Tenacibaculum soleae sp. nov., isolated from diseased sole (Solea senegalensis Kaup)

A novel Gram-negative, rod-shaped, gliding bacterial strain designated LL04 12.1.7^T was isolated from diseased sole (Solea senegalensis Kaup) in Galicia, Spain. Colonies were yellow-pigmented with uneven edges and did not adhere to the agar. The DNA G+C content of the strain was 29.8 mol%. 16S rRNA gene sequence similarity analysis indicated that strain LL04 12.1.7^T is a member of the genus Tenacibaculum in the family Flavobacteriaceae. Sequence similarities between the isolate and the type strains of other members of the genus were 96.7–94.8 %. The major fatty acids (>10 % of total fatty acids) were iso-C_{15 : 0} (23.1 %), iso-C_{15 : 0} 3-OH (10.6 %), C_{15 : 1}ω6c (12.2 %) and summed feature 3 (comprising C_{16 : 1}ω7c and/or iso-C_{15 : 0} 2-OH, 11.0 %). Genotypic and phenotypic data distinguished strain LL04 12.1.7^T from the 11 recognized Tenacibaculum species, indicating that it represents a novel species, for which the name Tenacibaculum soleae sp. nov. is proposed. The type strain is strain LL04 12.1.7^T (=CECT 7292^T =NCIMB 14368^T).

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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain LL04 12.1.7^T is AM746476.

The genus Tenacibaculum (Suzuki et al., 2001) in the family Flavobacteriaceae (Reichenbach, 1992a, b; Bernardet et al., 1996, 2002) currently comprises 11 species derived from different marine ecosystems and intensive aquaculture systems. Tenacibaculum maritimum and Tenacibaculum discolor were isolated from diseased fish (Wakabayashi et al., 1986; Piñeiro-Vidal et al., 2008); Tenacibaculum ovolyticum was isolated from fish eggs (Hansen et al., 1992); Tenacibaculum litopenaei (Sheu et al., 2007) and Tenacibaculum gallaicum (Piñeiro-Vidal et al., 2008) were from seawater of shrimp- and turbot-holding tanks, respectively; Tenacibaculum amylolyticum and Tenacibaculum mesophilum were from marine macroalgae and sponge, respectively (Suzuki et al., 2001); Tenacibaculum skagerrakense was from seawater (Frette et al., 2004); and Tenacibaculum lutimaris (Yoon et al., 2005), Tenacibaculum litoreum (Choi et al., 2006) and Tenacibaculum aestuarii (Jung et al., 2006) were from tidal flat sediment.

During the characterization of bacteria isolated from a diseased cultured sole (Solea senegalensis Kaup), strain LL04 12.1.7^T was recovered on plates of Flexibacter maritimus medium (FMM) (Pazos et al., 1996). Subcultivation was done on FMM or marine agar 2216 (MA; Difco) at 25 °C for 48 h. Strains were preserved at –80 °C in both marine broth 2216 (MB; Difco) supplemented with 15 % (v/v) glycerol and Microbank tubes (Prolab Diagnostics). Experimental infection assays have demonstrated that strain LL04 12.1.7^T is virulent for fingerlings of sole and turbot, but not for mice (data not shown).

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 MB culture 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 capacity of the strain to grow under anaerobic conditions was tested on MA using the GasPak anaerobic system (BBL). The optimal pH and the pH range for growth were determined in FMM broth adjusted to pH 4–10 according to Suzuki et al. (2001). Growth at various temperatures (8, 15, 18, 22, 25, 30, 37 and 44 °C) was determined on FMM agar plates. Tolerance to salinity was tested in FMM broth containing 10, 20, 30, 50, 70 or 100 % seawater or 0.8, 1, 3, 5, 7 or 10 % (w/v) NaCl. Indole and H₂S production were tested on FMM broth supplemented with 1 % (w/v) tryptone or 5 % (w/v) peptone, respectively. The Voges–Proskauer reaction was evaluated in seawater with 0.7 % (w/v) peptone and 0.5 % (w/v) (+)-D-glucose. The capacity of the strain to degrade casein (1 %), gelatin (1 %), starch (1 %) and Tween 80 (1 %) was evaluated in FMM medium (Suzuki et al., 2001). Utilization of carbon sources was tested on basal agar medium (0.2 g NaNO₃, 0.2 g NH₄Cl, 0.05 g yeast extract and 15 g agar in 1 l artificial seawater) containing 0.4 % carbon source [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 1 month of incubation was scored as a negative result. Other enzyme activities were evaluated in the API ZYM system (bioMérieux) following the manufacturer's instructions, except that sterile seawater was used as the suspension medium.

For analysis of fatty acid methyl esters, strain LL04 12.1.7^T was grown on MA plates for 48 h at 25 °C. Cell harvesting, saponification of lipids, methylation of fatty acids, extraction of fatty acid methyl esters, washing of extracts and GC analysis were performed according the standardized procedures of the Microbial Identification system (MIDI; Microbial ID) (Sasser, 1990).

Determination of the DNA G+C content and sequencing of the 16S rRNA gene of the isolate were carried out by the identification service of the DSMZ, Braunschweig, Germany. The 1506 bp sequence of strain LL04 12.1.7^T was automatically aligned using CLUSTAL W (Thompson et al., 1994) with those of the type strains of the 11 Tenacibaculum species and of other representative members of the family Flavobacteriaceae obtained from GenBank/EMBL. Phylogenetic trees were constructed by 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 Kimura's two-parameter model (Kimura, 1980). To evaluate phylogenetic trees, a bootstrap analysis with 1000 sample replications was performed with the SEQBOOT and CONSENSE programs in the PHYLIP 3.67 package (Felsenstein, 2007). The identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence similarity were achieved using the EzTaxon server (; Chun et al., 2007). Results of morphological, physiological and biochemical tests are given in Table 1 and in the species description.

(41K): Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing relationships between strain LL04 12.1.7T, other members of the genus Tenacibaculum and related genera of the family Flavobacteriaceae. Bootstrap values >50 % (based on 1000 replications) are shown at branching points. Solid circles indicate that the corresponding nodes are also recovered in the maximum-parsimony tree. Bar, 0.01 substitutions per site.

Table 1. Differential phenotypic characteristics of strain LL04 12.1.7T and the type strains of other Tenacibaculum species Strains: 1, T. soleae sp. nov. LL04 12.1.7T; 2, T. gallaicum DSM 18841T; 3, T. discolor DSM 18842T; 4, T. mesophilum MBIC1140T; 5, T. lutimaris KCTC 12302T; 6, T. skagerrakense ATCC BAA-458T; 7, T. amylolyticum MBIC4355T; 8, T. ovolyticum IAM 14318T; 9, T. maritimum NCIMB 2154T; 10, T. litoreum JCM 13039T; 11, T. aestuarii KCTC 12569T; 12, T. litopenaei BCRC 17590T. Data from Wakabayashi et al. (1986), Hansen et al. (1992), Suzuki et al. (2001), Bernardet et al. (2002), Frette et al. (2004), Yoon et al. (2005), Choi et al. (2006), Jung et al. (2006), Sheu et al. (2007), Piñeiro-Vidal et al. (2008) and this study. +, Positive; –, negative; W, weakly positive; ND, no data available; NG, no growth.

Fatty acid composition data for strain LL04 12.1.7^T and type strains of other Tenacibaculum species are detailed in Table 2. The main difference between the novel isolate and the other Tenacibaculum type strains was the high content of unsaturated fatty acids (13.9 %) in strain LL04 12.1.7^T. The cellular fatty acid profile of strain LL04 12.1.7^T was dominated by iso-C_{15 : 0} (23.1 %), iso-C_{15 : 0} 3-OH (10.6 %), iso-C_{16 : 0} 3-OH (8.4 %), C_{15 : 1}ω6c (12.2 %) and summed feature 3 (comprising C_{16 : 1}ω7c and/or iso-C_{15 : 0} 2-OH; 11.0 %) (Table 2).

Table 2. Cellular fatty acid compositions (%) of strain LL04 12.1.7T and the type strains of other Tenacibaculum species Strains: 1, T. soleae sp. nov. LL04 12.1.7T; 2, T. mesophilum MBIC1140T; 3, T. lutimaris KCTC 12302T; 4, T. skagerrakense ATCC BAA-458T; 5, T. maritimum NCIMB 2154T; 6, T. litoreum JCM 13039T; 7, T. aestuarii KCTC 12569T; 8, T. litopenaei BCRC 17590T. Data from Yoon et al. (2005), Jung et al. (2006), Choi et al. (2006), Sheu et al. (2007) and this study. –, Not detected; tr, trace (<1 %); ECL, equivalent chain-length. No data were available for T. ovolyticum, T. gallaicum or T. discolor. Fatty acids amounting to less than 1 % of the total fatty acids in all strains tested are not listed.

The DNA G+C content of strain LL04 12.1.7 ^T was 29.8 mol%, the lowest reported value within the genus Tenacibaculum. Comparison of the 16S rRNA gene sequence of strain LL04 12.1.7^T with those of the type strains of the 11 Tenacibaculum species and other members of the family Flavobacteriaceae demonstrated that strain LL04 12.1.7^T formed a robust cluster with the Tenacibaculum species. The phylogenetic tree based on 16S rRNA gene sequences is shown in Fig. 1. The closest relatives of strain LL04 12.1.7^T were the type strains of T. ovolyticum and T. aestuarii (96.7 % sequence similarity), T. lutimaris and T. mesophilum (96.4 %), T. gallaicum (96.2 %), T. amylolyticum (96.0 %), T. litoreum (95.9 %), T. discolor (95.8 %), T. skagerrakense (95.7 %), T. litopenaei (95.0 %) and T. maritimum (94.8 %). All these values are lower than the theoretical threshold (97 %) for the delineation of bacterial species based on 16S rRNA gene sequence similarity (Stackebrandt & Goebel, 1994; Stackebrandt & Ebers, 2006). According to the phenotypic and genetic data obtained in this study, it is concluded that strain LL04 12.1.7^T represents a novel species within the genus Tenacibaculum, for which the name Tenacibaculum soleae sp. nov. is proposed.

Description of Tenacibaculum soleae sp. nov.
Tenacibaculum soleae [so.le'ae. L. gen. n. soleae of a sole, in reference to the source of the isolate, a cultured sole (Solea senegalensis Kaup)].

Cells are Gram-negative rods, 0.5 µm in diameter and 2–25 µm in length, motile by gliding. Spherical cells are observed in ageing cultures. Colonies on FMM agar and MA 2216 (Difco) are flat and yellow with uneven edges and do not adhere to the agar. The yellow pigment does not belong to the flexirubin type. Strictly aerobic. Growth occurs in media containing 50–100 % seawater, but not in media supplemented with NaCl only. Growth occurs at 14–30 °C (optimum 22–25 °C) and pH 6.0–8.0. Catalase and cytochrome oxidase activities are present. Gelatin and casein are hydrolysed, but Tween 80 and starch are not. The Voges–Proskauer test is negative. No acid is produced from carbohydrates. H₂S and indole are not produced. L-Proline, L-glutamate, 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 and cystine arylamidase activities are present, but trypsin, α-chymotrypsin and all enzymes related to the metabolism of carbohydrates are absent.

The type strain is LL04 12.1.7^T (=CECT 7292^T =NCIMB 14368^T), isolated from a diseased sole (Solea senegalensis) reared in Galicia (north-western Spain).