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1 College of Agriculture and Natural Resources, Department of Soil and Environmental Sciences, National Chung Hsing University, Taichung 402, Taiwan, Republic of China
2 Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Institute of Marine Technology, Nan-Tzu, Kaohsiung 811, Taiwan, Republic of China
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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) belongs to the family Xanthomonadaceae in the order Xanthomonadales (Garrity & Holt, 2001). Members of this genus comprise aerobic, Gram-negative, non-spore-forming rods that are characterized chemotaxonomically by a fatty acid profile consisting of straight-chain saturated, unsaturated and branched-chain fatty acids of the iso/anteiso type as well as 3-OH fatty acids, in addition to the presence of a ubiquinone with eight isoprene units (Q-8) as the major respiratory quinone. At the time of writing, the genus Pseudoxanthomonas comprises ten recognized species: Pseudoxanthomonas broegbernensis, P. daejeonensis, P. japonensis, P. kalamensis, P. kaohsiungensis, P. koreensis, P. mexicana, P. suwonensis, P. taiwanensis and P. yeongjuensis. In this paper, we describe a bacterial strain (designated IMMIB AFH-5T) that was isolated from a sample of oil-contaminated soil. On the basis of phylogenetic and phenotypic evidence, it is proposed that this strain represents a novel species of the genus Pseudoxanthomonas. Strain IMMIB AFH-5T was isolated on nutrient agar from a soil sample collected from an oil-contaminated site located in Chyai County, Taiwan. The isolate was subsequently cultivated on brain-heart infusion (BHI) agar (no. 237100; Becton Dickinson) and tryptone soya agar (TSA) (CM 131; Oxoid) to determine its morphological characteristics. Pigment production was determined by growing the strains at 37 °C for 7 days and observations were made at 24 h intervals. Fermentation tests were performed using the API CORYNE, API 20 STREP and the API 20E systems (bioMérieux). Assimilation tests were performed using the API 20NE system (bioMérieux). Enzyme reactions and acid production from carbohydrates were assessed after 7 days incubation at 37 °C. Salinity tolerance was measured by growing the strain in tryptone soya broth supplemented with NaCl at final concentrations in the range 0.5–12 % (w/v).
Chemotaxonomic characteristics of strain IMMIB AFH-5T were determined by cultivating the organism at 37 °C in shake flasks containing BHI broth for 1 week. After a check for purity at maximum growth, the organism was killed with formaldehyde (1 %, v/v), harvested by centrifugation, washed with distilled water and freeze-dried. Lipids were extracted using acid methanolysis as described by Minnikin et al. (1980). Fatty acids were purified with preparative TLC as described by Yassin (1988), separated, identified and quantified using a GC-MS (QP2010; Shimadzu) equipped with a capillary column type HP-1 (Agilent). 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 MS (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.
DNA was isolated using an UltraClean microbial DNA isolation kit (Mo Bio) as described in the manufacturer's protocol. DNA G+C contents were determined by HPLC (Mesbah et al., 1989) using the 
phage as a reference. Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene sequence and purification of PCR products were carried out using procedures described previously (Rainey et al., 1996). The purified PCR products were sequenced using a Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems) as described by the manufacturer. A Genetic Analyzer (310; Applied Biosystems) was used for electrophoresis of the sequence reaction products. The 16S rRNA gene sequences of recognized species of the genus Pseudoxanthomonas (obtained from GenBank) were added to the ARB database (Ludwig et al., 2004) and aligned using the respective tool from 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) approaches. The evolutionary distance matrix for the neighbour-joining method was calculated using the corrections of Jukes & Cantor (1969). The topology of the neighbour-joining tree was evaluated using bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings.
Cells of strain IMMIB AFH-5T were found to be Gram-negative, curved rods that were motile by means of a single polar flagellum. On TSA and BHI agar, colonies were smooth, yellow and circular. Date-brown pigments were produced on TSA and BHI agar after 24 h incubation at 37 °C. The organism was able to grow at salinities in the range 0.5–3 % (w/v) NaCl in tryptone soya broth, with optimal growth occurring in the presence of 2 % (w/v) NaCl. The organism was aerobic, catalase-positive and oxidase-positive. It lacked nitrate reductase and urease activities. A comparison of the physiological characteristics (based on API 20NE tests) of strain IMMIB AFH-5T with those of related species of the genus Pseudoxanthomonas is shown in Table 1. In common with other recognized species of the genus Pseudoxanthomonas, strain IMMIB AFH-5T was unable to assimilate caprate, adipate or phenylacetate.
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. Strain IMMIB AFH-5T formed a distinct lineage within the genus Pseudoxanthomonas, a phyletic line that was supported by all of the tree-making algorithms. The isolate shared 16S rRNA gene sequence similarities within the range 95.2–96.3 % with members of the genus Pseudoxanthomonas. It is also apparent from Fig. 1 that the isolate is loosely associated with the type strains of P. kalamensis (96.3 % similarity) and P. yeongjuensis (96.3 % similarity), though these relationships are not supported by a high bootstrap percentage (75 %). The divergence values with respect to recognized Pseudoxanthomonas species (>3 %) show that isolate IMMIB AFH-5T represents a hitherto unknown species of this genus (Stackebrandt & Goebel, 1994). Tests that proved useful in distinguishing the novel species from some recognized Pseudoxanthomonas species are shown in Table 1
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7c (4.0 %), n-C14 : 0 (3.37 %), cyclo-C17 : 0 (2.83 %), iso-C17 : 19c (2.37 %), iso-C11 : 0 (2.25 %), anteiso-C15 : 0 (1.16 %), iso-C17 : 18c (0.91 %), anteiso-C13 : 0 (0.50 %), iso-C13 : 0 (0.49 %), n-C18 : 0 (0.42 %), n-C18 : 19c (0.39 %), n-C18 : 17c (0.39 %) and n-C17 : 0 (0.24 %) as the major non-hydroxylated cellular fatty acid methyl esters. The hydroxylated fatty acids included n-C12 : 0 3-OH (1.52 %), iso-C13 : 0 3-OH (0.63 %) and iso-C11 : 0 3-OH (0.40 %). Table 2 shows the fatty acid profile of isolate IMMIB AFH-5T in comparison with those of the type strains of Pseudoxanthomonas species. Mass spectral analysis of the main isoprenoid quinones isolated from strain IMMIB AFH-5T showed a strong peak at m/z 749.46 attributable to [M+Na]+ in the high-mass region. This corresponds to an ubiquinone with eight isoprene units (Q-8).
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). On the basis of both phenotypic and phylogenetic evidence, therefore, strain IMMIB AFH-5T represents a novel species within the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas spadix sp. nov. is proposed.
Description of Pseudoxanthomonas spadix sp. nov.
Pseudoxanthomonas spadix [spa'dix. L. fem. adj. spadix (date-brown) date-coloured, referring to the brown pigment produced by the organism].
Cells are Gram-negative, non-spore-forming, curved rods that are motile by means of a single polar flagellum. Aerobic, catalase-positive and oxidase-positive. On TSA and BHI agar, colonies are smooth and yellow with entire margins. Produces a diffusible, date-brown pigment that becomes more marked in older cultures. Grows at temperatures between 22 and 37 °C. Has the chemotaxonomic characteristics of members of the genus Pseudoxanthomonas. Possesses a ubiquinone with eight isoprene units (Q-8) as the major isoprenoid quinone. The fatty acid profile mainly consists of saturated and unsaturated acids with straight-chain and branched-chain fatty acids of the iso/anteiso type. The predominant non-hydroxylated fatty acids are iso-C15 : 0, iso-C16 : 0/n-C16 : 0, and anteiso-C17 : 0, whereas the major 3-OH fatty acids are iso-C11 : 0 3-OH, iso-C12 : 0 3-OH and iso-C13 : 0 3-OH. Does not produce acid from L-arabinose, amygdalin, D-glucose, glycogen, inositol, inulin, D-lactose, maltose, D-mannitol, D-melibiose, D-raffinose, L-rhamnose, D-ribose, D-sorbitol, sucrose, trehalose or xylose. Hydrolyses aesculin and gelatin, but not hippurate or starch. Possesses alkaline phosphatase and 
-glucosidase activities but is negative for arginine dihydrolase, 
-glucosidase, -galactosidase, -galactosidase, -glucuronidase, N-acetyl--glucosaminidase, leucine aminopeptidase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, pyrazinamidase, pyrrolidonyl arylamidase, nitrate reductase and urease. Positive for acetoin production, but negative for indole and H2S production. Assimilates D-glucose, but not L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate or phenylacetic acid. The DNA G+C content is 68.5±0.5 mol%.
The type strain, IMMIB AFH-5T (=DSM 18855T=CCUG 53828T), was isolated from a soil sample collected from an oil-contaminated site located in Chyai County, Taiwan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Yassin, A. F. (1988). Chemotaxonomische Untersuchungen zur vereinfachten Differenzierung und Identifizierung von aeroben Aktinomyzeten und Mykobakterien. Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematische-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn.
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