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1 Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural Development Administration (RDA), Suwon 441-707, Korea
2 Korean Agricultural Culture Collection (KACC), Genetic Resources Division, National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea
3 Department of Environmental Sciences, University of California, Riverside, CA 92521, USA
4 School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea
5 Environmental and Molecular Microbiology Lab, Department of Biological Sciences, KAIST (Korea Advanced Institute of Science and Technology), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea
Correspondence
Soon-Wo Kwon
swkwon{at}rda.go.kr
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 4M1T is AY927994.
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, is placed in the family Xanthomonadaceae, order Xanthomonadales, class Gammaproteobacteria and phylum Proteobacteria (Garrity & Holt, 2001) in the taxonomic hierarchy. This genus is closely related phylogenetically with the genera Xanthomonas, Xylella and Stenotrophomonas (Finkmann et al., 2000; Chen et al., 2002; Thierry et al., 2004; Yang et al., 2005). However, members of the genus Pseudoxanthomonas can be differentiated from members of these related genera by the reduction of nitrate but not nitrite and the lack of C13 : 0 iso 3-OH fatty acid (Yang et al., 1993; Finkmann et al., 2000; Assih et al., 2002). The genus Pseudoxanthomonas currently comprises six species, Pseudoxanthomonas broegbernensis (type species; Finkmann et al., 2000), P. taiwanensis (Chen et al., 2002), P. mexicana (Thierry et al., 2004), P. japonensis (Thierry et al., 2004), P. koreensis (Yang et al., 2005) and P. daejeonensis (Yang et al., 2005). In the course of studying the bacterial diversity associated with cotton waste composts, several strains were isolated, showing a yellow colony colour on CASO agar (DSMZ medium no. 220; http://www.dsmz.de/media/media.htm) incubated at 30 °C. Among them, three, 4M1T, 4M9 and 4M12, were phylogenetically distinct from the known species within the genus Pseudoxanthomonas.
Gram staining, KOH test and L-alanine aminopeptidase assay were performed by using a Gram stain kit (Difco), 3 % (w/v) KOH (Buck, 1982) and Bactident aminopeptidase (Merck), respectively. Motility was tested on motility medium of 0·1 % yeast extract, 0·01 % K2HPO4 and 0·2 % agar. Oxidative or fermentative utilization of glucose was determined in Hugh & Leifson medium (Hugh & Leifson, 1953). Catalase activity was tested by 3 % (v/v) H2O2 solution. Morphology was observed using oil immersion phase-contrast microscopy after 1 day incubation on CASO agar at 30 °C. Oxidase, hydrolysis of aesculin, cellulose, starch, casein, gelatin, Tweens 20, 40 and 80 and DNA and indole production were assessed according to the methods of Smibert & Krieg (1994). Hydrolysis of chitin (1 %, w/v) and tyrosine (0·5 %, w/v) was tested by the appearance of a clear zone around the colonies. Urease activity was determined by the method described by MacFaddin (2000). The pH range (pH 4·0–10·0 at intervals of 1·0 pH unit) for growth was determined on CASO agar that was buffered with citrate-phosphate buffer or Tris/HCl (Breznak & Costilow, 1994). Growth at various NaCl concentrations was investigated in CASO broth and growth at various temperatures (5–50 °C) was measured on CASO agar. Tests using the commercial systems API ZYM, API 20NE and API 50CH (bioMérieux) were generally performed according to the manufacturer's instructions. The API ZYM tests were read after 4 h incubation at 30 °C and the other API tests were read after 48 h at 30 °C. The Biolog GN identification system was also used as recommended by the manufacturer at 30 °C. Sensitivity to antibiotics was determined with the routine disc-diffusion plate method. For the nitrate and nitrite reduction test, each isolate was inoculated into three serum bottles (25 ml) containing 13 ml R2A medium, while nitrate and nitrite were added as KNO3 and NaNO2 at concentrations of 10 mM. The reduction of nitrate and nitrite was monitored by ion chromatograph (model 790 personal IC; Metrohm) equipped with a conductivity detector and anion-exchange column (Metrosep Anion Supp 4; Metrohm).
Fatty acid methyl esters were extracted and prepared by the standard protocol of the Microbial Identification system (MIDI; Microbial ID) after cells were grown on tryptic soy agar for 24 h at 30 °C. Isoprenoid quinones were analysed by HPLC as described previously (Groth et al., 1996). The DNA G+C content (mol%) was determined by HPLC analysis of deoxyribonucleosides as described by Mesbah et al. (1989) using a reverse-phase column (Supelcosil LC-18-S; Supelco).
The 16S rRNA genes were amplified (Kwon et al., 2003) and directly sequenced (Hiraishi, 1992). To establish the phylogenetic positions of the strains, the nearly complete 16S rRNA gene sequence of strain 4M1T comprising 1509 nt was determined and the phylogenetic tree was constructed together with the sequences of related taxa obtained from GenBank. The sequences were aligned by using the MEGALIGN program of DNASTAR. An evolutionary distance matrix was generated as described by Jukes & Cantor (1969). The evolutionary tree for the datasets was inferred from the neighbour-joining method of Saitou & Nei (1987) by using MEGA version 2.1 (Kumar et al., 2001). The stability of relationships was assessed by performing bootstrap analyses of the neighbour-joining data based on 1000 resamplings.
DNA–DNA hybridization was carried out as a filter-hybridization method described by Seldin & Dubnau (1985). Probe labelling was conducted by using the non-radioactive DIG-High prime system (Roche) and hybridized DNA was visualized using the DIG luminescent detection kit (Roche). DNA–DNA relatedness was quantified by using a densitometer (Bio-Rad).
The strains were aerobic, motile, Gram-negative rods, generally 0·3–0·5x1·2–3·0 µm in size (Supplementary Fig. S1 available in IJSEM Online). Colonies were round, yellow and convex with entire margins. These strains were positive for oxidase and catalase, KOH ring test, hydrolysis of aesculin, Tween 20, tyrosine and DNA. They showed negative reactions in tests for arginine dihydrolase, hydrolysis of casein, cellulose, chitin, Tween 40, starch and urea and indole production. Optimal growth of all the strains occurred at 30 °C and they grew in the range of 10–45 °C. They grew in the pH range of 6·0–8·0 and in the presence of 5 % (w/v) NaCl, but not at 7 % NaCl.
The hydrolysis of gelatin and Tween 80 showed discrepancies between the different test methods. Therefore, the conventional test results were used (Table 1). In API 50CH, none of the tested strains showed any reactions except for the hydrolysis of aesculin, indicating that this test kit was inappropriate for testing the metabolic properties of these strains. Other metabolic properties are shown in Supplementary Table S1 (available in IJSEM Online). In nitrate and nitrite reduction tests, two isolates, 4M1T and 4M9, revealed the reduction of nitrate to nitrite but no nitrite reduction, whereas isolate 4M12 showed nitrite reduction but no reduction of nitrate to nitrite (results are available in Supplementary Table S2 in IJSEM Online).
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). However, these three novel strains had trace amounts (0·1–0·2 %) of iso-C13 : 0 3-OH. The predominant isoprenoid quinone of the tested strains was Q-8. The DNA G+C content of the strains was 66·6–68·4 mol%.
Strain 4M1T was phylogenetically clustered in the genus Pseudoxanthomonas (Fig. 1) and the sequence similarity was 96·9–99·0 % with Pseudoxanthomonas species with validly published names. Strain 4M1T showed the highest sequence similarity, 99·0 and 98·6 %, respectively, with P. koreensis T7-09T and P. daejeonensis TR6-08T. The similarity of the 16S rRNA gene sequences of strain 4M1T to those of all other species within phylogenetically closely related genera such as Xanthomonas, Xylella and Stenotrophomonas was below 97 %.
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). The DNA–DNA hybridization values between strain 4M1T and type strains of other Pseudoxanthomonas species were: 52 (P. broegbernensis), 57 (P. japonensis), 62 (P. mexicana), 60 (P. taiwanensis), 58 (P. koreensis), 63 (P. daejeonensis) and 56 % (P. kaohsiungensis). Although strain 4M1T showed a relatively high degree of 16S rRNA gene sequence similarity, it could be differentiated from other species of the genus Pseudoxanthomonas by means of DNA–DNA hybridization and a few physiological tests. In conclusion, we propose the name Pseudoxanthomonas suwonensis sp. nov. for the three novel strains.
Description of Pseudoxanthomonas suwonensis sp. nov.
Pseudoxanthomonas suwonensis (su.won.en'sis. N.L. fem. adj. suwonensis referring to Suwon region, where the bacterium was first found).
Cells are aerobic, Gram-negative, motile rods, generally 0·3–0·5x1·2–3·0 µm in size, occurring singly. Colonies are round, yellow and convex with entire margins. Temperature range for growth is 10–45 °C and optimal temperature is 30 °C. Growth occurs in the pH range of 6·0–8·0 and in the presence of up to 5 % (w/v) NaCl. Positive for oxidase, catalase and hydrolysis of aesculin, Tween 20, tyrosine and DNA. Negative for arginine dihydrolase, hydrolysis of casein, cellulose, chitin, Tween 40, starch and urea and indole production. The predominant isoprenoid quinone is Q-8. Principal fatty acids (greater than 5 %) are iso-C15 : 0 (30·8 %), anteiso-C15 : 0 (13·2 %), iso-C16 : 0 (11·1 %), iso-C17 : 1
9c (10·6 %), iso-C11 : 0 (7·4 %) and iso-C11 : 0 3-OH (7·0 %). The DNA G+C content is 66·6–68·4 mol%.
The type strain, 4M1T (=KACC 11320T=DSM 17175T), was isolated from cotton waste composts in Korea.
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