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1 Departamento de Microbiología y Parasitología, Facultad de Biología, Universidad de Santiago de Compostela, Campus Sur 15782, Santiago de Compostela, Spain
2 Stoltseafarm S.A., Lira, Spain
Correspondence
Ysabel Santos
ysantos{at}usc.es
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ABSTRACT |
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, belongs to the family Flavobacteriaceae (Reichenbach, 1992a, b; Bernardet et al., 1996, 2002) and currently comprises nine species: Tenacibaculum maritimum from diseased fish (Wakabayashi et al., 1986), T. ovolyticum from fish eggs (Hansen et al., 1992), T. amylolyticum from the surfaces of marine macroalgae and T. mesophilum from sponges (Suzuki et al., 2001), T. skagerrakense isolated from seawater (Frette et al., 2004), T. lutimaris, T. litoreum, T. aestuarii from tidal flat sediment (Yoon et al., 2005; Choi et al., 2006; Jung et al., 2006) and T. litopenaei isolated from a shrimp mariculture pond (Sheu et al., 2007).
During the characterization of bacteria isolated from the kidney of a diseased sole (Solea senegalensis) and from seawater from holding tanks for turbot (Psetta maxima), strains LL04 11.1.1T and A37.1T were recovered on plates of Flexibacter maritimus medium (FMM; Pazos et al., 1996) at 25 °C (T. maritimum was previously misclassified within the genus Flexibacter). The diseased fish showed the typical signs observed in fish affected by marine flexibacteriosis caused by T. maritimum (i.e. eroded mouth, rotten fins, shallow skin lesions and paleness of internal organs). Pathogenicity assays demonstrated that the isolated bacteria were virulent for turbot and sole (Piñeiro-Vidal et al., 2007). The isolated bacteria were subcultured on FMM agar at 25 °C for 48 h and maintained at –80 °C both in FMM broth supplemented with 15 % (v/v) glycerol and in Microbank tubes (Prolab Diagnostics).
Morphological, physiological and biochemical tests were performed as described by Bernardet et al. (2002). The Gram reaction was tested by using the bioMérieux Gram-stain kit (according to the manufacturer's instructions) and the non-staining KOH method (Buck, 1982). Gliding motility was determined by phase-contrast microscopic examination of a fresh FMM broth culture and by the hanging drop technique as recommended by Bernardet et al. (2002). The presence of flexirubin-type pigments was determined by using the KOH test as described by Reichenbach (1989). Catalase and oxidase activities were determined as described by Cowan & Steel (1965). The ability of the strains to grow under anaerobic conditions was tested in FMM agar by using the GasPak anaerobic system (BBL). To assess their growth at different pH values, the strains were cultured in FMM broth adjusted to pH values ranging from 4 to 10. The temperature range for growth was determined on FMM agar plates incubated at 8, 15, 18, 22, 25, 30, 37 and 44 °C. Tolerance of salinity was tested in FMM broth containing seawater strengths of 10, 20, 30, 50, 70 and 100 % or at NaCl concentrations of 0.8, 1, 3, 5, 7 and 10 % (w/v). Indole production was tested in FMM broth supplemented with 1 % (w/v) tryptone, H2S production was determined in FMM broth supplemented with 5 % (w/v) peptone and the Voges–Proskauer reaction was determined in seawater supplemented with 0.7 % (w/v) peptone and 0.5 % (w/v) D-glucose. The ability of the novel strains to degrade casein (1 %), gelatin (1 %), starch (0.4 %) and Tween 80 (1 %) and to produce nitrate reductase were evaluated in FMM medium supplemented with substrate concentrations as reported previously (Suzuki et al., 2001). The utilization of carbon sources was tested on basal agar medium [containing, l–1 artificial seawater (Sigma), 0.2 g NaNO3, 0.2 g NH4Cl, 0.05 g yeast extract, 15 g agar] supplemented with 0.4 % carbon source [(+)-D-sucrose, (–)-D-ribose, (+)-D-galactose, (+)-D-glucose, L-proline, L-glutamate or L-tyrosine] as described by Suzuki et al. (2001). The absence of growth after incubation in the media for 1 month was scored as a negative result. Other enzymic activities were evaluated using the API ZYM system (bioMérieux) according to the manufacturer's instructions, except that sterile seawater was used as the suspension medium.
The results of the morphological, physiological and biochemical tests are given in the species descriptions and in Table 1.
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) with those of the type strains of the nine species of the genus Tenacibaculum and other representative members of the family Flavobacteriaceae obtained from GenBank. The 16S rRNA gene sequence similarities were determined using MEGA, version 3.1 (Kumar et al., 2004). Phylogenetic trees were constructed by using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) methods (Fig. 1). The evolutionary distance matrix for the neighbour-joining method was generated according to the Kimura two-parameter model (Kimura, 1980). The robustness of the phylogenetic trees was determined by means of bootstrap analyses based on 1000 replications. In both trees, strains LL04 11.1.1T and A37.1T formed a robust cluster with the species of the genus Tenacibaculum. Data from the sequence similarity analysis indicated that the closest relatives of strains LL04 11.1.1T and A37.1T were T. litoreum (99.4 and 98.4 %, respectively), T. mesophilum (97.4 and 96.7 %, respectively), T. aestuarii (97.3 % for both isolates), T. lutimaris (97.3 and 96.8 %, respectively), T. skagerrakense (96.4 and 95.9 %, respectively), and T. amylolyticum and (96.0 and 96.3 %, respectively). The 16S rRNA gene sequence similarity between strains LL04 11.1.1T and A37.1T was 98.4 %.
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. As the 16S rRNA gene sequence similarities between strains LL04 11.1.1T and A37.1T and between them and the type strain of T. litoreum were close to 99 %, DNA–DNA hybridization experiments were performed using the method described by De Ley et al. (1970), as modified by Huß et al. (1983). The experiment was performed at 45 °C in 2xSSC (1xSSC is 0.15 M sodium chloride plus 0.015 M sodium citrate at pH 7.0) using a Cary 100 Bio UV/visible spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature controller with an in situ temperature probe (Varian). The DNA G+C content determinations and the DNA–DNA hybridization experiments were performed by the Identification Service of the Deutsche Sammlung von Mikrooganismen und Zellkulturen GmbH (Braunschweig, Germany). The DNA G+C contents of strains LL04 11.1.1T and A37.1T were 32.1 and 32.7 mol%, respectively (Table 1
). The DNA–DNA relatedness between strains LL04 11.1.1T and A37.1T was 42.8 %, and the strains shared 39.6 and 40.2 % relatedness, respectively, with their closest relative, T. litoreum. Therefore, we conclude that strains LL04 11.1.1T and A37.1T represent two novel bacterial species within the genus Tenacibaculum, for which we propose the names Tenacibaculum discolor sp. nov. and Tenacibaculum gallaicum sp. nov., respectively.
Description of Tenacibaculum discolor sp. nov.
Tenacibaculum discolor (dis.co'lor. L. neut. adj. discolor of different colours, referring to the colours of the colonies).
Cells are Gram-negative rods that are 0.5 µm in diameter, 2–30 µm in length and motile by gliding. Spherical cells are rarely observed in ageing cultures. Colonies on FMM agar medium are flat, bright yellow, have uneven edges and do not adhere to the agar. On marine agar 2216 (Difco), colonies also appear bright yellow from the front, but appear bright green when viewed at an angle of 30–45 °C. The yellow pigment is not of the flexirubin type. Strictly aerobic. Growth occurs in media containing seawater at strengths in the range 30 to 100 % but not in media supplemented with NaCl alone. Growth occurs at 14–38 °C (optimum, 25–30 °C) and at pH 6.0–8.0. Catalase and cytochrome oxidase activities are present. Nitrate is reduced. Gelatin and casein are hydrolysed, but Tween 80 and starch are not. The Voges–Proskauer test gives a negative result. No acid is produced from carbohydrates. H2S and indole are not produced. L-Proline and L-glutamate are utilized, but (+)-D-sucrose, (–)-D-ribose, (+)-D-galactose, (+)-D-glucose and L-tyrosine are not utilized. In the API ZYM system, alkaline phosphatase, esterase, esterase lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase activities are present, but activities for all of the enzymes relating to the metabolism of carbohydrates are absent.
The type strain, LL04 11.1.1T (=NCIMB 14278T=DSM 18842T), was isolated from the kidney of a diseased sole (Solea senegalensis) in Galicia in north-west Spain.
Description of Tenacibaculum gallaicum sp. nov.
Tenacibaculum gallaicum (gal.lai'cum. L. neut. adj. gallaicum of Galicia, a north-western province of Spain, referring to the place of isolation).
Cells are Gram-negative rods that are 0.5 µm in diameter, 2–30 µm in length and motile by gliding. Spherical cells are rarely observed in ageing cultures. Colonies on FMM agar and marine agar 2216 media are flat, bright yellow, have uneven edges and do not adhere to the agar. Strictly aerobic. Growth occurs in media containing seawater strengths of 30–100 %, but not in media supplemented with NaCl alone. Growth occurs at 14–38 °C (optimum, 25–30 °C) and at pH 6.0–8.0. Catalase and cytochrome oxidase activities are present. Nitrate is reduced. Gelatin and casein are hydrolysed, but Tween 80 and starch are not hydrolysed. The Voges–Proskauer test gives a negative result. No acid is produced from carbohydrates. H2S and indole are not produced. L-Proline and L-glutamate are utilized but (+)-D-sucrose, (–)-D-ribose, (+)-D-galactose, (+)-D-glucose and L-tyrosine are not utilized. In the API ZYM system, alkaline phosphatase, esterase, esterase lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase are present, but all of the enzymes relating to the metabolism of carbohydrates are absent.
The type strain, A37.1T (=NCIMB 14147T=DSM 18841T), was isolated from seawater from a holding tank for turbot (Psetta maxima), in Galicia in north-west Spain.
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ACKNOWLEDGEMENTS |
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Suzuki, M., Nakagawa, Y., Harayama, S. & Yamamoto, S. (2001). Phylogenetic analysis and taxonomic study of marine Cytophaga-like bacteria: proposal for Tenacibaculum gen. nov. with Tenacibaculum maritimum comb. nov. and Tenacibaculum ovolyticum comb. nov., and description of Tenacibaculum mesophilum sp. nov. and Tenacibaculum amylolyticum sp. nov. Int J Syst Evol Microbiol 51, 1639–1652.[Abstract]
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