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1 Departamento de Microbiología, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C Concepción, Chile
2 BCCM/LMG Bacteria Collection, University of Ghent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
3 Institut für Mikrobiologie der Westfälischen, Wilhelms–Universität Münster, Corrensstrasse 3, D-48149 Münster, Germany
Correspondence
B. H. A. Rehm
rehm{at}uni-muenster.de
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ABSTRACT |
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7c), heptadecenoic acid (17 : 16c) and hexadecanoic acid (16 : 0). The results of DNA–DNA hybridization experiments and its physiological characteristics clearly distinguished the novel isolate from all known Sphingopyxis species and indicated that the strain represents a novel Sphingopyxis species. Therefore, the species Sphingopyxis chilensis sp. nov. is proposed, with strain S37T (=LMG 20986T =DSM 14889T) as the type strain. The transfer of Sphingomonas alaskensis to the genus Sphingopyxis as Sphingopyxis alaskensis comb. nov. is also proposed.
Published online ahead of print on 19 September 2002 as DOI 10.1099/ijs.0.02375-0.
The GenBank accession number for the 16S rDNA sequence of strain S37T is AF367204.
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. This genus was proposed as part of the splitting of the genus Sphingomonas (Yabuuchi et al., 1990), because the genus Sphingomonas represented a broad range of heterogeneous species with respect to physiology, phylogenetics and ecology. Thus, on the basis of phylogenetic evidence and some chemotaxonomic and phenotypic features that allow differentiation between the four clusters of Sphingomonas species, Takeuchi et al. (2001) proposed to emend the genus Sphingomonas (sensu stricto) and created three new genera: Sphingobium, Novosphingobium and Sphingopyxis. At the time of writing, the genus Sphingopyxis contains the species Sphingopyxis macrogoltabida and Sphingopyxis terrae. Bacteria belonging to this genus are Gram-negative, non-fermentative, aerobic, non-spore-forming, yellow-pigmented or whitish-brown, non-motile or motile, and are characterized chemotaxonomically by the presence of ubiquinone Q-10 and 2-hydroxymyristic acid (2-OH 14 : 0). Spermidine is the major polyamine component, sphingoglycolipids are present and the DNA G+C content is 63–65 mol%.
The intracellular accumulation of polyhydroxyalkanoates (PHAs) in the genus Sphingomonas has not been studied in much detail. These storage compounds are polyesters of commercial interest and represent a useful taxonomic criterion for differentiating bacterial genera (Kessler & Palleroni, 2000). Strain S37T, which degrades chlorophenols and is able to accumulate PHA, was isolated from sediments of a river polluted with chlorophenolic compounds (Godoy et al., 1999). On the basis of preliminary morphological and physiological study, strain 37T was identified as being related to Sphingomonas paucimobilis (Aranda et al., 1999; Yeber et al., 2000).
In this study, we describe the morphological, biochemical and phylogenetic characteristics of strain S37T, and also the analysis of reserve polymers of species belonging to the genera Sphingopyxis and Sphingomonas that are closely related to strain S37T. On the basis of phenotypic data, DNA–DNA hybridization data and the results of the 16S rDNA sequence analysis, we propose that strain S37T represents a novel species of the genus Sphingopyxis, Sphingopyxis chilensis sp. nov.
Bacterial strains and cultures
Strain S37T (=LMG 20986T=DSM 14889T) was isolated from the subsurface of a river (Biobío River in central Chile) polluted with chlorophenolic compounds as a 2,4,6-trichlorophenol-degrading bacterium (Godoy et al., 1999). Sphingomonas adhaesiva LMG 10922T, Sphingopyxis macrogoltabida LMG 17324T and Sphingomonas alaskensis LMG 18877T were obtained from the BCCM/LMG Bacteria Collection, Gent, Belgium. All strains were grown on R2A agar (Difco) at 25 °C unless indicated otherwise.
Morphological and biochemical characterization
The strains studied were characterized biochemically using API 20 NE test strips (bioMérieux). Phenotypic characterization using API 20 NE strips and the comparison with other strains from the genus Sphingopyxis are summarized in Table 1. Strain S37T is able to utilize glucose, mannose, maltose and fructose as sole carbon sources, whereas only weak growth was obtained with adipate, xylose and N-acetylglucosamine as sole carbon sources (Table 1). Additionally, growth on different fatty acids was examined by culturing strain S37T on mineral salts medium (Schlegel et al., 1961) in the presence of 1 % (w/v) propionate, butyrate, glutarate, gluconate, valerate, hexanoate, octanoate, fumarate or decanoate at 25 °C for 72 h. Strain S37T showed growth with fumarate as the sole carbon source, but was not able to grow with propionate, butyrate, glutarate, gluconate, valerate, hexanoate, octanoate or decanoate (all as sodium salts). Cells of strain S37T were Gram-negative, motile rods that grew aerobically. The strain produced small yellow colonies (2 mm) on R2A agar after 3 days incubation. The morphological, physiological and biochemical characteristics of strain S37T are consistent with the characteristics of the genus Sphingopyxis, as defined by Takeuchi et al. (2001) (Table 1). The phenotypic characteristics of strain S37T allow differentiation from Sphingomonas alaskensis and Sphingopyxis macrogoltabida. Unlike these other species, strain S37T is incapable of hydrolysing lactose (
-galactosidase activity). Strain S37T and Sphingopyxis macrogoltabida utilize D-mannose as a sole carbon source, whereas Sphingomonas alaskensis can not utilize this carbohydrate. In contrast to Sphingopyxis macrogoltabida, Sphingomonas alaskensis and strain S37T grow with maltose as a sole carbon source.
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. The 16S rDNA was amplified with a PCR, using a universal primer set corresponding to positions 8–27 (forward primer) and 1505–1525 (reverse primer) (Rainey et al., 1993). The PCR products were sequenced using the Taq Dye-Deoxy Terminator cycle sequencing kit (Applied Biosystems). Sequencing reactions were electrophoresed using an 4000L DNA (LI-COR) sequencer. The 16S rDNA sequence (1452 bp) of strain S37T was determined. Similarity values between 16S rDNA sequences were obtained using internet tools as described previously (Rehm, 2001). Initial investigations on the global 16S rDNA sequence-based position of the novel isolate used the ARB database (Ludwig & Strunk, 1996). On the basis of this work, the 16S rDNA sequences were compared with the existing 16S rDNA database for members of the phyletic group defined as members of the genera Sphingomonas, Novosphingobium, Sphingobium and Sphingopyxis. A phylogenetic tree was constructed as described previously (Hezayen et al., 2001; Rehm, 2001). A preliminary comparison against the GenBank database indicated that the strain is closely related to members of the genera Sphingopyxis and Sphingomonas. The sequence of strain S37T showed high similarity to sequences of Sphingomonas alaskensis LMG 18877T (98·8 %) and Sphingopyxis macrogoltabida LMG 17324T (98·2 %). Similarities of 92 and 95 % were found to the 16S rDNA sequences of Sphingomonas adhaesiva GIFU 11458T and Sphingopyxis terrae IFO 15098T, respectively. Construction of a 16S rDNA sequence-based phylogenetic tree indicated a phylogenetic position between Sphingomonas alaskensis and Sphingopyxis macrogoltabida (Fig. 1). For DNA base composition and DNA–DNA hybridization, cells were grown on trypticase soy agar (TSA; BBL) for 24 h at 28 °C. Isolation of genomic DNA and spectrophotometric determination of the G+C content were carried out according to Vancanneyt et al. (2001). The G+C content of the genomic DNA of strain S37T was 65·5 mol%. This value is within the range observed for members of the genus Sphingopyxis (Yabuuchi et al., 1990; Takeuchi et al., 2001). DNA–DNA hybridization was carried out by fluorometric hybridization in microdilution wells using biotinylated DNA (Ezaki et al., 1989). For the fluorescence measurements, an HTS7000 Bio Assay reader (Perkin-Elmer) was used. The hybridization temperature was 52 °C. The DNA–DNA reassociation values between strain S37T and Sphingomonas alaskensis LMG 18877T and Sphingopyxis macrogoltabida LMG 17324T were respectively 50 and 38 %. These values are below the threshold value (approx. 70 %) for possible relatedness at the species level (Wayne et al., 1987).
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). The whole-cell fatty acid compositions of strain S37T and related Sphingopyxis reference strains are given in Table 2. The major whole-cell fatty acids in strain S37T were octadecenoic acid (18 : 17c), heptadecenoic acid (17 : 16c), hexadecanoic acid (16 : 0) and summed feature 4 (see Table 2). Minor amounts of the 2-hydroxy fatty acids 14 : 0 2-OH, 15 : 0 2-OH and 16 : 0 2-OH were also present. The absence of 3-OH fatty acids and the presence of 2-OH myristic acid, hexadecenoic acid (16 : 1
5c) and octadecenoic acid (18 : 17c) as the major fatty acids and octadecenoic acid (18 : 17c 11-methyl) as a minor fatty acid in strain S37T are features characteristic of members of the genus Sphingopyxis (Table 2).
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) containing 0·05 % (w/v) NH4Cl and 0·1 M glucose at 25 °C. For analysis of PHA, 5 mg lyophilized cells was subjected to methanolysis in the presence of a solution containing 1 ml chloroform, 0·85 ml methanol and 0·15 ml sulfuric acid for 5 h at 100 °C. The resulting methyl esters were analysed by GC (Agilent 6850 series GC system) (Brandl et al., 1988). For qualitative analysis of methyl esters, a coupled GC/MS analysis was performed using an HP 6890 gas chromatograph with a model 5973 mass-selective detector (Hewlett Packard). The mass spectra obtained were compared with the NIST '98 mass spectrum library with the Windows Search program (version 1.6; National Institute of Standards and Technology, US Department of Commerce). Experiments with strain S37T, Sphingomonas adhaesiva, Sphingopyxis macrogoltabida and Sphingomonas alaskensis cultivated on mineral medium with 0·1 M glucose showed that these strains accumulated PHAs consisting of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (Table 3). The amount of PHA accumulated was variable among the species studied. The strongest PHA accumulation was obtained with Sphingopyxis macrogoltabida, in which it contributed 70·2 % of cellular dry weight. Sphingomonas alaskensis and strain S37T accumulated similar amounts, PHAs contributing 26·0 and 24·3 % of cellular dry weight, respectively, whereas PHA accumulation in Sphingomonas adhaesiva contributed only 2·9 % of cellular dry weight. Except for Sphingomonas adhaesiva, the major constituent of the PHA was 3-hydroxybutyric acid (range 97·2–99 mol%), and 3-hydroxyvaleric acid was the minor constituent (range 0·9–2·8 mol%).
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and the phylogenetic and phenotypic description given by Vancanneyt et al. (2001), Sphingomonas alaskensis should be reclassified in the genus Sphingopyxis. Phylogenetic data from the present study (Fig. 1
) confirm this. We therefore propose the reclassification of Sphingomonas alaskensis within the genus Sphingopyxis as Sphingopyxis alaskensis comb. nov. This polyphasic study also clearly demonstrates that strain S37T represents a novel species within the genus Sphingopyxis, for which we propose the name Sphingopyxis chilensis sp. nov.
Description of Sphingopyxis alaskensis comb. nov.
Basonym: Sphingomonas alaskensis Vancanneyt et al. 2001.
The description of this species is given by Vancanneyt et al. (2001). The type strain is strain RB2256T (=CCUG 45028T=CIP 106977T =DSM 13593T =LMG 18877T).
Description of Sphingopyxis chilensis sp. nov.
Sphingopyxis chilensis (chi.len'sis. N.L. adj. chilensis referring to Chile, where the type strain was isolated).
Cells are Gram-negative, motile, non-sporulating rods. Colonies are circular, low and convex with entire margins, yellow and 2 mm in diameter after 3 days incubation. The following enzyme activities are present: catalase, oxidase, hydrolysis of aesculin. No reactions are obtained for urease, production of indole, arginine dihydrolase, reduction of nitrate, hydrolysis of gelatin or lactose utilization. Glucose, D-mannose, maltose, malate and fumarate are assimilated. The following compounds are not assimilated: L-arabinose, D-mannitol, N-acetylglucosamine, caprate, citrate, phenylacetate, propionate, butyrate, glutarate, gluconate, valerate, hexanoate, octanoate and decanoate. The major fatty acids are 18 : 17c, 17 : 16c, 16 : 0 and summed feature 4; the presence of the 2-hydroxy fatty acids 14 : 0 2-OH, 15 : 0 2-OH and 16 : 0 2-OH is characteristic. The G+C content is 65·5 mol%. PHAs consisting of 3-hydroxybutyric acid and 3-hydroxyvaleric acid are accumulated in the presence of glucose as carbon source.
The type strain is S37T (=LMG 20986T =DSM 14889T), isolated from superficial sediment of a river contaminated with chlorophenolic compounds in Concepción, Chile.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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