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1 Netherlands Institute of Ecology (NIOO-KNAW), Centre for Terrestrial Ecology, Boterhoeksestraat 48, 6666 GA Heteren, The Netherlands
2 Laboratory for Microbiology, Ghent University, B-9000 Ghent, Belgium
3 Manchester Metropolitan University, The Heath Business & Technical Park, Runcorn, Cheshire, UK
Correspondence
W. de Boer
w.deboer{at}nioo.knaw.nl
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains LMG 23964T and LMG 23965T are AY281146 and AY281137, respectively. Those for other strains included in this study are listed in Table 1.
Detailed DNA–DNA hybridization results are available as supplementary material with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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, 2004). Collimonas strains have the interesting ability to grow at the expense of living fungal hyphae (mycophagy), albeit under laboratory conditions (De Boer et al., 2001, 2004). The taxonomy of these bacteria was examined using genomic fingerprinting (BOX-PCR), sequencing of 16S rRNA genes and physiological characterization, which revealed four clusters of strains (De Boer et al., 2004). So far, only cluster C strains have been formally classified, as the novel species Collimonas fungivorans. The present investigation was designed to establish the taxonomic position of the three other clusters and of 26 new Collimonas isolates from different types of soils in the Netherlands (Table 1).
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) (Table 1
). Based on colony morphology in combination with Collimonas-specific restriction fragment length polymorphism analysis of 16S rRNA genes, these 26 isolates were identified as Collimonas isolates (Höppener-Ogawa et al., 2007). All isolates were stored at –80 °C and maintained on 10-fold-diluted tryptone soy broth (TSB) agar for routine culturing. The 10-fold-diluted TSB agar contained (l–1) 1 g KH2PO4, 5 g NaCl, 3 g TSB (Oxoid) and 20 g agar. Media were adjusted to pH 6.5 with 1 M NaOH before autoclaving.
Repetitive sequence-based PCR profiles of the isolates were determined using the BOX-A1R primer, as described by Rademaker et al. (1997). Colony PCR was performed using fresh colonies that were taken from 10-fold-diluted TSB agar after 24 h of incubation (Rademaker et al., 1997). Visual comparison of the banding profiles and UPGMA clustering of strains using Pearson's product–moment correlation coefficients in the Bionumerics version 3.5 software package revealed that the majority (n=18) of the new isolates fell within clusters B and D described previously (De Boer et al., 2004). Only one of the new isolates fell in cluster A. The remaining seven isolates occupied distinct positions in the dendrogram (Fig. 1).
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). Analysis of the whole-cell protein patterns supported the clustering result of the BOX-PCR fingerprint patterns (results not shown).
Twenty isolates (Fig. 2) were analysed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) of intact cells. Bacterial cultures were grown and maintained on Columbia blood agar, containing 5 % (v/v) sheep blood. Incubation was standardized to 24 h and strains were grown aerobically at 28 °C. All strains were subcultured three times prior to MALDI-TOF analysis. Sample and target plate preparation, data acquisition using a M@LDI Linear TOF Mass Spectrometer [Waters Corporation (Micromass)] and data processing (with the aid of the MassLynx/MicrobeLynxTM software; Micromass) were performed as described previously (Keys et al., 2004). After cluster analysis of the spectral profiles (Fig. 2), 17 isolates formed four clusters, confirming results obtained by BOX-PCR fingerprinting and whole-cell protein electrophoresis (Fig. 1 and data not shown). The mass range m/z 2500–7500 Da contained the most discriminatory peaks, whereas the low (m/z 500–2500 Da) and high (m/z 7500–10 000 Da) mass ranges were very similar (data not shown). Strain R-35526, which occupied a distinct position in the BOX-PCR analysis (Fig. 1), and strains LMG 23973 (cluster B) and R-35529 (cluster D) represented a fifth cluster in the numerical analysis of the MALDI-TOF MS profiles.
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) as described by Willems et al. (2001). Genomic DNA was prepared as described by Marmur (1961). A hybridization temperature of 45 °C was used. Isolates with different DNA fingerprints (Fig. 1) were selected as representatives of each cluster and were subsequently hybridized with each other (Supplementary Table S1, available in IJSEM Online). The cluster B strains (LMG 23971 and LMG 23973) showed DNA–DNA hybridization values of 75 and 70 %, respectively, towards C. fungivorans LMG 21973T (cluster C). Strains LMG 23968 and R-35524 exhibited DNA–DNA hybridization values towards strain LMG 23965T of 75 and 87 %, respectively, indicating that these three cluster D strains represent a single genospecies. All DNA–DNA hybridizations between strains representing distinct clusters yielded low to intermediate values in the range of 31 to 64 % (Supplementary Table S1). These data indicate that the cluster B isolates belong to C. fungivorans, whereas the cluster A and D isolates represent two novel genospecies. The DNA G+C contents of C. fungivorans LMG 21973T and strains LMG 23964T, LMG 23965T and LMG 23971, as determined by HPLC (Mesbah et al., 1989), were 59, 57, 59 and 59 mol%, respectively.
Biochemical tests were performed for isolates representing clusters A (LMG 23964T, LMG 23966, LMG 23967, R-35550 and R-35551), B (LMG 23971, LMG 23972, LMG 23973, R-35508 and R-35509), C (LMG 21973T, R-35554, R-35555 and R-35556) and D (LMG 23965T, LMG 23968, R-35510, R-35511, R-35512, R-35516, R-35518, R-35524, R-35529 and R-35530). Strains were examined for catalase and oxidase activities (King et al., 1954). The ability to oxidize various carbon sources was tested using Biolog GN plates following the manufacturer's instructions (Table 2). Detection of enzyme activities was done using the API 20NE and API ZYM microtest systems (bioMérieux) according to the manufacturer's instructions (Table 2). The presence of the nifH gene was examined as described by Rosch & Bothe (2005). Test results and differential biochemical characteristics are listed in Table 2 and in the species descriptions.
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could not be used to distinguish clusters within the genus Collimonas (results not shown).
In summary, data from the present study demonstrate that cluster B isolates belong to C. fungivorans and that cluster A and D isolates represent two novel Collimonas species, which can be differentiated from each other and from C. fungivorans by means of genotypic (Fig. 1; De Boer et al., 2004) and phenotypic (Table 2) characteristics. We propose to classify the cluster A and D strains formally into the novel species Collimonas arenae sp. nov. and Collimonas pratensis sp. nov., respectively. The taxonomic status of seven isolates (Table 1) identified as members of the genus Collimonas by their ability to clear colloidal chitin and by their Collimonas-specific 16S rRNA restriction patterns (Höppener-Ogawa et al., 2007) needs further study. These strains may represent additional species within the genus Collimonas. The data obtained by MALDI-TOF MS indicate that this novel technology is useful for the rapid identification of Collimonas strains at the genus and species levels, although two of 20 isolates examined clustered aberrantly.
Description of Collimonas arenae sp. nov.
Collimonas arenae (a.re'nae. L. gen. n. arenae of sand, referring to the isolation of strains from sandy soil).
After 2 days of incubation at 20 °C on 10-fold-diluted TSB agar, colonies are flat, translucent and whitish with a yellowish central part and 3–7 mm in diameter with a granular-structured periphery (colony type II) (De Boer et al., 2004). Cells exhibit oxidase and weak catalase activity. The nifH gene, required for nitrogen fixation, is not detected by PCR-based methods. Carbon-source utilization is presented in Table 2. C. arenae can be differentiated from C. fungivorans and C. pratensis by the inability to assimilate trehalose and the lack of β-galactosidase activity.
The type strain is Ter10T (=LMG 23964T =CCUG 54727T). It has a DNA G+C content of 59 mol% and was isolated from (semi-)natural grassland in the Netherlands in 1998.
Description of Collimonas pratensis sp. nov.
Collimonas pratensis (pra.ten'sis. L. fem. adj. pratensis growing in a meadow, referring to the isolation of strains from grassland).
After 2 days of incubation at 20 °C on 10-fold-diluted TSB agar, colonies are small, glossy and whitish, 1–3 mm in diameter (colony type III) (De Boer et al., 2004). One isolate (R-35518) produces a purple pigment, which deviates from the general genus description (De Boer et al., 2004). Cells exhibit oxidase but no or weak catalase activity. The nifH gene required for nitrogen fixation is not detected by PCR-based methods. Carbon-source utilization and enzyme production are given in Table 2 and indicate a strong phenotypic flexibility within the species. C. pratensis can be differentiated from C. fungivorans by its colony morphology and its pronounced lipase activity. Additionally, comparison between the type strains of C. pratensis and C. fungivorans shows more differences in use of carbon substrates and production of enzymes (Table 2). Differentiation of C. pratensis from C. arenae is discussed above.
The type strain is Ter91T (=LMG 23965T =CCUG 54728T), which has a DNA G+C content of 59 mol% and was isolated from (semi-)natural grassland in the Netherlands.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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De Boer, W., Klein Gunnewiek, P. J. A., Kowalchuk, G. A. & Van Veen, J. A. (2001). Growth of chitinolytic dune soil β-subclass Proteobacteria in response to invading fungal hyphae. Appl Environ Microbiol 67, 3358–3362.
De Boer, W., Leveau, J. H. J., Kowalchuk, G. A., Klein Gunnewiek, P. J. A., Abeln, E. C. A., Figge, M. J., Sjollema, K., Janse, J. D. & Van Veen, J. A. (2004). Collimonas fungivorans gen. nov., sp nov., a chitinolytic soil bacterium with the ability to grow on living fungal hyphae. Int J Syst Evol Microbiol 54, 857–864.
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.
Höppener-Ogawa, S., Leveau, J. H. J., Smant, W., van Veen, J. A. & de Boer, W. (2007). Specific detection and real-time PCR quantification of potentially mycophagous bacteria belonging to the genus Collimonas in different soil ecosystems. Appl Environ Microbiol 73, 4191–4197.
Keys, C. J., Dare, D. J., Sutton, H., Wells, G., Lunt, M., McKenna, T., McDowall, M. & Shah, H. N. (2004). Compilation of a MALDI-TOF mass spectral database for the rapid screening and characterisation of bacteria implicated in human infectious diseases. Infect Genet Evol 4, 221–242.[CrossRef][Medline]
King, E. O., Ward, M. K. & Rainey, D. E. (1954). Two simple media for the demonstration of pyocyanin and fluorescein. J Lab Clin Med 44, 301–307.[Medline]
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
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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.
Rademaker, J. L. W., Louws, F. J. & de Bruijn, F. J. (1997). Characterization of the diversity of ecologically important microbes by rep-PCR genomic fingerprinting. In Molecular Microbial Ecology Manual, supplement 3, chapter 3.4.3, pp. 1–26. Dordrecht: Kluwer Academic.
Rosch, C. & Bothe, H. (2005). Improved assessment of denitrifying, N2-fixing, and total-community bacteria by terminal restriction fragment length polymorphism analysis using multiple restriction enzymes. Appl Environ Microbiol 71, 2026–2035.
Willems, A., Doignon-Bourcier, F., Goris, J., Coopman, R., de Lajudie, P., De Vos, P. & Gillis, M. (2001). DNA–DNA hybridization study of Bradyrhizobium strains. Int J Syst Evol Microbiol 51, 1315–1322.[Abstract]
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-D-glucosea*, L-asparagine, D-glucuronic acid, L-glutamic acid, D-mannitola, β-hydroxybutyric acid and malic acida. All strains were also positive for production of alkaline phosphataseb, leucine arylamidaseb, acid phosphataseb and naphthol-AS-BI-phosphohydrolaseb. All strains were negative for utilization of i-erythritol, melibiose, 
-hydroxybutyric acid. All strains were also negative for production of cystine arylamidaseb, β-glucuronidaseb,