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1 Departamento de Microbiología y Genética, Universidad de Salamanca, Spain
2 Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie en Microbiologie, Universiteit Gent KL, Ledeganckstraat 35, B-9000 Gent, Belgium
3 Instituto de Recursos Naturales y Agrobiología, IRNASA-CSIC, Salamanca, Spain
Correspondence
Raúl Rivas
raul{at}wwwedu-micro.usal.es
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8c and C16 : 0. The fatty acid C16 : 18c, present in strain MACL04T, was not detected in the recognized Alcanivorax species. The sequences of the large and short 16S–23S intergenic spacer regions showed similarities of 97.2 and 98.8 % (ungapped) with respect to A. dieselolei B-5T. Partial sequences of gyrB and alkb genes showed 94.0 % similarity between strain MACL04T and A. dieselolei B-5T. The G+C content of strain MACL04T was 62.8 mol%. The data from this polyphasic study indicate that strain MACL04T represents a novel species of the genus Alcanivorax, for which the name Alcanivorax balearicus sp. nov. is proposed. The type strain is MACL04T (=LMG 22508T=CECT 5683T).
The GenBank/EMBL/DDBJ accession numbers for strain MACL04T are AY686709 (16S rRNA gene), EF091805 (small ITS), EF091804 (large ITS), AY683535 (partial alkB gene) and EF091802 (partial gyrB gene).
Figures showing the colony morphologies of strain MACL04T and Alcanivorax dieselolei B-5T grown on media containing Tween 20, an electron micrograph of a cell of strain MACL04T grown on nutrient agar, and TP-RAPD profiles and phylogenetic trees based on 16S–23S ITS and partial alkB and gyrB gene sequences and a table detailing the fatty acid profiles of strain MACL04T and recognized species of the genus Alcanivorax are available as supplementary material with the online version of this paper.
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; Bruns & Berthe-Corti, 1999; Fernández-Martínez et al., 2003; Liu & Shao, 2005). Here we characterize a strain, designated MACL04T, which was isolated from water collected from Lake Martel, a subterranean saline lake in Mallorca (Balearic Islands), Spain. Strain MACL04T is phylogenetically close to A. dieselolei B-5T but, based on its phenotypic, chemotaxonomic and molecular characteristics, we propose that it should be considered to represent a novel species within the genus Alcanivorax. Strain MACL04T was isolated under aseptic conditions from water samples taken at a depth of 10 cm, during a bacterial biodiversity study of Lake Martel. Under sterile conditions, a 200 ml sample was filtered under vacuum through a membrane filter with a pore diameter of 45 µm (Millipore). The membrane was placed on a plate containing YED medium (0.5 % yeast extract, 0.7 % glucose and 2 % agar, w/v) supplemented with 1.5 % (w/v) NaCl and incubated at 28 °C.
Cells of strain MACL04T formed white–cream-coloured colonies on nutrient agar medium supplemented with 1.5 % NaCl. Optimum growth was observed on TPA medium (1 % v/v Tween 20, 1 % w/v peptone, 0.5 % w/v NaCl, 0.01 % w/v CaCl2 and 1.5 % w/v agar). On this medium, colonies of strain MACL04T were opaque and showed blue–green iridescence, whereas colonies of A. dieselolei B-5T were transparent and glistening, without coloured iridescence (see Supplementary Fig. S1 available in IJSEM Online). Gram staining was carried out by using the procedure described by Doetsch (1981). Cells were gently suspended in sterile water, stained with 0.2 % uranyl acetate and examined at 80 kV with a Zeiss EM 209 transmission electron microscope (Peix et al., 2003). Cells of strain MACL04T were Gram-negative, short rod-shaped (1.1–1.3x0.6–0.8 µm) and motile by means of a polar or subpolar flagellum (see Supplementary Fig. S2 available in IJSEM Online), whereas cells of A. dieselolei showed several lophotrichous flagella (see Table 1). The generation times of strain MACL04T and A. dieselolei B-5T were compared using turbidimetry in liquid TPA medium (Koch, 1981). The results showed that A. dieselolei B-5T had a generation time of approximately 5 h, whereas the generation time for strain MACL04T was approximately 2.5 h.
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). Amplification of the gene and sequencing were performed according to methods described previously (Rivas et al., 2002b). An almost complete 16S rRNA gene sequence was obtained and compared with sequences deposited in databases. The sequences were aligned using CLUSTAL_X software (Thompson et al., 1997) and distances were calculated according to the Kimura two-parameter method (Kimura, 1980). Phylogenetic trees were inferred using the neighbour-joining method (Saitou & Nei, 1987) and bootstrap analysis was performed, based on 1000 resamplings. The MEGA2 package (Kumar et al., 2001) was used for all analyses. Comparison with sequences held in GenBank indicated that strain MACL04T was very closely related to A. dieselolei (Figure 1). The 16S rRNA gene sequence of strain MACL04T showed nearly 100 % similarity to that of A. dieselolei B-5T. Despite this fact, taking into account the morphological and growth behaviour differences between strain MACL04T and A. dieselolei B-5T, we performed a polyphasic study to establish the taxonomic status of strain MACL04T.
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), using the primer pair 879F (5'-GCCTGGGGAGTACGGCCGCA-3') and 1522R (5'-AAGGAGGTGATCCANCCRCA-3'), which correspond to Escherichia coli positions 879–898 and 1509–1522, respectively. According to our previous results, strains showing the same pattern belong to the same species and different species display different TP-RAPD patterns (Rivas et al., 2001, 2004). Moreover, from results obtained previously, TP-RAPD patterns are not strain dependent (Rivas et al., 2001) and in some cases the profiles can be slightly different in subspecies of the same species (Rivas et al., 2002a). Therefore, we analysed the TP-RAPD pattern of strain MACL04T and compared it with those of the type strains of recognized species of the genus Alcanivorax (see Supplementary Fig. S3 available in IJSEM Online). The TP-RAPD profiles of all these strains were different and the differences observed between A. dieselolei B-5T and strain MACL04T indicated that they could belong to different species.
The 16S–23S intergenic spacer (ITS) region was amplified and sequenced as described by Liu & Shao (2005). PCR amplification of the ITS region yielded two products, as occurs for all Alcanivorax species. Phylogenetic analysis based on the ITS sequences showed similar groupings to those obtained using the 16S rRNA gene and confirmed the close phylogenetic relationship between strain MACL04T and A. dieselolei (see Supplementary Fig. S4 available in IJSEM Online). The sequences of the large and short ITS regions showed 97.2 and 98.8 % similarities (ungapped) with respect to A. dieselolei B-5T. As the genus Alcanivorax currently comprises only four species that are well separated phylogenetically, we could not come to any conclusions about the similarity among strains from different species. Nevertheless, the similarity values between strain MACL04T and A. dieselolei B-5T were similar to those found in different species of Pseudomonas that are phylogenetically closely related on the basis of 16S rRNA gene sequences. For example, the ITS regions of Pseudomonas graminis and Pseudomonas lutea (GenBank accession nos DQ023307 and EF091800, respectively) exhibit nearly 99 % similarity.
A fragment of the alkB gene was amplified and sequenced using the primers ALKBF (5'-TCAATACMGGDCAYGAG-3') and ALKBR (5'-TAGTTSACSAYCTCCAGC-3'), which were designed in this study based on the conserved regions of the codifying genes for alkane hydrolase sequences from Alcanivorax species. The PCR conditions used were: pre-heating at 95 °C for 9 min; 35 cycles of denaturation at 95 °C for 1 min, annealing at 50 °C for 1 min and extension at 72 °C for 1 min, and a final extension at 72 °C for 7 min. Strain MACL04T showed 94 % similarity with respect to A. dieselolei B-5T. Phylogenetic analysis based on the alkb gene yielded results similar to those obtained in the 16S and ITS analyses, showing that strain MACL04T and A. dieselolei B-5T form a group together with A. venustensis that is phylogenetically separate from the other species of the genus Alcanivorax (see Supplementary Fig. S5 available in IJSEM Online).
Partial sequences of the gyrB gene were obtained for strain MACL04T and A. dieselolei B-5T, by using primers designed on the basis of conserved sequences of a hypervariable zone of this gene in Alcanivorax and other phylogenetically related species sequenced previously by Okamoto et al. (2004): GYRBUNIVF (5'-GGBCTBCAYGGBGTRGG-3') and GYRBUNIVR (5'-CCCGCWGARTCACCCTCC-3'). The PCR conditions used were the same as those for the amplification of the alkb gene described above, except that the annealing temperature used was 52 °C. Strain MACL04T showed 94 % similarity with respect to strain A. dieselolei B-5T. Despite the fact that this value was higher than those obtained among other species of Alcanivorax, they are distant from a phylogenetic point of view and, as was observed in the case of the ITS region, similarity limits among the closely related species of this genus could not be established (see Supplementary Fig. S6 available in IJSEM Online). However, in the related genus Pseudomonas, similarity values greater than 95 % for gyrB have been reported for several species, such as Pseudomonas kilonensis, Pseudomonas brassicacearum and Pseudomonas thivervalensis (Cladera et al., 2006).
Analysis of the fatty acid composition was performed as described by Zimmermann et al. (1998) using the culture conditions recommended by Yakimov et al. (1998). This analysis was performed at the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) as was also the case for A. dieselolei. The cellular fatty acid pattern of strain MACL04T comprised 17 different fatty acids (see Supplementary Table S1 available in IJSEM Online). The major fatty acid of strain MACL04T was C19 : 0 cyclo 8c (29.33 %). The fatty acids C16 : 0 (24.40 %) and C18 : 17c (14.68 %) were also present in high amounts. Other fatty acids were present in smaller amounts, including C16 : 17c (7.90 %), C12 : 0 3-OH (5.84 %), C12 : 0 (5.46 %), C10 : 0 (2.56 %), C16 : 18c (2.10 %), C17 : 0 (1.49 %), C17 : 0 cyclo (1.45 %) and C12 : 0 2-OH (1.38 %). Fatty acids C11 : 0, C18 : 0, 11-methyl C18 : 17c, C20 : 26,9c, an unidentified fatty acid with an equivalent chain-length value of 12.484 and an unidentified fatty acid with an equivalent chain-length value of 11.799 were present in amounts of less than 1 %. This profile has several differences with respect to that of A. dieselolei (Liu & Shao, 2005). The major fatty acid of strain MACL04T, C19 : 0 cyclo 8c, was not the main fatty acid in A. dieselolei. Such a large amount of C19 : 0 cyclo 8c has not been reported previously for Alcanivorax species. The fatty acid C18 : 17c was one of the two major fatty acids in A. dieselolei, but not in strain MACL04T. The fatty acid C16 : 18c present in strain MACL04T was not detected in A. dieselolei or in the other Alcanivorax species. Quantitative differences were detected for C16 : 0, C16 : 17c and C12 : 0 3-OH between strain MACL04T and A. dieselolei B-5T.
DNA for base composition analysis was prepared according to Chun & Goodfellow (1995). The G+C content of the DNA was determined using the thermal denaturation method (Mandel & Marmur, 1968). The DNA G+C content of strain MACL04T was 62.8 mol%. DNA–DNA hybridization was carried out using the method of Ezaki et al. (1989), following the recommendations of Willems et al. (2001). The results obtained showed 67 % (±1 %, SD of three determinations) DNA–DNA relatedness between strain MACL04T and A. dieselolei B-5T.
The phenotypic characterization was performed as described by Yakimov et al. (1998), Bruns & Berthe-Corti (1999) and Liu & Shao (2005). In all the tests, A. dieselolei B-5T was used as a reference. Utilization of carbon sources was tested using SM1 medium (Yakimov et al., 1998), supplemented with 1.5 % (w/v) NaCl and 1 % (w/v) of the various carbon sources (Table 1
). The pH indicator Bromothymol blue (0.05 %, w/v) was included in the media. Assimilation of n-alkanes (decane, tetradecane, hexadecane and eicosane) was tested as described by Bruns & Berthe-Corti (1999). The physiological and biochemical characterizations were completed using API 20NE strips (bioMérieux), according to the manufacturer's instructions. The medium supplied with the API 20NE test strips was supplemented with 1.5 % (w/v) NaCl before use. The temperature range for growth was determined by incubating cultures in YED medium supplemented with 1.5 % (w/v) NaCl at 4–45 °C. The pH range was determined using YED medium, with final pH values of 4.0–10.0. Salt tolerance was studied using nutrient agar medium (Difco, Becton-Dickinson) containing 0–20 % (w/v) NaCl. Antibiotic susceptibility tests were performed as described by Liu & Shao (2005).
The phenotypic characteristics of strain MACL04T are given in Table 1. Unlike other species of the genus Alcanivorax, strain MACL04T did not require NaCl for growth. Strain MACL04T was unable to grow at 45 °C, whereas A. dieselolei can grow at this temperature. Utilization of glucose and L-arabinose by strain MACL04T was weakly positive, whereas A. dieselolei was unable to use carbohydrates as was reported previously by Liu & Shao (2005). Hydrolysis of Tween 20 was negative after 4 days incubation for strain MACL04T, whereas at this time A. dieselolei B-5T produced abundant lipases (tweenases). Strain MACL04T differed in the use of acetate, lactate and propionate as sole carbon sources. Use of pyruvate was positive in both strain MACL04T and A. dieselolei B-5T; however, acid pH was produced by strain MACL04T, whereas alkaline pH was produced by A. dieselolei B-5T.
The phenotypic differences together with differences observed in the chemotaxonomic and molecular analyses support the classification of strain MACL04T as representing a novel species of the genus Alcanivorax, for which the name Alcanivorax balearicus sp. nov. is proposed.
Description of Alcanivorax balearicus sp. nov.
Alcanivorax balearicus (ba.le.a'ri.cus. L. masc. adj. balearicus of the Balearic Islands, where the organism was isolated).
Gram-negative, aerobic, rod-shaped cells. Motile by means of a polar or subpolar flagellum. Colonies on nutrient agar supplemented with 1.5 % (w/v) NaCl are circular, smooth and white–cream-coloured; colonies are opaque and mucoid with blue–green iridescence on media containing Tween 20, and usually 2–4 mm in diameter within 5 days at 28 °C. Oxidase- and catalase-positive. Urease-negative. Phylogenetically related to members of the family Alcanivoracaceae. Halotolerant; ionic supplements are not required for growth. Major fatty acids are C19 : 0 cyclo 8c, C16 : 0 and C18 : 17c; fatty acids C16 : 17c, C12 : 0 3-OH, C12 : 0, C10 : 0, C16 : 18c, C17 : 0, C17 : 0 cyclo, C12 : 0 2-OH, C18 : 0, 11-methyl C18 : 17c, C20 : 26,9c and C11 : 0 are present in smaller amounts. Growth occurs in NaCl at concentrations up to 15 % (w/v), although NaCl is not required for growth. Temperature range for growth is 4–37 °C (optimal growth occurs at 28 °C). pH range for growth is 5.5–9 (optimal growth occurs at pH 7). Negative for production of lipase in media containing Tween 20 after 4 days incubation at 28 °C; weak and slow production is detected after incubation for 1 week. Aesculin, casein, gelatin and starch are not hydrolysed and arginine dihydrolase and 
-galactosidase activities and indole production and nitrate reduction are negative. Can grow using glucose and other carbohydrates as a carbon source. Decane, tetradecane, hexadecane, eicosane, adipate, caproate, citrate, malate, mannitol, phenylacetate, glutarate, quinate and pyruvate can be used as sole carbon sources. Weak growth occurs with acetate, propionate, glucuronate and L-arabinose. Does not grow with lactate, N-acetylglucosamine, gentiobiose, maltose, D-mannose, xylose or sucrose. Sensitive to neomycin and polymyxin B and resistant to cefuroxime, ampicillin, penicillin, erythromycin and ciprofloxacin. The DNA G+C content of the type strain is 62.8 mol%.
The type strain, MACL04T (=LMG 22508T=CECT 5683T), was isolated from water from Lake Martel, a subterranean saline lake in the Balearic Islands, Spain.
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ACKNOWLEDGEMENTS |
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