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1 Korean Agricultural Culture Collection (KACC), Genetic Resources Division, National Institute of Agricultural Biotechnology, 225th, Seodun-dong Gwonseon-gu, Suwon 441-707, Korea
2 Korean Agricultural Culture Collection (KACC), Metabolic Engineering Division, National Institute of Agricultural Biotechnology, 225th, Seodun-dong Gwonseon-gu, Suwon 441-707, Korea
4 Korean Agricultural Culture Collection (KACC), Molecular Physiology Division, National Institute of Agricultural Biotechnology, 225th, Seodun-dong Gwonseon-gu, Suwon 441-707, Korea
3 College of Agriculture and Life Sciences, 192-1, Hyoja2-Dong, Chunchon 200-701, Korea
Correspondence
Soon Wo Kwon
swkwon{at}rda.go.kr
| ABSTRACT |
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The GenBank accession numbers for the 16S rDNA sequences described in this work are AF468448AF468453, as indicated in Fig. 2
.
A 16S rDNA-based phylogenetic tree containing additional reference sequences is available as supplementary data in IJSEM Online (http://ijs.sgmjournals.org/).
| MAIN TEXT |
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-,
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-subclasses).
Members of the genus Pseudomonas are widely distributed in agricultural soils; they have a variety of functions related to the decomposition of organic matter and the promotion of plant growth and can also show pathogenic effects (Palleroni, 1993
). Most of the farming soils in Korea are characterized, chemically, by low soil pH values and in terms of cultivation methods, by rotation of upland and paddy land. A soil environment such as this, together with Korea's peninsular isolation, would tend to influence the population structure and evolution of Pseudomonas species and lead to the development of a bacterial population that differs from those of other regions. We isolated several groups of the genus Pseudomonas from Korean agricultural soils. As a result of 16S rRNA gene sequence analysis of these strains, we found three phylogenetic groups distinct from the established species of the genus Pseudomonas. In this study, the strains of the three groups were characterized using phenotypic and genomic characteristics to confirm their taxonomic status.
Four strains (Ps 1-2, Ps 1-10, Ps 5-5 and Ps 9-14T) from soils of the Goesan, Samchok and Umsong regions, three strains (Ps 2-22, Ps 3-1 and Ps 3-10T) from soil of Umsong Region and four strains (Pss 14, Pss 25, Pss 26T and Pss 27) from soil of Jinju Region in Korea were isolated using P1 agar medium (l-1: 1 g KH2PO4, 0·5 g MgSO4.7H2O, 0·2 g KCl, 5 g NaNO3, 1 g deoxycholic acid, 5 g betaine and 15 g agar; Kato & Itoh, 1983
). In general, all strains were cultured on trypticase soy agar (TSA) medium at 30 °C unless otherwise stated. Strains were preserved using two methods: deep-freezing with 15 % glycerol and freeze-drying with 15 % skim milk.
For observation of cell morphology by TEM, cells were grown on TSA and suspended in physiological saline solution. A small drop of suspension was placed on a carbon-coated copper grid and the cells were negatively stained with 0·5 % uranyl acetate for observation under the electron microscope (model 912AB; LEO). To investigate basic physiological and biochemical characteristics, we used the methods of Stanier et al. (1966)
and Schaad (1988)
for the following tests: Gram reaction, oxidase reaction, arginine dihydrolase, nitrate reduction, levan formation from sucrose and hydrolysis of gelatin, starch, Tween 80 and aesculin. Catalase was assayed with the SpotTest catalase test (Difco). Fluorescent pigment production was tested on King medium B (King et al., 1954
) and Pseudomonas agar F (PAF; Difco). Temperature tolerance was tested by checking growth at 4, 30, 37 and 41 °C and tolerance of salinity was tested with growth on trypticase soy broth supplemented with 1, 3, 5, 7 and 9 % (w/v) NaCl and solidified with 1·5 % (w/v) agar. Utilization of carbon sources was examined by using the Biolog identification system. All strains were tested three times with GN2 microplates (Biolog) according to the manufacturer's recommendations; the reactions were observed after 24 or 48 h. An API 20 NE test kit was also used for classical and phenotypic tests; examination was done after 48 h.
Genomic DNA was isolated by the method of Ausubel et al. (1987)
, except that the lysates were extracted twice with chloroform to remove residual phenol. The concentration of DNA was measured by using a spectrophotometer. The G+C content of genomic DNA was determined by HPLC (by the Laboratorium voor Microbiologie, Ghent, Belgium) as described previously (Mesbah et al., 1989
). To determine genomic relatedness, the filter hybridization method was performed according to Seldin & Dubnau (1985)
. Probe labelling was conducted by using the non-radioactive DIG-High prime system (Roche); hybridized DNA was visualized using the DIG luminescent detection kit (Roche). Reassociation was conducted at two temperatures, 60 and 65 °C. DNADNA relatedness was quantified by using a densitometer (Bio-Rad).
16S rDNAs were amplified by using universal primers fD1 and rP2 (Weisburg et al., 1991
) and their nucleotide sequences were determined with an Applied Biosystems 377 sequencer (Applied Biosystems). The 16S rDNA 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 the neighbour-joining program of MEGA (Kumar et al., 1993
). The stability of relationships was assessed by performing bootstrap analyses of the neighbour-joining data based on 1000 resamplings.
All strains of the three groups are Gram-negative and non-spore-forming rods. Cells are approximately 1 µm by 1·5 (strain Ps 3-10T) or 2 (Ps 9-14T and Pss 26T) µm in size and are motile by means of single (Ps 3-10T and Pss 26T) or multiple (Ps 9-14T) polar flagella (Fig. 1
). A comparison of physiological and biochemical characteristics among the Ps 9-14, Ps 3-10 and Pss 26 groups and other closely related Pseudomonas species is shown in Table 1
. The Ps 9-14 group is phenotypically related to the Ps 3-10 group, but can be clearly differentiated from it by the presence of multiple flagella, by the absence of nitrate reduction and by Tween 80 hydrolysis. The Ps 9-14 and Ps 3-10 groups also share phenotypic characteristics with Pseudomonas jessenii and Pseudomonas pavonaceae, but the two novel groups can be clearly distinguished from the two established species by virtue of several biochemical characteristics. Members of the Pss 26 group shows phenotypic relationships with Pseudomonas citronellolis, Pseudomonas nitroreducens and Pseudomonas alcaligenes. However, Pss 26 group members are generally differentiated from P. citronellolis by the absence of fluorescence and the absence of growth at 4 °C and can be distinguished from P. nitroreducens by the absence of fluorescence and by the ability to grow at 41 °C. Although the phenotypic differentiation of Pss 26 group members from P. alcaligenes is not clear, none of the strains in the Pss 26 group hydrolyses gelatin or Tween 80, whereas some strains of P. alcaligenes do (Table 1
). Many of the phenotypic traits of P. citronellolis were tested in this study using strain LMG 18378T because its taxonomic characterization was not fully determined previously (Seubert, 1960
). Phenotypic variability is observed among strains of each of the three groups. The absence of gelatin hydrolysis and lecithinase production, the presence of glucose acidification and the assimilation of D-galacturonic acid, D-glucuronic acid and
-ketobutyric acid differentiate strain Ps 1-2 from the other strains of the Ps 9-14 group, while the absence of L-pyroglutamic acid utilization and the presence of urease production are observed only for strain Ps 1-10. The strains of the Ps 3-10 group form a relatively homogeneous group and cannot be differentiated using the key phenotypic characteristics shown in Table 1
. However, on the basis of carbohydrate utilization, strain Ps 2-22 can be differentiated from strains Ps 3-1 and Ps 3-10T in that it utilizes D-arabitol, D-mannitol and D-psicose but does not use D-glucuronic acid or hydroxy-L-proline. The four strains of the Pss 26 group are also homogeneous since they shared most phenotypic characteristics, except for the assimilation of certain carbohydrates; strain Pss 14 uses hydroxy-L-proline and cannot use acetic acid and adipate, unlike the other three strains. The G+C contents of strains Ps 9-14T, Ps 3-10T and Pss 26T are respectively 60·7, 60·0 and 66·9 mol%, all of which fall within the expected range for the genus Pseudomonas (5870 mol%; Palleroni, 1984
) (Table 1
).
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To study DNADNA relatedness among the three novel groups and closely related Pseudomonas species, the genomic DNA of each of strains Ps 9-14T, Ps 3-10T and Pss 26T was labelled and hybridized to those of the Korean isolates and closely related Pseudomonas species at two hybridization temperatures (60 and 65 °C). The DNA relatedness values among strains within each of three novel groups were greater than 92 % (Table 2
). Genomic DNA relatedness between strain Ps 9-14T and other Pseudomonas species ranged from 14 to 56 % and from 7 to 42 % at hybridization temperatures of 60 and 65 °C, respectively, and strain Ps 9-14T showed high DNA relatedness to strain Ps 3-1 and Pseudomonas migulae CIP 105470T. Strain Ps 3-10T exhibited DNA relatedness of 948 % and 742 % at hybridization temperatures of 60 and 65 °C, respectively, and showed high levels of relatedness to strain Ps 9-14T and P. jessenii CIP 105274T. The level of DNA relatedness between strain Pss 26T and other Pseudomonas species varied from 17 to 54 % and from 6 to 38 % at 60 and 65 °C, respectively. Strain Pss 26T showed the highest value for DNA relatedness to P. citronellolis LMG 18378T (Table 2
).
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Tm value of 5 °C or less constitute a single species (Wayne et al., 1987In the light of the results presented here, we describe three novel species, Pseudomonas koreensis sp. nov., Pseudomonas umsongensis sp. nov. and Pseudomonas jinjuensis sp. nov.
Description of Pseudomonas koreensis sp. nov.
Pseudomonas koreensis (ko.re.en'sis. N.L. adj. koreensis pertaining to Korea).
Cells are Gram-negative, non-spore-forming rods, approximately 1x2 µm in size, motile by more than one polar flagellum. Colonies are circular and white-yellow on LuriaBertani (LB) agar and become mucoid after 2 days when cultured on TSA. Cells produce a fluorescent pigment on King B and PAF media. Catalase- and oxidase-positive and shows hydrolysis of arginine and Tween 80. Most strains liquefy gelatin, but do not show hydrolysis of starch and show no acidification of glucose. Reduction of nitrate to nitrite is negative. Most strains show positive lecithinase reaction. Urease reaction is variable among strains. Indole is not produced on tryptophan. Strains grow at 4 °C but not at 37 °C. Growth occurs in media supplemented with 5 % NaCl, but not at a salinity higher than 7 %. Results obtained with Biolog GN2 microplates indicate that strains utilize Tweens 40 and 80, N-acetyl-D-glucosamine, L-arabinose, D-arabitol, D-fructose, D-galactose,
-D-glucose, D-mannitol, D-mannose, methyl pyruvate, monomethyl succinate, acetic acid, cis-aconitic acid, citric acid, D-galactonic acid lactone, D-gluconic acid, D-glucosaminic acid,
-hydroxybutyric acid,
-hydroxybutyric acid,
-ketovaleric acid, DL-lactic acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, succinic acid, bromosuccinic acid, glucuronamide, alaninamide, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-glutamic acid, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-proline, L-serine, L-threonine,
-aminobutyric acid, urocanic acid, inosine, uridine, putrescine, 2-aminoethanol and glycerol. Utilization of D-galacturonic acid, D-glucuronic acid,
-ketobutyric acid and L-pyroglutamic acid is variable among strains. The other organic substrates included in Biolog GN2 microplates are not utilized. The test using the API 20 NE strip shows that strains assimilate glucose, arabinose, mannose, mannitol, N-acetylglucosamine, gluconate, caprate, malate and citrate. Strains do not assimilate maltose, adipate or phenylacetate. The G+C content of the DNA of strain Ps 9-14T is 60·7 mol%. The type strain is strain Ps 9-14T (=KACC 10848T =LMG 21318T).
Description of Pseudomonas umsongensis sp. nov.
Pseudomonas umsongensis (um.song.en'sis. N.L. adj. umsongensis referring to Umsong Region in Korea, where the bacteria were first found).
Cells are Gram-negative, non-spore-forming rods approximately 1x1·5 µm in size, motile by a single polar flagellum. Colonies are circular and white-yellow on LB agar. Cells produce a fluorescent pigment on King B and PAF media. Catalase- and oxidase-positive, shows hydrolysis of arginine and shows nitrate reduction, but does not hydrolyse gelatin, Tween 80 or starch. Does not show lecithinase, urease or
-galactosidase reactions. Indole is not produced on tryptophan and there is no acidification of glucose. Strains grow at 4 °C but not at 37 °C. Growth occurs in media supplemented with 5 % NaCl, but not at a salinity higher than 7 %. Results obtained with Biolog GN2 microplates indicate that strains utilize Tweens 40 and 80, L-arabinose, D-fructose, D-galactose,
-D-glucose, D-mannose, methylpyruvate, monomethyl succinate, acetic acid, cis-aconitic acid, citric acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid,
-hydroxybutyric acid,
-hydroxybutyric acid,
-ketobutyric acid,
-ketoglutaric acid,
-ketovaleric acid, DL-lactic acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, succinic acid, bromosuccinic acid, alaninamide, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-glutamic acid, L-histidine, L-leucine, L-ornithine, L-proline, L-pyroglutamic acid, L-serine, L-threonine, DL-carnitine,
-aminobutyric acid, urocanic acid, phenylethylamine, putrescine, 2-aminoethanol and glycerol. Utilization of D-arabitol, D-mannitol, D-psicose, D-glucuronic acid and hydroxy-L-proline is variable among strains. The other organic substrates included in the Biolog GN2 microplates are not utilized. The test using the API 20 NE strip shows that strains assimilate glucose, arabinose, mannose, gluconate, caprate, malate, citrate and phenylacetate. Assimilation of mannitol is variable among strains. The strains do not assimilate N-acetylglucosamine, maltose or adipate. The G+C content of the DNA of Ps 3-10T is 60·0 mol%. The type strain is strain Ps 3-10T (=KACC 10847T =LMG 21317T).
Description of Pseudomonas jinjuensis sp. nov.
Pseudomonas jinjuensis (jin.ju.en'sis. N.L. adj. jinjuensis referring to Jinju Region in Korea, where the bacteria were first found).
Cells are Gram-negative, non-spore-forming rods approximately 1x2 µm in size, motile by a single polar flagellum. Colonies are circular and white-yellow on LB agar. No fluorescent pigments are produced on King B or PAF media. Catalase- and oxidase-positive. Strains show denitrification and hydrolysis of arginine. There is no hydrolysis of gelatin, Tween 80 or starch and no acidification of glucose is observed. Indole is not produced from tryptophan. Lecithinase, urease and
-galactosidase reactions are negative. Strains grow at 41 °C but not at 4 °C. Growth occurs in media supplemented with 3 % NaCl, but not at a salinity higher than 5 %. Results obtained with Biolog GN2 microplates indicate that strains utilize Tweens 40 and 80,
-D-glucose, methylpyruvate, monomethyl succinate, cis-aconitic acid, citric acid, formic acid, D-gluconic acid,
-hydroxybutyric acid,
-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid,
-ketobutyric acid,
-ketoglutaric acid,
-ketovaleric acid, DL-lactic acid, propionic acid, quinic acid, sebacic acid, succinic acid, bromosuccinic acid, alaninamide, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, L-histidine, L-leucine, L-ornithine, L-proline, L-serine, L-threonine, DL-carnitine, urocanic acid, phenylethylamine, putrescine and glycerol. Utilization of acetic acid and hydroxy-L-proline is variable among strains. The other organic substrates included in the Biolog GN2 microplates are not utilized. The test using the API 20 NE strip shows that the strains assimilate glucose, gluconate, caprate, malate, citrate and phenylacetate. Assimilation of adipate is variable among strains. The strains do not assimilate arabinose, mannose, mannitol, N-acetylglucosamine or maltose. The G+C content of the DNA of strain Pss 26T is 66·9 mol%. The type strain is strain Pss 26T (=KACC 10760T =LMG 21316T).
| ACKNOWLEDGEMENTS |
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H.-Y. Weon, B.-Y. Kim, S.-H. Yoo, J.-H. Joa, S.-W. Kwon, and W.-G. Kim Andreprevotia chitinilytica gen. nov., sp. nov., isolated from forest soil from Halla Mountain, Jeju Island, Korea Int J Syst Evol Microbiol, July 1, 2007; 57(7): 1572 - 1575. [Abstract] [Full Text] [PDF] |
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B. Camara, C. Strompl, S. Verbarg, C. Sproer, D. H. Pieper, and B. J. Tindall Pseudomonas reinekei sp. nov., Pseudomonas moorei sp. nov. and Pseudomonas mohnii sp. nov., novel species capable of degrading chlorosalicylates or isopimaric acid Int J Syst Evol Microbiol, May 1, 2007; 57(5): 923 - 931. [Abstract] [Full Text] [PDF] |
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S.-W. Kwon, B.-Y. Kim, H.-Y. Weon, Y.-K. Baek, and S.-J. Go Arenimonas donghaensis gen. nov., sp. nov., isolated from seashore sand Int J Syst Evol Microbiol, May 1, 2007; 57(5): 954 - 958. [Abstract] [Full Text] [PDF] |
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U. Behrendt, A. Ulrich, P. Schumann, J.-M. Meyer, and C. Sproer Pseudomonas lurida sp. nov., a fluorescent species associated with the phyllosphere of grasses Int J Syst Evol Microbiol, May 1, 2007; 57(5): 979 - 985. [Abstract] [Full Text] [PDF] |
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S.-W. Kwon, B.-Y. Kim, K.-H. Lee, K.-Y. Jang, S.-J. Seok, J.-S. Kwon, W.-G. Kim, and H.-Y. Weon Pedobacter suwonensis sp. nov., isolated from the rhizosphere of Chinese cabbage (Brassica campestris) Int J Syst Evol Microbiol, March 1, 2007; 57(3): 480 - 484. [Abstract] [Full Text] [PDF] |
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O. Prakash, K. Kumari, and R. Lal Pseudomonas delhiensis sp. nov., from a fly ash dumping site of a thermal power plant Int J Syst Evol Microbiol, March 1, 2007; 57(3): 527 - 531. [Abstract] [Full Text] [PDF] |
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B.-Y. Kim, H.-Y. Weon, S.-H. Yoo, S.-B. Hong, S.-W. Kwon, E. Stackebrandt, and S.-J. Go Niabella aurantiaca gen. nov., sp. nov., isolated from a greenhouse soil in Korea Int J Syst Evol Microbiol, March 1, 2007; 57(3): 538 - 541. [Abstract] [Full Text] [PDF] |
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A. Stolz, H.-J. Busse, and P. Kampfer Pseudomonas knackmussii sp. nov. Int J Syst Evol Microbiol, March 1, 2007; 57(3): 572 - 576. [Abstract] [Full Text] [PDF] |
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S.-H. Yoo, H.-Y. Weon, B.-Y. Kim, J.-H. Kim, Y.-K. Baek, S.-W. Kwon, S.-J. Go, and E. Stackebrandt Pseudoxanthomonas yeongjuensis sp. nov., isolated from soil cultivated with Korean ginseng Int J Syst Evol Microbiol, March 1, 2007; 57(3): 646 - 649. [Abstract] [Full Text] [PDF] |
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B.-Y. Kim, H.-Y. Weon, S.-H. Yoo, S.-Y. Lee, S.-W. Kwon, S.-J. Go, and E. Stackebrandt Variovorax soli sp. nov., isolated from greenhouse soil Int J Syst Evol Microbiol, December 1, 2006; 56(12): 2899 - 2901. [Abstract] [Full Text] [PDF] |
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B.-Y. Kim, H.-Y. Weon, S.-H. Yoo, J.-S. Kim, S.-W. Kwon, E. Stackebrandt, and S.-J. Go Marinob |