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merda1,
ek2ová2
ková1
1 Department of Plant Biology, Faculty of Agronomy, Mendel University of Agriculture and Forestry in Brno, Zem
dlská 1, 613 00 Brno, Czech Republic
2 Czech Collection of Microorganisms, Faculty of Science, Masaryk University Brno, Tvrdého 14, 602 00 Brno, Czech Republic
3 Institute of Public Health in Ostrava, Partyzánské nám. 7, 702 00 Ostrava, Czech Republic
Correspondence
Jakub merda
Jsmerda{at}mail.muni.cz
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain C/2T is AF537343.
Fatty acid profiles, DNA relatedness data and an extended phylogenetic tree for strain C/2T and related Paenibacillus species are available as supplementary material in IJSEM Online.
Present address: Department of Botany, Faculty of Science, Masaryk University Brno, Kotlá
ská 2, 611 37 Brno, Czech Republic. 
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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) was applied to detect sources of contamination. An unknown bacterium, strain C/2T, was detected as being a source of contamination. This strain was isolated in February 2001, from surface-sterilized seeds of the garden pea. This finding is in agreement with the observation of Leifert et al. (1994), who reported micro-organisms surviving the surface sterilization of plant material. Elvira-Recuenco & Van Vuurde (2000) also proved that various bacteria are abundant in pea cultivars. Characterization of the 16S rRNA gene sequence indicated that strain C/2T is a member of the genus Paenibacillus (Ash et al., 1993). An analysis of 16S rRNA gene sequence similarity (<97 %) indicated that strain C/2T differs from all currently recognized species of the genus Paenibacillus.
Seeds of the garden pea, Pisum sativum ‘Caesar’, harvested in 1999 at Agritec (a seed-producing company in umperk, Czech Republic), were used in the experiment. A sample of 1 kg seeds was surface sterilized for 1 min with 70 % ethanol; this was followed by vigorous shaking in 15 % household bleach (Savo – 5 % sodium hypochlorite; Bochemie) for 15 min (Saettler et al., 1989). The seeds were than rinsed three times in sterile double-distilled water, ground and then soaked in sterile double-distilled water for 3 h. The supernatant was filtered through a 0·2 µm Supor membrane disc filter (Gelman). The filter discs were placed on nutrient agar plates containing 523 medium (Viss et al., 1991) and/or Leifert Waites sterility test medium (Leifert et al., 1994) and incubated at 27 °C in the dark for 1 week. Strain C/2T was isolated and purified from the colonies of micro-organisms for further study.
The isolate and reference cultures were cultivated on tryptone soy agar (Oxoid) at 30 °C for the investigation of morphological and physiological characteristics. Cells used for cellular fatty acid analysis were grown on trypticase soy broth (BBL) solidified with agar (Difco) at 28 °C for 24 h. For DNA extraction, all samples were cultivated on tryptone soy agar at 30 °C for 4 days.
Phenotypic characterization was performed using the methods of Gordon et al. (1973), with two exceptions: hydrolysis of Tween 80 and gelatin was performed according to the method of Páová & Kocur (1984) and, for oxidase and hippurate hydrolysis, the OXItest and the HIPPURATEtest (Pliva-Lachema) were used. Anaerobic growth was determined in BBL anaerobic agar (Becton Dickinson). Acid production from carbohydrates was determined using API 50 CH strips (bioMérieux), according to the manufacturer's directions, after incubation for up to 7 days. The distinguishing diagnostic traits of strain C/2T and closely related species are shown in Table 1.
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Whole-cell fatty acids from cultures of the isolate and from references cultures were extracted and analysed according to the instructions of the Microbial Identification System operating manual (MIDI). The major fatty acid present was anteiso-C15 : 0, which is as expected for the genus Paenibacillus (Shida et al., 1997). Strain C/2T differed from the others by having considerably larger amounts of iso-C15 : 0 (13·5 %) and iso-C17 : 0 (6·1 %) (Table 1
; details are available in Supplementary Table S1 in IJSEM Online).
Genomic DNA was isolated from the bacterial culture as described by Lambert et al. (1998). For measurement of the G+C content, the DNA was hydrolysed in 90 % formic acid for 30 min at 140 °C (Swarts et al., 1996). Hydrolysed DNA was analysed by HPLC using a Finnigan AQA mass spectrometer (ThermoQuest). Relative amounts were determined from peak areas and coefficients of relative molar absorption. The DNA G+C content was 50·8 mol%, which is in accordance with the overall content for the genus Paenibacillus (Shida et al., 1997).
Strain C/2T was characterized by using PCR amplification of the 16S rRNA gene with the Paenibacillus-specific forward primer PAEN515F (Shida et al., 1997) and universal reverse primer 1377R (Shida et al., 1996). The 16S rRNA gene was amplified with primers 8F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1516R (5'-GGTGATCCAGCCGCACCTTCC-3'), using a Progene thermal cycler (Techne). The PCR product was purified with SigmaSpin post-reaction columns (Sigma-Aldrich) and ligated into the vector pCR2.1 (Invitrogen). Competent cells of Escherichia coli TOP10F' were transformed with a TA cloning kit (Invitrogen) according to the manufacturer's instructions. Plasmids containing the insert were sequenced using an automatic DNA analyser (ABI PRISM 310; Perkin-Elmer).
The resultant 16S rRNA gene sequence was aligned with reference sequences obtained from GenBank with the CLUSTAL X 1.8 multiple alignment program (Thompson et al., 1997). Evolutionary distance matrices were calculated using the algorithm of Jukes & Cantor (1969). The phylogenetic tree was constructed by the neighbour-joining method (Saitou & Nei, 1987) using the software package TREECON for Windows (Van de Peer & De Wachter, 1994). The stability of the relationships was assessed statistically by using a bootstrap analysis based on 1000 resamplings (Felsenstein, 1985).
The phylogenetic position of the 16S rRNA gene sequence of strain C/2T is shown in a dendrogram (Fig. 1; an extended version of this tree is available as Supplementary Fig. S1 in IJSEM Online). Strain C/2T was clearly located within the genus Paenibacillus, close to the species Paenibacillus phyllosphaerae, Paenibacillus curdlanolyticus and Paenibacillus kobensis. The levels of 16S rRNA gene sequence similarity to these closely related species were 94·0, 94·9 and 95·7 %, respectively.
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, but with the following modifications. The genomic probe of strain C/2T was prepared using a DIG DNA labelling and detection kit (Roche) according to the manufacturer's instructions. A positively charged nylon membrane (Roche) and a BM-purple (Roche) colorimetric substrate were used. Dot blots were scanned with a CCD camera (Sony) and analysed using the software GDS 8000 (UVP). DNA relatedness values for strain C/2T with respect to closely related species ranged from 25 % with Paenibacillus kobensis to 17 % with Paenibacillus phyllosphaerae (see Supplementary Table S2).
According to Stackebrandt & Goebel (1994) and Wayne et al. (1987), the levels obtained for 16S rRNA gene similarity (
97 %) and DNA–DNA similarity (70 %), respectively, support the genomic distinction of strain C/2T from other species. On the basis of the phylogenetic, chemotaxonomic and phenotypic data, strain C/2T should be classified as the type strain of a novel species of the genus Paenibacillus, for which we propose the name Paenibacillus mendelii sp. nov.
Description of Paenibacillus mendelii sp. nov.
Paenibacillus mendelii (men.de'li.i. N.L. gen. n. mendelii of Mendel, to honour J. G. Mendel, the founder of genetics).
Cells are Gram-variable rods. Aerobic or facultatively anaerobic. Spores are oval with a subterminal position in a swollen sporangium. Optimal growth occurs between 25 and 30 °C. Colonies on nutrient agar are circular, smooth, flat, bright, translucent with entire edges and about 1–2 mm in diameter. Positive tests for catalase, oxidase, lecithinase, 
-galactosidase and hydrolysis of aesculin, for anaerobic growth and for growth at pH 8·5. Acid is produced from glycerol, D-arabinose, L-arabinose, ribose, D-xylose, methyl -xyloside, galactose, D-glucose, D-fructose, rhamnose, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, melezitose, D-raffinose, -gentiobiose, D-turanose, L-fucose and 5-ketogluconate. Negative tests for production of acetoin and indole, utilization of citrate, haemolysis, arginine dihydrolase and DNase, and for hydrolysis of casein, starch, hippurate, urea, gelatin, Tween 80 and tyrosine. Nitrate is not reduced and no growth occurs at 50 °C or in 5 % NaCl. Hydrolysis of agar is not observed. Acid is not produced from erythritol, L-xylose, adonitol, D-mannose, L-sorbose, dulcitol, inositol, mannitol, sorbitol, methyl 
-D-mannoside, methyl -D-glucoside, N-acetylglucosamine, amygdalin, arbutin, inulin, starch, glycogen, xylitol, D-lyxose, D-tagatose, D-fucose, D-arabitol, L-arabitol, gluconate or 2-ketogluconate. Sensitive to erythromycin, tetracycline, vancomycin, cephalothin and piperacillin and resistant to clindamycin and chloramphenicol. The DNA G+C content is 50·8 mol%. The major fatty acid is anteiso-C15 : 0.
Habitat not known; the type strain, C/2T (=CCM 4839T=LMG 23002T), was isolated from surface-sterilized pea seeds.
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ACKNOWLEDGEMENTS |
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Sobek for assistance with isolation and purification of strain C/2T and Dr Petr Dole
el for the HPLC analysis. This research was partially supported by the Ministry of Education of the Czech Republic (project no. MSM 021622416), the Ministry of Agriculture of the Czech Republic (project no. QF3072) and the National Research Centre: Signalling in Plants, Czech Republic (grant no. LN00A081). |
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