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1 Department of Marine Biotechnology, National Kaohsiung Marine University, Kaohsiung, Taiwan
2 Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd, Nan-Tzu, Kaohsiung City 811, Taiwan
3 Department of Soil Environmental Science, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, Taiwan
4 Department of Aquaculture, National Kaohsiung Marine University, Kaohsiung, Taiwan
Correspondence
Wen-Ming Chen
p62365{at}ms28.hinet.net
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7c and/or iso-C15 : 0 2-OH; 21.3 %), iso-C17 : 0 3-OH (12.7 %) and iso-C15 : 1 (8.7 %). The G+C content of the genomic DNA was 35.2 mol%. Hence, genotypic and phenotypic data demonstrate that strain B-IT should be classified within a novel species in the genus Tenacibaculum, for which the name Tenacibaculum litopenaei sp. nov. is proposed. The type strain is B-IT (=BCRC 17590T=LMG 23706T).
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is a member of the family Flavobacteriaceae (Reichenbach, 1992a, b; Bernardet et al., 1996, 2002). It currently contains eight species isolated from different marine sources. Tenacibaculum maritimum and T. ovolyticum [previously Flexibacter maritimus (Wakabayashi et al., 1986) and Flexibacter ovolyticus (Hansen et al., 1992)] were isolated from diseased red sea bream fingerling and halibut egg, respectively. Tenacibaculum mesophilum and T. amylolyticum were isolated from sponge homogenate and the surface of marine macroalgae, respectively (Suzuki et al., 2001). Tenacibaculum skagerrakense was isolated from seawater (Frette et al., 2004), while Tenacibaculum lutimaris, T. litoreum and T. aestuarii were isolated from tidal flat sediment (Yoon et al., 2005; Choi et al., 2006; Jung et al., 2006).
In August 2000, in the course of a study aiming at isolating chitinolytic bacteria, a surface water sample was collected from a shrimp (Litopenaeus vannamei) mariculture pond located in Pingtung County, southern Taiwan. The water sample was serially diluted and plated on chitin salt agar plates and incubated at 28 °C. Chitin salt medium contained (l–1) 5 g colloidal chitin, 30 g NaCl, 1.0 g MgSO4.7H2O, 0.2 g K2HPO4, 0.5 g CaCl2.2H2O and 1.0 ml trace element solution. The pH was adjusted to 7.5. Colloidal chitin was prepared from commercial chitin (Ohka Chemical) as described by Chang et al. (2004). The trace element solution contained (l–1) 2.8 g H3BO3, 2.0 g MnSO4.4H2O, 0.2 g ZnSO4.7H2O, 0.08 g CuSO4.5H2O, 0.1 g FeCl3 and 0.14 g Na2MoO4.2H2O (Chang et al., 2004). Agar plates were prepared by adding 1.5 % (w/v) Bacto agar (BD Difco) to the chitin salt medium. A bacterial strain that showed large clear zones around the colonies resulting from chitin degradation was isolated and designated B-IT. Strain B-IT was further subcultivated on marine agar 2216 (MA; BD Difco) and the organism was subjected to a polyphasic taxonomic study.
The morphology of bacterial cells was observed during the lag, exponential and stationary phases of growth under a phase-contrast microscope. Gliding motility was tested as described by Bowman (2000) and Bernardet et al. (2002). Flagellar staining was performed using the Spot Test flagella stain (BD Difco). The Gram reaction was assessed using the Gram stain set (BD Difco) and the Ryu non-staining KOH method (Powers, 1995). Accumulation of poly-
-hydroxybutyrate granules was observed by light microscopy after staining cells with Sudan black. Cellular pigments were extracted from cultures grown on MA by using an acetone/methanol mixture (7 : 2, v/v) and absorption spectra were determined with a scanning UV/visible spectrophotometer. The presence of flexirubin-type pigments was investigated as described by Reichenbach (1992a) and Bernardet et al. (2002). The pH range for optimum growth was examined in marine broth 2216 (MB) using appropriate biological buffers (pH 3–11) (Chung et al., 1995). The pH was adjusted prior to sterilization; post-sterilization controls revealed that only minor changes in pH had occurred. Requirement for NaCl was determined using nutrient broth containing no NaCl and 0.5 % and 1.0–10.0 % NaCl (w/v, at 1.0 % intervals). The temperature range for growth (4, 10, 15, 20, 25, 30, 35, 39 and 40 °C) was examined in MB adjusted to pH 7. Growth was examined by measuring the turbidity (OD600) of cultures grown at various pH, NaCl concentrations and temperatures. Anaerobic cultivation was performed on MA using the Oxoid AnaeroGen system.
Extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene were carried out as described by Chen et al. (2001). Sequence reaction fragments were separated using a DNA sequencer (ABI PRISM 310 instrument; Applied Biosystems) and sequences were assembled by using the Fragment Assembly System program from the Wisconsin package 9.1 (GCG, 1995). The resulting sequence was compared with available 16S rRNA gene sequences from the RDP2 and GenBank databases. Multiple-sequence alignments of strain B-IT and its closest relatives were performed using the BioEdit software (Hall, 1999). Phylogenetic trees were inferred using the maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) tree-making algorithms. An evolutionary distance matrix was generated for the neighbour-joining algorithm using the Jukes & Cantor (1969) distance model and bootstrap analysis (1000 resamplings).
A comparison of the nearly complete 16S rRNA gene sequence (1471 nt) of strain B-IT with those of representative members of the genera classified in the family Flavobacteriaceae showed that this organism fell within the evolutionary radiation of the genus Tenacibaculum (Fig. 1). Strain B-IT was most closely related (99.5–99.7 %) to ‘[Flexibacter] echinicida’, an unpublished bacterial species isolated from diseased sea urchin (Tajima et al., 1997a, b) and now considered as a member of Tenacibaculum (Masuda et al., 2004). Unfortunately, it has not been possible to obtain strains of ‘[Flexibacter] echinicida’ for comparison with strain B-IT. Among species with validly published names, strain B-IT was most closely related to T. mesophilum MBIC1140T and T. lutimaris CL-TF13T, sharing a 16S rRNA gene sequence similarity of 96.1 % with each. Strain B-IT shared lower 16S rRNA gene sequence similarity with the type strains of T. aestuarii (95.7 %), T. amylolyticum (95.5 %), T. litoreum (95.4 %), T. skagerrakense (95.0 %), T. ovolyticum (93.3 %) and T. maritimum (93.2 %). Similarity levels of strain B-IT to other bacterial species in the family Flavobacteriaceae were less than 93 %.
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. Strain B-IT showed low DNA–DNA hybridization values with T. mesophilum MBIC1140T (26±4 %) and T. lutimaris CL-TF13T (12±3 %), clearly indicating that it represents a novel species in the genus Tenacibaculum.
Fatty acid methyl esters were extracted from cells grown on MA for 48 h at 28 °C and prepared by the standard protocol of the Microbial Identification System (MIDI; Microbial ID). The fatty acid composition of strain B-IT was dominated by iso-C15 : 0 (22 %), summed feature 3 (C16 : 17c and/or iso-C15 : 0 2-OH; 21.3 %), iso-C17 : 0 3-OH (12.7 %) and iso-C15 : 1 (8.7 %). The detailed fatty acid compositions of strain B-IT and other Tenacibaculum species are shown in Table 1. The fatty acid profile of strain B-IT was in accordance with those of other Tenacibaculum species (Jung et al., 2006; Choi et al., 2006; Yoon et al., 2005), although distinguishable by the absence of C15 : 0 (Table 1).
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. The G+C content of strain B-IT was 35.2±1.0 mol%.
The commercially available API 20NE (bioMérieux), API ZYM (bioMérieux) and Biolog GN2 (Biolog) microtest systems were used according to the manufacturers' instructions to determine the biochemical properties, enzyme activities and carbohydrate utilization pattern of strain B-IT. The API ZYM test was read after 4 h incubation at 37 °C, while API 20NE and Biolog GN2 were read after 72 h at 28 °C. The three commercial systems were inoculated with a cell suspension in artificial seawater (NaCl, 24 g; MgCl2, 5.1 g; Na2SO4, 4 g; CaCl2, 1.1 g; KCl, 0.7 g; NaHCO3, 0.2 g; KBr, 0.1 g; H3BO3, 0.027 g; SrCl2, 0.024 g; NaF, 0.003 g; distilled water, 1 l; Lyman & Fleming, 1940). Sensitivity of strain B-IT to different antibiotics was analysed by the diffusion method on MA plates. The following antibiotic discs (Oxoid) were used: ampicillin (10 µg), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), novobiocin (30 µg), rifampicin (5 µg), penicillin G (10 U), streptomycin (10 µg) and tetracycline (30 µg). The effect of antibiotics on cell growth was assessed after 2 days of incubation at 28 °C.
Detailed results of phenotypic study are provided in Table 2 and in the species description. Strain B-IT could be distinguished from its phylogenetic relatives using a combination of phenotypic properties, especially nitrate reduction, hydrolysis of chitin, starch, gelatin and Tween 80 and utilization of various organic compounds (such as citrate, L-leucine, L-proline, L-glutamate, L-aspartate, D-glucose and sucrose).
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Description of Tenacibaculum litopenaei sp. nov.
Tenacibaculum litopenaei (li.to.pen.ae'i. N.L. n. Litopenaeus the scientific name of a genus of penaeid shrimp; N.L. gen. n. litopenaei of Litopenaeus, referring to the isolation of the type strain from a shrimp belonging to the genus Litopenaeus).
Cells are aerobic, Gram-negative, non-flagellated, non-spore-forming, straight rods, 0.3–0.5 µm in width and 2–10 µm in length and motile by gliding. Degenerative spherical cells are occasionally observed in ageing broth cultures. Poly--hydroxybutyrate granules are not accumulated. Forms yellow-coloured, circular, convex colonies with spreading margins after 48 h of incubation at 28 °C on MA. Flexirubin-type pigments are absent. Carotenoid pigments are produced with absorption peaks at 480, 454 and 425 nm. Grows at 10–39 °C, in the presence of 2–10 % NaCl and at pH 5–10. Optimum growth occurs at 28–37 °C, with 3 % NaCl and at pH 7.0–8.0. Positive for gelatin hydrolysis and for oxidase, alkaline phosphatase, C4 esterase, C8 lipase, C14 lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, 
-galactosidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase activities; negative for nitrate reduction, indole production, glucose fermentation, aesculin hydrolysis, catalase, urease, arginine dihydrolase, trypsin, -chymotrypsin and -glucuronidase activities (API 20NE and API ZYM). The following carbon sources are utilized (Biolog GN2): -D-glucose, acetic acid, -ketobutyric acid, -ketoglutaric acid, -ketovaleric acid, L-glutamic acid, hydroxy-L-proline, L-proline, L-serine, L-threonine and uridine. The following carbon sources are not utilized: -cyclodextrin, dextrin, glycogen, Tweens 40 and 80, N-acetyl-D-galactosamine, adonitol, arabinose, arabitol, cellobiose, i-erythritol, D-fructose, L-fucose, D-galactose, gentiobiose, myo-inositol, -D-lactose, lactulose, maltose, D-mannitol, D-mannose, melibiose, methyl -D-glucoside, D-psicose, D-raffinose, L-rhamnose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol, methyl pyruvate, monomethyl succinate, cis-aconitic acid, citric acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, p-hydroxyphenylacetic acid, -, - and 
-hydroxybutyric acids, itaconic acid, DL-lactic acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, glucuronamide, alaninamide, D- and L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-histidine, glycyl L-aspartic acid, glycyl L-glutamic acid, L-leucine, L-ornithine, L-phenylalanine, L-pyroglutamic acid, D-serine, DL-carnitine, -aminobutyric acid, urocanic acid, inosine, thymidine, phenyl ethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, glycerol, DL--glycerol phosphate, glucose 1-phosphate and glucose 6-phosphate. Resistant to ampicillin, gentamicin, kanamycin, penicillin G and streptomycin; sensitive to chloramphenicol, erythromycin, nalidixic acid, novobiocin, rifampicin and tetracycline. The major fatty acids are iso-C15 : 0, summed feature 3 (C16 : 17c and/or iso-C15 : 0 2-OH), iso-C17 : 0 3-OH and iso-C15 : 1. The G+C content of the DNA is 35.2 mol%.
The type strain, B-IT (=BCRC 17590T=LMG 23706T), was isolated from a water sample collected from a shrimp (Litopenaeus vannamei) mariculture pond located in Pingtung County, southern Taiwan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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