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Microbial Exopolysaccharide Research Group, Department of Microbiology, Faculty of Pharmacy, Campus Universitario de Cartuja s/n, University of Granada, 18071 Granada, Spain
Correspondence
Emilia Quesada
equesada{at}ugr.es
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ABSTRACT |
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7c, 16 : 0, 16 : 17c/15 : 0 iso 2-OH, 12 : 0 3-OH, 12 : 0, 11-methyl 18 : 17c and 10 : 0. The proposed name is Halomonas almeriensis sp. nov., with strain M8T (=CECT 7050T=LMG 22904T) as the type strain.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain M8T is AY858696.
A dendrogram based on the simple-matching coefficient and UPGMA method, a neighbour-joining tree showing the phylogenetic relationships between H. almeriensis and other Halomonas species and taxa of Gram-negative halophilic bacteria, and a transmission electron micrograph showing the morphology of strain M8T are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Mata et al., 2002; Ventosa et al., 1998; Vreeland et al., 1980). Taxonomically Halomonas is a heterogeneous bacterial group. On the basis of 16S and 23S rRNA gene sequences, Arahal et al. (2002) have established three clearly distinguishable phylogenetic groups, in addition to which another three groups can also be identified by phenotypic studies, according to their capacity to produce acids from glucose and their use of a variety of compounds as sole sources of carbon and energy (Mata et al., 2002). Some of the Halomonas species, including Halomonas eurihalina, Halomonas maura, Halomonas ventosae and Halomonas anticariensis, which were isolated and characterized by our research group (Quesada et al., 1990; Bouchotroch et al., 2001; Martínez-Cánovas et al., 2004a, b), produce extracellular polysaccharides with potential biotechnological applications (Calvo et al., 2002; Béjar et al., 1998; Martínez-Checa et al., 2002; Arias et al., 2003; Quesada et al., 2004). We describe here a novel exopolysaccharide-producing species belonging to the genus Halomonas, with the proposed name Halomonas almeriensis.
Strain M8T was isolated from a water sample taken from a saltern in the Cabo de Gata-Níjar wildlife reserve in the province of Almería in south-east Spain during a wide range of samplings made by our research group in 18 hypersaline habitats in Spain and Morocco (Martínez-Cánovas et al., 2004c). It was routinely kept and grown at 32 °C in MY medium (Moraine & Rogovin, 1966) with 7·5 % w/v marine salts (Rodríguez-Valera et al., 1981).
Phenotypic characterization, on the basis of 112 tests, was done as described by Mata et al. (2002). We compared the novel strain with Halomonas species using the software TAXAN (Information Resources Group, Maryland Biotechnology Institute, University of Maryland, College Park, USA) based on numerical analysis. The dendrogram obtained by the simple-matching coefficient (SSM) (Sokal & Michener, 1958) and UPGMA method (Sneath & Sokal, 1973) (see Supplementary Fig. S1 in IJSEM Online) shows that strain M8T was related to the non-acid-producing group of Halomonas species (Mata et al., 2002), although it shares less than 63 % similarity with them. This low similarity can be put down to the fact that strain M8T is extremely fastidious nutritionally. The main phenotypic differences between H. almeriensis (M8T) and its nearest phylogenetically related strains of the genus Halomonas are shown in Table 1.
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and Ferragut & Leclerc (1976). The G+C content of the DNA of the novel strain was 63·5 mol%, within the range proposed for Halomonas species of 52–68 mol% (Franzmann et al., 1988).
A partial fragment of the 16S rRNA gene was amplified by PCR using the protocol of Saiki et al. (1988). The forward primer, 16F27 (5'-AGAGTTTGATCATGGCTCAG-3'), annealed at positions 8–27 and the reverse primer, 16R1488 (5'-CGGTTACCTTGTTAGGACTTCACC-3') (both from Pharmacia), annealed at the complement of positions 1511–1488 (Escherichia coli numbering according to Brosius et al., 1978). To complete the sequence we designed an internal primer, 5'-GAGGATGATCAGCCACACTG-3', which annealed at positions 401–421. The PCR product was purified using the GFX PCR DNA and Gel Band purification kit (Amersham Biosciences). Direct sequence determinations of PCR-amplified DNAs were made with an ABI PRISM dye-terminator, cycle-sequencing, ready-reaction kit (Perkin-Elmer) and an ABI PRISM 377 sequencer (Perkin-Elmer) according to the manufacturer's instructions. The sequence obtained (1459 bp) was compared with 16S rRNA reference gene sequences retrieved from the GenBank and EMBL databases by BLAST search. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 3.0 (Kumar et al., 2004) after multiple alignment of the data by CLUSTAL_X (Thompson et al., 1997). Distances and clustering were determined using the neighbour-joining and maximum-parsimony algorithms, and a bootstrap analysis (1000 replications) was made to determine the stability of the clusters. The neighbour-joining tree is available as Supplementary Fig. S2 in IJSEM Online. A similar result (not shown) was obtained using the maximum-parsimony algorithm. The taxa included in the tree shown in Fig. 1 represent only the nearest neighbours. Our analyses confirmed that the novel strain belongs to the genus Halomonas, is located within group 1 of Halomonas species described by Arahal et al. (2002) and shares 95·8 % 16S rRNA gene sequence similarity with Halomonas halmophila (Dobson et al., 1990). The 16S rRNA gene fragment analysed contains the 15 signature nucleotides defined for the family Halomonadaceae (Dobson & Franzmann, 1996).
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with the modifications introduced by Ziemke et al. (1998) and Bouchotroch et al. (2001). The result shows the low hybridization (10·1 %) with H. halmophila, which was chosen on the basis of our phylogenetic study as being the most closely related Halomonas species.
The fatty acids were analysed at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) by high-resolution GLC using a moist pellet of the cells obtained from a culture in MY medium supplemented with 7·5 % w/v sea-salt mixture. Strain M8T shows a combination of fatty acids found in other species of Halomonas (Dobson & Franzmann, 1996) (see species description), although it also contains a relatively high proportion of 10 : 0 (2·11 %), 12 : 0 (1·22 %) and 11-methyl 18 : 17c (2·75 %).
A transmission electron micrograph showing the morphology and cell size of strain M8T and the presence of an extracellular polymer that is released into the external medium is available as Supplementary Fig. S3 in IJSEM Online. The TEM method used is fully described by Bouchotroch et al. (2001).
On the basis of phylogeny, DNA–DNA hybridization, fatty acid composition and phenotypic differences between the novel and previously described species within the genus Halomonas, we consider that strain M8T represents a novel species, for which we propose the name Halomonas almeriensis sp. nov.
Description of Halomonas almeriensis sp. nov.
Halomonas almeriensis (al.me.ri.en'sis. N.L. fem. adj. almeriensis denizen of the province of Almería in south-east Spain, where the strain was isolated).
Cells are Gram-negative, non-motile rods, 2–2·5x0·75 µm, occurring singly or in pairs. They accumulate poly-
-hydroxyalkanoates and produce exopolysaccharide. Colonies are round, convex, creamy-white and mucoid. Their growth pattern is uniform in a liquid medium. It is moderately halophilic, capable of growth in salt concentrations (mixture of sea salts) of 5–25 % w/v. It grows at 15–37 °C and pH 6–10. It is chemo-organotrophic. Its metabolism is respiratory with oxygen as the terminal electron acceptor. The cells do not grow anaerobically in the presence of nitrate, nitrite or fumarate. Catalase is produced but not oxidase. It does not produce acids from sugars. Indole, methyl red and Voges–Proskauer are negative. It does not hydrolyse starch, aesculin, gelatin, casein, Tween 20, Tween 80, DNA or tyrosine. It produces phosphatase and grows on MacConkey agar, but does not produce phenylalanine deaminase, urease, ONPG or lecithinase. Gluconate is oxidized. It does not produce pigment from tyrosine, H2S from L-cysteine, grow on cetrimide agar or lyse blood. D-Gluconate is acceptable as a sole carbon energy source, whereas aesculin, L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannose, D-melezitose, salicin, starch, D-trehalose, D-xylose, acetate, citrate, formate, fumarate, malonate, propionate, succinate, adonitol, ethanol, glycerol, myo-inositol, D-mannitol and sorbitol are not. L-alanine and L-serine are used as sole sources of carbon, nitrogen and energy, whereas L-histidine, DL-isoleucine, L-lysine, L-methionine and L-valine are not. It is susceptible to amoxicillin (25 µg), ampicillin (10 µg), carbenicillin (100 µg), cefotaxime (30 µg), cefoxitin (30 µg), chloramphenicol (30 µg), erythromycin (15 µg), kanamycin (30 µg), nitrofurantoin (300 µg), rifampicin (30 µg), streptomycin (10 µg), tobramycin (10 µg) and trimethoprim/sulfamethoxazole (1·25/23·75 µg). It is resistant to nalidixic acid (30 µg), polymyxin B (300 IU) and sulphamide (250 µg). Principal fatty acids (greater than 1 %) are 18 : 17c (50·66 %); 11-methyl 18 : 17c (2·75 %); 16 : 0 (21·08 %); 16 : 17c/15 : 0 iso 2-OH (14·16 %); 12 : 0 3-OH (5·64 %); 12 : 0 (1·22 %) and 10 : 0 (2·11 %). The DNA G+C content of the type strain is 63·5 mol% (Tm method).
The type strain, M8T (=CECT 7050T=LMG 22904T), was isolated from a hypersaline water sample taken from a saltern at Cabo de Gata (Almería, south-east Spain).
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ACKNOWLEDGEMENTS |
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