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Int J Syst Evol Microbiol 55 (2005), 787-791; DOI  10.1099/ijs.0.63210-0
© 2005 International Union of Microbiological Societies

Pseudoxanthomonas koreensis sp. nov. and Pseudoxanthomonas daejeonensis sp. nov.

Deok-Chun Yang1, Wan-Taek Im2, Myung Kyum Kim2 and Sung-Taik Lee2

1 Department of Oriental Medicinal Material and Processing, College of Life Science, Kyung Hee University, 1 Seocheon, Kihung Yongin, Kyunggi 449-701, South Korea
2 Environmental and Molecular Microbiology Laboratory, Department of Biological Sciences, KAIST (Korea Advanced Institute of Science and Technology), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, South Korea

Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr


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Gram-negative, non-spore-forming, rod-shaped bacteria, T7-09T and TR6-08T, were isolated from soil from a ginseng field in South Korea and characterized to determine their taxonomic position. 16S rRNA gene sequence analysis showed that the two isolates shared 99·5 % sequence similarity. Strains T7-09T and TR6-08T were shown to belong to the Proteobacteria and showed the highest levels of sequence similarity to Pseudoxanthomonas broegbernensis DSM 12573T (98·1 %), Pseudoxanthomonas mexicana AMX 26BT (97·4–97·5 %), Pseudoxanthomonas japonensis 12-3T (96·5–96·6 %), Pseudoxanthomonas taiwanensis ATCC BAA-404T (95·7 %) and Xanthomonas campestris ATCC 33913T (96·3–96·5 %). The sequence similarity values with respect to any species with validly published names in related genera were less than 96·5 %. The detection of a quinone system with Q-8 as the predominant compound and a fatty acid profile with C15 : 0 iso as the predominant acid supported the assignment of the novel isolates to the order ‘Xanthomonadales’. The two isolates could be distinguished from the established species of the genus Pseudoxanthomonas by the presence of quantitative unsaturated fatty acid C17 : 1 iso {omega}9c and by their unique biochemical profiles. The results of DNA–DNA hybridization clearly demonstrated that T7-09T and TR6-08T represent separate species. On the basis of these data, it is proposed that T7-09T (=KCTC 12208T=IAM 15116T) and TR6-08T (=KCTC 12207T=IAM 15115T) be classified as the type strains of two novel Pseudoxanthomonas species, for which the names Pseudoxanthomonas koreensis sp. nov. and Pseudoxanthomonas daejeonensis sp. nov., respectively, are proposed.

Published online ahead of print on 22 October 2004 as DOI 10.1099/ijs.0.63210-0.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains T7-09T and TR6-08T are AY550263 and AY550264, respectively.


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The order ‘Xanthomonadales’, the nomenclature of which was suggested in the most recent edition of Bergey's Manual of Systematic Bacteriology (Garrity & Holt, 2001Go), is one of 14 orders within the class ‘Gammaproteobacteria’ and contains only one family, the ‘Xanthomonadaceae’. The genus Pseudoxanthomonas, which belongs to the family ‘Xanthomonadaceae’, was first described by Finkmann et al. (2000)Go. At the time of writing, the genus Pseudoxanthomonas consists of two species with validly published names, Pseudoxanthomonas broegbernensis (Finkmann et al., 2000Go) and Pseudoxanthomonas taiwanensis (Chen et al., 2002Go). Phylogenetically, the genus Pseudoxanthomonas is most closely related to the genera Xanthomonas, Stenotrophomonas, Xylella and Luteimonas in the family ‘Xanthomonadaceae’.

In this study, two strains were isolated from soil from a ginseng field in Daejeon, a city in South Korea, and were characterized by using polyphasic approaches. The polyphasic taxonomic analyses indicated that these bacteria represented novel species within the genus Pseudoxanthomonas.

16S rRNA gene sequence studies
16S rRNA gene sequences were analysed as described by Yoon et al. (1997Go, 1998)Go. The 16S rRNA gene sequences of T7-09T and TR6-08T consisted of 1419 and 1462 nt, respectively. The 16S rRNA gene sequences of related taxa were obtained from GenBank. The two isolates shared 99·5 % sequence similarity. Strains T7-09T and TR6-08T were shown to belong to the Proteobacteria; the highest sequence similarities were found with respect to P. broegbernensis DSM 12573T (98·1 %), Pseudoxanthomonas mexicana AMX 26BT (97·4–97·5 %), Pseudoxanthomonas japonensis 12-3T (96·5–96·6 %), P. taiwanensis ATCC BAA-404T (95·7 %) and Xanthomonas campestris ATCC 33913T (96·3–96·5 %). The sequence similarities from any species with validly published names in related genera were less than 96·5 %. Multiple alignments were performed with the CLUSTAL_X program (Thompson et al., 1997Go), gaps were edited using the BioEdit program (Hall, 1999Go) and evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983Go). The phylogenetic tree was constructed by using a neighbour-joining method (Saitou & Nei, 1987Go) in the MEGA 2.0 program (Kumar et al., 2001Go), with bootstrap values based on 100 replications (Felsenstein, 1985Go). Phylogenetically, the two isolates were located on the Pseudoxanthomonas lineage, and the degree of branching was supported by high bootstrap values (Fig. 1Go).



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  		Fig. 1. Phylogenetic tree, based on 16S rRNA gene sequences, showing the phylogenetic relationships of Pseudoxanthomonas species and related genera. The tree was constructed by using the neighbour-joining method, using 16S rRNA gene sequences. Bootstrap values, expressed as percentages of 100 replications, are shown at the branch points. Bar, 0·01 substitution per nucleotide position.

 
DNA studies
The DNA G+C contents of strains T7-09T and TR6-08T were analysed according to the methods of Mesbah et al. (1989)Go and Tamaoka & Komagata (1984)Go and were determined to be 69·5±0·5 mol% and 68·7±0·4 mol%, respectively. DNA–DNA hybridization experiments were carried out as described previously (Ezaki et al., 1989Go). Strain T7-09T hybridized with P. broegbernensis DSM 12573T and TR6-08T at 2·8 and 3·4 %, respectively, while strain TR6-08T hybridized with P. broegbernensis DSM 12573T and T7-09T at 28·9 and 7·6 %, respectively. The results demonstrated that strain T7-09T and strain TR6-08T represent species that are separate from P. broegbernensis.

Chemotaxonomic characteristics
The results of chemotaxonomic analyses are given in the species description. The quinone systems of both isolates, which were analysed according to the procedures of Collins & Jones (1981)Go and Shin et al. (1996)Go, contained Q-8 as the predominant ubiquinone. The quinone system is in accordance with the characteristics of species of the genera Pseudoxanthomonas and Xanthomonas.

Cells used for analysis of cellular fatty acids were cultivated on trypticase soy agar (TSA) at 30 °C for 48 h, then saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). Fatty acids, analysed using a Hewlett Packard 6890 gas chromatograph, were identified by the Microbial Identification software package (Sasser, 1990Go). Both isolates had fatty acid profiles similar to those of P. broegbernensis DSM 12573T and P. taiwanensis ATCC BAA-404T, which contained C15 : 0 iso as the predominant acid (Table 1Go). Both isolates also had another major fatty acid, C17 : 1 iso {omega}9c, which differentiates them from other reference species of the genus Pseudoxanthomonas. Moderate amounts of C15 : 0 anteiso, C16 : 0 iso, C16 : 1 {omega}9c, C17 : 0 iso and unknown 12·075 were also detected.


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  		Table 1. Cellular fatty acid profiles of strains T7-09T and TR6-08T and other species of the genus Pseudoxanthomonas

Strains: 1, P. koreensis T7-09T; 2, P. daejeonensis TR6-08T; 3, P. broegbernensis DSM 12573T (Chen et al., 2002Go); 4, P. taiwanensis ATCC BAA-404T (Chen et al., 2002Go); 5, P. mexicana AMX 26BT (Thierry et al., 2004Go); 6, P. japonensis 12-3T (Thierry et al., 2004Go). Strains T7-09T and TR6-08T were cultured on TSA at 30 °C for 48 h and compared with other type strains under the same conditions. The values shown are percentages of total fatty acids. Unknown fatty acids (with equivalent chain-lengths) are shown.

 
Phenotypic characteristics
During the characterization of organisms isolated from soil from the ginseng field, strains T7-09T and TR6-08T were cultured on R2A agar (Difco) at 30 °C, on which they appeared as white (T7-09T) or yellow (TR6-08T) colonies. On this agar, both isolates were able to grow at 30–37 °C, but not at 4 or 60 °C. Growth at 30 °C was also observed on nutrient agar and TSA.

Gram reactions were tested by using the non-staining method described by Buck (1982)Go. Cell morphology and motility were observed under a Nikon light microscope (x1000), with cells grown for 3 days at 30 °C on R2A agar. The results of physiological characterization are given in the species description. Utilization of substrates as sole carbon sources and other physiological characteristics were determined with the API 32GN and API 20NE galleries, according to the instructions of the manufacturer (bioMérieux).

Reduction of nitrate and nitrite was confirmed by inoculating cells, in each case, into three serum bottles (25 ml) containing 13 ml R2A medium; nitrate or nitrite was added (as KNO3 or NaNO2) at concentrations up to 10 mM. The reduction of nitrate and nitrite was monitored by using an ion chromatograph (model 790 personal IC; Metrohm) equipped with a conductivity detector and an anion-exchange column (Metrosep Anion Supp 4; JL Science); gas production was observed by using small tubes turned upside down in the serum bottles and by using GC.

Taxonomic considerations
16S rRNA gene sequence comparisons and chemotaxonomic characteristics of the isolates suggest the assignment of strains T7-09T and TR6-08T to the genus Pseudoxanthomonas. The DNA–DNA hybridization results, together with the unique physiological profiles of the isolates, demonstrated that strain T7-09T and strain TR6-08T each represent a separate species within the genus Pseudoxanthomonas. These two strains showed very low levels of DNA–DNA hybridization with P. broegbernensis, and were also distant from P. taiwanensis, P. mexicana and P. japonensis in the phylogenetic tree. On the basis of these data, T7-09T (=KCTC 12208T=IAM 15116T) and TR6-08T (=KCTC 12207T=IAM 15115T) should be classified as the type strains of two novel Pseudoxanthomonas species, for which the names Pseudoxanthomonas koreensis sp. nov. and Pseudoxanthomonas daejeonensis sp. nov., respectively, are proposed.

Description of Pseudoxanthomonas koreensis sp. nov.
Pseudoxanthomonas koreensis (ko.re.en'sis. N.L. fem. adj. koreensis pertaining to Korea, the location of the soil sample from which the type strain was isolated).

Cells are Gram-negative, oxidase-positive with an oxidative metabolism, catalase-positive, non-spore-forming, non-motile rods. Good growth occurs aerobically on R2A agar, nutrient agar and TSA at 30 °C; white, semi-translucent, non-shiny colonies with entire edges form within 3 days, having diameters of approximately 1·5–3 mm. The quinone system consists of Q-8. The fatty acid profile is largely composed of C15 : 0 iso and C17 : 1 iso {omega}9c. The G+C content of the genomic DNA is high (69·5±0·5 mol%). Differential phenotypic characteristics are summarized in Table 2Go.


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  		Table 2. Differential phenotypic characteristics for strains T7-09T and TR6-08T and related species

Strains: 1, P. koreensis T7-09T; 2, P. daejeonensis TR6-08T; 3, P. broegbernensis DSM 12573T (Chen et al., 2002Go); 4, P. taiwanensis ATCC BAA-404T (Chen et al., 2002Go); 5, P. mexicana AMX 26BT (Thierry et al., 2004Go); 6, P. japonensis 12-3T (Thierry et al., 2004Go). Symbols: +, positive; –, negative; +/–, weakly positive; V, variable; ND, no data. Strains T7-09T and TR6-08T are negative for assimilation of 3-hydroxybenzoate, 4-hydroxybenzoate, 2-ketogluconate, 5-ketogluconate, adipate, caprate, L-fucose, gluconate, itaconate, DL-lactate, malonate, phenyl acetate, propionate, suberate, valerate, D-ribose, mannitol, L-histidine, glycogen and salicin.

 
The type strain, T7-09T (=KCTC 12208T=IAM 15116T), was isolated from soil from a ginseng field in Daejeon, South Korea.

Description of Pseudoxanthomonas daejeonensis sp. nov.
Pseudoxanthomonas daejeonensis (dae.je.on.en'sis. N.L. fem. adj. daejeonensis pertaining to Daejeon in Korea, the location of the soil sample from which the type strain was isolated).

Cells are Gram-negative, oxidase-positive with an oxidative metabolism, catalase-positive, non-spore-forming, motile rods. Good growth occurs aerobically on R2A agar, nutrient agar and TSA at 30 °C; yellow, semi-translucent and non-shiny colonies with entire edges form within 3 days, having diameters of approximately 1·5–3 mm. The quinone system consists of Q-8. The fatty acid profile is largely composed of C15 : 0 iso and C17 : 1 iso {omega}9c. The G+C content of the genomic DNA is high (68·7±0·4 mol%). Differential phenotypic characteristics are summarized in Table 2Go.

The type strain, TR6-08T (=KCTC 12207T=IAM 15115T), was isolated from soil from a ginseng field in Daejeon, South Korea.


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Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]

Finkmann, W., Altendorf, K., Stackebrandt, E. & Lipski, A. (2000). Characterization of N2O-producing Xanthomonas-like isolates from biofilters as Stenotrophomonas nitritireducens sp. nov., Luteimonas mephitis gen. nov., sp. nov. and Pseudoxanthomonas broegbernensis gen. nov., sp. nov. Int J Syst Evol Microbiol 50, 273–282.[Abstract]

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