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1 Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie en Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
2 BCCM/LMG Bacteria Collection, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
3 Department of Biology and Ecology, University of Ostrava, Bráfova 7, CS-70103 Ostrava 1, Czech Republic
Correspondence
Jean Swings
jean.swings{at}rug.ac.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Published online ahead of print on 19 September 2002 as DOI 10.1099/ijs.0.02316-0.
The GenBank/EMBL accession numbers for the 16S rDNA sequences of strains LMG 18551T, LMG 18561T and LMG 19434T are AJ289162, AJ289164 and AJ493583.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Although several species were described, the genus was not included in the Approved Lists of Bacterial Names (Skerman et al., 1980) and thus lost its standing in nomenclature in 1980. In 1986, the genus Cellvibrio was revived (Blackall et al., 1986), upon the isolation of novel strains from Australian soils (Blackall et al., 1985) that showed the phenotype of authentic strains of ‘Cellvibrio’ Winogradsky 1929. A single species, Cellvibrio mixtus, and two subspecies, C. mixtus subsp. mixtus and C. mixtus subsp. dextranolyticus, were described. During the review process of the present paper, a manuscript was submitted (P. Kämpfer, personal communication) that described a novel species, Cellvibrio japonicus, and revived the species names Cellvibrio vulgaris and Cellvibrio fulvus, each on the basis of a single strain (Humphry et al., 2003).
In a search for bacterial cultures that are able to degrade cellulosic plant fibres rapidly in vitro, we previously isolated 77 cellulolytic bacteria from Belgian and Czech soils and characterized them by fatty acid analysis (Lednická et al., 2000). Among the isolates, a group of 34 strains met the phenotypic description of the genus Cellvibrio (Blackall et al., 1985). On the basis of principal-component analysis of their fatty acid profiles, they were assigned to two subclusters and two strains remained single. By 16S rDNA sequence analysis, Lednická et al. (2000) confirmed that representative strains were phylogenetically related to strain ACM 2603, an authentic strain of C. mixtus (94·9–98·9 % sequence similarity), with Pseudomonas asplenii (
92·5 % sequence similarity) as their nearest neighbour from other genera.
The purpose of this study was to clarify the taxonomic relationships between the Cellvibrio isolates of Lednická et al. (2000) and authentic Cellvibrio strains, using genomic and phenotypic analysis. Three novel species, Cellvibrio ostraviensis sp. nov., Cellvibrio fibrivorans sp. nov. and Cellvibrio gandavensis sp. nov., are proposed, and additional strains of C. vulgaris are described.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, as well as C. mixtus subsp. mixtus ACM 2601T and ACM 2603, C. fulvus LMG 2847T and C. vulgaris LMG 2848T were investigated [the latter two strains were both classified in C. mixtus subsp. mixtus by Blackall et al. (1985)]. Strain designations and origins are listed in Table 1.
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. In addition, API ZYM tests were carried out as described by the manufacturer (bioMérieux) and read after 4 h incubation at 28 °C. The results were coded semi-quantitatively as described by Mergaert et al. (1984). The data were analysed numerically using the Gower similarity coefficient (SG) and a dendrogram was constructed using UPGMA (Sneath & Sokal, 1973) with the Bionumerics software package (Applied Maths).
Fatty acid analysis.
The fatty acid compositions are based on the data generated by Lednická et al. (2000) or obtained as described by the same authors. Principal-component analysis was performed as described by Lednická et al. (2000).
DNA preparation.
DNA was prepared from cells grown on trypticase soy broth (BBL) supplemented with 1·5 % (w/v) Bacto agar (Difco) (TSBA) at 28 °C, according to the method of Pitcher et al. (1989), with the modifications described by Logan et al. (2000). Alternatively, the method of Marmur (1961) was applied.
Determination of DNA base composition.
DNA base composition was determined using an HPLC method. DNA was enzymically degraded into nucleosides as described by Mesbah et al. (1989). The nucleoside mixture obtained was then separated by HPLC using a Waters Symmetry Shield C8 column thermostatted at 37 °C. The solvent was 0·02 M NH4H2PO4, pH 4·0, with 1·5 % acetonitrile. Non-methylated lambda phage DNA (Sigma) was used as the calibration reference.
DNA–DNA hybridizations.
DNA–DNA hybridizations were carried out with photobiotin-labelled probes in microplate wells as described by Willems et al. (2001), using an HTS7000 BioAssay reader (Perkin Elmer) for the fluorescence measurements. The hybridization temperature was 40 °C. Reciprocal experiments were performed for every pair of strains and the results given are the means.
REP-PCR genomic fingerprinting.
Using purified DNA as template, REP-PCR (based on primers targeting the repetitive extragenic palindromic sequence) genomic fingerprints were obtained using the primers REP1R-I and REP2-I (Versalovic et al., 1991), as described by Rademaker & de Bruijn (1997). Numerical analysis was carried out using Pearson's correlation coefficient and UPGMA clustering (Sneath & Sokal, 1973) with the Bionumerics software package, as described by Rademaker et al. (2000).
16S rDNA sequencing.
DNA preparation and 16S rDNA sequence analysis of strain LMG 19434T were carried out as described previously (Lednická et al., 2000). The closest related sequences were found using the program FASTA. Phylogenetic analysis was performed using the Bionumerics software package, taking into account homologous nucleotide positions after discarding all unknown bases and gaps. Using the same software, a neighbour-joining dendrogram (Saitou & Nei, 1987) was constructed based on global alignment of the sequence similarities.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). C. vulgaris LMG 2848T (profile F) had a profile very similar to those of two isolates of Lednická et al. (2000). Six strains had unique profiles, including C. fulvus LMG 2847T and C. mixtus ACM 2603.
In order to investigate the genomic relatedness between strains of the different REP-PCR clusters, representatives were compared by DNA–DNA hybridization and their G+C contents were determined. The results, given in Table 2, reveal the existence of eight DNA hybridization groups, within each of which strains show more than 82 % relative DNA binding to each other and 3–49 % between strains from different DNA hybridization groups. Strains with identical REP-PCR profiles showed 90–100 % relative DNA binding to each other. C. mixtus strains ACM 2601T and ACM 2603 showed 98 % DNA binding to each other. DNA from the strains of DNA hybridization groups (HGs) 1 to 6 had 47·3–48·9 mol% G+C, and those from HGs 7 and 8 had 44·2–44·7 mol% G+C.
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88 % and seven phenons were delineated at SG>94 %. Each phenon corresponded to a single DNA hybridization group, except the one that contained the strains from HG 1 and C. fulvus LMG 2847T (HG 4).
Fatty acid analysis
The data on fatty acid compositions of the strains generated by Lednická et al. (2000), including the reference strains, were re-examined by principal-component analysis. Using principal components 1 and 2, essentially the same groups as delineated by Lednická et al. (2000) were recognized. Cluster C1 contained the strains from HG 7 and cluster C2 consisted of the strains of HGs 1, 2, 4, 5 and 6. Strains LMG 18453 and LMG 18563, forming HGs 3 and 8, were separated from each other and from clusters C1 and C2. The fatty acid compositions of the strains are summarized in Table 4. The main fatty acids were 15 : 0 iso 2-OH and/or 16 : 1
7c (summed in feature 3), 16 : 0, 18 : 07c and 10 : 0 3-OH.
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and the nearest related sequences available from the EMBL database. In the neighbour-joining dendrogram, shown in Fig. 1, the sequences from the Cellvibrio strains as well as ‘Pseudomonas cellulosa’ NCIMB 10462 (which has been reclassified as C. japonicus NCIMB 10462T; Humphry et al., 2003) formed a clear-cut cluster, with 94·8–99·9 % pairwise sequence similarity and less than 93·1 % similarity to other entries from the EMBL database.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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isolated 34 strains that were phenotypically very similar and phylogenetically highly related to C. mixtus ACM 2603. Two of these strains (LMG 18453 and LMG 18561) were successfully applied in standardized tests for the assessment of the degradation of several natural and chemically modified cellulosic fibres (Lednická et al., 2000; Frisoni et al., 2001). The purpose of the present study was to characterize 31 of these strains in more detail in order to determine their taxonomic relationships.
REP-PCR allows the rapid grouping of strains that are genomically highly related (Versalovic et al., 1991; Rademaker et al., 2000) and the selection of representative strains for further study by DNA–DNA hybridization and G+C content determination. The results show that the Cellvibrio collection investigated is genomically very heterogeneous, with at least 11 different REP-PCR fingerprints. Each enrichment, set up with soils taken at different locations, different dates and using different types of fibres, yielded different REP-PCR groups. Taking 70 % DNA relatedness as the threshold for species delineation (Wayne et al., 1987), eight DNA hybridization groups, each containing strains representing one or more REP-PCR groups, were found, and the strains fell within two ranges of G+C content. Four strains, ACM 2601T, ACM 2603, LMG 2847T and LMG 2848T, that were assigned to C. mixtus subsp. mixtus by Blackall et al. (1985), belonged to three different DNA hybridization groups. C. mixtus ACM 2601T and ACM 2603 have almost identical 16S rDNA sequences, show a high degree of DNA relatedness, have similar fatty acid profiles and show an almost identical phenotype. During the review process of this manuscript, a proposal was submitted to revive the species C. fulvus and C. vulgaris, with strains LMG 2847T and LMG 2848T, respectively, as the type strains (Humphry et al., 2003; P. Kämpfer, personal communication), and this proposal is supported by our data. Our isolates contained in HG 5 belong to the revived species C. vulgaris. HGs 1, 2, 3, 7 and 8 form separate novel species according to the definition by Wayne et al. (1987), as they show less than 70 % DNA relatedness to the type species C. mixtus and the revived species C. fulvus and C. vulgaris. They are also different from C. japonicus NCIMB 10462T, as they exhibit 16S rDNA sequence similarities to this strain of less than 95 % (Stackebrandt & Goebel, 1994).
In order to describe the DNA hybridization groups on a phenotypic basis, 52 morphological, physiological and biochemical traits were studied and analysed numerically. In addition, fatty acid profiles of the strains, obtained by Lednická et al. (2000) or determined by the authors of the present paper, were re-examined for their relevance in differentiating the DNA hybridization groups. The results allowed us to classify the different approaches according to increasing taxonomic resolution within Cellvibrio in the following order: G+C content < fatty acid analysis < phenotypic analysis < DNA–DNA hybridization < REP-PCR fingerprinting.
Phenotypic analysis showed that the hybridization groups form distinct phenotypic entities. The only exception was C. fulvus LMG 2847T (HG 4), which grouped among the strains from HG 1 by numerical analysis. The latter HG 1 was phenotypically the most heterogeneous group, but strain LMG 2847T differed from strains included in HG 1 as it was able to grow on mannose and did not show acid phosphatase or valine arylamidase activity (Table 4). Other phenotypic features that differentiate the different DNA hybridization groups are API ZYM tests for 
-galactosidase, 
-galactosidase, -glucosidase, cystine arylamidase and naphthol-AS-BI-phosphohydrolase, utilization of mannose, reduction of nitrate to nitrite, growth at 4 and 37 °C and mucoid growth on TSA (Table 3). Although HGs 1, 2, 4, 5 and 6 could not be separated clearly by principal-component analysis of the fatty acid compositions (see above), the data showed small but significant differences between the different DNA hybridization groups, especially with regard to the presence and abundance of hydroxylated dodecanoic (12 : 0 2-OH, 12 : 0 3-OH) and hydroxylated dodecenoic (12 : 1 3-OH) acids and tetradecanoic acid (14 : 0) (Table 4).
On the basis of the results, we propose the creation of three novel Cellvibrio species. Cellvibrio ostraviensis sp. nov., Cellvibrio fibrivorans sp. nov. and Cellvibrio gandavensis sp. nov., with strains LMG 19434T, LMG 18561T and LMG 18551T as the type strains, for the strains of HGs 1, 2 and 7, respectively. Strains of HG 5 belong to C. vulgaris. Strains LMG 18563 (HG 3), C. fulvus LMG 2847T (HG 4) and LMG 18543 (HG 8) differed by some phenotypic features, fatty acid composition and G+C content from each other and from the other groups. Pending the isolation of additional genomically related strains, we classify strains LMG 18543 and LMG 18563 as Cellvibrio sp.
Description of Cellvibrio ostraviensis sp. nov.
Cellvibrio ostraviensis (os.tra.vi.en'sis. M.L. masc. adj. ostraviensis pertaining to Ostravia, the Latin name of Ostrava, the town in the Czech Republic where the strains were isolated).
Non-pigmented or pale-yellow-pigmented, low-convex, translucent colonies with a diameter of 2–3 mm and with entire margins are formed on TSBA plates after 4 days at 28 °C. Cells are straight rods, 2–4 µm long and 0·7 µm wide, with single polar flagella. Physiological and biochemical characteristics are as for the genus (Blackall et al., 1985). In addition, nitrate is reduced to nitrite. The strains are positive for valine arylamidase, -glucosidase, alkaline and acid phosphatases and naphthol-AS-BI-phosphohydrolase and are negative for -galactosidase. Growth occurs at 4 °C but not at 37 °C. Additional features are given in Table 3. Typical fatty acids in extracts from cells grown on TSBA include 2-hydroxydodecanoic acid (12 : 0 2-OH) and tetradecanoic acid (14 : 0). Isolated from soil. The G+C contents of the DNA of two strains are 47·4 and 48·4 mol%. The type strain is LMG 19434T (=ACM 5173T).
Description of Cellvibrio fibrivorans sp. nov.
Cellvibrio fibrivorans (fi.bri.vo'rans. L. n. fibra fibre; L. v. voro to devour; N.L. part. adj. fibrivorans fibre-devouring).
Non-pigmented or pale-yellow-pigmented, low-convex, translucent colonies with a diameter of 2–3 mm and with entire margins are formed on TSBA plates after 4 days at 28 °C. Cells are straight rods, 2–4 µm long and 0·7 µm wide, with single polar flagella. Physiological and biochemical characteristics are as for the genus (Blackall et al., 1985). In addition, strains grow on maltose, arabinose, mannose and N-acetylglucosamine. Nitrate is reduced to nitrite. The strains are positive for -glucosidase and alkaline and acid phosphatases and negative for valine arylamidase, -galactosidase and naphthol-AS-BI-phosphohydrolase. No growth occurs at 37 °C. Additional features are given in Table 3. Typical fatty acids in extracts from cells grown on TSBA include 2-hydroxydodecanoic acid (12 : 0 2-OH), 3-hydroxydodecanoic acid (12 : 0 3-OH) and tetradecanoic acid (14 : 0). Isolated from soil. The G+C content of the DNA of two strains is 48·0 mol%. The type strain is LMG 18561T (=ACM 5172T).
Description of Cellvibrio gandavensis sp. nov.
Cellvibrio gandavensis (gan.da.ven'sis. M.L. masc. adj. gandavensis pertaining to Gandavum, the Latin name for Gent, the town in Belgium where the strains were isolated).
Non-pigmented, low-convex, opaque, shiny colonies with a diameter of 2–4 mm and with entire margins are formed on TSBA plates after 4 days at 28 °C. Confluent, very mucoid growth is produced. Cells are straight rods, 2–4 µm long and 0·7 µm wide, with single polar flagella. Physiological and biochemical characteristics are as for the genus (Blackall et al., 1985). In addition, strains grow on maltose, arabinose and mannose. Nitrate is not reduced. Strains are positive for -galactosidase and alkaline phosphatase and negative for -glucosidase and naphthol-AS-BI-phosphohydrolase. Growth occurs at 4 °C but not at 37 °C. Additional features are given in Table 3. Typical fatty acids in extracts from cells grown on TSBA include 3-hydroxydodecanoic acid (12 : 0 3-OH) and 3-hydroxydodecenoic acid (12 : 1 3-OH). Isolated from soil. The G+C content of the DNA of four strains is 44·2–44·6 mol%. The type strain is LMG 18551T (=ACM 5174T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Blackall, L. L., Hayward, A. C. & Sly, L. I. (1986). Cellvibrio nom. rev. In Validation of the Publication of New Names and New Combinations Previously Effectively Published Outside the IJSB, List no. 20. Int J Syst Bacteriol 36, 354–356.
Frisoni, G., Baiardo, M., Scandola, M., Lednická, D., Cnockaert, M. C., Mergaert, J. & Swings, J. (2001). Natural cellulose fibers: heterogeneous acetylation kinetics and biodegradation behavior. Biomacromolecules 2, 476–482.[CrossRef][Medline]
Humphry, D. R., Black, G. W. & Cummings, S. P. (2003). Reclassification of ‘Pseudomonas fluorescens subsp. cellulosa’ NCIMB 10462 (Ueda et al. 1952) as Cellvibrio japonicus sp. nov. and revival of Cellvibrio vulgaris sp. nov., nom. rev. and Cellvibrio fulvus sp. nov., nom. rev. Int J Syst Evol Microbiol 53, 393–400.
Lednická, D., Mergaert, J., Cnockaert, M. C. & Swings, J. (2000). Isolation and identification of cellulolytic bacteria involved in the degradation of natural cellulosic fibres. Syst Appl Microbiol 23, 292–299.[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.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Mergaert, J., Verdonck, L., Kersters, K., Swings, J., Boeufgras, J.-M. & De Ley, J. (1984). Numerical taxonomy of Erwinia species using API systems. J Gen Microbiol 130, 1893–1910.
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.
Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151–156.
Rademaker, J. L. W. & de Bruijn, F. J. (1997). Characterization and classification of microbes by rep-PCR genomic fingerprinting and computer assisted pattern analysis. In DNA Markers: Protocols, Applications and Overviews, pp. 151–171. Edited by G. Caetano-Anollés & P. M. Gresshoff. New York: Wiley.
Rademaker, J. L. W., Hoste, B., Louws, F. J., Kersters, K., Swings, J., Vauterin, L., Vauterin, P. & de Bruijn, F. J. (2000). Comparison of AFLP and rep-PCR genomic fingerprinting with DNA–DNA homology studies: Xanthomonas as a model system. Int J Syst Evol Microbiol 50, 665–677.[Abstract]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Skerman, V. B. D., McGowan, V. & Sneath, P. H. A. (editors) (1980). Approved lists of bacterial names. Int J Syst Bacteriol 30, 225–420.
Sneath, P. H. A. & Sokal, R. R. (1973). Numerical Taxonomy. The Principles and Practice of Numerical Classification. San Francisco: W. H. Freeman.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Versalovic, J., Koeuth, T. & Lupski, J. R. (1991). Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 19, 6823–6831.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
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]
Winogradsky, S. (1929). Etudes sur la microbiologie du sol. Sur la dégradation de la cellulose dans le sol. Ann Inst Pasteur 43, 529–633.
This article has been cited by other articles:
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  D. R. Humphry, G. W. Black, and S. P. Cummings Reclassification of 'Pseudomonas fluorescens subsp. cellulosa' NCIMB 10462 (Ueda et al. 1952) as Cellvibrio japonicus sp. nov. and revival of Cellvibrio vulgaris sp. nov., nom. rev. and Cellvibrio fulvus sp. nov., nom. rev. Int J Syst Evol Microbiol, March 1, 2003; 53(2): 393 - 400. [Abstract] [Full Text] [PDF]
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