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1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, P. R. China
2 Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100080, P. R. China
Correspondence
Xiuzhu Dong
dongxz{at}sun.im.ac.cn
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains Z-8T and Z-1 are AY956788 and AY956791, respectively.
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), especially pickles and preserved meat. In the food industry, they have been used as starter cultures in some kinds of sausage fermentation. However, pediococci can also cause quality problems for brewers when present in beer and wines (Gardner et al., 2001; Lonvaud-funel, 1999).
To date seven Pediococcus species have been described: Pediococcus acidilactici, Pediococcus claussenii (Dobson et al., 2002), Pediococcus damnosus, Pediococcus dextrinicus, Pediococcus inopinatus, Pediococcus parvulus and Pediococcus pentosaceus. A species that was previously designated as belonging to the genus Pediococcus, Pediococcus urinaeequi, was recently reclassified as Aerococcus urinaeequi (Felis et al., 2005). On the basis of 16S rRNA gene similarity, the genus Pediococcus was grouped in the Lactobacillus casei branch of the lactic acid bacteria (Collins et al., 1991). Within the genus Pediococcus, P. damnosus, P. inopinatus and P. parvulus formed a closely related cluster, whereas P. acidilactici, P. claussenii and P. pentosaceus formed a second cluster, and P. dextrinicus, a distinct Pediococcus species, was clustered with some Lactobacillus species.
Chinese liquor is a kind of wine produced by solidified fermentation of grain. During an investigation of lactic acid bacteria in Chinese wine breweries, we isolated several dozen Pediococcus strains. Two strains from a brewery in Hebei Province, China, exhibited phenotypic characteristics that differed from those of all existing Pediococcus species. Phylogenetic analysis based on 16S rRNA gene sequence similarity and DNA–DNA hybridization also indicated that the two strains represented a novel species of the genus Pediococcus.
P. damnosus LMG 11484T, P. inopinatus LMG 11409T and P. parvulus LMG 11486T were kindly provided by the Laboratory of Microbiology, Ghent University (BCCM/LMG, Ghent, Belgium). Strains Z-1 and Z-8T were originally isolated from the walls of a distilled-spirit-fermenting cellar in Hebei Province, China. They were isolated and purified by streaking the samples on de Man–Rogosa–Sharpe (MRS) agar (de Man et al., 1960). The strains were routinely grown aerobically on the same medium at 37 °C unless stated otherwise. The end-products of glucose fermentation in tryptone-peptone-yeast extract-glucose (TPYG) liquid were detected by using a gas chromatograph (GC-14B; Shimadzu). Isomers of lactate formed from glucose were determined by using a D-/L-lactic acid test kit (Roche Diagnostics). Catalase activity was determined by adding 15 % (v/v) hydrogen peroxide to the fresh cultures. Temperature profiles were determined by using a water bath with a temperature gradient. Growth at various pH values was determined in TPYG liquid adjusted with HCl or NaOH. Tolerance of ethanol was determined by monitoring growth in TPYG liquid with different concentrations of ethanol (v/v). The generation time was determined by monitoring the OD600 of an MRS liquid culture at 37 °C for 24 h. Sugar fermentation patterns were determined by using both conventional methods (Garvie, 1986) and the API 50 CH system (bioMérieux) after 24 h incubation at 37 °C. All of the tests were performed in duplicate.
Genomic DNA was extracted and purified as described by Marmur (1961). The 16S rRNA gene was amplified by PCR using the genomic DNA as template, and sequenced using an ABI PRISM 377XL DNA sequencer. The 16S rRNA gene sequences of the most-related species were retrieved from GenBank and aligned, and similarity analysis was performed using the CLUSTAL_X program (Thompson et al., 1997). A phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987), implemented in the MEGA2 program (Kumar et al., 2001). The stability of the clustering of the tree was evaluated by bootstrap analysis of 1000 datasets. DNA G+C content was determined by using the thermal denaturation method (Marmur & Doty, 1962). DNA–DNA relatedness was determined on the basis of DNA–DNA liquid reassociation rate (De Ley et al., 1970), using a DU-800 spectrophotometer (Beckman) with a thermal controller.
Cells of the two isolates were Gram-positive, non-spore-forming, non-motile cocci, occurring in pairs or tetrads. Catalase activity was negative and the strains were facultatively anaerobic. These characteristics indicated that the two isolates were members of the genus Pediococcus. Product determination showed that the two strains produced lactic acid [about 50 % (–)-D-/50 % (+)-L-] exclusively from glucose fermentation without gas. By using both conventional methods and the API 50 CH system, the two strains were shown to use rhamnose, ribose, D-xylose and L-xylose as sole carbon and energy sources, which differentiated them from phylogenetically closely related species (Table 1). Fermentation of D-arabinose, L-arabinose and methyl 
-D-glucoside was variable in the two strains. Both strains hydrolysed aesculin but not arginine. In addition, the two strains tolerated 10 % ethanol (v/v), in accordance with their original habitat. The mean generation time of the two strains was 1·09±0·06 h when cultured aerobically, and 1·01±0·06 h when cultured anaerobically.
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). It was shown that strain Z-8T clustered within the P. damnosus–P. inopinatus–P. parvulus group, with 16S rRNA gene sequence similarities ranging from 98·3 to 98·5 %. The two novel isolates had a partial 16S rRNA gene sequence similarity of 99·5 %. The mean DNA G+C content of the two novel strains was 38±1 mol% (37 mol% for strain Z-8T and 39 mol% for strain Z-1), which was within the range for the genus Pediococcus (34–42 mol%). DNA–DNA relatedness between the novel strains and their phylogenetically closest related species was determined at a hybridization temperature of 67 °C. The DNA–DNA relatedness between the two novel isolates, Z-1 and Z-8T, was 91 %, indicating a homogeneous genetic group. However, the DNA–DNA relatedness between strain Z-8T and P. damnosus LMG 11484T, P. inopinatus LMG 11409T and P. parvulus LMG 11486T was 19±4, 35±5 and 31±5 %, respectively, lower than the DNA–DNA relatedness threshold (70 %) for a species.
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). Based on the phenotypic characteristics, phylogenetic relationship and DNA–DNA relatedness, a novel species of the genus Pediococcus, Pediococcus cellicola sp. nov., is proposed.
Description of Pediococcus cellicola sp. nov.
Pediococcus cellicola (cel.li.co'la. L. n. cella a storeroom for wine and food; L. suff. -cola from L. n. incola an inhabitant, dweller; N.L. n. cellicola an inhabitant of a storeroom, indicating that the strains were originally isolated from a distilled-spirit-fermenting cellar).
Gram-positive, non-spore-forming, non-motile, spherical cells, 0·6–1 µm in diameter after 24 h incubation in aerobic MRS liquid at 37 °C. Cells occur in pairs or tetrads. Colonies are greyish white, low-convex, circular, entire, opaque and about 1 mm in diameter after 24 h cultivation on MRS plates at 37 °C. Catalase-negative. Facultatively anaerobic. Lactic acid [about 50 % (–)-D-/50 % (+)-L-] is produced from glucose fermentation, but not gas. Growth occurs at 13–44 °C, with an optimum at 30–37 °C. Optimum pH for growth is 6·0–6·8; no growth occurs at pH 3·8 and 8·2. Grows in 10 % ethanol (v/v) but not in 10 % NaCl (w/v). Aesculin but not arginine is hydrolysed. Acid is produced from galactose, glucose, fructose, mannose, methyl -D-mannoside, methyl -D-glucoside, N-acetylglucosamine, arbutin, salicin, cellobiose, gentiobiose, D-tagatose, 2-ketogluconate, rhamnose, ribose, D-xylose, L-xylose, lactose, maltose, trehalose and sucrose. Acid is not produced from glycerol, erythritol, adonitol, methyl 
-D-xyloside, sorbose, dulcitol, inositol, mannitol, sorbitol, amygdalin, aesculin, inulin, melezitose, raffinose, starch, glycogen, xylitol, D-turanose, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 5-ketogluconate or dextrin. Fermentation of D-arabinose, L-arabinose and methyl -D-glucoside is variable. Strain Z-8T ferments D-arabinose and L-arabinose but not methyl -D-glucoside, whereas the reverse applies for strain Z-1. Mean DNA G+C content is 38±1 mol% (37 mol% for strain Z-8T).
The type strain is Z-8T (=AS 1.3787T=LMG 22956T), which was isolated from a distilled-spirit-fermenting cellar in Hebei Province, China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Collins, M. D., Rodriguez, U., Ash, C., Aguirre, M., Farrow, J. A. E., Martinez-Murcia, A., Philips, B. A., Williams, A. M. & Wallbanks, S. (1991). Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16S rRNA. FEMS Microbiol Lett 77, 5–12.[CrossRef]
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de Man, J. C., Rogosa, M. & Sharpe, M. E. (1960). A medium for the cultivation of lactobacilli. J Appl Bacteriol 23, 130–135.
Dobson, C. M., Deneer, H., Lee, S., Hemmingsen, S., Glaze, S. & Ziola, B. (2002). Phylogenetic analysis of the genus Pediococcus, including Pediococcus claussenii sp. nov., a novel lactic acid bacterium isolated from beer. Int J Syst Evol Microbiol 52, 2003–2010.[Abstract]
Felis, G. E., Torriani, S. & Dellaglio, F. (2005). Reclassification of Pediococcus urinaeequi (ex Mees 1934) Garvie 1988 as Aerococcus urinaeequi comb. nov. Int J Syst Evol Microbiol 55, 1325–1327.
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