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1 Environmental and Molecular Microbiology Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, South Korea
2 National University of Uzbekistan, Students town, Tashkent, 700-174, Uzbekistan
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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8c and C18 : 0. The G+C content of genomic DNA of strain Y9T was 65.1 mol%. The results of DNA–DNA hybridization in combination with chemotaxonomic and physiological data demonstrated that strain Y9T represents a novel species within the genus Pleomorphomonas, for which the name Pleomorphomonas koreensis sp. nov. is proposed. The type strain is Y9T (=KCTC 12246T=NBRC 100803T)
Present address: Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, South Korea. 
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), the order Rhizobiales is the biggest group within the 
-2 subgroup of the Proteobacteria and constitutes a heterogeneous group from both a physiological and an ecological perspective. According to Bergey's Manual, the order Rhizobiales is divided into ten families on the basis of 16S rRNA gene phylogenetic analyses: Bartonellaceae, Beijerinckiaceae, Bradyrhizobiaceae, Brucellaceae, Hyphomicrobiaceae, Methylobacteriaceae, Methylocystaceae, Phyllobacteriaceae, Rhizobiaceae and Rhodobiaceae. The order Rhizobiales is characterized chemotaxonomically by its major respiratory ubiquinone (Q-10) and by the presence of octadecenoic acid (C18 : 1) as a major cellular fatty acid (like other members of the Alphaproteobacteria), and it contains plant and animal pathogens associated with eukaryotic cells (e.g. the families Bartonellaceae and Brucellaceae), plant endosymbionts (Rhizobiaceae), methane-oxidizing bacteria (Methylobacteriaceae and Methylocystaceae) and, in particular, a large number of nitrogen-fixing species (distributed throughout almost all of its families). In this study, a nitrogen-fixing, rod-shaped strain (Y9T) was isolated from a contaminated culture of the phototrophic bacterium Rhodopseudomonas palustris. On the basis of 16S RNA gene sequence data, strain Y9T was found to be closely related to Pleomorphomonas oryzae (order Rhizobiales of the Alphaproteobacteria) and was subjected to a taxonomic investigation. The aim of this study was to determine the taxonomic position of strain Y9T by using chemotaxonomic, physiological and DNA–DNA hybridization analyses. The results provided evidence that Y9T is a representative of a novel bacterial species.
Strain Y9T was isolated from a contaminated culture of the phototrophic bacterium R. palustris by direct plating on R2A agar (Difco). For analysis of quinones and DNA extraction, cell biomass was obtained from a culture grown in R2A broth at 30 °C. Strain Y9T was cultivated on a horizontal shaker at 150 r.p.m., and the purity was checked using a light microscope prior to harvesting by centrifugation. Unless indicated otherwise, the strain was grown on R2A agar or broth incubated at 30 °C and maintained as a glycerol suspension (20 %, w/v) at –70 °C.
Cell morphology and motility were observed under a Nikon light microscope (x1000 magnification) using cells grown for 2 days at 30 °C on R2A agar. The Gram reaction was performed using the non-staining method described by Buck (1982). Tests for catalase and oxidase were performed by using the procedures outlined by Cappuccino & Sherman (2002). Growth at various temperatures (4, 15, 25, 30, 37 and 42 °C) was investigated on R2A agar. Growth at 30 °C on nutrient agar, trypticase soy agar and MacConkey agar was also examined. Utilization of various substrates as sole carbon sources was investigated, together with some physiological characteristics, using the API 32GN and API 20NE galleries according to the instructions of the manufacturer (bioMérieux).
Nitrogen-fixing ability was determined by growing strain Y9T in 50 ml nitrogen-free medium (DSMZ medium no. 3; Deutsche Sammlung von Mikroorganismen und Zellkulturen) contained in a 125 ml serum bottle. The medium contained the following (in 1 l distilled water): 5.0 g glucose, 5.0 g mannitol, 0.1 g CaCl2.2H2O, 0.1 g MgSO4.7H2O, 5.0 mg Na2MoO4.2H2O, 0.9 g K2HPO4, 0.1 g KH2PO4, 0.01 g FeSO4.7H2O, 5.0 g CaCO3 and 1 ml trace element mixture. The trace element mixture (SL-6, in DSMZ medium no. 27) contained the following (l–1 distilled water): 0.1 g ZnSO4.7H2O, 0.03 g MnCl2.4H2O, 0.3 g H3BO3, 0.2 g CoCl2.6H2O, 0.01 g CuCl2.2H2O and 0.02 g NiCl2.6H2O. Acetylene reduction was performed for all liquid cultures by injecting purified acetylene into appropriate containers closed with rubber stoppers to yield 15 % acetylene (v/v); this was followed by incubation for up to 24 h. Ethylene was measured using a Hewlett Packard 5890A gas chromatograph equipped with a flame-ionization detector and a prepacked column (HayeSep N; Supelco). Additionally, the presence of the nifH gene was determined by a PCR with primers and reaction conditions as described by Poly et al. (2001).
The G+C content of the chromosomal DNA of strain Y9T was determined as described by Mesbah et al. (1989), using reversed-phase HPLC. Quinones were extracted from cells grown on nutrient broth (Difco), and analysed as described by Shin et al. (1996), using reversed-phase HPLC. Cellular fatty acids were analysed in organisms grown on trypticase soy agar (Difco) for 2 days. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acids, analysed by GC (6890; Hewlett Packard), were identified using the Microbial Identification software package (Sasser, 1990). DNA–DNA hybridization was performed fluorometrically by using the method of Ezaki et al. (1989), with photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values are quoted as DNA relatedness values.
PCR-mediated amplification of the 16S rRNA gene and sequencing of the purified PCR product were carried out according to Kim et al. (2005). The complete 16S rRNA gene sequence was compiled using SeqMan software (DNASTAR). 16S rRNA gene sequences of related taxa were obtained from the GenBank database. Multiple alignments were performed using the CLUSTAL X program (Thompson et al., 1997). Gaps were edited in the BioEdit program (Hall, 1999). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). The phylogenetic tree was constructed by using a neighbour-joining method (Saitou & Nei, 1987) and maximum parsimony (Fitch, 1972) in the MEGA 3 program (Kumar et al., 2004), with bootstrap values based on 1000 replications (Felsenstein, 1985).
Strain Y9T consisted of Gram-negative, non-spore-forming, non-motile rods. The cells were 0.3–0.5 µm in diameter and 1.5–3.0 µm in length. After 2 days incubation on R2A agar, colonies were smooth, low-convex, circular, translucent, non-shiny and white in colour. Strain Y9T grew at 15–42 °C on R2A agar but not at 4 °C or above 45 °C. Strain Y9T was able to reduce acetylene to ethylene at a mean rate of 57.5 µmol min–1 (mg wet weight)–1 at an incubation temperature of 30 °C. An internal region of nifH (about 360 bp) was clearly amplified, and strain Y9T grew well in a nitrogen-free liquid medium. Other physiological characteristics of strain Y9T are summarized in the species description, and some characteristics that differentiate it from P. oryzae F-7T are presented in Table 1.
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). Strain Y9T and P. oryzae formed a monophyletic clade with a high bootstrap value (100 %) that was supported by both types of tree-making method employed in this study.
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7c/9t/12t (63.5 %), C16 : 0 (13.3 %), C19 : 0 cyclo 8c (11.8 %), C14 : 0 3-OH/C16 : 1 iso (8.1 %) and C18 : 0 (3.3 %) (Table 2). At first sight, the two species appeared to have very different fatty acid profiles. However, the sum of the total amount of C19 : 0 cyclo 8c and C18 : 17c/9t/12t, which are interconvertible, was similar. The G+C content of the genomic DNA of strain Y9T was 65.1 mol%. The phylogenetic, phenotypic and chemotaxonomic results described in this study support classification of strain Y9T within the genus Pleomorphomonas (Xie & Yokota, 2005).
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). Strain Y9T exhibited <29 % DNA–DNA relatedness with the type strain of P. oryzae. This level is sufficiently low to permit the classification of strain Y9T in a distinct species with respect to previously described type strains (Wayne et al., 1987). On the basis of morphological, physiological and chemotaxonomic characteristics, together with data from the 16S rRNA gene sequence comparisons described above, strain Y9T should be placed within a novel species, for which we propose the name Pleomorphomonas koreensis sp. nov.
Description of Pleomorphomonas koreensis sp. nov.
Pleomorphomonas koreensis (ko.re.en'sis. N.L. fem. adj. koreensis pertaining to Korea, where the type strain was isolated and researched).
Cells are Gram-negative, oxidase-positive showing an oxidative metabolism, catalase-positive, non-spore-forming, non-motile rods, 0.3–0.5 µm in diameter and 1.5–3.0 µm in length. Growth occurs on R2A agar, nutrient agar and trypticase soy agar at 30 °C and pH 6.0–8.0. Able to assimilate L-fucose, N-acetylglucosamine and D-melibiose as sole carbon sources. Unable to assimilate 4-hydroxybutylate, caprate, valerate, adipate, suberate, glycogen, 5-ketogluconate or 2-ketogluconate as sole carbon sources. Other substrate-utilization data and details of enzyme production, acid production and physiological characteristics are indicated in Table 1
. Predominant quinone is Q-10. Fatty acids consist largely of C18 : 1, C16 : 0 and C19 : 0 cyclo 8c. The G+C content of the genomic DNA of the type strain is 65.1 mol% (determined by HPLC).
The type strain, Y9T (=KCTC 12246T=NBRC 100803T), was isolated from a contaminated culture of Rhodopseudomonas palustris in Daejeon, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cappuccino, J. G. & Sherman, N. (2002). Microbiology: a Laboratory Manual, 6th edn. Menlo Park, CA: Benjamin Cummings Science Publishing.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
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Kim, M. K., Im, W.-T., Ohta, H., Lee, M. & Lee, S.-T. (2005). Sphingopyxis granuli sp. nov., a 
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Shin, Y. K., Lee, J.-S., Chun, C. O., Kim, H.-J. & Park, Y.-H. (1996). Isoprenoid quinone profiles of the Leclercia adecarboxylata KCTC 1036T. J Microbiol Biotechnol 6, 68–69.
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Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Xie, C.-H. & Yokota, A. (2005). Pleomorphomonas oryzae gen. nov., sp. nov., a nitrogen-fixing bacterium isolated from paddy soil of Oryza sativa. Int J Syst Evol Microbiol 55, 1223–1237.
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