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1 Microbial Resources Division and Brazilian Collection of Environmental and Industrial Micro-organisms (CBMAI), CPQBA, UNICAMP, CP 6171, Brazil
2 Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel
3 Laboratory of Microbiology, Research Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate 041-8611, Japan
4 Laboratory of Microbiology and BCCMTM/LMG Bacteria Collection, Ghent University, K.L. Ledeganckstraat 35, Ghent 9000, Belgium
Correspondence
F. L. Thompson
Fabiano.Thompson{at}terra.com.br
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Thalassomonas loyana sp. nov. CBMAI 722T is AY643537.
Additional phylogenetic trees and an electron micrograph of cells of Thalassomonas loyana sp. nov. are available as supplementary figures in IJSEM online.
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Global climate changes, sea water pollution from aquaculture, oil spills and urban sewage, coral bleaching and other infectious diseases have been deemed to be the main causes of the decline of coral reefs (Hoegh-Guldberg, 2004; Hughes et al., 2003; Knowlton & Rohwer, 2003; Rosenberg & Ben-Haim, 2002; Sutherland et al., 2004). Recent reports suggest that bacteria may also play a role in the development of coral tumours (Breitbart et al., 2005). Coral reefs may well serve as indicators of both local environmental degradation and global climate changes.
In the present study, we analysed the taxonomic position of a novel isolate from the coral Favia favus suffering from white plague in the Eilat coral reef (Barash et al., 2005). Pure cultures of the strain caused the coral disease in controlled aquarium experiments (Barash et al., 2005). 16S rRNA gene sequence data positioned the novel isolate in the neighbourhood of the genus Thalassomonas (Macian et al., 2001). The two currently recognized species of this genus, Thalassomonas viridans and Thalassomonas ganghwensis, have been isolated from oysters in the Mediterranean sea and flat tide sediments in Korea, respectively, but have not, to date, ever been implicated in coral disease. Our data suggest that strain CBMAI 722T represents a novel species of the genus Thalassomonas.
Strain CBMAI 722T was isolated from diseased Favia favus by crushing the coral in 10 ml sterile artificial sea water with the aid of a mortar and pestle. The diseased Favia favus was collected by SCUBA diving at Eilat in the Gulf of Aqaba, Red Sea. Strain CBMAI 722T was grown on ZoBell 2216E agar medium (MA; Oppenheimer & ZoBell, 1952) at 20 °C for 48 h unless otherwise stated. Colony morphology was examined on cultures grown on MA by using a stereoscopic microscope. Cell morphology was examined on wet mounts via a phase-contrast microscope. Exponentially growing cells in marine broth (MB) medium were negatively stained with 1 % (w/v) uranyl acetate for electron microscopy (840A; JEOL). Sequences for 16S rRNA genes were generated on a DNA sequencer (ABI Prism 3100; Applied Biosystems) and analysed as described by Thompson et al. (2001). The consensus sequences were assembled and phylogenetic trees were constructed based on the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony and maximum-likelihood methods using KODON 2.0 software (Applied Maths). The DNA G+C content was determined by HPLC (Tamaoka & Komagata, 1984).
Phenotypic characterization of the isolate was performed using API 20NE (bioMérieux) and Biolog GN2 metabolic fingerprinting kits following the manufacturers' instructions with some modifications. The bacterial inocula were suspended in saline solution (3 % NaCl) containing 10 % MB for Biolog tests, whereas cell suspensions (2 % NaCl) were used for API 20NE tests. Reactions were recorded after 48 h at 30 °C. Carbon source utilization was also checked with standard basal medium as described previously (Baumann et al., 1984; Leifson, 1963; Oppenheimer & ZoBell, 1952). Salt tolerance was evaluated in a medium consisting of 0·5 g yeast extract and 2·5 g peptone per litre of sterile distilled water (Hidaka & Sakai, 1968). Alginate hydrolysis activity was determined using previously described methods (Sawabe et al., 1995). Antibiograms were determined using the disc diffusion method of Acar & Goldstein (1996) using commercial discs (Oxoid). Analysis of fatty acid methyl esters was carried out as described by Huys et al. (1994). For fatty acid analysis, cells were grown on MA for 48 h at 28 °C.
According to our 16S rRNA gene sequence analysis, strain CBMAI 722T is a member of the family Colwelliaceae (Ivanova et al., 2004). The closest phylogenetic neighbours of the novel isolate were T. ganghwensis KCTC 12041T and T. viridans CECT 5083T, with 95 and 94 % 16S rRNA gene sequence similarity, respectively (Fig. 1). This low level of similarity suggests that strain CBMAI 722T represents a novel branch within the family Colwelliaceae. Strain CBMAI 722T consistently grouped with Thalassomonas species even when different tree-building methods were used (see Supplementary Fig. S1 in IJSEM Online).
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). For instance, strain CBMAI 722T does not produce oxidase, does not possess pigment and does not grow at 15 or 37 °C, all of which are characteristics common to the other Thalassomonas species. It produces alginase, in contrast with the other Thalassomonas species. Strain CBMAI 722T is able to utilize cellobiose and L-arginine, but T. ganghwensis is not. Previously recognized Thalassomonas species contain the fatty acid 15 : 0, but this fatty acid is not found in strain CBMAI 722T. In addition, strain CBMAI 722T has a higher content of fatty acid 14 : 0 than the other Thalassomonas species. The 16S rRNA sequence analysis appears to suggest that strain CBMAI 722T may represent a new genus, but this remains to be determined in future studies with a broader collection of isolates of this novel taxon. We conclude that strain CBMAI 722T represents a novel species for which we propose the name Thalassomonas loyana sp. nov.
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Cells are 0·5–0·8 µm in width and 1–2 µm in length. They form translucent, convex, smooth-rounded colonies with an entire margin that are cream-coloured and 3 mm in size on MA after 3 days incubation at 25 °C. No growth occurs on 0 or 
10·0 % NaCl. No growth occurs below 15 °C or at 37 °C or above. Tests negative for indole, arginine dihydrolase, oxidase and urease activities. Weakly positive for gelatinase activity and nitrate reduction. Does not ferment glucose, but hydrolyses aesculin. According to Biolog tests, is able to utilize 
-cyclodextrin, dextrin, glycogen, N-acetyl-D-glucosamine, D-arabitol, D-cellobiose, i-erythritol, D-galactose, -D-glucose, maltose, D-raffinose, turanose, acetate, citrate, D-gluconic acid, D-glucosaminic acid, -ketoglutaric acid, DL-lactate, malonate, propionate, succinate, L-alanylglycine, L-glutamate, glycyl-L-aspartate, glycyl-L-glutamate, L-histidine, L-proline, DL-carnitine, -D-glucose 1-phosphate and D-glucose 6-phosphate as sole carbon sources. Does not utilize Tween 40, Tween 80, L-arabinose, L-fucose, gentiobiose, myo-inositol, -D-lactose, lactulose, D-mannitol, methyl 
-D-glucoside, D-psicose, L-rhamnose, D-sorbitol, D-trehalose, xylitol, pyruvic acid methyl ester, succinic acid monomethylester, cis-aconitic acid, D-galactonic acid lactone, D-galacturonic acid, D-glucuronic acid, -hydroxybutyric acid, -hydroxybutyric acid, 
-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, -ketobutyric acid, -ketovaleric acid, quinic acid, D-saccharic acid, sebacic acid, bromosuccinic acid, succinamic acid, glucuronamide, L-alaninamide, L-asparagine, L-aspartic acid, hydroxy-L-proline, L-leucine, L-ornithine, L-phenylalanine, L-pyroglutamic acid, D-serine, L-serine, L-threonine, -aminobutyric acid, urocanic acid, inosine, uridine, thymidine, phenyethylamine, putrescine, 2,3-butanediol, glycerol or DL--glycerol phosphate as sole carbon source. Weakly positive for utilization of N-acetyl-D-galactosamine, D-fructose, D-mannose, D-melibiose, sucrose, formic acid, D-alanine, L-alanine and 2-aminoethanol according to the Biolog system. The major fatty acids are summed feature 3 (31·3 %; comprising 16 : 1
7c and/or 15 iso 2-OH), 14 : 0 (13·1 %), 17 : 18c (11·7 %), 12 : 0 3-OH (6·3 %), 18 : 17c (6·6 %), 16 : 19c (5·7 %), 16 : 0 (4. 6 %), 15 : 18c (3·1 %), 11 : 0 3-OH (2·5 %), 12 : 0 (2·3 %), 18 : 19c (1·9 %), 13 : 0 (1·2 %), 14 : 15c (1·2 %) and 15 : 16c (1·2 %). Produces amylase, alginase, DNase and -galactosidase, but not agarase or 
-carragenase. Sensitive to (µg per disc) erythromycin (10), kanamycin (10), gentamicin (10), ampicillin (10) and tetracycline (10). The DNA G+C content of the type strain is 39·3 mol%.
The type strain of this species, CBMAI 722T (=LMG 22536T), was isolated from diseased coral in Eilat, Israel.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Barash, Y., Sulam, R., Loya, Y. & Rosenberg, E. (2005). Bacterial strain BA-3 and a filterable factor cause a white plague-like disease in corals from the Eilat coral reef. Aquat Microb Ecol 40, 183–189.
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Huys, G., Vancanneyt, M., Coopman, R., Janssen, P., Falsen, E., Altwegg, M. & Kersters, K. (1994). Cellular fatty-acid composition as a chemotaxonomic marker for the differentiation of phenospecies and hybridization groups in the genus Aeromonas. Int J Syst Bacteriol 44, 651–658.
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-aminobutyrate, L-tyrosine, D-sorbitol, DL-malate, 2-oxoglutarate, xylose, trehalose, glucuronate, putrescine, propionate, arabinose, myo-inositol, D-raffinose, rhamnose, D-ribose, salicin, sarcosine, tartrate, L-alanine, L-asparagine, L-citrulline, glycine, L-leucine, L-ornithine and L-serine.