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Departamento de Microbiología y Genética, Edificio Departamental, Campus Miguel de Unamuno, Universidad de Salamanca, 37007 Salamanca, Spain
Correspondence
Raúl Rivas
raul{at}wwwedu-micro.usal.es
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ABSTRACT |
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An expanded phylogenetic tree for strain PALXIL08T and representative strains of the genus Paenibacillus is available as a supplementary figure in IJSEM Online.
Present address: Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie en Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium. 
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Morales et al., 1995; Hespell, 1996; Nielsen & Sorensen, 1997; Ay et al., 1998; Lee et al., 2000; Sánchez et al., 2003; Velázquez et al., 2004; Rivas et al., 2005a, b). Paenibacillus species capable of hydrolysing vegetal polymers have been frequently isolated from soil and plant related sources, and a species has been isolated from the phyllosphere (Rivas et al., 2005b). This ecosystem is colonized by complex microbial populations; nevertheless, most species present in the phyllosphere remain undescribed because they have not been isolated in pure culture (Yang et al., 2001). In the present work, we describe the isolation and identification of a novel xylan-degrading bacterium from the bract phyllosphere of Phoenix dactylifera.
Strain PALXIL08T was isolated as described previously (Rivas et al., 2005b) on XED medium (xylan, 0.7 %; yeast extract, 0.3 %; agar, 2.5 %, w/v) after 7 days incubation at 28 °C and a pure culture was maintained in a glycerol suspension (25 %, v/v) at –80 °C.
Strain PALXIL08T was grown in nutrient agar for 48 h to check for motility by phase-contrast microscopy with the hanging drop method. Gram staining was carried out by using the procedure described by Doetsch (1981). The flagellation type was determined by electron microscopy after 48 h incubation of strain PALXIL08T in nutrient agar at 22 °C. The cells were gently suspended in sterile water and then stained with 0.2 % uranyl acetate and examined at 80 kV with a Zeiss EM 209 transmission electron microscope. Strain PALXIL08T was found to be Gram-variable and motile by means of peritrichous flagella.
For sequencing of the 16S rRNA gene, DNA extraction was carried out as described previously (Rivas et al., 2001). Amplification and sequencing of the 16S rRNA gene were performed using a previously described method (Rivas et al., 2003). An almost-complete 16S rRNA gene sequence was obtained and compared with those deposited in databases. Sequences were aligned using CLUSTAL X software (Thompson et al., 1997). Distances were calculated according to the models of Jukes & Cantor (1969), Kimura (1980), Tajima & Nei (1984) and Tamura & Nei (1993). Phylogenetic trees were inferred using the neighbour-joining method (Saitou & Nei, 1987), minimum evolution (Rzhetsky & Nei, 1993) and parsimony analysis (Felsenstein, 1983). Bootstrap analysis was based on 1000 resamplings. The MEGA2 package (Kumar et al., 2001) was used for all analyses. As no significant topological differences were found among the phylogenetic trees obtained by the different methods used, only those trees constructed by using the Kimura model and the neighbour-joining method are shown. Comparison of the almost-complete 16S rRNA gene sequence of strain PALXIL08T with sequences held in GenBank indicated that this organism is phylogenetically related to members of the genus Paenibacillus. Fig. 1 shows the phylogenetic tree obtained with the neighbour-joining method (an expanded tree is available as Supplementary Fig. S1 in IJSEM Online). The most closely related type strains are Paenibacillus kobensis DSM 10249T and Paenibacillus curdlanolyticus DSM 10247T (98.9 and 97.9 % sequence similarity, respectively). We included strains PALXIL02, PALXIL05 and PALXIL07 in the phylogenetic analysis because they were isolated from the same ecosystem, and because they belong to the same group, on the basis of 16S rRNA gene sequences. However, preliminary data on hybridization between strain PALXIL08T and strains PALXIL02, PALXIL05 and PALXIL07 produced values below 30 %.
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. An unsaturated menaquinone with seven isoprene units (MK-7) was the predominant isoprenoid quinone found in strain PALXIL08T. The major cellular fatty acid in strain PALXIL08T, Paenibacillus curdlanolyticus and Paenibacillus kobensis was anteiso-branched C15 : 0 (see Table 1), which is the dominant cellular fatty acid in all members of the genus Paenibacillus (Shida et al., 1997b). The fatty acid profile of PALXIL08T was similar to those of the type strains of related Paenibacillus species (Shida et al., 1997a), but it differed in the proportions of some fatty acids. The main differences with respect to the most closely related species were in the amounts of iso-C16 : 0, iso-C15 : 0, iso-C17 : 0, C16 : 0 and anteiso-C17 : 0 (for the novel isolate and Paenibacillus kobensis) and the amounts of iso-C16 : 0, iso-C17 : 0, C16 : 0, C17 : 0 and anteiso-C17 : 0 (for the novel isolate and Paenibacillus curdlanolyticus).
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. The value for strain PALXIL08T was determined using the thermal denaturation method (Mandel & Marmur, 1968) and was found to be 51 mol%, which is similar to those obtained for Paenibacillus curdlanolyticus and Paenibacillus kobensis (Shida et al., 1997a).
DNA–DNA hybridization was carried out as described by De Ley et al. (1970), with the modifications described by Huß et al. (1983) and Escara & Hutton (1980). DNA was isolated as described by Cashion et al. (1977). The DNA relatedness values for strain PALXIL08T versus Paenibacillus kobensis DSM 10249T and Paenibacillus curdlanolyticus DSM 10247T were 27.4 and 17.6 %; respectively; this suggested that the novel isolate was not closely related to the known species, according to the current concept of bacterial species (Wayne et al., 1987).
Phenotypic characterization was performed according to the standard methods described by Claus & Berkeley (1986) and Logan & Berkeley (1984) and by using the API 20NE and API 20E systems (bioMérieux) according to the manufacturer's instructions. Acid production from glucose, xylose, mannitol and L-arabinose, gas production from glucose, acetoin production, the ability to grow in the presence of 2, 5 and 7 % NaCl, nitrate reduction, anaerobic growth, and phenylalanine deaminase, catalase, caseinase and oxidase activities were analysed as described by Claus & Berkeley (1986). Amylases and cellulases were analysed as described previously (Rivas et al., 2003). Growth at temperatures ranging from 4 to 45 °C was determined in YED medium (0.5 % yeast extract, 0.7 % glucose and 2 % agar). Growth at pH 5.7 and 6.8 was tested as described by Claus & Berkeley (1986), growth at pH 7–8 was tested in YED medium containing 100 mM Na2HPO4/NaH2PO4 and growth at pH 9 and 10 was tested in the same medium containing 100 mM NaHCO3/Na2CO3. The differentiating characteristics of strain PALXIL08T and phylogenetically related species are shown in Table 2. The other characteristics determined are given in the species description. Strain PALXIL08T differs from both Paenibacillus kobensis and Paenibacillus curdlanolyticus in terms of nitrate reduction, oxidase, acetylmethylcarbinol and cellulase production and assimilation of mannose, rhamnose and N-acetylglucosamine. It also differs from Paenibacillus kobensis in terms of acid production from raffinose.
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Description of Paenibacillus cellulosilyticus sp. nov.
Paenibacillus cellulosilyticus (cel.lu.lo.si.ly'ti.cus. N.L. n. cellulosum cellulose; Gr. adj. lutikos able to loose, able to dissolve; N.L. adj. lyticus -a -um dissolving; N.L. masc. adj. cellulosilyticus cellulose-dissolving).
Spore-forming rods, 0.8–0.9 µm wide and 4.0–4.2 µm long. Gram-variable. Motile by means of peritrichous flagella. Ellipsoidal spores are formed in swollen sporangia and occupy a subterminal position in the cell. Aerobic or facultatively anaerobic, chemo-organotrophic and xylanolytic bacteria. Colonies on YED are circular, flat, white/cream, opaque and usually 1–3 mm in diameter after 48 h at 28 °C. The major quinone is MK-7. Main fatty acids are anteiso-C15 : 0 and iso-C16 : 0. Growth occurs at 10–37 °C (optimal growth occurs at 28 °C). Optimum pH is 7. Oxidase- and catalase-positive. In Voges–Proskauer broth, the pH is 6.5. Acetoin is produced. Grows in the presence of 2 % NaCl but not in 5 % NaCl. Nitrate is not reduced to nitrite. Negative for growth in anaerobic agar. Cellulases, xylanases, amylases and 
-galactosidase are actively produced, but gelatinase, caseinase, arginine dihydrolase, indole, lysine decarboxylase, ornithine decarboxylase, urease, tryptophan deaminase, phenylalanine deaminase and hydrogen sulfide are not produced. Aesculin is hydrolysed. Gas is not produced from D-glucose. D-Glucose, L-arabinose, mannose, maltose, xylose, rhamnose, sucrose, melibiose and gluconate are assimilated. Acid is produced from glucose, xylose, L-arabinose and raffinose. Negative for the assimilation of mannitol, inositol, sorbitol, N-acetylglucosamine, amygdalin, caprate, propionate, adipate, malate, citrate and phenylacetate. The DNA G+C content of the type strain is 51 mol%.
The type strain, PALXIL08T (=LMG 22232T=CECT 5696T), was isolated from the bract phyllosphere of Phoenix dactylifera in Palma de Mallorca (Spain).
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ACKNOWLEDGEMENTS |
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