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1 Departamento de Microbiología y Genética, Universidad de Salamanca, Spain
2 Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie en Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
3 Instituto de Recursos Naturales y Agrobiología, IRNA-CSIC, Salamanca, Spain
Correspondence
Encarna Velázquez
evp{at}usal.es
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain ALA10B2T is DQ855376, those for the atpD sequences of strains ALA10B2T and ALA38.2 are AM286426 and AM286429, respectively, and those for the recA sequences of strains ALA10B2T and ALA38.2 are AM286427 and AM286428, respectively.
A demonstration of cellulase activity, TP-RAPD profiles, an extended 16S rRNA gene-based neighbour-joining tree, atpD-, recA- and nodD-based neighbour-joining trees, images of nodules and plasmid profiles are available as supplementary material in IJSEM Online.
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), most of them have been isolated from nodules. Decaying wood is a good source of polysaccharide-hydrolysing micro-organisms, and we have previously isolated several species, mainly actinomycetes, able to hydrolyse xylan and cellulose from similar sources (Rivas et al., 2003, 2004a, b, c, d). In this work, from pulverized decaying wood of Populus alba, we isolated two Gram-negative strains, ALA10B2T and ALA38.2, that actively hydrolyse CM-cellulose in vitro. The 16S rRNA gene sequences of these strains allowed their classification in the genus Rhizobium, near to Rhizobium galegae and Rhizobium huautlense, two species that do not produce cellulases under the conditions used in this study. A polyphasic study of these strains, including phenotypic and molecular taxonomic approaches, showed that they belong to a novel species of the genus Rhizobium. For isolation of strains ALA10B2T and ALA38.2, a sample of pulverized decaying wood was collected aseptically from a cavity in the trunk of a healthy Populus alba tree in Salamanca, Spain. From this sample, 1 g was ground, placed in 9 ml sterile water and stirred for 60 min. Aqueous portions (100 µl of the mixture) were spread on CEA medium containing 0.7 % CM-cellulose as the only carbon source, 0.3 % yeast extract and 2.5 % agar. Colonies were picked from these plates and inoculated on the same medium. Cellulase activity was detected after 5 days incubation at 28 °C after staining with a 1 % aqueous Congo red solution. Two bacterial strains, ALA10B2T and ALA38.2, showing strong cellulase activity, were isolated (see Supplementary Fig. S1, available in IJSEM Online).
These strains were subjected to two-primers randomly amplified polymorphic DNA (TP-RAPD) pattern analysis, performed as described previously (Rivas et al., 2002a) using primers 879F (5'-GCCTGGGGAGTACGGCCGCA-3') and 1522R (5'-AAGGAGGTGATCCANCCRCA-3'), which respectively correspond to Escherichia coli positions 879–898 and 1509–1522. The patterns obtained contain a band that corresponds to the fragment of the 16S rRNA gene amplified with these primers and several others produced by random amplification of the total DNA (Rivas et al., 2001, 2002a). According to our previous results, bacterial strains showing identical TP-RAPD patterns belong to the same species, and strains with different TP-RAPD patterns belong to different species (Rivas et al., 2001, 2002a; Zurdo-Piñeiro et al., 2004). The TP-RAPD patterns of strains ALA10B2T and ALA38.2 (Supplementary Fig. S2, lanes 1 and 2) were the same, but were different from those of strains of R. galegae (lane 3) and R. huautlense (lane 4), suggesting that they belong to the same species, but to a different species from their closest phylogenetic relatives.
Nearly complete 16S rRNA gene sequences of the strains isolated in this study were obtained according to a previously described method (Rivas et al., 2002b). The sequences of strains ALA10B2T and ALA38.2 exhibit 100 % similarity and therefore only the sequence of the strain ALA10B2T is included in the phylogenetic tree. The sequence was compared with those held in GenBank using the BLASTN program (Altschul et al., 1990). 16S rRNA gene sequences were aligned using the CLUSTAL X software (Thompson et al., 1997) and distances were calculated according to the model of Kimura (1980) and the neighbour-joining method (Saitou & Nei, 1987). Bootstrap analysis was based on 1000 resamplings. The MEGA2 package (Kumar et al., 2001) was used for all analyses. The resulting neighbour-joining tree is shown in Fig. 1 (a phylogenetic tree including a wider selection of reference strains is available as Supplementary Fig. S3). The results indicate that the strains from this study are phylogenetically related to members of the genus Rhizobium within the family Rhizobiaceae. According to 16S rRNA gene sequence comparisons, the closest relatives of strain ALA10B2T are R. galegae USDA 4128T, showing 98.2 % similarity, followed by R. huautlense S02T, showing 98.1 % similarity.
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; Willems et al., 2003). Phylogenetic sequence analysis was performed with the Bionumerics software (Applied Maths), including sequences of close relatives available from EMBL. Distances were calculated and clustering was performed with the neighbour-joining algorithm as described above. Bootstrap analysis was performed using 500 replications. The two strains had the same sequence for each of these genes. recA gene sequence analysis supports the separate phylogenetic position of the strains from this study within the genus Rhizobium, with Rhizobium mongolense USDA 1844T (87.9 %), R. galegae LMG 6214T (87.8 %) and R. huautlense LMG 18254T and Rhizobium etli USDA 9032T (both 87.2 %) as the most closely related strains (Supplementary Fig. S4). atpD sequence analysis revealed similarly low sequence similarities between strain ALA10B2T and several species from the genera Rhizobium and Ensifer. The most similar strains were ‘Sinorhizobium morelense’ LMG 21331 (91.3 %), Ensifer adhaerens STM 2072T (91.1 %), R. galegae USDA 4128T (90.1 %) and R. huautlense S02T (88.9 %) (Supplementary Fig. S5). The low similarities found between the recA and atpD sequences of the strains from this study and those of R. galegae and R. huautlense, their closest neighbours from the 16S rRNA gene sequence analysis, indicate that these strains may represent a separate Rhizobium species.
The white poplar tree from which the strains from this study were isolated was located in a soil in which indigenous plants of Medicago sativa constituted the main herbaceous legume plant. Therefore we included this plant in nodulation tests, performed as described previously (Velázquez et al., 2005), together with other plants of wide host range such as Phaseolus vulgaris or Macroptilium atropurpureum. The results obtained showed that the strains were able to nodulate Medicago sativa only, forming ineffective nodules (Supplementary Fig. S6).
The plasmid content was determined by the method of Plazinski et al. (1985), with the modifications described by Rivas et al. (2002b), using the strain Ensifer meliloti GR4 as a reference (Toro & Olivares, 1986). The symbiotic plasmid was identified by Southern hybridization analysis. Plasmid DNA was transferred to a nylon membrane and immobilized by baking at 80 °C for 2 h. Oligonucleotide primers were designed to amplify fragments of the nodD and nifH genes conserved among members of the family Rhizobiaceae as described previously (Rivas et al., 2002b). PCR-amplified fragments of nodD were labelled with the DIG DNA Labelling kit (Roche Diagnostics) following the manufacturer's instructions and used as a probe. Hybridization was detected with the DIG nucleic acid detection kit (Boehringer Mannheim), using BCIP and NBT as substrates for alkaline phosphatase, according to the manufacturer's instructions. The two strains from this study showed identical plasmid profiles (represented in Supplementary Fig. S7, lane 2), with two plasmids of about 40 and 250 kb. The plasmid of 250 kb was identified as the pSym after hybridization with the nodD probe (Supplementary Fig. S7, lane 4).
A partial nodD gene fragment (512 bp) from strain ALA10B2T was amplified and sequenced as described previously (Rivas et al., 2002b). Comparison against sequences held in GenBank showed that the closest relative to the nodD gene from strain ALA10B2T was that of Ensifer meliloti 1021, with 99.6 % similarity (Supplementary Fig. S8). The nifH gene could not be amplified using the conditions reported by Rivas et al. (2002b). The results of nodulation tests in alfalfa showed the formation of ineffective nodules in this legume by the strains from this study (Supplementary Fig. S6).
For base composition analysis, DNA was prepared according to Chun & Goodfellow (1995). The G+C content of DNA from strain ALA10B2T was determined as 57 mol% using the thermal denaturation method (Mandel & Marmur, 1968). DNA–DNA hybridization was carried out by using the method of Ezaki et al. (1989), as described by Willems et al. (2001). The two novel strains showed 10–22 % relatedness to R. galegae USDA 4128T and 14–25 % relatedness to R. huautlense S02T (Supplementary Table S1). These results indicate that strains ALA10B2T and ALA38.2 do not belong to either of these species when the recommendation of a threshold value of 70 % DNA–DNA relatedness for species definition is considered (Wayne et al., 1987).
Phenotypic characterization of the strains from this study was based on growth with different carbon sources and resistance to different antibiotics, as described by Wang et al. (1998), and by using API 20NE according to the manufacturer's instructions (bioMérieux). The temperature range for growth was determined by incubating cultures in YMA medium at 4 to 40 °C. The pH range was determined in the same medium with a final pH between 4.0 and 10.0. Salt tolerance was studied in YMA medium containing 0–5 % (w/v) NaCl. Cellulase activity was detected after 5 days incubation at 28 °C on plates containing CEA medium as explained above. The type strains of R. galegae and R. huautlense were used as references.
Slight phenotypic differences were observed between the two strains from this study, as shown in Table 1, which also includes results for R. galegae and R. huautlense. The strains from this study differ from R. galegae in cellulase production, growth in the presence of 3 % NaCl, use of L-methionine as a carbon source and resistance to erythromycin and ampicillin. They differ from R. huautlense in cellulase production, growth at 4 °C and pH 9, growth in 3 % NaCl and resistance to kanamycin and erythromycin. Therefore, the new group can be differentiated genotypically and phenotypically from previously described species and we therefore propose to name it Rhizobium cellulosilyticum sp. nov.
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Gram-negative rods, as for other species of the genus. Colonies are small and pearl white in YMA at 28 °C, the optimal growth temperature. The optimum pH is 7–7.5. Grows at 4–37 °C, pH 6–8 and 0–4 % (w/v) NaCl. Strains of this species actively produce cellulases on media containing CM-cellulose as substrate. Nitrate reduction is negative. Produces 
-galactosidase and hydrolyses aesculin in API 20NE. Production of urease is positive in strain ALA10B2T but weak in strain ALA38.2. Does not produce indole, arginine dihydrolase or gelatinase in the same system. Utilizes glucose, L-arabinose, mannose, mannitol, N-acetylglucosamine and malate as carbon sources. Maltose is used as a carbon source, but growth of strain ALA38.2 is weak. Grows on glutamate and methionine as carbon and nitrogen sources. Does not grow on gentiobiose, caprate, adipate, citrate or phenylacetate. Known strains are resistant to 5 µg ampicillin ml–1 and 50 µg chloramphenicol ml–1 and sensitive to 5 µg erythromycin and kanamycin ml–1. The G+C content of the type strain is 57 mol%.
The type strain ALA10B2T (=LMG 23642T=DSM 18291T=CECT 7176T) and reference strain ALA38.2 (=LMG 23643) were isolated from sawdust of Populus alba in Spain.
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ACKNOWLEDGEMENTS |
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