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1 College of Agriculture and Natural Resources, Department of Soil and Environmental Sciences, National Chung Hsing University, Taichung, 402, Taiwan, ROC
2 Laboratory of Microbiology, Department of Sea Food Science, National Kaohsiung Institute of Marine Technology, Nan-Tzu, Kaohsiung, 811, Taiwan, ROC
3 Institut für Medizinische Mikrobiologie und Immunologie der Universität Bonn, 53127 Bonn, Germany
Correspondence
A. F. Yassin
yassin{at}mibi03.meb.uni-bonn.de
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accommodates strictly aerobic, chemo-organotrophic, yellow- to whitish-brown-pigmented, Gram-negative, rod-shaped bacteria. Members of the genus can be characterized chemotaxonomically by having a fatty acid profile that contains C18 : 1
7c as the major fatty acid and C14 : 0 2-OH as the main hydroxylated fatty acid. They contain ubiquinone Q-10 as the main respiratory quinone and spermidine as the major polyamine. The polar lipid pattern consists of diphosphatidylglycerol, phosphatidylglycerol, sphingoglycolipid, phosphatidylethanolamine, phosphatidyldimethylethanolamine and phosphatidylcholine (Busse et al., 1999; Stolz et al., 2000; Pal et al., 2005; Prakash & Lal, 2006; Wittich et al., 2007). Members of the genus Sphingobium represent environmental isolates that play an important role in the bioremediation and biodegradation of pollutants. At the time of writing, the genus Sphingobium comprised 12 recognized species. In this paper, the taxonomic characterization of a yellow-pigmented isolate, IMMIB HF-1T, preliminarily identified as a member of the genus Sphingobium, is presented. Strain IMMIB HF-1T was isolated on nutrient agar (NA) from oil-contaminated soil near the oil refinery located in Kaohsiung County, Taiwan. The isolate was subsequently cultivated on brain-heart infusion (BHI) agar (Becton Dickinson no. 237100) and tryptone soy agar (TSA; Oxoid CM 131) to determine its morphological characteristics. Fermentation and enzymic tests were performed using the API Coryne, API 20 Strep and API 20E systems and assimilation tests were performed using the API 20NE and API 50CH systems according to the manufacturer's instructions (bioMérieux), except for the time of incubation. All strips were read after 7 days incubation at 37 °C. Salt tolerance was determined by cultivating the organism in tryptone soy broth supplemented with NaCl at final concentrations of 0.0–12.0 %.
The chemotaxonomic characteristics of strain IMMIB HF-1T were determined by cultivating the organism at 37 °C in shake flasks containing BHI broth for 1 week. At maximum growth, the purity of the culture was checked and the organism was killed with formaldehyde (1 %, v/v), harvested by centrifugation, washed with distilled water and freeze-dried. Lipids were extracted by acid methanolysis as described by Minnikin et al. (1980). Fatty acids were analysed as described recently by Yassin et al. (2007). Polar lipids were determined using TLC, as described by Yassin et al. (1993). Respiratory quinones were extracted and purified according to Collins et al. (1977). Mass spectral analyses of the quinones were recorded in positive ion mode on a Q-TOF 2 mass spectrometer (Micromass) equipped with a nanospray source as described by Yassin & Hupfer (2006). For the compounds under study, the major ions observed with electrospray were protonated pseudo-molecular ions, [M+Na]+. The identity of the ubiquinone was verified by observing the diagnostic ion at m/z 197, which represents the benzylium ion. For polyamine analysis, strain IMMIB HF-1T was grown on PYE medium (0.3 % peptone from casein, 0.3 % yeast extract, pH 7.2), harvested at approximately 70 % of the maximum optical density and, after lyophilization, polyamines were extracted and analysed as described by Busse & Auling (1988) and Stolz et al. (2007).
Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA and the purification of PCR products were carried out according to the methods of Rainey et al. (1996). Purified PCR products were sequenced using a Taq DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems) using the manufacturer's protocol. A genetic analyzer (310; Applied Biosystems) was used for electrophoresis of reaction products. The 16S rRNA gene sequences of species of the genus Sphingobium with validly described names retrieved from GenBank were added to the ARB database (Ludwig et al., 2004) and aligned using the respective tools of the ARB package. The resulting alignment was corrected manually and evolutionary trees were inferred using maximum-parsimony (Fitch, 1971), neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, 1981) methods. An evolutionary distance matrix was calculated using the correction of Jukes & Cantor (1969). The topologies of the resultant tree were evaluated in bootstrap analyses (Felsenstein, 1985) of the neighbour-joining method based on 1000 resamplings.
The almost complete 16S rRNA gene sequence (1471 nt) of strain IMMIB HF-1T was determined in this study. A tree depicting the phylogenetic affinity of this isolate within the genus Sphingobium is shown in Fig. 1. It is evident from the tree that strain IMMIB HF-1T formed a distinct subline within the genus Sphingobium, branching proximal to the base of a subcluster of species that included Sphingobium cloacae, Sphingobium yanoikuyae and Sphingobium amiense. However, bootstrap resampling showed that the association of strain IMMIB HF-1T with this subcluster of species was not statistically significant and, from the tree construction analysis, it is evident that strain IMMIB HF-1T does not exhibit a significant affinity with any recognized species. Highest sequence similarities were shown with the type strains of S. amiense (97.0 %), Sphingobium xenophagum (96.7 %) and S. cloacae (96.5 %); other species showed lower levels of similarity. However, sequence divergence values of 3 % or greater are considered to be strong evidence that organisms are not related at the species level (Stackebrandt & Goebel, 1994). Support for the distinctiveness of strain IMMIB HF-1T was also evident from phenotypic analyses (Table 1).
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7c (46.23 %) as major hydroxylated and non-hydroxylated fatty acids, respectively. The detailed fatty acid profiles of strain IMMIB HF-1T and other Sphingobium species are shown in Table 2. Polar lipid analysis showed that the isolate contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine and sphingoglycolipid as characteristic polar lipids (data not shown). Mass spectral analysis of the main isoprenoid components isolated from strain IMMIB HF-1T showed a strong peak at m/z 885.6, attributable to [M+Na]+ in the high mass region. This corresponds to ubiquinone with ten isoprene units (Q-10). The polyamine pattern of strain IMMIB HF-1T contained spermidine as the predominant compound [23.0 µmol (g dry weight)–1] and only trace amounts of 1,3-diaminopropane, putrescine and spermine [
0.1 µmol (g dry weight)–1]. This polyamine pattern is in agreement with those observed for members of the genus Sphingobium (Busse et al., 1999; Takeuchi et al., 2001).
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-glucosidase, pyrrolidonyl arylamidase, leucine aminopeptidase and pyrazinamidase activities. It had no urease activity and was unable to reduce nitrate to nitrite. The detailed biochemical properties of strain IMMIB HF-1T are given in the species description below. Differential biochemical characteristics of strain IMMIB HF-1T and recognized species of the genus Sphingobium are given in Table 1
. From the above, based on both phenotypic and phylogenetic criteria, it is suggested that strain IMMIB HF-1T represents a novel species within the genus Sphingobium, for which the name Sphingobium olei sp. nov. is proposed.
Description of Sphingobium olei sp. nov.
Sphingobium olei (o'le.i. L. gen. neut. n. olei of/from oil, as the organism was isolated from oil-contaminated soil).
Cells are Gram-negative and rod-shaped. On NA, TSA and BHI agar, colonies are yellow and slightly elevated with entire margins. Aerobic, catalase- and oxidase-positive, grows at temperatures of 22–37 °C and tolerates 1.0 % NaCl. Possesses the salient chemotaxonomic characteristics of the genus Sphingobium. The detailed fatty acid profile is presented in Table 2. The respiratory quinone consists of ubiquinone with ten isoprene units (Q-10). Contains diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine, sphingoglycolipid and an unknown phospholipid. Major cellular polyamine is spermidine [23.0 µmol (g dry weight)–1] and trace amounts of 1,3-diaminopropane, putrescine and spermine [<0.1 µmol (g dry weight)–1] are also present. Hydrolyses gelatin, hippurate and aesculin. Assimilates D-galactose, gentiobiose, D-glucose, glycerol, L-arabinose, arbutin, maltose, potassium gluconate, malic acid, starch, sucrose, trehalose and D-xylose, but not D-adonitol, adipic acid, amygdalin, D-arabinose, D- or L-arabitol, phenylacetic acid, capric acid, D-cellobiose, trisodium citrate, dulcitol, erythritol, fructose, D- or L-fucose, N-acetylglucosamine, glycogen, inositol, inulin, D-lactose, D-lyxose, D-mannose, D-mannitol, D-melezitose, D-melibiose, D-raffinose, ribose, salicin, L-sorbose, D-tagatose, turanose, L-xylose or xylitol. Produces acid from starch, arbutin, D-fucose, gentiobiose, D-glucose, maltose, sucrose, trehalose and D-xylose, but not from amygdalin, D- or L-arabinose, D- or L-arabitol, D-cellobiose, dulcitol, erythritol, D-fructose, L-fucose, N-acetylglucosamine, glycerol, glycogen, inositol, inulin, D-lactose, D-lyxose, D-mannitol, D-mannose, D-melezitose, D-melibiose, D-raffinose, L-rhamnose, D-ribose, salicin, L-sorbose, D-sorbitol, D-tagatose, turanose or xylitol. Negative for oxidative fermentation of amygdalin, L-arabinose, D-glucose, inositol, D-mannitol, D-melibiose, L-rhamnose, D-sorbitol and sucrose. Positive for alkaline phosphatase, -glucosidase, pyrrolidonyl arylamidase, leucine aminopeptidase and pyrazinamidase activities, but negative for arginine dihydrolase, -galactosidase, 
-galactosidase, -glucuronidase, N-acetyl--glucosaminidase, 2-nitrophenyl -D-galactopyranoside, 4-nitrophenyl -D-galactopyranoside, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, nitrate reductase and urease activities. Produces acetoin (Voges–Proskauer reaction), but not indole or H2S.
The type strain, IMMIB HF-1T (=DSM 18999T=CCUG 54329T), was isolated from oil-contaminated soil near the oil refinery located in Kaohsiung County, Taiwan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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