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1 Observatoire Océanologique, Laboratoire d'Océanographie Biologique de Banyuls, Université Pierre et Marie Curie (Paris VI), Institut National des Sciences de l'Univers (INSU), CNRS UMR 7621, BP44, 66651 Banyuls-sur-Mer Cedex, France
2 Laboratoire de Microbiologie des Environnements Extrêmes, UMR 6197, IFREMER, Centre de Brest, BP70, 29280 Plouzané, France
3 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany
Correspondence
Philippe Lebaron
lebaron{at}obs-banyuls.fr
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ABSTRACT |
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Graphs showing the growth of strain 13IX/A01/164T at various temperatures, salt concentrations and pH values are available as supplementary material in IJSEM Online.
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). However, although the majority of thermophilic and extremely halophilic micro-organisms belong to the Archaea, members of the Bacteria with similar characteristics and properties have been isolated. For example, the orders Thermales (Rainey & da Costa, 2001) and Thermotogales (Huber & Stetter, 1992) encompass 13 genera of thermophilic strains and the genera Halorhodospira (Imhoff & Süling, 1996) and Salinibacter (Antón et al., 2002) are composed of extremely halophilic species. Some recently described genera, such as Rhodothermus and Thermonema, belong to the family Crenotrichaceae within the Bacteroidetes. This family is remarkable as all its members described to date exhibit characteristics that are regarded as extremophilic. Species of the genera Rhodothermus (Alfredsson et al., 1988; Sako et al., 1996) and Thermonema (Hudson et al., 1989; Tenreiro et al., 1997) are thermophilic and have optimal growth temperatures between 60 and 80 °C. Isolates of the genus Salinibacter are extremely halophilic and require at least 150 g salt l–1 for growth. In this paper, a novel strain of the family Crenotrichaceae, isolated from the north-western Mediterranean Sea, is described; this strain represents the first member of this family that does not exhibit the extreme characteristics that are found in other members of the family.
Samples were collected in September 2001 in the bay of Banyuls-sur-Mer (42° 29' N 3° 08' E) by submerging a sterile bottle and opening it at a depth of 0.5 m (Agogué et al., 2004). Subsamples were spread on marine agar plates (MA 2216; Difco) and incubated at 25 °C for 2 weeks. Colonies were picked and purified after at least three subcultures. Among these colonies, an isolate that formed orange-coloured colonies was obtained and referenced as strain 13IX/A01/164T (Agogué et al., 2005).
Microscopic observations (Olympus AX70) indicated that cells of strain 13IX/A01/164T were motile rods, approximately 2.5±0.4 µm long and 0.2±0.06 µm wide. No gliding motility was observed. Cells were negatively stained for transmission electron microscopy (Raguénès et al., 1997). A polar flagellum was observed (Fig. 1). The Ryu KOH reaction (Powers, 1995) led to immediate cell lysis that was confirmed by phase-contrast microscopy (Olympus AX70). This positive reaction indicated that cells were Gram-negative.
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. Growth was observed at 10–40 °C, with optimum growth at 30 °C (see Supplementary Fig. S1a available in IJSEM Online). The strain grew at NaCl concentrations of 0–70 g l–1; an optimum concentration for growth could be defined at 10 g l–1, but growth rates were quite similar at 5 and 20–40 g l–1 (Supplementary Fig. S1b). Growth occurred at pH 5.0–10.0, with a clear optimum at pH 8.0. A linear increase in the growth rate was observed from pH 5.0 to 8.0; at pH 9.0, growth decreased dramatically to 60 % of the value obtained at pH 8.0 (Supplementary Fig. S1c). The shortest generation time observed was 23 min. The ability to use different substrates was investigated with Biolog GN2 MicroPlates (De Groote et al., 1999) according to the manufacturer's instructions except for the incubation time (measurements were performed every hour for 24 h). Positive reactions are indicated in Table 1. Weak positive reactions were observed for cellobiose, lactose and 
-hydroxybutyrate.
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; a negative reaction was observed.
Fatty acid methyl esters were extracted and prepared by the standard protocol of the Microbial Identification System (MIDI; Microbial ID) using cells grown in MB. As cells did not grow on the medium recommended by the MIDI system (trypticase soy broth), the fatty acids obtained could not be compared directly to those of the MIDI database. Extracts were analysed using a Hewlett Packard model HP6890A GC equipped with an FID as described previously (Kämpfer & Kroppenstedt, 1996). The fatty acid composition of strain 13IX/A01/164T was as follows: 15 : 0 iso (23.5 %), 15 : 0 iso 2-OH (11.3 %), 17 : 1
8c (11 %), 15 : 0 (10.7 %), 15 : 16c (10.6 %), 13 : 0 iso (6.5 %), 17 : 19c iso (6.5 %), 15 : 0 anteiso (4.1 %), 17 : 16c (3.2 %), 14 : 0 iso (2.3 %), 16 : 0 iso (2.3 %), 17 : 0 iso (1.5 %), 16 : 0 (1.4 %), 17 : 0 (1 %), 15 : 18c (1 %), and 16 : 15c (1 %). Although fatty acid data are missing for species of Salinibacter, those of Rhodothermus (Silva et al., 2000) have significantly different compositions, with major amounts (>20 % each) of 17 : 0 anteiso, 15 : 0 anteiso and 17 : 0 iso.
Genomic DNA was extracted as described by Wery et al. (2001a). The G+C content was determined by thermal denaturation using the method of Marmur & Doty (1962) and conditions reported by Raguénès et al. (1997). The G+C content of the genomic DNA of strain 13IX/A01/164T was 41.8±1.1 mol%. The 16S rRNA gene was amplified and sequenced as described by Agogué et al. (2005). The sequence was compared to those available in GenBank using BLAST (Altschul et al., 1997). Alignments and similarities were obtained by the CLUSTAL X method. The phylogenetic reconstruction was produced using PHYLO_WIN (Galtier et al., 1996) with the Jukes–Cantor correction for determination of the distance matrix, followed by neighbour joining (Saitou & Nei, 1987) for determination of the best phylogenetic tree. Maximum-likelihood methods (Felsenstein, 1981) were also employed for phylogenetic analysis. Bootstrap values were determined according to Felsenstein (1985). Strain 13IX/A01/164T was phylogenetically affiliated to the family Crenotrichaceae within the phylum Bacteroidetes (Fig. 2) and was most closely related to Rhodothermus marinus NR-32T (16S rRNA gene sequence similarity of 87 %). Strain 13IX/A01/164T was also closely related to strains of Salinibacter ruber, Thermonema rossianum and Thermonema lapsum.
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). Nevertheless, strain 13IX/A01/164T exhibited no extreme characteristics, it is neither thermophilic nor halophilic, and the environment from which it was isolated clearly differed from those where the most closely related species were found. No filamentous cells, such as those observed with Thermonema strains, were observed. The DNA G+C content of strain 13IX/A01/164T was significantly lower (by 5–24 mol%) than those of related species. Based on phenotypic and genotypic differences between strain 13IX/A01/164T and its nearest relatives, it is proposed that strain 13IX/A01/164T should be assigned as a representative of a novel species in a new genus belonging to the Crenotrichaceae. Because of the geographical origin of strain 13IX/A01/164T and its lack of original characteristics compared with the extremophilic behaviour of the most closely related species, the name Balneola vulgaris gen. nov., sp. nov. is proposed.
Description of Balneola gen. nov.
Balneola (Bal.ne'o.la. M.L. fem. n. Balneola the ancient name of Banyuls, referring to the area of isolation of the first characterized strain).
Aerobic, motile, Gram-negative rod growing optimally at 30 °C and pH 8.0. Catalase-positive and oxidase-negative. The major fatty acids are 15 : 0 iso (23.5 %), 15 : 0 iso 2-OH (11.3 %), 17 : 18c (11 %), 15 : 0 (10.7 %), 15 : 16c (10.6 %), 13 : 0 iso (6.5 %) and 17 : 19c iso (6.5 %). Phylogenetically affiliated with the phylum Bacteroidetes within the family Crenotrichaceae. The type species is Balneola vulgaris.
Description of Balneola vulgaris sp. nov.
Balneola vulgaris (vul.ga'ris. L. fem. adj. vulgaris common, referring to the lack of specific characteristics).
Forms orange colonies on MA medium. Grows at 10–40 °C (optimum 30 °C), pH 5.0–10.0 (optimum pH 8.0) and a salinity range of 0–50 g l–1 (optimum 20 g l–1). Positive reactions with Biolog GN2 plates are obtained for N-acetylgalactosamine, adonitol, arabinose, arabitol, erythritol, fructose, fucose, glucose, lactulose, maltose, methyl glucoside, sorbitol, acetate, hydroxyphenylacetate and propionate. Positive reactions with the API ZYM system are obtained for alkaline phosphatase, leucine arylaminidase, valine arylaminidase, trypsin, chymotrypsin and acid phosphatase. Other characteristics are given in Table 1.
The type strain is 13IX/A01/164T (=DSM 17893T=CIP 109092T=OOB 256T), isolated from a water column in the bay of Banyuls-sur-Mer (42° 29' N 3° 08' E). The DNA G+C content of strain 13IX/A01/164T is 42 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Agogué, H., Casamayor, E. O., Bourrain, M., Obernosterer, I., Joux, F., Herndl, G. J. & Lebaron, P. (2005). A survey on bacteria inhabiting the sea surface microlayer of coastal ecosystems. FEMS Microbiol Ecol 54, 269–280.
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