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merda1,
ek2ová2í3
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, 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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type. The DNA G+C content (50 mol%) and the major fatty acid (anteiso-C15 : 0) of strain LMG 19508T were also consistent with its affiliation to the genus Paenibacillus. DNA–DNA hybridization distinguished strain LMG 19508T from other phylogenetically related Paenibacillus species. Therefore, the isolate represents a novel species, for which the name Paenibacillus sepulcri sp. nov. is proposed. The type strain is CCM 7311T (=LMG 19508T).
A extended neighbour-joining phylogenetic tree based on almost-complete 16S rRNA gene sequences is available as a supplementary figure in IJSEM Online.
Present address: Institute of Botany and Zoology, Faculty of Science, Masaryk University, Kotlá
ská 2, 611 37 Brno, Czech Republic. 
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and its description was emended by Shida et al. (1997). The genus Paenibacillus is under intensive taxonomic investigation, with ten species having been formally described in the last year. Heyrman & Swings (2001) isolated a strain, originally classified as Paenibacillus sp. LMG 19508, from biodeteriorated mural paintings in the Servilia tomb at the Roman necropolis of Carmona in Seville, Spain. Thereafter, the strain was deposited in the Czech Collection of Microorganisms as CCM 7311 and used for biodiversity studies. The phylogenetic position and characteristics of this strain were investigated using a polyphasic approach. The sequence of its 16S rRNA gene showed that the closest phylogenetic neighbour is Paenibacillus mendelii (merda et al., 2005). On the basis of phylogenetic, DNA relatedness, chemotaxonomic and phenotypic analyses, we propose the classification of strain CCM 7311T as the type strain of a novel species within the genus Paenibacillus.
Strain CCM 7311T and the reference cultures listed in Table 1 were grown on tryptone soya agar (Oxoid) at 30 °C to investigate their morphological and physiological characteristics. For cellular fatty acid analysis, cells were grown on trypticase soya broth (BBL) solidified with agar (Difco) at 28 °C for 24 h. For DNA extraction, all samples were cultivated on tryptone soya agar (Oxoid) at 30 °C for 4 days.
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. The method of Páová & Kocur (1984) was used to test for the hydrolysis of Tween 80 and gelatin, and OXItest and the HIPPURATEtest (Pliva-Lachema) were used for oxidase and hippurate hydrolysis tests. Anaerobic growth was determined in BBL anaerobic agar tubes (Becton Dickinson) according to the manufacturer's instructions. Acid production from carbohydrates was determined using API 50 CH strips (bioMérieux), according to the manufacturer's instructions, after incubation for up to 7 days. The distinguishing phenotypic traits of strain CCM 7311T and closely related species are shown in Table 1
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Whole-cell fatty acids from the isolate and reference cultures were extracted and analysed according to the instructions in the manual for the Microbial Identification System (MIDI, 2001). The major fatty acid in strain CCM 7311T is anteiso-C15 : 0, which corresponds with that of members of the genus Paenibacillus (Shida et al., 1997). The fatty acid profile of the isolate is similar to those of the related reference strains (Table 2). It was possible to differentiate the novel strain from its closest phylogenetic neighbour, P. mendelii CCM 4839T on the basis of the considerably smaller amounts of C15 : 0 (2.3 %), iso-C15 : 0 (5.0 %) and iso-C17 : 0 (1.8 %) in strain CCM 7311T or by the absence of C16 : 1
11c. In comparison with other paenibacilli, strain CCM 7311T differed, for example, by containing a larger proportion of anteiso-C15 : 0 (59.3 %). Only Paenibacillus kobensis CCM 4537T had a comparable fatty acid pattern.
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type, containing meso-diaminopimelic acid as the diagnostic diamino acid. Analysis of the respiratory quinones was carried out by the DSMZ Identification Service and Dr Brian Tindall (DSMZ). MK-7 was the only menaquinone present in strain CCM 7311T.
Genomic DNA was isolated from the bacterial culture as described by Lambert et al. (1998). To determine the G+C content, DNA was hydrolysed in 90 % formic acid for 30 min at 140 °C (Swarts et al., 1996). The hydrolysed DNA was analysed by HPLC using a Finnigan AQA mass spectrometer (ThermoQuest). The relative amounts were determined from the peak areas and coefficients of relative molar absorption. The G+C content was 50 mol%, in accordance with the overall content for members of the genus Paenibacillus (Shida et al., 1997).
DNA–DNA hybridization was performed according to Jahnke (1994), with the modifications of merda et al. (2005). The levels of DNA–DNA hybridization for strain CCM 7311T with respect to P. mendelii CCM 4839T, Paenibacillus phyllosphaerae CCM 7310T, Paenibacillus curdlanolyticus CCM 4536T, Paenibacillus glycanilyticus JCM 11221T and P. kobensis CCM 4537T were 16, 15, 20, 24 and 27 %, respectively. According to Wayne et al. (1987), these levels of DNA–DNA relatedness (
70 %) support the view that strain CCM 7311T is genomically distinct from these other species.
Amplification and sequencing of the 16S rRNA gene were performed as described by merda et al. (2005). 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 with 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 performing a bootstrap analysis based on 1000 resamplings (Felsenstein, 1985).
The phylogenetic position of the 16S rRNA gene sequence of the strain is shown in the dendrogram (Fig. 1; see also Supplementary Fig. S1 available in IJSEM Online). The closest phylogenetic neighbours of the strain CCM 7311T are P. mendelii CCM 4839T, P. phyllosphaerae CCM 7310T, P. curdlanolyticus CCM 4536T and P. kobensis CCM 4537T. The binary similarity values of strain CCM 7311T and the related species ranged between 96.7 % (P. mendelii CCM 4839T) and 93.5 % (P. glycanilyticus JCM 11221T). According to Stackebrandt & Goebel (1994), these levels of 16S rRNA gene similarity (97 %) indicate that strain CCM 7311T is genomically distinct from these other species.
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Description of Paenibacillus sepulcri sp. nov.
Paenibacillus sepulcri (se.pul'cri. L. gen. neut. n. sepulcri from a tomb, pertaining to the place of isolation of the type strain).
Cells are Gram-variable, facultatively anaerobic rods. Spores are oval and are positioned subterminally in swollen sporangia. Moderately psychrotolerant. The temperature range for growth is 10–30 °C, but no growth occurs at 37 °C; the optimum is 25 °C. Grows at pH 6.0–8.0; the optimum is between pH 7.2 and 7.4. Tolerates up to 2 % NaCl, but not 3 % NaCl. Colonies on nutrient agar are circular, smooth, slightly convex with complete edges and colourless. Tests positive for catalase, oxidase, lecithinase and 
-galactosidase and tests negative for arginine dihydrolase, DNase, haemolysis and citrate utilization. Hydrolyses aesculin and hippurate but not casein, starch, urea, gelatin, Tween 80 or tyrosine. Acid is produced from D-arabinose, L-arabinose, D-xylose, methyl -D-xyloside, galactose, D-glucose, D-mannose, rhamnose, mannitol, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, melezitose, D-raffinose, glycogen, -gentiobiose, D-turanose, L-fucose and 5-ketogluconate, but not from glycerol, erythritol, ribose, L-xylose, adonitol, D-fructose, L-sorbose, dulcitol, inositol, sorbitol, methyl 
-D-mannoside, methyl -D-glucoside, N-acetylglucosamine, amygdalin, arbutin, inulin, starch, xylitol, D-tagatose, D-fucose, D-arabitol, L-arabitol, gluconate or 2-ketogluconate. Does not reduce nitrate to nitrite and is negative for the production of acetoin and indole. Hydrolysis of agar is not observed. The DNA G+C content is 50 mol%. The major fatty acid is anteiso-C15 : 0. Cell-wall analysis reveals meso-diaminopimelic acid as the diagnostic diamino acid in the peptidoglycan. The predominant menaquinone is MK-7.
The natural habitat is unknown. The type strain, CCM 7311T (=LMG 19508T), was isolated from biodeteriorated mural paintings in the Servilia tomb at the Roman necropolis of Carmona in Seville, Spain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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