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Laboratory of Microbiology, Department of Biochemistry, Physiology and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium
Correspondence
Bram Vanparys
bram.vanparys{at}ugent.be
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain LMG 22951T is AJ786801.
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MAIN TEXT |
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, and at present the genus comprises two species, Devosia riboflavina (Nakagawa et al., 1996), formerly Pseudomonas riboflavina (Foster, 1944), comprising one soil isolate, and Devosia neptuniae (Rivas et al., 2003), comprising two isolates from a dinitrogen-fixing root-nodule from the aquatic legume Neptunia natans in India. Furthermore, a hitherto uncultured endosymbiont of the marine ciliated protozoon Euplotes magnicirratus was shown to belong to Devosia and given the provisional name ‘Candidatus Devosia euplotis' (Vannini et al., 2004). Three closely related sequences of this provisional species are available in the EMBL database.
Among organisms isolated from an enriched nitrifying community, strain LMG 22951T was recovered on a mineral salts medium (Stanier et al., 1966) with 1·69 mM succinate incubated at 22 °C for 3 weeks. Enrichment of the autotrophic nitrifying community from activated sludge was stimulated by adding a daily load of nitrogen [58·7 g total ammoniacal nitrogen (TAN) l–1 day–1 as ammonium chloride and 29·3 g 
N l–1 day–1 as sodium nitrite] to a 500 litre reactor. The purpose of the enriched nitrifying community is to enhance removal of ammonia and nitrite in aquaria and aquacultures. For more detailed information refer to Grommen et al. (2002).
The nearly complete 16S rRNA gene sequence of LMG 22951T was determined as described below. DNA was extracted according to Pitcher et al. (1989) as modified by Heyndrickx et al. (1996). PCR amplification was performed as described by Heyrman & Swings (2001). The PCR-amplified 16S rRNA gene products were purified using the Nucleofast 96 PCR system (Machery-Nagel). For each sequence reaction a mixture of 3 µl purified product, 1 µl BigDye Termination RR mix version 3.1 (Perkin Elmer), 1·5 µl BigDye buffer (5x) and 3 µl (20 ng µl–1) of one of the six sequencing primers was used. MilliQ water was added to a total volume of 10 µl. The primers and PCR program were as described by Heyrman & Swings (2001). The sequence was determined using an Applied Biosystems 3100 DNA Sequencer following the manufacturer's protocols (Perkin-Elmer). Sequence assembly was performed with the software KODON version 2.0 (Applied Maths). Phylogenetic analysis using BIONUMERICS version 3.5 (Applied Maths) showed that the closest related sequences of LMG 22951T were D. neptuniae (97·4 % 16S rRNA gene sequence similarity) and D. riboflavina (97·0 %). Furthermore, LMG 22951T showed 97·5 % similarity to the three sequences of ‘Candidatus D. euplotis' (Fig. 1).
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) according to the further specifications given by Logan et al. (2000), was 61·9 mol%. This is within the range of 61–63 mol% given in the emended genus description of Devosia (Rivas et al., 2003). DNA–DNA hybridization experiments were performed between LMG 22951T and the type strains of D. neptuniae and D. riboflavina using a modification of the microplate method of Ezaki et al. (1989) as described by Willems et al. (2001). A hybridization temperature of 45 °C (calculated with correction for the presence of 50 % formamide) was used. LMG 22951T showed DNA–DNA relatedness of 24 % to D. neptuniae LMG 21357T and 17 % to D. riboflavina LMG 2277T, suggesting that the strain represents a novel genospecies.
After incubation for 48 h at 28 °C on trypticase soy agar (TSA), a loopful of well-grown cells was harvested and fatty acid methyl esters were prepared as described by Vandamme et al. (1992), separated and then identified using the Sherlock Microbial Identification System (version 3.0; MIDI). Dominant fatty acids of LMG 22951T were C18 : 1
7c (36·32 %), 11-methyl-C18 : 17c (30·42 %), C16 : 0 (11·34 %) and C18 : 0 (9·68 %). LMG 22951T differs from the other Devosia species in containing smaller amounts of C16 : 0 and larger amounts of C18 : 0 and C18 : 17c (Table 1). Neighbour-joining clustering (data not shown) with the Canberra metric distance coefficient using BIONUMERICS version 3.5 (Applied Maths) indicated 76·3 % similarity of LMG 22951T to D. neptuniae LMG 21357T and 70·3 % to D. riboflavina LMG 2277T.
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. Densitometric analysis, normalization and interpolation of the protein profiles were performed using GELCOMPAR v4.2 (Applied Maths). UPGMA clustering of the protein profiles (data not shown) revealed a similarity below 70 % between LMG 22951T and D. neptuniae LMG 21357T and D. riboflavina LMG 2277T. This observation supports the conclusion that LMG 22951T belongs to a novel species within the genus Devosia.
Cell morphology was investigated by light microscopy at x1000 magnification of cells grown on TSA for 48 h at 28 °C. Cells were Gram stained and examined for catalase and oxidase activity. The presence of the nitrogen-fixing genes nodD and nifH was tested using the primers and PCR conditions described by Rivas et al. (2002) after plasmid DNA extraction with a Fast Plasmid Mini kit (Eppendorf). Strains were tested for the ability to denitrify by anaerobic incubation at 22 or 37 °C in trypticase soy broth (TSB) supplemented with 10 mM 
as KNO3 and 10 µM phenol red. The presence of nitrite and nitrate was tested after 24 and 48 h and 7 days by using the procedure described by Smibert & Krieg (1994). Escherichia coli LMG 2029T was included as a control. Utilization of carbon sources and enzyme production (Table 2) were tested using API 20NE, API 50CH, API ZYM strips (bioMérieux) and Biolog MicroPlates according to the manufacturers' instructions. D. riboflavina LMG 2277T and D. neptuniae LMG 21357T were included as controls. Strain LMG 22951T produced negative results for all API 20NE test strip carbon assimilation tests. The same result was achieved using a mineral salts medium (Stanier et al., 1966) as well as using denser cell suspensions, extended the incubation time (up to 14 days) and the addition of 0·01 % yeast extract. API 50CH strips and the Biolog system did not give reproducible results, even with prolonged incubation (up to 7 days). Antimicrobial susceptibilities (Table 2) were determined with the disc diffusion method using an ST6090 Disc Dispenser (Oxoid). Antibiograms were determined according to the conventional Kirby–Bauer method (Bauer et al., 1966). Isolates were classified into three categories based on the quantitative interpretation criteria recommended by the NCCLS (1993).
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Description of Devosia limi sp. nov.
Devosia limi (li'mi. L. gen. n. limi of sludge).
After 48 h of incubation at 28 °C on TSA or NA, colonies are circular with smooth margins, light yellow to light brown and 1–4 mm in diameter. Cells are rod-shaped (0·5–1 µm wide and 1–3 µm long), Gram-negative, obligately aerobic, non-spore-forming and non-motile. No formation of filaments is observed. Cells are oxidase and catalase positive. Enzyme activities, carbon source utilization and resistance to antibiotics are given in Table 2
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The type strain, LMG 22951T (=DSM 17137T), was isolated from a commercial nitrifying inoculum in Gent, Belgium.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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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.
Foster, J. W. (1944). Microbiological aspects of riboflavin. I. Introduction. II. Bacterial oxidation of riboflavin to lumichrome. J Bacteriol 47, 27–41.
Grommen, R., Van Hauteghem, I., Van Wambeke, M. & Verstraete, W. (2002). An improved nitrifying enrichment to remove ammonium and nitrite from freshwater aquaria systems. Aquaculture 211, 115–124.[CrossRef]
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Heyrman, J. & Swings, J. (2001). 16S rDNA sequence analysis of bacterial isolates from biodeteriorated mural paintings in the Servilia tomb (necropolis of Carmona, Seville, Spain). Syst Appl Microbiol 24, 417–422.[CrossRef][Medline]
Logan, N. A., Lebbe, L., Hoste, B. & 7 other authors (2000). Aerobic endospore-forming bacteria from geothermal environments in northern Victoria Land, Antarctica, and Candlemas Island, South Sandwich archipelago, with the proposal of Bacillus fumarioli sp. nov. Int J Syst Evol Microbiol 50, 1741–1753.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Nakagawa, Y., Sakane, T. & Yokota, A. (1996). Transfer of "Pseudomonas riboflavina" (Foster 1944), a Gram-negative, motile rod with long-chain 3-hydroxy fatty acids, to Devosia riboflavina gen. nov., sp. nov., nom. rev. Int J Syst Bacteriol 46, 16–22.
NCCLS (1993). Performance standards for antimicrobial disk susceptibility tests, 5th edn. Approved standard M2-A5. Vilanove, PA: National Committee for Clinical Laboratory Standards.
Pitcher, D. G., Saunders, L. A. & Owen, N. A. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151–156.
Pot, B., Vandamme, P. & Kersters, K. (1994). Analysis of electrophoretic whole organism protein fingerprints. In Chemical Methods in Prokaryotic Systematics, pp. 493–521. Edited by M. Goodfellow & A. G. O'Donnell. Chichester: Wiley.
Rivas, R., Velázquez, E., Willems, A., Vizcaíno, N., Subba-Rao, N. S., Mateos, P. F., Gillis, M., Dazzo, F. B. & Martínez-Molina, E. (2002). A new species of Devosia that forms a unique nitrogen-fixing root-nodule symbiosis with the aquatic legume Neptunia natans (L.f.) Druce. Appl Environ Microbiol 68, 5217–5222.
Rivas, R., Willems, A., Subba-Rao, N. S., Mateos, P. F., Dazzo, F. B., Kroppenstedt, R. M., Martínez-Molina, E., Gillis, M. & Velázquez, E. (2003). Description of Devosia neptuniae sp. nov. that nodulates and fixes nitrogen in symbiosis with Neptunia natans, an aquatic legume from India. Syst Appl Microbiol 26, 47–53.[CrossRef][Medline]
Smibert, R. M. & Krieg, N. R. (1994). In Methods for General and Molecular Bacteriology, p. 649. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
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Vannini, C., Rosati, G., Verni, F. & Petroni, G. (2004). Identification of the bacterial endosymbionts of the marine ciliate Euplotes magnicirratus (Ciliophora, Hypotrichia) and proposal of ‘Candidatus Devosia euplotis’. Int J Syst Evol Microbiol 54, 1151–1156.
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-galactosidase, 
-glucosidase, indole production, glucose fermentation, arginine dihydrolase and gelatinase. In contrast to previous publications, all strains gave negative results for nitrate reductase and urease. All strains were negative for assimilation of gluconate, caprate, adipate, malate, citrate and phenylacetate. R, Resistant; S, sensitive; I, intermediate. All strains were resistant to erythromycin (15 µg), ciprofloxacin (5 µg), polymyxin B (300 U), gentamicin (10 µg), chloramphenicol (30 µg), rifampicin (5 µg), amikacin (30 µg) and kanamycin (30 µg). All strains were sensitive to ampicillin (10 µg) and vancomycin (30 µg).