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1 Institut für Medizinische Mikrobiologie und Immunologie der Universität Bonn, 53127 Bonn, Germany
2 College of Agriculture and Natural Resources, Department of Soil & Environmental Sciences, National Chung Hsing University, Taichung 402, Taiwan, ROC
3 Kekulé-Institut für Organische Chemie und Biochemie der Universität Bonn, 53121 Bonn, Germany
Correspondence
A. F. Yassin
yassin{at}mibi03.meb.uni-bonn.de
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) as currently defined is a member of the family Gordoniaceae (Stackebrandt et al., 1997) and accommodates actinomycetes with atypical cell morphology that are unable to grow at temperatures above 30 °C, and possesses mycolic acids with carbon chain length of C50 : 0–C56 : 0. At the time of writing, the genus Williamsia comprised four recognized species, Williamsia deligens (Yassin & Hupfer, 2006) isolated from human blood, Williamsia muralis (Kämpfer et al., 1999) isolated from indoor building material of a children's day-care centre in Finland, Williamsia maris (Stach et al., 2004) isolated from deep sediments of the Sea of Japan and Williamsia marianensis (Pathom-Aree et al., 2006) isolated from a deep-sea sediment. These species form a distinct clade within the evolutionary radiation occupied by mycolic-acid-containing actinomycetes, i.e. by organisms classified in the suborder Corynebacterineae (Stackebrandt et al., 1997). In the present paper we describe an actinobacterial strain, designated IMMIB SR-4T, which was isolated from an oil-contaminated soil sample. Based on phylogenetic and phenotypic evidence, it is proposed that this isolate should be classified as representing a novel species of the genus Williamsia.
Isolate IMMIB SR-4T was isolated from a soil sample collected from an oil-contaminated site located in Chyai County, Taiwan. The organism was cultured on Columbia agar supplemented with 5 % sheep blood and brain heart infusion (BHI) agar to determine its morphological characteristics. Air-dried smears were stained via Gram's method in order to determine the Gram reaction and cell morphology. Ziehl-Neelsen's method was used to determine acid-fastness. Growth temperatures were determined by incubating the organism at 22, 28, 30, 37 and 42 °C. The physiological properties of the strain were determined by using tests for hydrolysis of complex substrates as described by Gordon (1966, 1967) and Gordon & Mihm (1957) as well as tests for carbon source utilization according to Yassin et al. (1995). The isomeric form of diaminopimelic acid was determined according to the method of Becker et al. (1964) and whole-cell sugars were identified by using the method of Lechevalier (1968). Lipids were extracted by using acid methanolysis and mycolic acids were detected with TLC as described by Minnikin et al. (1980); pyrolysis GC of the mycolate was performed according to Yassin et al. (1993a). Fatty acids were purified, identified and quantified by GC as described by Yassin (1988). Phospholipids were extracted, purified and identified as described by Yassin et al. (1993b). Menaquinones were extracted and purified according to Collins et al. (1977). Mass spectra of the menaquinones 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).
Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and purification of PCR products were carried out according to the procedures of Rainey et al. (1996). Purified PCR products were sequenced by using a Taq DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems) as described in the manufacturer's protocol. An Applied Biosystems 310 DNA Genetic Analyzer was used for electrophoresis of the sequence reaction products. The 16S rRNA gene sequences of recognized species of the genus Williamsia retrieved from GenBank were added to the ARB database (Ludwig et al., 2004) and were aligned by using the respective tool within the ARB package. The resulting alignment was corrected manually and evolutionary trees were inferred using maximum-parsimony (Kluge & Farris, 1969), neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, 1981) methods. The evolutionary distance matrix was calculated using the correction of Jukes & Cantor (1969). The topology of the resultant tree was evaluated by bootstrap analyses (Felsenstein, 1985) of the neighbour-joining data based on 1000 resamplings by using the ARB package.
The almost-complete 16S rRNA gene sequence of strain IMMIB SR-4T was determined [1479 nt; 95.9 % of the Escherichia coli sequence (Brosius et al., 1978)]. 16S rRNA gene sequence comparison clearly indicated that strain IMMIB SR-4T was a member of the suborder Corynebacterineae (Stackebrandt et al., 1997), and the sequence contained all signature nucleotides expected for this suborder as well as those described recently (Yassin & Hupfer, 2006) for the family Gordoniaceae. Furthermore, the new isolate contained the signature nucleotides at positions 293–304 (G–C) and 307 (C) shared with other members of the genus Williamsia. However, strain IMMIB SR-4T differed from recognized Williamsia species in that it possessed C–G rather than G–C at positions 1007 : 1022. Therefore, the current pattern of signature nucleotides for members of the genus Williamsia may need to be updated as novel species are described.
The phylogenetic tree constructed (Fig. 1) shows the position of strain IMMIB SR-4T within the radiation of representative phylogenetic groups of the suborder Corynebacterineae. It is evident from the tree that strain IMMIB SR-4T not only falls within the phylogenetic radiation occupied by the genus Williamsia but forms a distinct phyletic line that is loosely associated (bootstrap value of 55 %) with the type strains of W. maris and W. deligens. Strain IMMIB SR-4T showed 16S rRNA gene sequence similarities of 97.0 and 98.1 % to W. maris SJS0289/JS1T and W. deligens IMMIB RIV-956T, respectively, and 95.5 and 95.6 % to W. marianensis MT8T and W. muralis MA140/96T, respectively. Therefore, the distinctiveness of strain IMMIB SR-4T from W. maris, W. marianensis and W. muralis is underpinned by exhibiting 
3 % sequence divergence (Stackebrandt & Goebel, 1994) from these three species. It is known that the type strains of W. marianensis and W. muralis share high 16S rRNA gene sequence similarity (99.5 % or 7 nt differences out of 1443 positions) but belong to genomic species with a mean DNA–DNA relatedness value of 11 % (Pathom-Aree et al., 2006). It is therefore evident that the high 16S rRNA gene sequence similarity found between strain IMMIB SR-4T and the type strain of W. deligens (98.1 %, corresponding to 30 nt differences) indicates that the test strain represents a distinct taxonomic entity within the genus Williamsia. It is also clear from Table 1 that strain IMMIB SR-4T can be distinguished readily from the type strain of W. deligens based on a set of biochemical properties.
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). One-dimensional TLC of the whole-cell acid methanolysate of the organism revealed the presence of two lipid spots on the chromatogram. The lower spot corresponded to mycolic acids as identified by its Rf value (0.55) and the higher spot corresponded to non-hydroxylated fatty acids. Pyrolysis GC of the purified mycolic acid methyl esters from strain IMMIB SR-4T released fatty acid methyl esters of C16 : 0 (55.0 % of the total cleavage products) and C18 : 0 (45.0 %) as pyrolysis cleavage products. GC analyses of the non-hydroxylated fatty acid methyl esters revealed the presence of tetradecanoate (1.7 % of the total fatty acids), pentadecanoate (0.15 %), cis-hexadecenoate (0.35 %), hexadecanoate (15.6 %), 10-methyl hexadecanoate (0.52 %), heptadecanoate (0.16 %), octadecenoate (4.9 %), octadecanoate (7.4 %), tuberculostearic acid (10-methyl octadecanoate; 17.1 %), eicosenoate (1.2 %), eicosanoate (0.57 %), docosanoate (15.8 %), tetracosenoate (1.3 %) and tetracosanoate (34.3 %) as the major cellular fatty acid methyl esters. Polar lipid analysis showed that the organism contained phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and diphosphatidylglycerol as characteristic phospholipids (i.e. phospholipid type PII sensu Lechevalier et al., 1977). Mass spectral analysis of the main respiratory quinone components from strain IMMIB SR-4T revealed a strong peak at m/z 809.5 attributable to [M+Na]+ in the high-mass region. This corresponds to a dihydrogenated menaquinone with nine isoprene units, MK-9(H2). The second band revealed a strong peak at m/z 741.58 attributable to [M+Na]+ in the high-mass region. This corresponds to a dihydrogenated menaquinone with eight isoprene units, MK-8(H2).
Strain IMMIB SR-4T was examined for a range of phenotypic markers. The organism showed morphological properties consistent with its assignment to the genus Williamsia. It was aerobic, and built smooth, orange- to orange–red-pigmented colonies on BHI agar and Columbia agar supplemented with 5 % sheep blood. Cells were Gram-positive rods and were non-acid–alcohol-fast. The organism was able to grow at 22 and 30 °C but not at higher temperatures. It is evident from Table 1
that strain IMMIB SR-4T can be readily differentiated from the type strains of the four recognized Williamsia species. It is of particular note that only isolate IMMIB SR-4T was able to hydrolyse gelatin and to utilize acetamide and serine as simultaneous nitrogen and carbon sources for energy. The biochemical properties of strain IMMIB SR-4T are given in the species description below. It is apparent from the genotypic and phenotypic data presented that strain IMMIB SR-4T represents a novel species of the genus Williamsia, for which the name Williamsia serinedens sp. nov. is proposed.
Description of Williamsia serinedens sp. nov.
Williamsia serinedens (se.ri.ne'dens. N.L. n. serinum serine; L. part. pres. edens eating; N.L. part. adj. serinedens eating serine).
Forms smooth, orange- to orange–red-pigmented colonies on agar media. Cells are Gram-positive rods and are non-acid–alcohol-fast. Grows at 22–30 °C, but not at higher temperatures. Contains the salient chemotaxonomic characteristics of the genus Williamsia. Its mycolic acids were cleaved on pyrolysis to release C16 : 0 and C18 : 0 fatty acids as the major products. The fatty acid profile consists mainly of straight-chain saturated, unsaturated and 10-methyl-branched fatty acids. Hydrolyses gelatin and urea but not adenine, casein, elastin, aesculin, guanine, hypoxanthine, testosterone, tyrosine or xanthine. Assimilates acetate, adonitol, L-arabinose, 2,3-butandiol, citrate, meso-erythritol, galactose, glucose, p-hydroxybenzoate, lactate, maltose, mannitol, paraffin, 1,2-propandiol, sucrose, sorbitol, trehalose and xylose as carbon sources, but not adipate, isoamyl alcohol, cellobiose, gluconate, m-hydroxybenzoate, myo-inositol, lactose, melezitose, raffinose or rhamnose. Utilizes acetamide, L-alanine and serine as simultaneous carbon and nitrogen sources, but not arginine, gelatin, ornithine or proline.
The type strain, IMMIB SR-4T (=DSM 45037T=CCUG 53151T), was isolated from an oil-contaminated soil sample in Chyai County, Taiwan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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