SGM Journals
Bacteroidetes

Euzebyella saccharophila gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae

Teresa Lucena, Javier Pascual, Assunta Giordano, Agata Gambacorta, Esperanza Garay, David R. Arahal, M. Carmen Macián, María J. Pujalte

  • 1Colección Española de Cultivos Tipo (CECT) y Departamento de Microbiología y Ecología, Universidad de Valencia, Spain
  • 2Istituto di Chimica Biomolecolare, CNR, Pozzuoli, Italy
  • Correspondence
    María J. Pujalte
    maria.j.pujalte{at}uv.es

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(12):2871–2876 · https://doi.org/10.1099/ijs.0.020875-0

    View at publisher PubMed

    Abstract

    Strain 7SM30T, an aerobic marine, Gram-negative, heterotrophic and yellow- to orange-pigmented bacterium isolated from seawater from Castellón, Spain, was characterized using a polyphasic approach. Analysis of the 16S rRNA gene sequence showed that the isolate represented a novel lineage within the family Flavobacteriaceae. The most closely related genera were Pseudozobellia, Zobellia and Kriegella. Cells of strain 7SM30T were non-motile rods that required sea salts for growth, used a wide variety of carbohydrates as sole carbon and energy sources and, unlike species of the genera Pseudozobellia and Zobellia, did not possess flexirubin-type pigment or hydrolyse agar. Strain 7SM30T contained MK6 as the sole respiratory quinone. Phosphatidylethanolamine (PE) was the only identifiable polar lipid, although other lipids were also detected. The predominant cellular fatty acids were saturated C15 and monounsaturated C15. The DNA G+C content was around 40 mol%. On the basis of extensive phenotypic and phylogenetic comparative analysis, it is concluded that the new strain represents a novel genus and species, for which the name Euzebyella saccharophila gen. nov., sp. nov., is proposed. The type strain of the type species is 7SM30T (=CECT 7477T=KCTC 22655T).

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 7SM30T is FN554868.

    • Images of colonies of strain 7SM30T and additional phylogenetic trees constructed using the maximum-likelihood and neighbour-joining methods are available as supplementary figures with the online version of this paper. A supplementary table detailing the phenotypic characteristics of the type strains included in the study is also available.

    The number of genera belonging to the phylum ‘Bacteroidetes’ has increased considerably in recent years. The family Flavobacteriaceae (Reichenbach, 1989; Reichenbach, 1992; emended by Bernardet et al., 1996, 2002) is one of the major phylogenetic lineages within this phylum, with almost 90 recognized genera by the end of 2009. In the present study, a novel flavobacterium is described that showed low levels of 16S rRNA gene sequence similarity with members of the genus Zobellia and their relatives.

    Strain 7SM30T was isolated from a seawater sample obtained from a depth of 10 m off the coast of Vinaroz, Castellón, Spain, in July 1989. The seawater was decimally diluted with sterile seawater, plated on marine agar (MA; Difco) and incubated at 20–22 °C for up to 10 days. Isolated colonies on the plates corresponding to the highest dilutions were streaked in the same medium and conditions until pure cultures were obtained. Strains were maintained by suspending cells in marine broth (MB; Difco) supplemented with 20 % glycerol at −80 °C (Ortigosa et al., 1994).

    The novel strain was recovered recently as a part of a project to characterize a collection of unidentified marine isolates kept at the Department of Microbiology and Ecology, University of Valencia, Spain, and was subjected to partial 16S rRNA gene sequencing. A blast search of public databases indicated that the sequence of strain 7SM30T had less than 95 % similarity to the sequence of any recognized species of bacteria, with Pseudozobellia thermophila as the closest relative. Significant molecular and phenotypic differences found between the new strain and its relatives supported the proposal of a new genus to accommodate strain 7SM30T.

    Phenotypic characterization of strain 7SM30T followed previously reported methods (Macián et al., 2001, 2005). For comparative purposes, the following reference strains were used: P. thermophila CECT 7618T, Kriegella aquimaris CECT 7617T, Zobellia russellii CECT 7505T and Zobellia uliginosa CECT 4277T. Strains were routinely grown in MA or MB for 48–72 h at 25–28 °C for inocula or sample preparation. Unless otherwise indicated, media other than MA and MB were supplemented with half-strength artificial seawater (ASW; 400 mM NaCl, 100 mM MgSO4 . 7H2O, 20 mM KCl and 20 mM CaCl2 . 2H2O) or marine cations supplement (MCS, Farmer & Hickman-Brenner, 2006). Suspension media for microscopic observations or miniaturized testing were also performed in salt-supplemented solutions.

    Cell morphology and gliding motility were observed on wet mounts under phase-contrast microscopy. Z. russellii CECT 7505T was used as a positive control for motility.

    Strain 7SM30T was Gram-negative and lysed readily when suspended in 3 % (w/v) KOH solution. It did not show swarming motility, although growth on some sole carbon and energy sources (d-fructose, trehalose) showed rhizoid margins.

    When grown on MA (28 °C, 48 h), strain 7SM30T formed regular, rounded colonies with entire edges and a yellow–orange, non-diffusible pigmentation (see Supplementary Fig. S1 in IJSEM Online). The pigment did not show the colour shift characteristic for flexirubin-type pigments when colonies were flooded with 20 % KOH solution (Reichenbach, 1989; Bernardet et al., 2002). The ability to grow at different temperatures was tested on MA incubated at 4 °C and 15 °C (7 days) and 28, 37, and 40 °C (48 h). The strain was able to grow at temperatures from 15 to 37 °C, but no growth was seen at at 4 °C or 40 °C. Ionic requirements were tested on salt tolerance agar (STA), containing 1 % peptone, 0.3 % yeast extract and 1.2 % purified agar. This medium was supplemented with the following salt combinations (w/v): (i) with and (ii) without 2 % NaCl, (iii) with 2 % NaCl and 0.9 % MgCl2 . 6H2O, (iv) with 2 % NaCl and 0.2 % CaCl2 . 2H2O, (v) with 2 % NaCl, 0.9 % MgCl2 . 6H2O and 0.2 % CaCl2 . 2H2O. Strain 7SM30T did not grow without NaCl or in STA supplemented with only NaCl or Na and Ca chlorides, but showed good growth when both NaCl and MgCl2 were added to the medium. This indicated that the strain had a specific requirement for both sodium and magnesium, but not for calcium or potassium ions. The four reference strains were tested in parallel: P. thermophila showed the same ionic requirements as strain 7SM30T, K. aquimaris required Na, Mg and Ca ions (but no potassium), while the two species of the genus Zobellia tested required Na and one of the divalent cations, either magnesium or calcium, but not potassium (see Supplementary Table S1 in IJSEM Online). The salinity range for growth was tested on MA as already reported (Macián et al., 2005). Strain 7SM30T showed good growth from 0.35 % to 10 % total salinity and no growth with 15 % or higher salinities.

    Strain 7SM30T was oxidase- and catalase-positive, unable to ferment d-glucose, d-galactose, l-arabinose, sucrose, amygdalin, melibiose or lactose in O/F medium (with MCS) and unable to grow on MA in anaerobic conditions (GENbag anaer, bioMérieux). It did not reduce nitrate to nitrite in nitrate broth or with API 20NE strips. Other activities are given in the species description and in Supplementary Table 1. Extracellular hydrolysis of casein, DNA, starch, alginate and Tween 80 were tested after a week of incubation on MA-based media, with negative results for Tween 80, alginate and agar hydrolysis, and a weak positive result for DNase. On casein and starch, a very faint and small halo could be seen after 7 and 4 days incubation, respectively.

    The results for strain 7SM30T in API ZYM, API 20NE, API 50 CHE and Biolog GN microplate tests, performed in duplicate, are reported in the species description.

    Carbon sources used for growth were tested on Baumann’s basal medium [50 mM Tris/HCl, pH 7.5; 19 mM NH4Cl; 0.33 mM K2HPO4 . 3H2O; 0.1 mM FeSO4 . 7H2O and 1.3 % (w/v) purified agar (Oxoid) on half-strength ASW; Baumann & Baumann, 1981]. The strain grew readily in basal medium supplemented with 0.5 % yeast extract (positive control) and with several carbohydrates. The growth was mucous in the latter case. Carbon sources that supported growth are indicated at the species description.

    Analyses of respiratory quinones, polar lipids, cellular fatty acids and DNA G+C mol% content were carried out by the Identification Service of the DSMZ and Dr B. J. Tindall (DSM, Braunschweig, Germany). Major cellular fatty acids were determined at the DSMZ by GLC after 48 h incubation on MA at 28 °C using a previously described method (Kämpfer & Kroppenstedt, 1996). Independently, analysis of polar lipids, fatty acids and DNA G+C mol% content was also performed at the Istituto di Chimica Biomolecolare, CNR, Pozzuoli, Italy. Polar lipids and fatty acids were determined according to Romano et al. (2001).

    Strain 7SM30T had an MK6 menaquinone system. Analysis of polar lipids detected PE and four unidentified lipids (one unknown phospholipid). Non-saturated C15 and monosaturated C15 were the most abundant fatty acids. The major hydroxylated fatty acid was iso-C17 : 0 3-OH, as shown in Table 1⇓. Analysis of the DNA G+C content resulted in a value of 39.5 mol% (HPLC) or 41.2 mol% using the method described by Poli et al. (2009). The difference between both determined values of G+C mol% content (1.7 % units) could be attributed to the different methodologies. It was concluded that strain 7SM30T had a G+C content of around 40 mol%, which was in accordance with the descriptions of other members of the family Flavobacteriaceae.

    Table 1.

    Chemotaxonomic characterization of strain 7SM30T and P. thermophila, K. aquimaris and species of the genus Zobellia

    Taxa: 1, strain 7SM30T (data from this study); 2, P. thermophila (Nedashkovskaya et al., 2009); 3, K. aquimaris (Nedashkovskaya et al., 2008); 4, Zobellia (five species) (Nedashkovskaya et al., 2004, 2009). All taxa possessed MK6 as the respiratory quinone. Cellular fatty acid composition was determined according to the Microbial Identification System (Microbial ID). –, Not detectable; tr, trace amount (<1 %); nd, not determined.

    The almost complete 16S rRNA gene sequence of strain 7SM30T was obtained by direct PCR amplification. This was followed by sequencing using primers 616V (forward) and 699R (reverse) as described in Arahal et al. (2008) for a stretch of around 1000 nt close to the 5′ end, and primers P609D (forward) and P1525R (reverse) as described in Lucena et al. (2010) for a segment of about 750 nt close to the 3′ end. The assembled and manually corrected sequence was compared with public sequences in NCBI gene databases using the blast program (National Center for Biotechnology Information; ). Related sequences were analysed further using the arb program package (Ludwig et al., 2004; ). Sequence alignments were further corrected manually using the arb_edit sequence editor. Phylogenetic analysis using alternative treeing methods (maximum-parsimony, maximum-likelihood and neighbour-joining) and data subsets were performed using the appropriate arb tools (Ludwig et al., 1998).

    Analysis of the 16S rRNA gene sequence of strain 7SM30T (1450 nt) revealed its phylogenetic position within the family Flavobacteriaceae, having K. aquimaris and the recently described species P. thermophila as the closest relatives. The sequence of strain 7SM30T showed maximum gene sequence similarity to that of P. thermophila (94.8 %) followed by K. aquimaris (93.3 %) and three species of the genus Zobellia, Z. rusellii, Z. galactinivorans and Z. uliginosa, with similarities of 94.3, 93.1 and 92.3 %, respectively. Less than 92 % 16S rRNA gene sequence similarity was found with other species of the family. The maximum-parsimony-based phylogenetic tree is presented in Fig. 1⇓; additional information about the robustness of the tree topology, bootstrap values and node stability was obtained by using additional treeing methods (see Supplementary Figs S2 and S3 in IJSEM Online). Taking together the tree topology and 16S rRNA gene sequence similarities, it was concluded that strain 7SM30T represented a new taxon, probably a novel genus, in the vicinity of the genera Pseudozobellia, Kriegella and Zobellia. The G+C mol% content, of around 40 mol%, was similar to the values described for the genera Kriegella and Zobellia, but was distinct from the value for the genus Pseudozobellia (Table 1⇑). The quinone composition was in agreement with genera described within the family Flavobacteriaceae (Bernardet et al., 2002). The cellular fatty acid composition was also somewhat similar to those of the closely related genera. However, given the low 16S rRNA gene sequence similarity values and several phenotypic differences, this new taxon should be considered to represent a novel genus. The absence of flexirubin-type pigment, agarase activity and gliding motility enabled the differentiation of strain 7SM30T from members of the genera Pseudozobellia, Kriegella and Zobellia. Differences in the enzymic activities, temperature range for growth, salinity ranges and ionic requirements also differentiated strain 7SM30T from closely related species (Table 2⇓). Thus, it is concluded that strain 7SM30T represents a new genus and novel species for which the name Euzebyella saccharophila gen. nov., sp. nov. is proposed.

    Figure image not available in archive
    Fig. 1.

    Maximum-parsimony phylogenetic tree based on almost complete 16S rRNA gene sequences of strain 7SM30T and closely related species. GenBank accession numbers are given in parentheses. Bootstrap values >70 % are shown at branching points (percentage of 1000 resamplings). Nodes that were also obtained in the maximum-likelihood and neighbour-joining trees are indicated by open and filled circles, respectively. Bar, 1 substitution per 100 nt positions.

    Table 2.

    Differential characteristics between strain 7SM30T, P. thermophila, K. aquimaris and species of the genus Zobellia

    Taxa: 1, strain 7SM30T (this study); 2, P. thermophila (Nedashkovskaya et al., 2009); 3, K. aquimaris (Nedashkovskaya et al., 2008); 4, Zobellia (five species) (Nedashkovskaya et al., 2004, 2009). +, Positive; –, negative; v, variable among species.

    Description of Euzebyella gen. nov.

    Euzebyella (Eu.ze.by′el.la. N.L. fem. n. Euzebyella named after the French microbiologist J. P. Euzéby, for his outstanding contribution to the nomenclature of Prokaryotes and to bacterial taxonomy in general).

    Gram-negative, aerobic, mesophilic, chemo-organotrophic, slightly halophilic, pigmented bacteria. Cells are non-motile, straight rods. No growth can be observed without seawater or without the addition of marine cations to the medium. Flexirubin-type pigment is not produced. Oxidase- and catalase-positive, unable to reduce nitrates. Respiratory quinone is MK6. Predominant fatty acids are iso-C15 : 0, iso-C15 : 1 G and the major hydroxylated fatty acid is iso-C17 : 0 3-OH. Polar lipids include PE and other unidentified lipids. The DNA G+C content is around 40 mol%. The type species is Euzebyella saccharophila.

    Description of Euzebyella saccharophila sp. nov.

    Euzebyella saccharophila [sac.cha.ro′phi.la. Gr. n. saccharon sugar; N.L. adj. philus -a -um (from Gr. adj. philos -ê -on) friend, loving; N.L. fem. adj. saccharophila sugar-loving].

    In addition to the characteristics given for the genus, the species displays the following traits: cells are approximately 0.4 μm in width and 1.4–5 μm in length. Growth occurs on MA as regular colonies with a non-diffusible yellow–orange pigmentation. Growth is mucous on sugar-containing media. Temperature range for growth is 15–37 °C, no growth at 4 or 40 °C. Good growth is obtained in media with 3.5–100 g l−1 marine salts, no growth occurs at 150 g l−1. Requires sodium and magnesium ions for growth. Does not produce arginine dihydrolase, lysine or ornithine decarboxylases, urease or indole from tryptophan. Hydrolyses aesculin and DNA (weakly), but does not hydrolyse gelatin, casein, alginate, agar, starch or Tween 80. Does not produce acid in O/F medium (with MCS) from d-glucose, d-galactose, sucrose, l-arabinose, amygdalin or melibiose, either in aerobic or anaerobic conditions. Carbon sources that sustain abundant growth in basal medium are preferentially carbohydrates: l-arabinose, d-xylose, d-glucose, d-fructose, d-galactose, trehalose, d-mannose, l-rhamnose (weak), maltose, cellobiose, sucrose, lactose, melibiose, salicin, d-gluconate and N-acetyl-d-glucosamine. The following organic acids, amino acids and carbohydrates are not used: acetate, pyruvate, propionate, butyrate, citrate, t-aconitate, 2-oxoglutarate, succinate, fumarate, malate, lactate, 3-hydroxybutyrate, glycine, l-leucine, l-serine, l-threonine, l-glutamate, l-alanine, l-arginine, l-tyrosine, l-ornithine, l-citrulline, 4-aminobutyrate, l-aspartate, l-lysine, l-histidine, l-sarcosine, putrescine, d-ribose, amygdalin, d-glucuronate, d-galacturonate, d-glycerol, d-mannitol, d-sorbitol, myo-inositol, d-glycerate and d-saccharate. The following activities with API ZYM tests are positive: alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl β-glucosaminidase and α-mannosidase. Esterase (C4), trypsin and naphthol-AS-BI-phosphohydrolase are weak. Lipase (C14), cystine arylamidase, α-chymotrypsin, β-glucuronidase and α-fucosidase are negative. API 20NE strips give positive responses for aesculin hydrolysis, β-galactosidase and assimilation of d-glucose, l-arabinose, N-acetylglucosamine and maltose. Gluconate and pyruvate give a weak response. Nitrate reduction, indole production, d-glucose fermentation, arginine dihydrolase, urease, gelatin hydrolysis and assimilation of d-mannitol, caprate, adipate, malate, citrate and phenylacetate are negative. The following carbohydrates are acidified in API 50 CHE tests (48 h): d-arabinose, l-arabinose, d-xylose, d-glucose, d-fructose, d-mannose, l-rhamnose, methyl α-d-mannopyranoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose and sucrose. A slight acidification is noted on d-ribose, methyl β-d-xylopyranoside, d-galactose, l-sorbose, methyl α-d-glucopyranoside, melibiose, trehalose, inulin, raffinose, starch and d-lyxose. A negative response is obtained with glycerol, erythritol, l-xylose, d-adonitol, dulcitol, inositol, d-mannitol, d-sorbitol, melezitose, glycogen, xylitol, gentiobiose, turanose, d-tagatose, d-fucose, l-fucose, d-arabitol, l-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. Substrates oxidized in Biolog GN microplates after 48 h incubation are: α-cyclodextrin, dextrin, glycogen, Tween 40, N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, l-arabinose, cellobiose, d-fructose, d-galactose, gentiobiose, α-d-glucose, α-lactose, lactulose, maltose, d-mannose, melibiose, methyl β-d-glucosamine, d-psicose, raffinose, d-sorbitol, sucrose, trehalose, turanose, d-gluconic acid and glucose 1-phosphate. Weak responses are obtained with the following substrates: adonitol, myo-inositol, l-rhamnose, xylitol, d-galacturonic acid, α-ketoglutaric acid, succinamic acid, l-asparagine, l-aspartic acid, l-glutamic acid, glycyl l-glutamic acid, l-ornithine, inosine, uridine, dl-α-glycerol phosphate and glucose 6-phosphate. Negative responses are obtained with: Tween 80, d-arabitol, i-erythritol, l-fucose, d-mannitol, methylpyruvate, monomethyl succinate, acetic acid, cis-aconitic acid, citric acid, formic acid, d-galactonic acid lactone, d-glucosaminic acid, d-glucuronic acid, α-hydroxybutyric acid, β-hydroxybutyric acid, γ-hydroxybutyric acid, p-hydroxyphenyl acetic acid, itaconic acid, α-ketobutyric acid, α-ketovaleric acid, dl-lactic acid, malonic acid, propionic acid, quinic acid, d-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, glucuronamide, alaninamide, d-alanine, l-alanine, l-alanyl glycine, glycyl l-aspartic acid, l-histidine, hydroxy l-proline, l-leucine, l-phenylalanine, l-proline, l-pyroglutamic acid, d-serine, l-serine, l-threonine, dl-carnitine, γ-aminobutyric acid, urocanic acid, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol and glycerol.

    The type strain, 7SM30T (=CECT 7477T=KCTC 22655T), was isolated from seawater from the western Mediterranean Sea. The DNA G+C % content of type strain is 39.5 mol% (HPLC) or 41.2 mol% (thermal denaturation).

    Acknowledgments

    This work has been supported by the Spanish Ministerio de Educación y Ciencia, project CGL2005-02292, to M. J. P. M. C. M. is the recipient of a contract granted by the ‘support personnel’ program of the Spanish Ministry of Education and Science. J. P. is the recipient of a PhD fellowship from the Spanish Ministry of Education and Science.

    References

    1. Arahal, D. R., Sánchez, E., Macián, M. C. & Garay, E. (2008). Value of recN sequences for species identification and as a phylogenetic marker within the family “Leuconostocaceae”. Int Microbiol 11, 33–39.
    2. Baumann, P. & Baumann, L. (1981). The marine gram-negative eubacteria: Genera Photobacterium, Beneckea, Alteromonas, Pseudomonas and Alcaligenes. In The Prokaryotes, vol. II, pp. 1302–1331. Edited by Starr, M. P., Stolp, H., Trüper, H. G., Balows, A. & Schleger, H.. Berlin, Heidelberg. : Springer-Verlag.
    3. Bernardet, J.-F., Segers, P., Vancanneyt, M., Berthe, F., Kersters, K. & Vandamme, P. (1996). Cutting a Gordian knot: emended classification and description of the genus Flavobacterium, emended description of the family Flavobacteriaceae, and proposal of Flavobacterium hydatis nom. nov. (basonym, Cytophaga aquatilis Strohl and Tait 1978). Int J Syst Bacteriol 46, 128–148.
    4. Bernardet, J. F., Nakagawa, Y. & Holmes, B. (2002). Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. Int J Syst Evol Microbiol 52, 1049–1070.
    5. Farmer, J. J., III & Hickman-Brenner, F. W. (2006). The genera Vibrio and Photobacterium. In The Prokaryotes: a Handbook on the Biology of Bacteria, 3rd edn, vol. 6, pp. 508–563. Edited by Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K. H. & Stackebrandt, E.. New York. : Springer.
    6. Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005.
    7. Lucena, T., Pascual, J., Garay, E., Arahal, D. R., Macián, M. C. & Pujalte, M. J. (2010). Haliea mediterranea sp. nov., a new marine gammaproteobacterium. Int J Syst Evol Microbiol 60, 1844–1848.
    8. Ludwig, W., Strunk, O., Klugbauer, S., Klugbauer, N., Weizenegger, M., Neumaier, J., Bachleitner, M. & Schleifer, K.-H. (1998). Bacterial phylogeny based on comparative sequence analysis. Electrophoresis 19, 554–568.
    9. Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai, T., Steppi, S. & other authors (2004). arb: a software environment for sequence data. Nucleic Acids Res 32, 1363–1371.
    10. Macián, M. C., Ludwig, W., Aznar, R., Grimont, P. A. D., Schleifer, K.-H., Garay, E. & Pujalte, M. J. (2001). Vibrio lentus sp. nov., isolated from Mediterranean oysters. Int J Syst Evol Microbiol 51, 1449–1456.
    11. Macián, M. C., Arahal, D. R., Garay, E., Ludwig, W., Schleifer, K.-H. & Pujalte, M. J. (2005). Thalassobacter stenotrophicus gen. nov., sp. nov., a novel marine α-proteobacterium isolated from Mediterranean sea water. Int J Syst Evol Microbiol 55, 105–110.
    12. Nedashkovskaya, O. I., Makoto, S., Vancanneyt, M., Cleenwerck, I., Lysenko, A. M., Mikhailov, V. V. & Swings, J. (2004). Zobellia amurskyensis sp. nov., Zobellia laminariae sp. nov. and Zobellia russellii sp. nov., novel marine bacteria of the family Flavobacteriaceae. Int J Syst Evol Microbiol 54, 1643–1648.
    13. Nedashkovskaya, O. I., Suzuki, M., Lee, J. S., Kim, S. B. & Mikhailov, V. V. (2008). Kriegella aquimaris gen. nov., sp. nov., isolated from marine environments. Int J Syst Evol Microbiol 58, 2624–2628.
    14. Nedashkovskaya, O. I., Suzuki, M., Lee, J. S., Lee, K., Shevchenko, L. S. & Mikhailov, V. V. (2009). Pseudozobellia thermophila gen. nov., sp. nov., a bacterium of the family Flavobacteriaceae, isolated from the green alga Ulva fenestrata. Int J Syst Evol Microbiol 59, 806–810.
    15. Ortigosa, M., Garay, E. & Pujalte, M. J. (1994). Numerical taxonomy of aerobic, Gram-negative bacteria associated with oysters and surrounding seawater of the Mediterranean coast. Syst Appl Microbiol 17, 589–600.
    16. Poli, A., Romano, I., Cordella, P., Orlando, P., Nicolaus, B. & Ceschi Berrini, C. (2009). Anoxybacillus thermarum sp. nov., a novel thermophilic bacterium isolated from thermal mud in Euganean hot springs, Abano Terme, Italy. Extremophiles 13, 867–874.
    17. Reichenbach, H. (1989). Order I. Cytophagales Leadbetter 1974. In Bergey's Manual of Systematic Bacteriology, vol. 3, pp. 2011–2013. Edited by Staley, J. T., Bryant, M. P., Pfennig, N. & Holt, J. G.. Baltimore. : Williams & Wilkins.
    18. Reichenbach, H. (1992). Flavobacteriaceae fam. nov. In Validation of the Publication of New Names and New Combinations Previously Effectively Published Outside the IJSB, List no. 41. Int J Syst Bacteriol 42, 327–328.
    19. Romano, I., Nicolaus, B., Lama, L., Trabasso, D., Caracciolo, G. & Gambacorta, A. (2001). Accumulation of osmoprotectants and lipid pattern modulation in response to growth conditions by Halomonas pantelleriense. Syst Appl Microbiol 24, 342–352.