Taxonomic study of Cellvibrio strains and description of Cellvibrio ostraviensis sp. nov., Cellvibrio fibrivorans sp. nov. and Cellvibrio gandavensis sp. nov.

Gram-negative, aerobic, cellulolytic bacteria with polar flagella were described as early as 1929 and assigned to the genus Cellvibrio (Winogradsky, 1929). 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 (94·998·9 % 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.

Strains investigated.
Thirty-one strains, belonging to cluster C of Lednická et al. (2000), as well as C. mixtus subsp. mixtus ACM 2601^T and ACM 2603, C. fulvus LMG 2847^T and C. vulgaris LMG 2848^T 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.

Table 1. Strains investigated in this study Abbreviations: ACM (formerly UQM), Australian Collection of Microorganisms, Brisbane, Queensland, Australia; LMG, BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie, Gent, Belgium; NCIMB, National Collection of Industrial and Marine Bacteria, Aberdeen, UK. Numbers prefixed by R refer to strains from the research collection of the Laboratorium voor Microbiologie, Universiteit Gent, Belgium, as given by Lednická et al. (2000). HG, DNA hybridization group; NT, not tested.

Biochemical tests.
Phenotypic characterization was carried out as described by Lednická et al. (2000). 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 (S_G) 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.

DNADNA hybridizations.
DNADNA 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 19434^T 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.

Genomic fingerprinting and DNA relatedness
REP-PCR fingerprints were prepared for all strains, except C. mixtus subsp. mixtus ACM 2601^T. On the basis of numerical and visual comparison, 11 clusters, grouping strains with almost identical profiles, could be distinguished (Table 1). C. vulgaris LMG 2848^T (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 2847^T and C. mixtus ACM 2603.

In order to investigate the genomic relatedness between strains of the different REP-PCR clusters, representatives were compared by DNADNA 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 349 % between strains from different DNA hybridization groups. Strains with identical REP-PCR profiles showed 90100 % relative DNA binding to each other. C. mixtus strains ACM 2601^T and ACM 2603 showed 98 % DNA binding to each other. DNA from the strains of DNA hybridization groups (HGs) 1 to 6 had 47·348·9 mol% G+C, and those from HGs 7 and 8 had 44·244·7 mol% G+C.

Table 2. DNADNA relatedness and G+C contents of Cellvibrio strains Abbreviations: HG, DNA hybridization group; RP, REP-PCR profile (see Table 1 for details); , not determined. DNA reassociation values are means of reciprocal values.

Phenotypic characterization
Using phase-contrast microscopy, cells of representative strains are straight rods, about 24 µm long and 0·7 µm wide. The cells show single polar flagella, and no mixed flagellation was observed. Biochemical and physiological characteristics are given in Table 3.

Table 3. Phenotypic characteristics of Cellvibrio strains Tests were read after 48 h incubation at 28 °C, except when mentioned otherwise. Strains from each DNA hybridization group were tested as follows: HG 1, C. ostraviensis sp. nov., eight strains; HG 2, C. fibrivorans sp. nov., six strains; HG 3, Cellvibrio sp. LMG 18563; HG 4, C. fulvus LMG 2847T; HG 5, C. vulgaris, four strains; HG 6, C. mixtus, two strains; HG 7, C. gandavensis sp. nov., 12 strains; HG 8, Cellvibrio sp. LMG 18453. Tests are scored as: +, positive; -, negative; d, strain-dependent. For strain-dependent reactions, reactions for type strains are given in parentheses. All strains investigated were positive for oxidase, catalase, hydrolysis of cellulose, starch and pectate, motility, forming of curdlan-type polysaccharide, API 20NE tests for aesculin hydrolysis and p-nitrophenyl-β-galactosidase and API ZYM tests for alkaline phosphatase, leucine arylamidase, C4 and C8 esterases and N-acetyl-β-D-glucosaminidase. All strains were negative for Gram stain, API 20NE tests for N2 from NO3, indole, acid from glucose, arginine dihydrolase, urease, gelatin liquefaction and growth on mannitol, gluconate, caprate, adipate, malate, citrate and phenylacetate and API ZYM tests C14 lipase, trypsin, chymotrypsin, β-glucuronidase, α-mannosidase and α-fucosidase.

The reactions of the strains in 52 phenotypic tests were coded semi-quantitatively and used to construct a dendrogram by numerical analysis (not shown). All strains grouped at S_G88 % and seven phenons were delineated at S_G>94 %. Each phenon corresponded to a single DNA hybridization group, except the one that contained the strains from HG 1 and C. fulvus LMG 2847^T (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 : 1ω7c (summed in feature 3), 16 : 0, 18 : 0ω7c and 10 : 0 3-OH.

Table 4. Fatty acid compositions of Cellvibrio strains Values are percentages of total fatty acids; the range is given and the number of strains showing the feature, if not all, is given in parentheses. See Table 3 for strains analysed. , Not detected in any strain; tr, trace (1·0 % of total). Also in trace amounts in some strains: 11 : 0, 11 : 0 2-OH, 11 : 0 iso 3-OH, 13 : 0, 13 : 0 2-OH, 15 : 1ω6c, 17 : 1ω6c, 11-methyl 18 : 1ω7c, 18 : 0 3-OH. Unknown fatty acids are designated by their equivalent chain-length (ECL), relative to the chain lengths of known straight-chain, saturated fatty acids. Summed feature 3 comprises 15 : 0 iso 2-OH, 16 : 1ω7c or both. Summed feature 5 comprises 18 : 2ω6,9c, 18 : 0 anteiso or both.

16S rDNA sequence analysis
To investigate the phylogenetic affiliation of the strains from HG 2, the 16S rRNA gene of strain LMG 19434^T was sequenced and compared with the sequences generated earlier by Lednická et al. (2000) 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 10462^T; Humphry et al., 2003) formed a clear-cut cluster, with 94·899·9 % pairwise sequence similarity and less than 93·1 % similarity to other entries from the EMBL database.

(25K): Fig. 1. Neighbour-joining dendrogram based on 16S rDNA sequences showing the estimated phylogenetic relationships of existing and novel members of Cellvibrio (including P. cellulosa, now C. japonicus). P. asplenii was the nearest phylogenetic neighbour and was included as an outgroup. Bootstrap values of branches are shown as percentages of 1000 replicates, when less than 100 %. Bar, 5 % sequence divergence.

In the scope of a biodegradability study of cellulosic fibres, Lednická et al. (2000) 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 DNADNA 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 2601^T, ACM 2603, LMG 2847^T and LMG 2848^T, that were assigned to C. mixtus subsp. mixtus by Blackall et al. (1985), belonged to three different DNA hybridization groups. C. mixtus ACM 2601^T 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 2847^T and LMG 2848^T, 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 10462^T, 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 < DNADNA hybridization < REP-PCR fingerprinting.

Phenotypic analysis showed that the hybridization groups form distinct phenotypic entities. The only exception was C. fulvus LMG 2847^T (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 2847^T 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 α-galactosidase, β-galactosidase, α-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 19434^T, LMG 18561^T and LMG 18551^T 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 2847^T (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 23 mm and with entire margins are formed on TSBA plates after 4 days at 28 °C. Cells are straight rods, 24 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, α-glucosidase, alkaline and acid phosphatases and naphthol-AS-BI-phosphohydrolase and are negative for β-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 19434^T (=ACM 5173^T).

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 23 mm and with entire margins are formed on TSBA plates after 4 days at 28 °C. Cells are straight rods, 24 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 α-glucosidase and alkaline and acid phosphatases and negative for valine arylamidase, β-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 18561^T (=ACM 5172^T).

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 24 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, 24 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 β-galactosidase and alkaline phosphatase and negative for α-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 44·244·6 mol%. The type strain is LMG 18551^T (=ACM 5174^T).

This work has been carried out within the scope of FAIR project CT98-3919, financed by the European Commission. D. L. is also indebted to the Ministerie van de Vlaamse Gemeenschap, Departement Onderwijs, Belgium, for specialization grants. J. G. and P. D. V. are indebted to the Ministerie van de Vlaamse Gemeenschap, Bestuur Wetenschappelijk Onderzoek, Belgium, for grant Concerted Research Action 12050797. P. D. V. and J. S. are also indebted to the Fonds voor Wetenschappelijk Onderzoek Vlaanderen for research grant G.0156.02. C. mixtus strains ACM 2603 and ACM 2601^T were kindly provided by L. I. Sly and S. P. Cummings, respectively.