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NODAI Culture Collection Center, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan
Correspondence
Akihito Endo
pegaman{at}hotmail.co.jp
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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Maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences of the novel strains and related species are available as supplementary figures in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Shochu is a traditional Japanese distilled spirit made from rice, sweet potato, barley or other starchy materials; Aspergillus niger and Saccharomyces cerevisiae play roles in saccharification of the starch and its fermentation into alcohol. Concomitantly, five strains of lactic acid bacteria were isolated from the compost and were separated into two groups on the basis of their 16S rRNA gene sequences: they formed two subclusters, the members of which were related to the obligately heterofermentative lactobacilli Lactobacillus buchneri, L. diolivorans, L. hilgardii, L. kefiri, L. parabuchneri and L. parakefiri. The DNA–DNA relatedness results showed clear separation of the isolates into two groups that correlated well with the two subclusters generated from a phylogenetic analysis. Moreover, the levels of DNA–DNA relatedness showed clear separation of the two groups from the phylogenetic relatives. This report provides a taxonomic analysis of the five isolates, and proposes two novel Lactobacillus species. A sample of compost was prepared from a distilled shochu residue in Miyazaki Prefecture in the southern Kyushu region of Japan. The sample was serially diluted with sterile saline before being plated on MRS (Oxoid) agar containing (l–1) 10 mg cycloheximide, 10 mg sodium azide, 5.0 g calcium carbonate and 15 g agar. Plates were incubated for 5 days at 30 °C. After incubation, colonies that produced clear zones were picked into MRS broth and maintained in the same medium. Of the isolates, five (NRIC 0676T, NRIC 0677T, NRIC 0678, NRIC 0679 and NRIC 0680) were used in the present study. Strains NRIC 0677T and NRIC 0680 grew more slowly than the other three isolates in MRS broth; these isolates took 2–3 days to reach stationary phase, whereas NRIC 0676T, NRIC 0678 and NRIC 0679 took 1–2 days. L. buchneri NRIC 1040T, L. diolivorans NRIC 0695T, L. hilgardii NRIC 1060T, L. kefiri NRIC 1693T, L. parabuchneri NRIC 1780T and L. parakefiri NRIC 0217T were obtained from the NODAI Culture Collection Center, Tokyo University of Agriculture (Tokyo, Japan) and were used as reference strains in the present study. The reference strains were also maintained in MRS broth.
The 16S rRNA gene sequences of the five isolates were determined as described previously (Endo & Okada, 2005). The closest known relatives of the isolates were determined by performing DataBase searches, and the sequences of closely related species were retrieved from the DDBJ database. Multiple alignments of the sequences were carried out with the program CLUSTAL_X, version 1.18 (Thompson et al., 1997). Distance matrices for the aligned sequences were calculated by using the two-parameter method of Kimura (1980). The neighbour-joining method was used to construct a phylogenetic tree (Saitou & Nei, 1987). The robustness of individual branches was estimated by using bootstrapping with 1000 replicates (Felsenstein, 1985). Phylogenetic trees were also constructed by using the maximum-likelihood (Cavalli-Sforza & Edwards, 1967) and maximum-parsimony (Kluge & Farris, 1969) methods with PHYLIP version 3.65 (Felsenstein, 2005). The 16S rRNA gene sequences of the isolates were compared with one another, and the sequences of strains NRIC 0676T and NRIC 0677T were used to search for sequence similarity using DataBase. Approximately 1390 bp of the 16S rRNA gene sequences of the isolates and related species were used to construct the phylogenetic tree. After the sequence comparisons, the isolates were separated into two groups. The 16S rRNA gene sequence similarities between strains NRIC 0676T, NRIC 0678 and NRIC 0679 ranged from 99.9 to 100 %, and the sequence similarity between NRIC 0677T and NRIC 0680 was 100 %. The sequence similarity between strains NRIC 0676T and NRIC 0677T was 97.6 %. The highest levels of sequence similarity between NRIC 0676T and strains of known species of lactic acid bacteria were obtained with the type strains of L. hilgardii, L. buchneri, L. diolivorans, L. parabuchneri, L. kefiri and L. parakefiri (98.2, 97.5, 97.3, 96.8, 96.7 and 96.7 %, respectively); for NRIC 0677T the highest values were with respect to the type strains of L. hilgardii, L, buchneri, L. diolivorans, L. parabuchneri, L. kefiri and L. parakefiri (98.0, 97.7, 97.6, 97.8, 97.5 and 97.1 %, respectively). The strains in the two groups formed two subclusters, the members of which were closely related to obligately heterofermentative lactobacilli (L. buchneri, L. diolivorans, L. hilgardii, L. kefiri, L. parabuchneri and L. parakefiri) in the neighbour-joining analysis (Fig. 1). Identical tree topologies were obtained by using the maximum-likelihood and maximum-parsimony methods (see Supplementary Figs S1 and S2 available in IJSEM Online). L. buchneri, L. hilgardii and L. kefiri were classified within the L. buchneri phylogenetic group, together with Lactobacillus fructivorans, L. lindneri and L. sanfransiscensis, by Schleifer & Ludwig (1995). Our phylogenetic trees showed that the L. buchneri phylogenetic group was divided into three subclusters: the L. buchneri subcluster (L. buchneri, L. diolivorans, L. hilgardii, L. kefiri, L. parabuchneri, L. parakefiri and the five novel isolates), the Lactobacillus brevis subcluster (Lactobacillus acidifarinae, L. brevis, L. parabrevis, L. spicheri and L. zymae) and the L. fructivorans subcluster (L. fructivorans, L. homohiochii, L. lindneri and L. sanfransiscensis) (Fig. 1). The sequence similarities of the members of the L. buchneri subcluster ranged from 96.7 to 99.1 %, and the sequence similarities between the members of the L. buchneri subcluster and the members of the L. brevis and L. fructivorans subclusters were 92.6–95.2 % and 90.7–94.0 %, respectively. The sequence similarities among the members of the L. brevis subcluster were 96.1–99.7 %, and those between the members of the L. brevis and L. fructivorans subclusters were 90.7–94.0 %. The sequence similarities among the members of the L. fructivorans subcluster ranged from 93.6 to 99.2 %.
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). The DNA G+C contents of the novel isolates were also determined. Extraction and isolation of bacterial DNA were performed by using the method of Marmur (1961), as modified by Ezaki et al. (1983). DNA–DNA hybridization was carried out by using the microdilution-well technique, with photobiotin for labelling of the DNA (Ezaki et al., 1989). The G+C contents of the strains tested were determined by HPLC as described by Tamaoka & Komagata (1984). The levels of DNA–DNA relatedness showed clear separation of the isolates into two groups. Strains NRIC 0676T, NRIC 0678 and NRIC 0679 shared high levels of DNA–DNA relatedness (87–100 %), and strains NRIC 0677T and NRIC 0680 shared 95 % DNA–DNA relatedness. In contrast, strain NRIC 0676T showed low levels of DNA–DNA relatedness (42–46 %) with respect to NRIC 0677T and NRIC 0680, and NRIC 0677T also showed low levels of DNA–DNA relatedness (43–48 %) with respect to NRIC 0676T, NRIC 0678 and NRIC 0679. Therefore, we concluded that the two groups were genetically distinguishable from each other; these groups correlated well with the two subclusters obtained using phylogenetic analysis. The levels of DNA–DNA relatedness for NRIC 0676T with respect to L. hilgardii NRIC 1060T, L. diolivorans NRIC 0695T, L. buchneri NRIC 1040T, L. parabuchneri NRIC 1780T, L. kefiri NRIC 1693T and L. parakefiri NRIC 0217T were 47, 35, 30, 29, 23 and 20 %, respectively; those for NRIC 0677T with respect to these reference strains were 37, 32, 25, 25, 30 and 21 %, respectively. The G+C contents of strains NRIC 0676T, NRIC 0678, NRIC 0679 ranged from 40 to 41 mol% and that of strains NRIC 0677T and NRIC 0680 was 40 mol%.
Randomly amplified polymorphic DNA (RAPD) fingerprinting was performed to differentiate the five isolates by a method described previously (Endo & Okada, 2006). Primers D (5'-GAGGACAAAG), E (5'-GGCGTCGGTT) and F (5'-GGCCACGGAA) were used in this study. The fingerprinting indicated that the isolates were separated into two groups. The fingerprints of strains NRIC 0676T, NRIC 0678 and NRIC 0679 differed slightly, and those of strains NRIC 0677T and NRIC 0680 were similar to each other (Fig. 2). This demonstrated that the isolates were separated at the strain level within each group. The two groups produced fingerprints that were quite different. The groups correlated well with the separate groups generated by phylogenetic analysis or levels of DNA–DNA relatedness.
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); MRS broth was used as a basal medium. The characteristics of the isolates (described in detail in the species descriptions below) were compared with those of closely related species (Table 1). Members of the L. brevis and L. fructivorans subclusters were not used for comparisons with the isolates because of the low levels of 16S rRNA gene sequence similarity to the novel strains. The isolates were separated into two groups on the basis of phenotypic characteristics, and these groups correlated well with the two groups separated on the basis of phylogenetic analysis, levels of DNA–DNA relatedness and RAPD fingerprinting. Strains NRIC 0676T, NRIC 0677T and NRIC 0678 grew at 45 °C and grew slowly at 15 °C, but strains NRIC 0677T and NRIC 0680 did not grow at 15 or 45 °C. Growth at 45 °C was not found in other species of the L. buchneri subcluster (Table 1). However, as the major characteristics of the strains in the two groups were similar to those of the phylogenetic relatives, precise identification among the two groups and their phylogenetic relatives based on phenotypic features is difficult. Therefore, determinations of DNA–DNA relatedness are needed for precise identification of the species in the L. buchneri subcluster. The same has been observed for the species in the Lactobacillus acidophilus group (Fujisawa et al., 1992; Naser et al., 2006).
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Description of Lactobacillus farraginis sp. nov.
Lactobacillus farraginis (far.ra'gi.nis. L. gen. n. farraginis of mash, pertaining to shochu mash, an ingredient of a compost material from which the type strain was isolated).
Cells are Gram-positive, non-motile rods, measuring 0.8x3–6 µm. Cells occur singly or in pairs and chains. Facultatively anaerobic and catalase-negative. Colonies on MRS agar are beige, smooth and approximately 2 mm in diameter after incubation for 4 days. Heterofermentative and produces lactic acid, carbon dioxide and ethanol or acetic acid from D-glucose. D- and L-lactate are produced in a ratio of 1 : 1. Nitrate is not reduced. Acid is produced from D-glucose, D-fructose, D-galactose, L-arabinose, D-ribose, maltose, melibiose, sucrose, raffinose and D-melezitose, weakly from D-gluconate, but not from D-mannose, D-xylose, L-rhamnose, cellobiose, lactose, D-salicin, D-trehalose, D-mannitol, D-sorbitol or starch. Dextran is not formed from sucrose. Cells grow at temperatures between 15 and 45 °C but not at 10 or 50 °C. All strains grow at pH 4.0–8.5; some strains grow at pH 9.0. No growth is observed in MRS broth containing 5 % (w/v) NaCl. Cells do not contain meso-diaminopimelic acid in their peptidoglycan. The DNA G+C content ranges from 40 to 41 mol%.
The type strain is NRIC 0676T (=JCM 14108T=DSM 18382T), and has a G+C content of 41 mol%. All three known strains were isolated from a compost of distilled shochu residue collected at a shochu distillery in Miyazaki Prefecture, southern Kyushu region, Japan, in 2003.
Description of Lactobacillus parafarraginis sp. nov.
Lactobacillus parafarraginis (pa.ra.far.ra'gi.nis. Gr. prep. para beside; L. gen. n. farraginis of mash, the specific epithet of Lactobacillus farraginis; N.L. gen. n. parafarraginis beside farraginis, pertaining to the close relationship to L. farraginis).
Cells are Gram-positive, non-motile rods, measuring 0.8x2–4 µm. Cells occur singly or in pairs and chains. Facultatively anaerobic and catalase-negative. Colonies on MRS agar are beige, smooth and approximately 1–2 mm in diameter after incubation for 4 days. Heterofermentative and produces lactic acid, carbon dioxide and ethanol or acetic acid from D-glucose. D- and L-lactate are produced in the ratio 3 : 2. Nitrate is not reduced. Acid is produced from D-glucose, D-fructose, D-galactose, L-arabinose, D-ribose, D-xylose, maltose, melibiose, sucrose, raffinose and D-melezitose, weakly from D-gluconate, but not from D-mannose, L-rhamnose, cellobiose, lactose, D-salicin, D-trehalose, D-sorbitol or starch. Acid production from D-mannitol is variable depending on the strain. Dextran is not formed from sucrose. Cells grow at temperatures between 20 and 37 °C but not at 15 or 45 °C. All strains grow at pH 4.0–8.5. Growth is observed in MRS broth containing 5 % (w/v) NaCl. Cells do not contain meso-diaminopimelic acid in their peptidoglycan. The DNA G+C content is 40 mol%.
The type strain is NRIC 0677T (=JCM 14109T=DSM 18390T), and has a G+C content of 40 mol%. Both known strains were isolated from a compost of distilled shochu residue collected at a shochu distillery in Miyazaki Prefecture, southern Kyushu area, Japan, in 2003.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Endo, A. & Okada, S. (2005). Lactobacillus satsumensis sp. nov., isolated from mashes of shochu, a traditional Japanese distilled spirit made from fermented rice and other starchy materials. Int J Syst Evol Microbiol 55, 83–85.
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