in Table 2. The results of phenotypic examination of the strains studied, including the ability of strains LMG T The analysis of fatty acid methyl esters was carried out 22550 to LMG 22555 to form acid from D-sucrose and according to the standard protocol of the Microbial mannitol and to utilize mannitol, in combination with the Identification System (Microbial ID). The main cellular molecular distinctiveness allow the differentiation of strains fatty acids of the strains studied were 15 : 0 iso, 15 : 1 iso G, LMG 22550T to LMG 22555 from their closest relative, [C.] 17 : 0 iso 3-OH, iso 17 : 1v9c and summed feature 3 (see marinoflava, at the species level. However, on the basis of Table 1). [C.] marinoflava is distinguished from the novel the phenotypic analysis, the cellular fatty acid composition isolates by a significantly larger amount of 17 : 0 iso 3-OH and the phylogenetic positions of the strains examined, we and smaller amount of iso 17 : 1v9c. Furthermore, the propose that LMG 22550T to LMG 22555 and [C.] marino- presence of minor amounts of 15 : 0 3-OH and the absence flava LMG 1345T represent two distinct species of the of anteiso 17 : 1v9c and summed feature 4 (Table 1) fatty same novel genus. The main phenotypic characteristics that acids in the [C.] marinoflava whole-cell fatty acid profile differentiate the strains studied from other relatives in the support its differentiation from strains LMG 22550T to family Flavobacteriaceae are listed in Table 2. LMG 22555. Isoprenoid quinones were extracted from lyophilized cells and analysed as described previously We conclude that the bacteria studied could not be assigned (Nedashkovskaya et al., 2003d). The major respiratory to any of the existing genera or species currently included quinone was MK-6. in the family Flavobacteriaceae. Consequently, we propose that strains LMG 22550T to LMG 22555 be placed in a novel genus, Leeuwenhoekiella gen. nov., as Leeuwenhoekiella aequorea sp. nov., and that [C.] marinoflava be reclassified Table 1. Fatty acid content (mean percentage of total) of as Leeuwenhoekiella marinoflava comb. nov. members of the genus Leeuwenhoekiella gen. nov. Those fatty acids for which the amount (for all taxa) is less than Description of Leeuwenhoekiella gen. nov. 1 % are not given. tr, Trace amount (less than 1 %); n, number of Leeuwenhoekiella [Leeu.wen.hoe.ki.el9la. N.L. fem. dim. n. strains studied. Leeuwenhoekiella of , named in honour of the famous Dutchman (1632– Fatty acid L. aequorea L. marinoflava ] (n=6) LMG 1345T 1723), discoverer of micro-organisms .

15 : 0 2-OH 1?2±0?21?4 Rod-shaped cells, motile by gliding. Gram-negative. Endo- 15 : 0 3-OH – 1?3 spores are not formed. Strictly aerobic. Produces non- 15 : 0 anteiso 4?5±0?42?7 diffusible yellow pigments. No flexirubins are formed. 15 : 0 iso 18?2±1?116?0 Chemo-organotrophic. Cytochrome oxidase-, catalase-, 15 : 1 iso G 7?6±1?610?4 b-galactosidase- and alkaline phosphatase-positive. The 15 : 0 iso 3-OH 2?1±0?23?9 major respiratory quinone is MK-6. The dominant cellular 16 : 0 iso 3?8±1?21?1 fatty acids (>5 %) are 15 : 0 iso, 15 : 1 iso G, 17 : 0 iso 16 : 1 iso H 1?4±0?4tr3-OH, iso 17 : 1v9c and summed feature 3 (see Table 1). 16 : 0 iso 3-OH 3?4±0?92?5 According to 16S rRNA gene sequence phylogenetic 17 : 0 2-OH 4?2±0?64?4 analysis, the genus Leeuwenhoekiella is a member of the 17 : 0 iso 3-OH 12?7±1?022?1 family Flavobacteriaceae. The type species is Leeuwen- 17 : 1v6c 2?1±0?41?8 hoekiella marinoflava. 17 : 1v8c tr 1?3 anteiso 17 : 1v9c 1?6±0?4–Description of Leeuwenhoekiella marinoflava iso 17 : 1v9c 18?8±2?912?1 comb. nov. Summed feature 3* 9?4±1?610?9 Leeuwenhoekiella marinoflava (ma.ri.no.fla9va. L. adj. mar- Summed feature 4* 1?3±0?2– inus marine; L. adj. flavus golden yellow; N.L. fem. adj. ECL 13?565D tr 1?3 marinoflava marine and yellow-pigmented). ECL 16?582D tr 1?2 Basonym: Cytophaga marinoflava (ex Colwell et al. 1966) *Summed features consist of one or more fatty acids that could not Reichenbach 1989. be separated by the Microbial Identification System. Summed feature 3: 15 : 0 iso 2-OH, 16 : 1v7c and/or 16 : 1v7t. Summed feature 4: The main characteristics are those as given for the genus 17 : 1 iso I and/or 17 : 1 anteiso B. and by Reichenbach (1989). In addition, growth is observed DUnidentified fatty acids (ECL, equivalent chain length). at 4–37 uC. Optimal temperature for growth is 21–23 uC.

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Table 2. Differential characteristics of the genus Leeuwenhoekiella and other related genera of the family Flavobacteriaceae

Genera: 1, Leeuwenhoekiella gen. nov.; 2, Arenibacter;3,Zobellia;4,Muricauda;5,Vitellibacter;6,Aequorivita;7,Maribacter. Data were taken from Barbeyron et al. (2001), Bruns et al. (2001), Ivanova et al. (2001), Bowman & Nichols (2002), Nedashkovskaya et al. (2003b, c, 2004b, c) and this study. Abbreviations: 2, negative; +, positive; V, variable; ND, not determined.

Characteristic 1 2 3 4 5 6 7

Anaerobic growth 222+ 222 Gliding motility + 2 ++22+ Oxidase/catalase +/++/++/++/2 +/+ 2/++/+ Flexirubin pigments 22+ 2 + 22 + Requirement for Na for growth 2 ++++ V + Acid formation from carbohydrates +++ND 22 V Growth at/in: 37 uC ++ V ++ 22 42 uC 2 VV2 + 22 15 % NaCl + 222222 Hydrolysis of: Agar 22+ 222 V Casein + 2 VND+ V 2 Gelatin + V + 2 ++ V Starch + 2 V 222 V DNA 2 VVND+ 2 V Nitrate reduction 2 ++222 V DNA G+C content (mol%) 35–38 37–40 36–44 41 41 33–39 35–39

Growth occurs at 0–15 % NaCl, with optimal growth 2–4 mm in diameter, circular with entire edges and bright at 1–3 % NaCl. Nitrate is not reduced. Indole, H2S and yellow in colour. Growth is observed at 4–37 uC. Optimal acetoin (Voges–Proskauer reaction) are not produced. temperature for growth is 23–25 uC. Growth occurs at Decomposes casein, gelatin, Tweens 20, 40 and 80 and 0–15 % NaCl, with optimal growth at 0–5 % NaCl. Nitrate starch. Does not hydrolyse DNA, urea, cellulose (CM- is not reduced. Indole, H2S and acetoin (Voges–Proskauer cellulose and filter paper) or chitin. Forms acid from reaction) are not produced. Decomposes casein, gelatin, D-galactose and glycerol, but not from L-arabinose, D- starch and Tweens 20, 40 and 80. Does not hydrolyse cellobiose, L-fucose, D-glucose, D-lactose, D-maltose, D- agar, DNA, urea, cellulose (CM-cellulose and filter paper) melibiose, L-raffinose, L-rhamnose, L-sorbose, D-sucrose, or chitin. Forms acid from D-galactose, D-sucrose, glycerol D-trehalose, DL-xylose, N-acetylglucosamine, citrate, acet- and mannitol, but not from L-arabinose, D-cellobiose, ate, fumarate, malate, adonitol, dulcitol, inositol or L-fucose, D-glucose, D-lactose, D-maltose, D-melibiose, L- mannitol. Utilizes L-arabinose, D-glucose, D-lactose, raffinose, L-rhamnose, L-sorbose, DL-xylose, N-acetylglucos- D-mannose and D-sucrose, but not inositol, sorbitol, amine, acetate, citrate, fumarate, malate, adonitol, dulcitol mannitol, malonate or citrate. Susceptible to benzylpeni- or inositol. Can oxidize D-trehalose. Utilizes L-arabinose, cillin, carbenicillin, lincomycin, doxycycline, erythromycin D-glucose, D-lactose, D-mannose, D-sucrose and mannitol, and chloramphenicol. The G+C content of the DNA is but not inositol, sorbitol, malonate or citrate. The G+C 38 mol%. content of the DNA is 35–36 mol%.

The type strain is LMG 1345T (=ATCC 19326T). Isolated The type strain is LMG 22550T (=CCUG 50091T), which from sea water collected in the North Sea off Aberdeen, was isolated from Antarctic sea water. Strain LMG 22555 Scotland, UK. was isolated from the sea urchin Strongylocentrotus inter- medius found in the Sea of Japan. Description of Leeuwenhoekiella aequorea sp. nov. Acknowledgements 9 Leeuwenhoekiella aequorea (ae.quo.re a. L. fem. adj. aequo- This research was supported by grants from the Federal Agency for rea of the sea, marine). Sciences and Innovations of the Ministry for Education and Sciences of the Russian Federation (no. 2-2.16), the Russian Foundation for The main characteristics are as given for the genus. In Basic Research (no. 05-04-48211) and the Program of Fundamental addition, cells range from 0?5to0?6 mm in width and from Investigations of the Presidium of the Russian Academy of Sciences 1?6to2?3 mm in length. On marine agar 2216, colonies are ‘Molecular and Cell Biology’.

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References Kushner, D. J. (1978). Life in high salt and solute concentrations: halophilic bacteria. In Microbial Life in Extreme Environments. Edited Barbeyron, T., L’Haridon, S., Corre, E., Kloareg, B. & Potin, P. by D. J. Kushner. London: Academic Press. (2001). Zobellia galactanovorans gen. nov., sp. nov., a marine species McCammon, S. A. & Bowman, J. P. (2000). of Antarctic of Flavobacteriaceae isolated from a red alga, and classification of Flavobacterium species: description of Flavobacterium gillisiae [Cytophaga] uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as sp. nov., Flavobacterium tegetincola sp. nov. and Flavobacterium Zobellia uliginosa gen. nov., comb. nov. Int J Syst Evol Microbiol 51, xanthum sp. nov., nom. rev., and reclassification of [Flavobacterium] 985–997. salegens as Salegentibacter salegens gen. nov., comb. nov. Int J Syst Bernardet, J.-F., Nakagawa, Y. & Holmes, B. (2002). Proposed Evol Microbiol 50, 1055–1063. minimal standards for describing new taxa of the family Flavo- Mergaert, J., Verhelst, A., Cnockaert, M. C., Tan, T. L. & Swings, J. bacteriaceae and emended description of the family. Int J Syst Evol (2001). Characterization of facultative oligotrophic bacteria from Microbiol 52, 1049–1070. polar seas by analysis of their fatty acids and 16S rDNA sequences. Bowman, J. P. & Nichols, D. S. (2002). Aequorivita gen. nov., a Syst Appl Microbiol 24, 98–107. member of the family Flavobacteriaceae isolated from terrestrial and Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise marine Antarctic habitats. Int J Syst Evol Microbiol 52, 1533–1541. measurement of the G+C content of deoxyribonucleic acid by Bowman, J. P., McCammon, S. A., Brown, J. L., Nichols, P. D. high-performance liquid chromatography. Int J Syst Bacteriol 39, & McMeekin, T. A. (1997). Psychroserpens burtonensis gen. nov., 159–167. sp. nov., and Gelidibacter algens gen. nov., sp. nov., psychrophilic Nakagawa, Y. & Yamasato, K. (1996). Emendation of the genus bacteria isolated from Antarctic lacustrine and sea ice habitats. Int Cytophaga and transfer of Cytophaga agarovorans and Cytophaga J Syst Bacteriol 47, 670–677. salmonicolor to Marinilabilia gen. nov.: phylogenetic analysis of Bowman, J. P., McCammon, S. A., Lewis, T., Skerratt, J. H., Brown, the Flavobacterium-Cytophaga complex. Int J Syst Bacteriol 46, J. L., Nichols, D. S. & McMeekin, T. A. (1998). Psychroflexus torquis 599–603. gen. nov., sp. nov., a psychrophilic species from Antarctic sea ice, Nakagawa, Y., Hamana, K., Sakane, T. & Yamasato, K. (1997). and reclassification of Flavobacterium gondwanense (Dobson et al. Reclassification of Cytophaga aprica (Lewin 1969) Reichenbach 1989 1993) as Psychroflexus gondwanense gen. nov., comb. nov. Micro- in Flammeovirga gen. nov. as Flammeovirga aprica comb. nov. biology 144, 1601–1609. and Cytophaga diffluens (ex Stanier 1940; emend. Lewin 1969) Bruns, A., Rohde, M. & Berthe-Corti, L. (2001). Muricauda ruestring- Reichenbach 1989 in Persicobacter gen. nov. as Persicobacter diffluens ensis gen. nov., sp. nov., a facultatively anaerobic, appendaged comb. nov. Int J Syst Bacteriol 47, 220–223. bacterium from German North Sea intertidal sediment. Int J Syst Nedashkovskaya, O. I., Suzuki, M., Vysotskii, M. V. & Mikhailov, Evol Microbiol 51, 1997–2006. V. V. (2003a). Reichenbachia agariperforans gen. nov., sp. nov., a Cleenwerck, I., Vandemeulebroecke, K., Janssens, D. & Swings, J. novel marine bacterium in the phylum Cytophaga–Flavobacterium– (2002). Re-examination of the genus Acetobacter, with descriptions Bacteroides. Int J Syst Evol Microbiol 53, 81–85. of Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. Nedashkovskaya, O. I., Suzuki, M., Vysotskii, M. V. & Mikhailov, Int J Syst Evol Microbiol 52, 1551–1558. V. V. (2003b). Vitellibacter vladivostokensis gen. nov., sp. nov., a Dobson, S. J., Colwell, R. R., McMeekin, T. A. & Franzmann, P. D. new member of the phylum Cytophaga–Flavobacterium–Bacteroides. (1993). Direct sequencing of the polymerase chain reaction-amplified Int J Syst Evol Microbiol 53, 1281–1286. 16S rRNA gene of Flavobacterium gondwanense sp. nov. and Nedashkovskaya, O. I., Suzuki, M., Vysotskii, M. V. & Mikhailov, Flavobacterium salegens sp. nov., two new species from a hypersaline V. V. (2003c). Arenibacter troitsensis sp. nov., isolated from marine Antarctic lake. Int J Syst Bacteriol 43, 77–83. bottom sediment. Int J Syst Evol Microbiol 53, 1287–1290. Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric Nedashkovskaya, O. I., Kim, S. B., Han, S. K. & 7 other authors deoxyribonucleic acid-deoxyribonucleic acid hybridization in micro- (2003d). Mesonia algae gen. nov., sp. nov., a novel marine bacterium dilution wells as an alternative to membrane filter hybridization in of the family Flavobacteriaceae isolated from the green alga which radioisotopes are used to determine genetic relatedness among Acrosiphonia sonderi (Ku¨tz) Kornm. Int J Syst Evol Microbiol 53, bacterial strains. Int J Syst Bacteriol 39, 224–229. 1967–1971. Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998). Nedashkovskaya, O. I., Kim, S. B., Han, S. K., Rhee, M. S., Lysenko, Evaluation of a microplate DNA-DNA hybridization method A. M., Falsen, E., Frolova, G. M., Mikhailov, V. V. & Bae, K. S. compared with the initial renaturation method. Can J Microbiol (2004a). Ulvibacter litoralis gen. nov., sp. nov., a novel member of 44, 1148–1153. the family Flavobacteriaceae isolated from the green alga Ulva Gosink, J. J., Woese, C. R. & Staley, J. T. (1998). Polaribacter gen. fenestrata. Int J Syst Evol Microbiol 54, 119–123. nov., with three new species, P. irgensii sp. nov., P. franzmannii Nedashkovskaya, O. I., Kim, S. B., Han, S. K., Lysenko, A. M., sp. nov. and P. filamentus sp. nov., gas vacuolate polar marine Mikhailov, V. V. & Bae, K. S. (2004b). Arenibacter certesii sp. nov., a bacteria of the Cytophaga–Flavobacterium–Bacteroides group and novel marine bacterium isolated from the green alga Ulva fenestrata. reclassification of ‘Flectobacillus glomeratus’asPolaribacter glomeratus Int J Syst Evol Microbiol 54, 1173–1176. comb. nov. Int J Syst Bacteriol 48, 223–235. Nedashkovskaya, O. I., Suzuki, M., Vancanneyt, M., Cleenwerck, I., Ivanova, E. P., Nedashkovskaya, O. I., Chun, J. & 7 other authors Lysenko, A. M., Mikhailov, V. V. & Swings, J. (2004c). Zobellia (2001). Arenibacter gen. nov., a new genus of the family Flavo- amurskyensis sp. nov., Zobellia laminariae sp. nov. and Zobellia bacteriaceae and description of a new species, Arenibacter latericius russellii sp. nov., novel marine bacteria of the family Flavo- sp. nov. Int J Syst Evol Microbiol 51, 1987–1995. bacteriaceae. Int J Syst Evol Microbiol 54, 1643–1648. Johansen, J. E., Nielsen, P. & Sjøholm, C. (1999). Description of Nedashkovskaya, O. I., Kim, S. B., Lysenko, A. M., Frolova, G. M., Cellulophaga baltica gen. nov., sp. nov. and Cellulophaga fucicola Mikhailov, V. V., Lee, K. H. & Bae, K. S. (2005). Description of gen. nov., sp. nov. and reclassification of [Cytophaga] lytica to Aquimarina muelleri gen. nov., sp. nov., and proposal of the Cellulophaga lytica gen. nov., comb. nov. Int J Syst Bacteriol 49, reclassification of [Cytophaga] latercula Lewin 1969 as Stanierella 1231–1240. latercula gen. nov., comb. nov. Int J Syst Evol Microbiol 55, 225–229.

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Reichenbach, H. (1989). Genus I. Cytophaga Winogradsky 1929, Gunnerus and Astrid Ridges, Antarctic Ocean. Arch Hydrobiol Spec 577,AL emend. In Bergey’s Manual of Systematic , vol. 3, Issues Advanc Limnol 54, 237–253. pp. 2015–2050. Edited by J. T. Staley, M. P. Bryant, N. Pfennig & Vancanneyt, M., Mengaud, J., Cleenwerck, I. & 7 other authors J. C. Holt. Baltimore: Williams & Wilkins. (2004). Reclassification of Lactobacillus kefirgranum Takizawa et al. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new 1994 as Lactobacillus kefiranofaciens subsp. kefirgranum subsp. nov. method for reconstructing phylogenetic trees. Mol Biol Evol 4, and emended description of L. kefiranofaciens Fujisawa et al. 1988. 406–425. Int J Syst Evol Microbiol 54, 551–556. Suzuki, M., Nakagawa, Y., Harayama, S. & Yamamoto, S. (2001). Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). Phylogenetic analysis and taxonomic study of marine Cytophaga-like International Committee on Systematic Bacteriology. Report of the bacteria: proposal for Tenacibaculum gen. nov. with Tenacibaculum ad hoc committee on reconciliation of approaches to bacterial maritimum comb. nov. and Tenacibaculum ovolyticum comb. nov., systematics. Int J Syst Bacteriol 37, 463–464. and description of Tenacibaculum mesophilum sp. nov. and Tenaci- Wilson, K. (1987). Preparation of genomic DNA from bacteria. baculum amylolyticum sp. nov. Int J Syst Evol Microbiol 51, 1639–1652. In Current Protocols in Molecular Biology, pp. 2.4.1–2.4.5. Edited Tan, T. L. (1997). Biolog metabolic fingerprints for clustering by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. marine oligotrophic bacteria from polar regions. In Microbial Seidman, J. A. Smith & K. Struhl. New York: Green Publishing and Communities – Functional Versus Structural Approaches, pp. 161– Wiley-Interscience. 170. Edited by H. Insam & A. Rangger. Berlin: Springer. Winogradsky, S. (1929). E´ tudes sur la microbiologie du sol – sur Tan, T. L., Joiris, C. R., Glansdorff, N. & Ru¨ ger, H.-J. (1999). la de´gradation de la cellulose dans le sol. Ann Inst Pasteur 43, 549– Dominance of oligotrophic bacteria in surface waters above 633 (in French).

Downloaded from www.microbiologyresearch.org by 1038 International Journal of Systematic and Evolutionary Microbiology 55 IP: 193.191.134.1 On: Mon, 24 Oct 2016 11:11:10