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State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, P. R. China
Correspondence
Xiuzhu Dong
dongxz{at}sun.im.ac.cn
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ABSTRACT |
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-gentiobiose. The mean DNA G+C content of the two strains was 46·9±0·8 mol% (46·4 mol% for the type strain, C-5-1T). Phylogenetic analysis based on 16S rRNA gene sequence similarity showed that strain C-5-1T was clustered in the Lactobacillus casei/Pediococcus phylogenetic group and was closely related to Pediococcus dextrinicus JCM 5887T (97·9 % similarity), followed by Lactobacillus algidus JCM 10491T (93·9 % similarity). The DNA–DNA relatedness between the type strain C-5-1T and P. dextrinicus JCM 5887T and L. algidus JCM 10491T was only 5·4 and 4·3 %, respectively. Therefore, based on phenotypic, genotypic and phylogenetic analyses, a novel Lactobacillus species, Lactobacillus concavus sp. nov., is proposed. The type strain is C-5-1T (=AS 1.5017T=LMG 22739T).
Published online ahead of print on 10 June 2005 as DOI 10.1099/ijs.0.63509-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Lactobacillus concavus sp. nov. AS 1.5017T is AY683322.
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; Skerman et al., 1980), nearly 120 species have been recognized. The lactobacilli are widely distributed in the environment and are frequently found in fermented food, vegetables and in the intestines of man and animals (Omar et al., 2000; Gardner et al., 2001; Satokari et al., 2003). Lactobacilli also play important roles in the wine-making process; they are responsible for malolactic fermentation, which follows alcoholic fermentation by yeasts (Lonvaud-Funel, 1999). In recent years, some novel lactic acid bacteria, such as Lactobacillus ferintoshensis (Simpson et al., 2001), Lactobacillus nagelii (Edwards et al., 2000), Lactobacillus amylolyticus (Bohak et al., 1998) and Pediococcus claussenii (Dobson et al., 2002), have been isolated from malt whisky, grape wine, beer malt and beer, respectively. Chinese liquors are a type of wine produced through a solid fermentation of grain. During this process, grains of wheat or other cereals are mixed with water and sealed in a fermenting cellar for several months. The grains are fermented by the microorganisms present in the mud wall of the cellar. During an investigation of lactic acid bacteria in Chinese wine breweries, we isolated two Lactobacillus strains from a brewery in Hebei province, China, which exhibited phenotypic characteristics that distinguished them from all the Lactobacillus species with validly published names. Phylogenetic analysis based on 16S rRNA gene sequence similarity and DNA–DNA hybridization indicated that the strains could represent a novel species of the genus Lactobacillus.
Lactobacillus algidus JCM 10491T (Kato et al., 2000) was provided by JCM (Japan Collection of Microorganisms). Strains C-5-1T and HB5 were originally isolated from wall samples from a distilled spirit fermenting cellar in Hebei province, China. The samples were cultured anaerobically in MRS liquid (de Man et al., 1960) at 37 °C for 2 days and then purified on MRS agar. The strains were routinely grown anaerobically on the same medium at 37 °C unless otherwise stated. The end products of glucose fermentation in tryptone peptone yeast-extract glucose (TPYG) medium (Scardovi, 1986) were detected using a gas chromatograph (GC-14B; Shimadzu). Isomers of lactate formed from glucose were determined using a D-L-lactic acid test kit (Roche Diagnostics). Catalase activity was determined by adding 15 % (v/v) hydrogen peroxide to a fresh culture on a glass slide. The temperature profile was determined by using a water bath with a temperature gradient. Growth at various pH values was investigated using TPYG liquid medium adjusted with hydrochloric acid or NaOH solution. Tolerance to ethanol was measured by monitoring growth in TPYG liquid medium containing various concentrations (v/v) of ethanol. Generation time was measured by monitoring the OD600 of the MRS culture at 37 °C for 72 h. The sugar fermentation pattern was determined using the API 50 CH system (bioMérieux) and 24 h incubation at 37 °C. The cell-wall peptidoglycan was of the diaminopimelic acid (DAP) type as determined using a method modified from Becker et al. (1964). All of the tests were performed in duplicate.
Genomic DNA was extracted and purified using a modified method of Marmur (1961) and Dong et al. (2000). The 16S rRNA gene was amplified by PCR using genomic DNA as template. PCR products were sequenced by using a Dye terminator cycle sequencing ready reaction kit with an ABI PRISM 377XL DNA sequencer. The closest matching sequences were retrieved from the GenBank database and aligned. 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 MEGA2 (Kumar et al., 2001). The stability of the tree was evaluated by bootstrap analysis of 1000 datasets. DNA G+C content was determined by the thermal denaturation method (Marmur & Doty, 1962). DNA–DNA relatedness was determined on the basis of the DNA–DNA liquid hybridization rate (De Ley et al., 1970) at 63 °C using a spectrophotometer with a thermal controller (DU-800; Beckman).
The two isolates were Gram-positive, non-spore-forming, non-motile curved rods. They had no catalase activity, were facultatively anaerobic and produced lactic acid exclusively [about 5 % (–)-D and 95 % (+)-L] without gas from glucose fermentation. The strains grew on acetate agar (pH 5·4) and did not produce H2S. These characters demonstrated that they were members of the genus Lactobacillus. By using the API 50 CH system, the two novel strains were found to ferment only a few types of sugars (Table 1 and species description). Strains C-5-1T and HB5 showed different reactions for the fermentation of D-fructose and amygdalin. The mean generation time of the two strains was 2·3±0·1 h when cultured anaerobically and 3·6±0·3 h when cultured aerobically. As expected from their habitat, the two strains were able to grow in concentrations of 8 % (v/v) ethanol.
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) rooted with Bacillus subtilis NCDO 1769T was constructed using the neighbour-joining method. Strain C-5-1T was found to be clustered in the L. casei/Pediococcus phylogenetic group (Collins et al., 1991) with 16S rRNA gene sequence similarity ranging from 87·3 to 97·9 %. Strain C-5-1T had the highest 16S rRNA gene sequence similarity to Pediococcus dextrinicus JCM 5887T (97·9 %). The highest similarity to a Lactobacillus species, L. algidus JCM 10491T, was only 93·9 %. The stability of the clustering in the tree (Fig. 1) was supported by high bootstrap values (
80 %). Therefore, strain C-5-1T may represent a novel Lactobacillus species belonging to the L. casei/Pediococcus phylogenetic group.
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). The DNA–DNA hybridization rate between strains C-5-1T and HB5 was determined to be 100 %, indicating that they formed a homogeneous genetic group. However, the DNA–DNA relatedness between strain C-5-1T and the phylogenetically closest species, L. algidus JCM 10491T and P. dextrinicus JCM 5887T, was 4·3 and 5·4 %, respectively; much lower than the DNA–DNA relatedness threshold of 70 % suggested to delineate a species.
Although strain C-5-1T shared the highest 16S rRNA gene sequence similarity to P. dextrinicus JCM 5887T (97·9 %), the following characteristics verified that the two novel strains constituted a separate species. Strain C-5-1T was a curved rod, while P. dextrinicus was spherical and occurred in tetrads. P. dextrinicus fermented D-fructose, amygdalin, galactose, D-mannose and gluconate (acid and gas) but strain C-5-1T did not (Table 1
). Strain C-5-1T and P. dextrinicus showed low values of DNA–DNA relatedness and had a DNA G+C content which differed by about 6 mol%. The two novel strains could be easily differentiated from the closely related Lactobacillus species, L. algidus, a psychrophilic bacterium isolated from refrigerated beef, as they were able to grow above 30 °C, were unable to ferment ribose, galactose, L-arabinose or D-mannose and had a 10 mol% difference in DNA G+C content (Table 1).
Collins et al. (1991) showed that P. dextrinicus formed a peripheral branch of the genus Pediococcus with low 16S rRNA gene sequence similarity (92·6–93 %), but that it was intermixed in the 16S rRNA phylogenetic tree with species of Lactobacillus with even higher 16S rRNA gene sequence similarity (92·0–94·8 %). This implied that P. dextrinicus could be a specific lactic acid coccus and that its taxonomic position might need to be reconsidered taking into account its ability to ferment different substrates, such as dextrin and starch, when compared with other Pediococcus species. Recently, it has been found that bacterial cell shape is controlled by only a few genes, such as mreB and fstZ, and an alteration in cell shape can be induced by the inactivation of a single gene in this group (Jones et al., 2001). Hence, it may not be appropriate to use cell shape as a taxonomic marker in some groups of bacteria such as Lactobacillus and Pediococcus.
On the basis of a combination of phenotypic characteristics, phylogenetic relationships and DNA–DNA relatedness, a novel species of the genus Lactobacillus, Lactobacillus concavus sp. nov., is proposed.
Description of Lactobacillus concavus sp. nov.
Lactobacillus concavus (con.ca'vus. L. adj. concavus curved, referring to the curved shape of the strains).
Cells are Gram-positive, non-spore-forming, non-motile curved rods, about 0·5–0·6x2·0–2·5 µm in size after 24 h incubation in anaerobic MRS liquid medium. Colonies are white, convex and smooth with an entire margin. Colonies are about 1 mm in diameter after 24 h cultivation on anaerobic MRS plates. Catalase-negative. Facultatively anaerobic. Lactic acid [about 5 % (–)-D and 95 % (+)-L], but no gas, is produced from glucose and gluconate fermentation. The diagnostic diamino acid in the cell-wall peptidoglycan is meso-DAP. The optimum temperature for growth is 30–37 °C; the temperature ranges for growth for C-5-1T and HB5 are 10–42 °C and 10–39 °C, respectively. Optimum pH is 6·0–6·4; the pH ranges for growth of C-5-1T and HB5 are 3·8–8·1 and 4·3–8·1, respectively. Growth can occur in 8 % (v/v) ethanol, but not in 6·5 % (w/v) NaCl. Aesculin is hydrolysed but arginine is not hydrolysed. Voges–Proskauer test is negative. Acid is produced from D-glucose, N-acetylglucosamine, arbutin, salicin, cellobiose, maltose, trehalose and -gentiobiose. Acid is not produced from glycerol, erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl -xyloside, galactose, D-mannose, L-sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl 
-D-mannoside, methyl -D-glucoside, lactose, melibiose, sucrose, inulin, melezitose, D-raffinose, starch, glycogen, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. Fermentation of D-fructose and amygdalin is variable. The DNA G+C content is 46·9±0·8 mol% (46·4 mol% for the type strain, C-5-1T).
The type strain, C-5-1T (=AS 1.5017T=LMG 22739T), was isolated from the walls of a distilled spirit fermenting cellar in Hebei province, China.
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ACKNOWLEDGEMENTS |
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