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1 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain
2 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100080 Beijing, People's Republic of China
3 Department of Biotechnology, University of the Western Cape, Bellville 7535, Cape Town, South Africa
4 Genencor International BV, Archimedesweg 30, 2333 CN Leiden, The Netherlands
5 Department of Infection, Immunity and Inflammation, University of Leicester, Leicester LE1 9HN, UK
Correspondence
M. C. Márquez
cmarquez{at}us.es
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ABSTRACT |
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7c, C16 : 0 and C12 : 0, and its polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, a phosphoglycolipid and six unidentified phospholipids. On the basis of the polyphasic evidence from this study, strains CG12T and CG13 represent a novel genus and species, for which the name Aquisalimonas asiatica gen. nov., sp. nov. is proposed. The type strain of Aquisalimonas asiatica is CG12T (=CCM 7368T=CECT 7151T=CGMCC 1.6291T=DSM 18102T).
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). In nature, salinity often goes together with alkalinity. Alkaline, saline lakes represent unique habitats with high pH and variable (up to saturation) salt concentrations. In September 2003, two novel, alkalitolerant, moderately halophilic, Gram-negative bacteria (CG12T and CG13) were isolated from water samples taken during an expedition to Lake Chagannor, an alkaline, saline lake in Inner Mongolia, China; these strains are described here.
Lake Chagannor is located at 43° 21' N 113° 08' E; the temperature of the water was 17 °C, the pH 9.5 and the conductivity 21.3 mS cm–1. During studies on the diversity of moderately halophilic bacteria from Lake Chagannor, water samples were diluted with sterile salt solution, plated on alkaline saline media and incubated aerobically at 37 °C; strains CG12T and CG13 were isolated. The isolation medium contained the following (g l–1): glucose, 10.0; peptone (Difco) 5.0; yeast extract (Difco), 5.0; KH2PO4, 1.0; MgSO4.7H2O, 0.2; NaCl, 80; Na2CO3, 20. NaCl and Na2CO3 were autoclaved separately and added to the medium prior to incubation (Duckworth et al., 1996). Solid media contained 2.0 % (w/v) agar; the strains were maintained on this solid medium.
To characterize these isolates phenotypically, standard tests were performed. The Gram-stain reaction was carried out using the method described by Dussault (1955). For the determination of cellular morphology and motility, a culture from exponentially grown liquid medium was examined by phase-contrast light microscopy. The morphology of the colonies, their pigmentation and their size were observed on the maintenance medium (with different salt concentrations) after 4 days incubation. Growth at different concentrations of salts was determined on the isolation medium, supplemented with 0, 0.5, 1, 3, 5, 7, 10, 15, 20, 25 or 30 % (w/v) total salts. The pH range for growth was determined on the isolation medium at pH values ranging from 5.0 to 11, achieved using the appropriate biological buffers (Na2HPO4/NaH2PO4 below pH 8.0, Na2CO3/NaHCO3 for pH 8.0–10.0 and Na2HPO4/NaOH for pH 11.0), as described previously (Gomori, 1955). The pH was readjusted after sterilization and growth was scored using optical density at 600 nm. The temperature range for growth was determined by testing temperatures from 15 to 55 °C. Catalase activity was assayed by adding 3 % H2O2 to culture plates. The oxidase reaction was performed on filter paper moistened with a 1 % (w/v) aqueous solution of N,N,N',N'-tetramethyl-p-phenylenediamine. Other tests (shown in Table 1 or included in the species description) were carried out using methods described previously (Ventosa et al., 1982; Quesada et al., 1984; García et al., 1987). Unless otherwise indicated, the testing was carried out using maintenance medium and incubation at 37 °C in sealed containers.
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Strains CG12T and CG13 were found to be Gram-negative, motile and strictly aerobic. The cells were rod-shaped, 0.7–0.9 µm wide and 2.0–10.0 µm in length. No endospore production was observed. When grown for 2 days at 37 °C on maintenance medium, the colonies were non-pigmented, circular with entire margins and 1 mm in diameter. Liquid nutrient broth also supported growth, although the cells had a strong tendency to clump as the cultures aged. The two isolates were moderately halophilic and alkalitolerant, growing in media containing 1–20 % (w/v) salts and optimally in media containing 7–10 % (w/v) salts. No growth was observed in the absence of NaCl. The isolates grew at pH 6.0–10.8 and optimally in media at pH 7.5–8.5. The strains were able to grow anaerobically with nitrate but could not grow without nitrate. Other phenotypic features are included in the species description.
Genomic DNA from strains CG12T and CG13 was obtained using the method described by Marmur (1961). The 16S rRNA gene of each strain was PCR-amplified using forward primer 16F27 and reverse primer 16R1488 (Mellado et al., 1995). Direct sequence determination of the PCR amplicons was carried out using an automated DNA sequencer (model 3100; Applied Biosystems). The 16S rRNA gene sequence analyses were performed using the ARB software package (Ludwig et al., 2004). The 16S rRNA gene sequences were aligned and the alignment confirmed and checked against both primary and secondary structures of the 16S rRNA molecule using the alignment tool of the ARB software package. Phylogenetic trees were constructed using three different methods, namely the maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Fitch, 1971) and neighbour-joining (Saitou & Nei, 1987) algorithms integrated in the ARB software for phylogenetic inference. The 16S rRNA gene sequences used for the phylogenetic comparisons were obtained from the GenBank database: their strain designations and accession numbers are shown on the maximum-parsimony phylogenetic tree presented in Fig. 1.
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). The application of maximum-likelihood, maximum-parsimony and distance methods resulted in highly similar tree topologies, so only the maximum-parsimony results are shown. The 16S rRNA gene sequences of strains CG12T and CG13 share 99.9 % similarity and are on the same phylogenetic branch, but they emerge as a separate entity with respect to the other members of the family Ectothiorhodospiraceae. The nearest known relatives of these strains are Alkalispirillum mobile DSM 12769T (94.4–94.6 % 16S rRNA gene sequence similarity) and Alkalilimnicola halodurans DSM 13718T (93.2–93.4 % similarity). No other known bacterium shared more than 93 % 16S rRNA gene sequence similarity with the two isolates. On the basis of sequence divergence, it was evident that the novel isolates could not be assigned to any of the described genera in this cluster.
The genotypic relatedness between the two isolates was determined by using DNA–DNA hybridization. These studies were carried out by following the competition procedure of Johnson (1994). Competitor DNAs were sonicated (Braun Melsungen) at 50 W for two periods of 15 s. Membrane filters 0.5 cm in diameter (HAHY; Millipore) and containing reference DNA (25 µg cm–2), were placed in 5 ml screw-capped vials (Greiner) containing labelled, sheared, denatured DNA and denatured, sheared competitor DNA. The ratio of the concentration of the competitor DNA to that of the labelled DNA was at least 150 : 1. The final volume and concentration were adjusted to 140 µl, 2x SSC and 30 % formamide. The optimal hybridization temperatures were 57.8 and 58.0 °C, which is within the limits of validity for the filter method (De Ley & Tijtgat, 1970). The vials were shaken gently for 18 h in a water bath (Grant Instruments); these procedures were done in triplicate. After hybridization, the filters were washed in 2x SSC at the optimal renaturation temperatures given above. The radioactivity bound to the filters was measured in a liquid scintillation counter (Beckman Instruments) and the percentage relatedness was calculated according to Johnson (1994). At least two independent determinations were carried out for each experiment and the reported results are the mean values. The percentages of DNA–DNA hybridization between strains CG12T and CG13 were 100 and 99 % (the latter being for the reverse hybridization). These data clearly support the conclusion that the two strains can be considered as representing members of the same species (Stackebrandt et al., 2002).
The G+C contents of the genomic DNAs were determined from the midpoint value (Tm) of each of the thermal denaturation profiles (Marmur & Doty, 1962) by using the equation of Owen & Hill (1979). The G+C contents of the genomic DNAs of strains CG12T and CG13 were 63.6 and 64.0 mol%, respectively.
For chemotaxonomic characterization, strain CG12T was selected as a representative of the two isolates. Fatty acid analysis was performed using standard methods and compared with the fatty acids in the database of the MIDI Sherlock Microbial Identification system (Microbial ID). The cells were cultured in maintenance medium at 37 °C for 24 h. The major fatty acids of strain CG12T were found to be C18 : 17c (38 %), C16 : 0 (17 %) and C12 : 0 (15 %), being different in composition from those described for Alkalispirillum mobile DSM 12769T and Alkalilimnicola halodurans DSM 13718T (the closest species on the basis of the 16S rRNA gene sequence comparison) (Rijkenberg et al., 2001; Yakimov et al., 2001). To complete the chemotaxonomic characterization of strain CG-12T and Alkalispirillum mobile DSM 12769T, analysis of polar lipids of these two strains was carried out by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). For strain CG12T, 11 polar lipids were detected, namely phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, a phosphoglycolipid and six different unknown phospholipids. In the case of Alkalispirillum mobile DSM 12769T, the polar lipids identified were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, a single phospholipid and an aminolipid (Fig. 2). These two polar lipid profiles are clearly different. In addition, neither has a lipid composition similar to that described for Alkalilimnicola halodurans DSM 13718T, which is dominated by phosphatidylglycerols and cardiolipins, although some phosphatidylethanolamines and phosphatidylcholines are also present (Yakimov et al., 2001).
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). For example, strain CG12T is moderately halophilic, growing in media containing 1–20 % (w/v) salts and exhibiting optimal growth in media containing 7–10 % (w/v) salts, whereas Alkalispirillum mobile DSM 12769T and Alkalilimnicola halodurans DSM 13718T are able to grow in media without NaCl. Both of the latter species can be considered as extremely halotolerant, although Alkalispirillum mobile has been described as a moderate halophile (Rijkenberg et al., 2001). Strain CG12T is alkalitolerant, growing in media with pH values between 6.0 and 10.8 and optimally at pH 7.5–8.5. However, Alkalispirillum mobile DSM 12769T and Alkalilimnicola halodurans DSM 13718T are obligate alkaliphiles, showing optimal growth above pH 9.0 and 8.5, respectively. In addition, the genomic DNA G+C content of strain CG12T is lower than those of Alkalispirillum mobile DSM 12769T and Alkalilimnicola halodurans DSM 13718T (66.2 and 65.6 mol%, respectively) (Rijkenberg et al., 2001; Yakimov et al., 2001). Finally, significant differences between the cellular fatty acid and polar lipid compositions of strain CG12T and those of the type strains of the genera Alkalispirillum and Alkalilimnicola also support the separation of the novel isolates into a novel taxon. As strains CG12T and CG13 cannot be assigned to any currently known genus of the family Ectothiorhodospiraceae, we suggest that they represent a novel genus and species, for which the name Aquisalimonas asiatica gen. nov., sp. nov. is proposed.
Description of Aquisalimonas gen. nov.
Aquisalimonas (A.qui.sa.li.mo'nas. L. n. aqua water; L. n. sal, salis salt; L. fem. n. monas a unit, a monad; N.L. fem. n. Aquisalimonas a bacterium living in salted water).
Gram-negative, rod-shaped cells occurring singly, in pairs or in long chains. Motile. Non-spore-forming. Catalase- and oxidase-positive. Moderately halophilic; does not grow in media without salt. Alkalitolerant. Nitrate, but not nitrite, is reduced. Cellular fatty acids mainly consist of C18 : 17c, C16 : 0 and C12 : 0. The genus is a member of the class Gammaproteobacteria within the family Ectothiorhodospiraceae. The G+C content of the genomic DNA of the known strains of the type species are 63.6 and 64.0 mol% (Tm). Only one species, Aquisalimonas asiatica, is described so far, and this species has been designated as the type species.
Description of Aquisalimonas asiatica sp. nov.
Aquisalimonas asiatica (a.si.a'ti.ca. L. fem. adj. asiatica Asian).
Displays the following properties in addition to those given in the genus description. Cells are 0.7–0.9x2.0–10.0 µm. Colonies are 1 mm in diameter, non-pigmented, circular and entire on the maintenance medium after 2 days cultivation at 37 °C. In liquid nutrient broth, usually grows with a strong tendency to form clumps at the bottom. Grows over a wide range (1–20 %, w/v) of salt concentrations, with optimal growth at 7–10 % (w/v) salts. Grows at 20–50 °C (optimally at 37 °C) and pH 6.0–10.8 (optimally at pH 7.5–8.5). Strictly aerobic. Grows anaerobically with nitrate. Nitrate is reduced to nitrite. Aesculin, casein, DNA, gelatin and starch are not hydrolysed; Tween 80 is hydrolysed. H2S is produced. Indole and phenylalanine deaminase tests are negative. Phosphatase test is positive. As determined using Biolog GN plates, the following compounds are oxidized: dextrin, glycogen, Tweens 40 and 80, L-arabinose, D-cellobiose, 
-D-glucose, maltose, L-rhamnose, turanose, pyruvic acid methyl ester, succinic acid methyl ester, acetic acid, cis-aconitic acid, citric acid, formic acid, -ketobutyric acid, DL-lactic acid, propionic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, glucuronamide, L-glutamic acid, L-proline, 
-aminobutyric acid and glycerol. The following compounds are not oxidized: -cyclodextrin, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, D-arabitol, i-erythritol, D-fructose, L-fucose, D-galactose, gentiobiose, myo-inositol, -D-lactose, lactulose, D-mannitol, D-mannose, D-melibiose, methyl 
-D-glucoside, D-psicose, D-raffinose, D-sorbitol, sucrose, D-trehalose, xylitol, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, p-hydroxyphenylacetic acid, itaconic acid, -ketoglutaric acid, -ketovaleric acid, malonic acid, quinic acid, D-saccharic acid, L-alaninamide, D- and L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, hydroxy-L-proline, leucine, L-ornithine, L-phenylalanine, L-pyroglutamic acid, D- and L-serine, L-threonine, DL-carnitine, urocanic acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, DL--glycerol phosphate, -D-glucose 1-phosphate and D-glucose 6-phosphate. Susceptible to chloramphenicol (30 µg) and erythromycin (15 µg). Resistant to bacitracin (10 U), cephalothin (30 µg), kanamycin (30 µg), nalidixic acid (30 µg), neomycin (10 µg), penicillin G (10 U), rifampicin (30 µg), streptomycin (30 µg), tetracycline (30 µg) and vancomycin (30 µg). The polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, a phosphoglycolipid and six different unidentified phospholipids. Additional characteristics of the strains are listed in Table 1. Known strains were isolated from water of an alkaline, saline lake.
The type strain, CG12T (=CCM 7368T=CECT 7151T=CGMCC 1.6291T=DSM 18102T), was isolated from Lake Chagannor in Inner Mongolia, China.
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ACKNOWLEDGEMENTS |
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