| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Korea Research Institute of Bioscience and Biotechnology, 52 Oeundong, Yusong, Daejeon 305-333, Republic of Korea
2 Division of Applied Life Science, EB-NCRC, PMBBRC, Gyeongsang National University, Jinju 660-701, Republic of Korea
3 Yeongnam Agricultural Research Institute, 1085 Neidong, Milyang 627-803, Republic of Korea
Correspondence
Chang-Jin Kim
changjin{at}kribb.re.kr
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
7c and/or iso-C15 : 0 2-OH). The genomic DNA G+C content was 56 mol% and the major isoprenoid quinone was Q-8. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain HW7T forms a distinct lineage with respect to closely related genera within the class Betaproteobacteria and that the levels of 16S rRNA gene sequence similarity with respect to the type species of related genera are less than 93 %. On the basis of the physiological and phylogenetic data, strain HW7T represents a novel genus and species of the Betaproteobacteria, for which the name Leeia oryzae gen. nov., sp. nov. is proposed. The type strain is HW7T (=KCTC 12585T=DSM 17879T).
Figures showing the general morphology and the polar lipids of cells of strain HW7T are available as supplementary material with the online version of this paper.
These authors contributed equally to this work. 
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
; Eller et al., 2005). Thus there have been many studies on anaerobic microbial communities in anoxic rice-field soils, and many anaerobic and methane-producing bacteria have been isolated from rice paddies (Akasaka et al., 2003; Dianou et al., 2001; Ueki et al., 2006). However, research has been relatively scant in relation to aerobic bacterial communities in rice fields. Therefore, in the course of screening aerobic bacteria from rice fields, we isolated a novel aerobic Gram-negative bacterium and investigated its physiological and molecular characteristics. Here we describe its taxonomic characteristics, which identify it as representing a novel genus and species within the Betaproteobacteria.
Strain HW7T was isolated from rice-paddy soil associated with the roots of Oryzae sativa growing in the Milyang area of Korea. We sampled rice roots from a rice paddy in June and removed soil debris from the roots by tapping. The roots were washed and the washings then diluted serially using 0.9 % (w/v) saline. The diluted solutions were spread on nutrient agar (NA; Difco) and incubated for 3 days at 30 °C. The isolated strain (HW7T) was routinely grown aerobically on NA for 2 days at 35 °C, except where indicated otherwise. Gram staining was determined using the bioMérieux Gram stain kit according to the manufacturer's instructions. Cell morphology and motility were studied using phase-contrast microscopy and transmission electron microscopy as described by Jeon et al. (2005). Growth was tested at different temperatures (4–45 °C) and at different pH values (5.0–10.0). NA media with different pH values were prepared as described previously (Gomori, 1955). Oxidase activity was tested by assessing the oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck), and catalase activity was determined by assessing the production of oxygen bubbles in a 3 % (v/v) aqueous hydrogen peroxide solution. The hydrolysis of L-tyrosine, aesculin, gelatin, starch, Tween 80 and urea and the reduction of nitrate were tested on NA according to previously described methods (Lanyi, 1987; Smibert & Krieg, 1994). Acid production from carbohydrates was tested as described by Leifson (1963). Additional enzyme activities were determined by using the API ZYM kit at 30 °C, as recommended by the manufacturer (bioMérieux). On NA, strain HW7T formed cream, circular/slightly irregular and slightly convex colonies when grown at 35 °C for 2 days. Growth was observed at temperatures between 10 and 40 °C (optimum, 32–35 °C) and at pH 5.0–8.5 (optimum, pH 6.0). The cells were rods (0.5–0.8 µm wide and 1.1–1.8 µm long) and were motile by means of single polar flagella (see Supplementary Fig. S1, available in IJSEM Online). Anaerobic growth was not observed over 5 days at 35 °C on NA. Other phenotypic features of strain HW7T are presented in the descriptions of the novel genus and species.
Analysis of fatty acid methyl esters was performed according to the instructions of the Microbial Identification System (MIDI; Microbial ID). Isoprenoid quinones and polar lipids were determined using the methods described by Komagata & Suzuki (1987). The DNA G+C content was determined using HPLC apparatus fitted with a reversed-phase column (GROM-SIL 100 ODS-2FE; Grom), according to the method of Tamaoka & Komagata (1984). The major cellular fatty acids of strain HW7T were C16 : 0 (42.66 %) and summed feature 3 (C16 : 17c and/or iso-C15 : 0 2-OH; 33.49 %) (Table 1). Strain HW7T contained phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol (see Supplementary Fig. S2, available in IJSEM Online). Unfortunately, polar lipids have not been reported for the genera Microvirgula and Laribacter and hence it is not clear whether strain HW7T can be distinguished from these two genera on the basis of polar lipid profiles. The genomic DNA G+C content of strain HW7T was 56 mol% and the predominant isoprenoid quinone was Q-8. The typical phenotypic characteristics of strain HW7T are summarized, and compared with those of the type strains of closely related taxa, in Table 2. Some of them are in accordance with characteristics of members of the Betaproteobacteria, whereas some others facilitate the differentiation of strain HW7T from closely related members (Bazylinski et al., 1986; Grimes et al., 1997; Patureau et al., 1998; Coenye et al., 2000; Yuen et al., 2001).
|
|
). The 16S rRNA gene sequence (1416 nt) of strain HW7T was compared with 16S rRNA gene sequences available from GenBank, using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/) to determine the approximate phylogenetic affiliation and using CLUSTAL W software (Thompson et al., 1994) to align the sequences of closely related organisms. Phylogenetic trees were constructed using three different methods, namely the neighbour-joining, maximum-likelihood and maximum-parsimony algorithms available in PHYLIP software, version 3.6 (Felsenstein, 2002). Similarity calculations were obtained by using the FASTA3 program of the European Bioinformatics Institute (Hinxton, Cambridge, UK; http://www.ebi.ac.uk) to compare the 16S rRNA gene sequence of the novel strain with those of related organisms. A bootstrap analysis was performed according to the algorithm of the Kimura two-parameter model (Kimura, 1980) of the neighbour-joining method in the PHYLIP package. The phylogenetic analysis based on 16S rRNA gene sequences showed that strain HW7T forms a distinct phylogenetic lineage with respect to the closely related genera Microvirgula and Laribacter within the Betaproteobacteria (Fig. 1). The topologies of the phylogenetic trees constructed using the maximum-likelihood and maximum-parsimony algorithms also supported the notion that the isolate could be a distinct phylogenetic genus (data not shown). A comparative analysis of 16S rRNA gene sequences showed that the levels of sequence similarity between strain HW7T and the closely related strains Microvirgula aerodenitrificans SGLY2T and Laribacter hongkongensis HKU1T were 92.8 and 91.0 %, respectively. Therefore, on the basis of the physiological, biochemical and phylogenetic data, strain HW7T represents a novel genus and species within the Betaproteobacteria, for which the name Leeia oryzae gen. nov., sp. nov. is proposed.
|
Cells are Gram-negative, non-spore-forming short rods. Oxidase- and catalase-positive. Cells are aerobic and are motile by means of single polar flagella. Nitrate is reduced to nitrite. The major isoprenoid quinone is Q-8. Polar lipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The predominant cellular fatty acids are C16 : 0 and summed feature 3 (C16 : 17c and/or iso-C15 : 0 2-OH). Phylogenetically, the genus belongs to the class Betaproteobacteria. The type species is Leeia oryzae.
Description of Leeia oryzae sp. nov.
Leeia oryzae (o.ry'zae. L. gen. n. oryzae of rice, referring to the rice-paddy fields where the strain was isolated).
Exhibits the following properties in addition to those given in the genus description. Colonies are cream, circular/slightly irregular and slightly convex on NA. Cells are approximately 0.5–0.8 µm wide and 1.1–1.8 µm long. Growth occurs at 10–40 °C (optimum, 32–35 °C) and pH 5.0–8.5 (optimum, pH 6.0). L-Tyrosine is hydrolysed. Hydrolysis of aesculin, gelatin, starch, Tween 80 and urea is not observed. Acid is produced from L-arabinose, D-glucose, D-mannose and D-ribose, but not from maltose, D-trehalose, D-xylose, glycerol, sucrose, cellobiose, D-fructose, 
-D-lactose or adonitol. API ZYM kit gives positive results for esterase (C4), leucine arylamidase and acid phosphatase but negative results for lipase, valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. Weak enzyme activities are observed for alkaline phosphatase, esterase lipase (C8) and naphthol-AS-BI-phosphohydrolase. Cellular fatty acids are C16 : 0 (42.66 %), summed feature 3 (C16 : 17c and/or iso-C15 : 0 2-OH) (33.49 %), C17 : 0 cyclo (7.86 %), C12 : 0 (2.50 %), C15 : 0 (2.13 %), C10 : 0 (1.62 %), C14 : 0 (1.29 %), C18 : 17c (1.18 %), C15 : 16c (1.12 %), C17 : 0 (1.03 %), C10 : 0 3-OH (0.99 %), iso-C15 : 0 (0.72 %), C18 : 0 (0.59 %) and C14 : 15c (0.54 %). The DNA G+C content of the type strain is 56 mol% (HPLC).
The type strain, HW7T (=KCTC 12585T=DSM 17879T), was isolated from rice-field soil in Korea.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Bazylinski, D. A., Palome, E., Blakemore, N. A. & Blakemore, R. P. (1986). Denitrification by Chromobacterium violaceum. Appl Environ Microbiol 52, 696–699.
Chin, K. J. & Janssen, P. H. (2002). Propionate formation by Opitutus terrae in pure culture and in mixed culture with a hydrogenotrophic methanogen and implications for carbon fluxes in anoxic rice paddy soil. Appl Environ Microbiol 68, 2089–2092.
Cleenwerck, I., De Wachter, M., Hoste, B., Janssens, D. & Swings, J. (2003). Aquaspirillum dispar Hylemon et al. 1973 and Microvirgula aerodenitrificans Patureau et al. 1998 are subjective synonyms. Int J Syst Evol Microbiol 53, 1457–1459.
Coenye, T., Falsen, E., Hoste, B., Ohlén, M., Goris, J., Govan, J. R. W., Gillis, M. & Vandamme, P. (2000). Description of Pandoraea gen. nov. with Pandoraea apista sp. nov., Pandoraea pulmonicola sp. nov., Pandoraea pnomenusa sp. nov., Pandoraea sputorum sp. nov. and Pandoraea norimbergensis comb. nov. Int J Syst Evol Microbiol 50, 887–899.[Abstract]
Dianou, D., Miyaki, T., Asakawa, S., Morii, H., Nagaoka, K., Oyaizu, H. & Matsumoto, S. (2001). Methanoculleus chikugoensis sp. nov., a novel methanogenic archaeon isolated from paddy field soil in Japan, and DNA–DNA hybridization among Methanoculleus species. Int J Syst Evol Microbiol 51, 1663–1669.[Abstract]
Eller, G., Kruger, M. & Frenzel, P. (2005). Comparing field and microcosm experiments: a case study on methano- and methylo-trophic bacteria in paddy soil. FEMS Microbiol Ecol 51, 279–291.[CrossRef][Medline]
Felsenstein, J. (2002). PHYLIP (phylogeny inference package), version 3.6a. Department of Genome Sciences, University of Washington, Seattle, USA.
Gomori, G. (1955). Preparation of buffers for use in enzyme studies. Methods Enzymol 1, 138–146.[CrossRef]
Grimes, D. J., Woese, C. R., MacDonell, M. T. & Colwell, R. R. (1997). Systematic study of the genus Vogesella gen. nov. and its type species, Vogesella indigofera comb. nov. Int J Syst Bacteriol 47, 19–27.
Jeon, C. O., Lim, J.-M., Lee, J.-M., Xu, L.-H., Jiang, C.-L. & Kim, C.-J. (2005). Reclassification of Bacillus haloalkaliphilus Fritze 1996 as Alkalibacillus haloalkaliphilus gen. nov., comb. nov. and the description of Alkalibacillus salilacus sp. nov., a novel halophilic bacterium isolated from a salt lake in China. Int J Syst Evol Microbiol 55, 1891–1896.
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Komagata, K. & Suzuki, K. (1987). Lipid and cell wall analysis in bacterial systematics. Methods Microbiol 19, 161–207.
Krieg, N. R. (2005). Genus XIII. Vogesella Grimes, Woese, Macdonell and Colwell 1997, 25VP. In Bergey's Manual of Systematic Bacteriology, vol. 2, part C, pp. 858–859. Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer.
Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, pp. 115–175. Edited by E. Stackebrandt & M. Goodfellow. Chichester, UK: Wiley.
Lanyi, B. (1987). Classical and rapid identification methods for medically important bacteria. Methods Microbiol 19, 1–67.
Lau, H.-T., Faryna, J. & Triplett, E. W. (2006). Aquitalea magnusonii gen. nov., sp. nov., a novel Gram-negative bacterium isolated from a humic lake. Int J Syst Evol Microbiol 56, 867–871.
Leifson, E. (1963). Determination of carbohydrate metabolism of marine bacteria. J Bacteriol 85, 1183–1184.
Logan, N. A. (1989). Numerical taxonomy of violet-pigmented, gram-negative bacteria and description of Iodobacter fluviatile gen. nov., comb. nov. Int J Syst Bacteriol 39, 450–456.
Patureau, D., Godon, J.-J., Dabert, P., Bouchez, T., Bernet, N., Delgenes, J. P. & Moletta, R. (1998). Microvirgula aerodenitrificans gen. nov., sp. nov., a new Gram-negative bacterium exhibiting co-respiration of oxygen and nitrogen oxides up to oxygen-saturated conditions. Int J Syst Bacteriol 48, 775–782.
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt. Washington, DC: American Society for Microbiology.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.[CrossRef]
Tanaka, K., Nakamura, K. & Mikami, E. (1991). Fermentation of S-citramalate, citrate, mesaconate, and pyruvate by a Gram-negative strictly anaerobic non-spore-former, Formivibrio citricus gen. nov., sp. nov. Arch Microbiol 155, 491–495.[CrossRef]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Ueki, A., Akasaka, H., Suzuki, D. & Ueki, K. (2006). Paludibacter propionicigenes gen. nov., sp. nov., a novel strictly anaerobic, Gram-negative, propionate-producing bacterium isolated from plant residue in irrigated rice-field soil in Japan. Int J Syst Evol Microbiol 56, 39–44.
Yang, H.-C., Im, W.-T., An., D.-S., Park, W.-S., Kim, I. S. & Lee, S.-T. (2005). Silvimonas terrae gen. nov., sp. nov., a novel chitin-degrading facultative anaerobe belonging to the ‘Betaproteobacteria’. Int J Syst Evol Microbiol 55, 2329–2332.
Yuen, K.-Y., Woo, P. C. Y., Teng, J. L. L., Leung, K.-W., Wong, M. K. M. & Lau, S. K. P. (2001). Laribacter hongkongensis gen. nov., sp. nov., a novel gram-negative bacterium isolated from a cirrhotic patient with bacteremia and empyema. J Clin Microbiol 39, 4227–4232.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
