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-proteobacterium isolated from a subterranean saline lake
1 Departamento de Microbiología y Genética, Lab. 209, Edificio Departamental de Biología, Universidad de Salamanca, Campus M. Unamuno, 37007 Salamanca, Spain
2 Instituto de Recursos Naturales y Agrobiología, CSIC, Salamanca, Spain
Correspondence
Raúl Rivas
raul{at}wwwedu-micro.usal.es
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8c, C18 : 17c, C16 : 0 and 11-methyl C18 : 17c. The G+C content of the DNA was 57·4 mol%. Oxidase and catalase activities were present. Growth with many different carbohydrates as the sole carbon source was observed. The data from this polyphasic study suggest that this bacterium belongs to a novel genus of the order ‘Rhizobiales’ and is not associated with any of the known families of this order. It is proposed that isolate MACL11T should be classified in a novel genus and species, Martelella mediterranea gen. nov., sp. nov., with MACL11T (=LMG 22193T=CECT 5861T) as the type strain.
The GenBank/EMBL/DDBJ accession number for the almost-complete 16S rRNA gene sequence of strain MACL11T is AY649762.
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), Aurantimonas (Denner et al., 2003) and Fulvimarina (Cho & Giovannoni, 2003). They were all retrieved from marine environments; none of them was isolated from saline lakes. Here, we describe the characterization of a strain belonging to a novel genus of the order ‘Rhizobiales’ that was isolated from water collected at Lake Martel, a subterranean saline lake in Mallorca, Spain. Lake Martel is 177 m long, approximately 30 m wide and approximately 5–12 m deep and contains semi-salt water. The temperature of the water remains constant at 18 °C and its pH is about 7·5. Strain MACL11T was isolated under aseptic conditions from water samples taken from Lake Martel at a depth of 10 cm. A 200 ml sample was filtered under vacuum in sterile conditions through a membrane filter (Millipore) with a pore diameter of 45 µm. The membrane was placed on a plate containing YED medium (0·5 % yeast extract, 0·7 % glucose, 2 % agar) supplemented with 5 % (w/v) NaCl and was incubated at 28 °C. The colony morphology was examined in cultures grown on YED medium supplemented with 5 % (w/v) NaCl.
Strain MACL11T was grown on YED medium (0·5 % yeast extract, 0·7 % glucose, 1·5 % agar) for 48 h to check for motility by phase-contrast microscopy (Axioskop 2; Zeiss). Gram staining was carried out using the procedure described by Doetsch (1981). Cells were gently suspended in sterile distilled water, stained with 0·2 % uranyl acetate and examined at 80 kV using a Zeiss EM 209 transmission electron microscope (Peix et al., 2003).
Cells of strain MACL11T were Gram-negative, non-motile short rods (1·0–1·1x1·3–1·5 µm) (Fig. 1). Colonies were white- to cream-coloured on YED medium. They were smooth and mostly flat. Morphologically, strain MACL11T was different from related genera (Table 1).
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). The temperature range for growth was determined by incubating cultures in YED medium between 4 and 45 °C. The pH range was determined in YED medium with a final pH between 4·0 and 10·0. Salt tolerance was studied in YED medium containing 0–20 % (w/v) NaCl.
Differentiating phenotypic characteristics of strain MACL11T with respect to closely related genera are shown in Table 1
. According to these data, this strain differs from members of the genus Rhizobium as regards growth at 40 °C, hydrolysis of aesculin and growth with 5 % NaCl, D-arabinose, citrate, D-mannose, D-sorbitol and sucrose. Strain MACL11T is different from members of the genus Aurantimonas in terms of growth without NaCl, hydrolysis of aesculin and growth with D-arabinose, citrate, mannitol, D-mannose, D-sorbitol and sucrose. Finally, isolate MACL11T differs from the genus Fulvimarina in terms of growth at 40 °C, hydrolysis of aesculin and growth with 10 % NaCl, D-arabinose, D-mannose and sucrose. Strain MACL11T grew optimally at 28 °C (growth range 4–37 °C; no growth at 40 °C) and pH 7·0 (pH range 5–8·5). Strain MACL11T grew optimally at NaCl concentrations of up to 2 % and it also grew in the presence of NaCl concentrations up to 5 % (w/v), although salt was not essential for growth.
Determinations of lipoquinones and cellular fatty acid composition were performed as described by Zimmermann et al. (1998).
Ubiquinones were the only respiratory lipoquinones detected: Q-10 predominated (98 %), although Q-9 was present in minor amounts (2 %). This quinone profile is characteristic of most species within the class ‘Alphaproteobacteria’ (Collins & Jones, 1981; Yokota et al., 1992; Busse et al., 1999).
The cellular fatty acid profile of MACL11T was characterized by 15 different fatty acids. The major fatty acids detected in strain MACL11T were C19 : 0 cyclo8c (41·44 %), C18 : 17c (21·66 %), C16 : 0 (12·04 %) and 11-methyl C18 : 17c (8·84 %). Additional fatty acids detected included summed feature 2 (7·68 %; comprising C14 : 0 3-OH, iso-C16 : 1 I, an unidentified fatty acid with an equivalent chain-length of 10·928 and/or C12 : 0 ALDE), C18 : 0 (4·29 %), 10-methyl C19 : 0 (1·08 %), C20 : 26,9c (0·70 %), C18 : 0 3-OH (0·68 %), summed feature 3 (0·63 %; comprising C16 : 17c and/or iso-C15 : 0 2-OH), C16 : 0 3-OH (0·52 %) and an unidentified fatty acid with an equivalent chain-length of 14·959 (0·44 %). According to published data, significant amounts of a C19 : 0 cyclo8c fatty acid are typical of members of the order ‘Rhizobiales’ (Wilkinson, 1988; Moreno et al., 1990; Jarvis et al., 1996; Dunfield et al., 1999; Kämpfer et al., 1999; Tighe et al., 2000). The fatty acid C20 : 36,9,12c, which is detected in all species of the genus Rhizobium, is not present in strain MACL11T. Another difference between strain MACL11T and members of the genus Rhizobium is the presence of 11-methyl C18 : 17c in the former. The fatty acids 11-methyl C18 : 17c and 10-methyl C19 : 0 detected in strain MACL11T are not present in members of the genera Aurantimonas and Fulvimarina. Moreover, strain MACL11T was clearly differentiated from members of the genera Rhizobium, Aurantimonas and Fulvimarina in terms of the proportions of several fatty acids (Tighe et al., 2000; Cho & Giovannoni, 2003; Denner et al., 2003).
DNA for G+C content determination was prepared according to the method of Chun & Goodfellow (1995). The G+C content of the DNA (mol%) was determined using the thermal denaturation method (Mandel & Marmur, 1968): the value for strain MACL11T was found to be 57·4 mol%. This value is similar to those obtained for members of the genera Fulvimarina and Rhizobium.
For 16S rRNA gene sequencing, DNA extraction was carried out as described previously (Rivas et al., 2001). 16S rRNA gene amplification and sequencing were performed according to the methods described previously (Rivas et al., 2003). An almost-complete 16S rRNA gene sequence was obtained and then compared with those deposited in the GenBank database. Sequences were aligned using CLUSTAL X software (Thompson et al., 1997). Distances were calculated according to the methods of Jukes & Cantor (1969), Kimura (1980), Tamura & Nei (1984) and Tajima & Nei (1993). Phylogenetic trees were inferred using the neighbour-joining method (Saitou & Nei, 1987), minimum evolution (Rzhetsky & Nei, 1993) and parsimony analysis (Felsenstein, 1983). Bootstrap analysis was based on 1000 resamplings. The MEGA2 package (Kumar et al., 2001) was used for all analyses.
On the basis of the 16S rRNA gene sequence analysis, the organism under study belongs to the order ‘Rhizobiales’ of the class ‘Alphaproteobacteria’. A comparison against the sequences held in GenBank indicated that the strain was most closely related to the genera Rhizobium (93·3 % similarity to Rhizobium rhizogenes IFO 13257T), Aurantimonas (90·4 % similarity to Aurantimonas coralicida DSM 14790T) and Fulvimarina (90·3 % similarity to Fulvimarina pelagi HTCC2506T). Fig. 2 shows the phylogenetic placement of strain MACL11T within the order ‘Rhizobiales’. The same results were obtained when the phylogenetic distances were calculated using the Jukes–Cantor one-parameter, the Kimura two-parameter, the Tamura-Nei three-parameter and the Tajima-Nei four-parameter methods. The three methods for obtaining phylogenetic trees (using the four above-mentioned methods with each to calculate phylogenetic distances), i.e. neighbour-joining, minimum evolution and parsimony analyses, also afforded the same results (data not shown). As shown in the phylogenetic tree (Fig. 2), strain MACL11T formed a separate branch within the order ‘Rhizobiales’ that was not significantly associated with any of the known families of this order; this relationship was supported by a high bootstrap value (98 %). Thus, phylogenetically, this novel genus probably belongs a novel family within the order ‘Rhizobiales’.
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Description of Martelella gen. nov.
Martelella (Mar.tel'ell.a. N.L. fem. dim. n. Martelella in honour of the French explorer E. Martel, who, in 1896, discovered Lake Martel inside the caves of Drach in Mallorca, the site where this micro-organism was isolated).
Cells are Gram-negative, non-spore-forming short rods. Strictly aerobic. Oxidase- and catalase-positive. Phylogenetically related to members of the order ‘Rhizobiales’. The only respiratory lipoquinones present are ubiquinones, Q-10 predominating. The most abundant fatty acids are C19 : 0 cyclo8c, C18 : 17c, C16 : 0 and 11-methyl C18 : 17c. The other minor fatty acids are C18 : 0, 10-methyl C19 : 0, C20 : 26,9c, C18 : 0 3-OH and C16 : 0 3-OH. The DNA G+C content of the type species is 57·4 mol%. The type species of the genus is Martelella mediterranea.
Description of Martelella mediterranea sp. nov.
Martelella mediterranea [me.di.ter.ra.ne'a. L. fem. adj. mediterranea inland (used to refer to the Mediterranean Sea), referring to the fact that the type strain was isolated from a Mediterranean island].
Displays the following properties in addition to those given in the genus description. Cells are non-motile coccobacillary rods, 1·0–1·1x1·3–1·5 µm in size. Colonies on YED medium supplemented with 5 % (w/v) NaCl are circular, smooth, opaque, white- to cream-coloured pigmented colonies that are usually 1–3 mm in diameter within 5 days at 28 °C. Grows in the presence of NaCl concentrations up to 5 % (w/v), although salt is not essential for growth. The temperature range for growth is 4–37 °C (optimal growth occurs at 28 °C) and the pH range for growth is 5–8·5 (optimal growth occurs at pH 7). Urease-positive. Indole is not produced. Aesculin is hydrolysed. Positive in the Voges–Proskauer reaction and for nitrate reduction. The type strain utilizes gluconate, glucose, malate, maltose and mannitol as sole carbon sources, and does not grow in adipate, amygdalin, arabinose, caproate, citrate, inositol, mannose, melibiose, N-acetylglucosamine, phenylacetate, rhamnose, sorbitol or sucrose. The type strain actively produces 
-galactosidase, alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, 
-galactosidase, -glucuronidase, -gluconidase, -gluconidase and N-acetyl--glucosaminidase. It does not produce gelatinase, caseinase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, chymotrypsin, naphthol-AS-BI-phosphohydrolase, -mannosidase, -fucosidase or H2S.
The type strain is MACL11T (=LMG 22193T=CECT 5861T), isolated from a water sample from a subterranean saline lake on Mallorca, Spain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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