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1 Laboratory of Microbial Resources and Ecology, Graduate School of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo 060-8589, Japan
2 Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo 060-8589, Japan
3 Research Institute of Biological Resources, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan
4 Department of Biology, Faculty of Science, Chiang Mai University, Huay Kaew Road, Muang District, Chiang Mai 50200, Thailand
5 Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand
Correspondence
Atsushi Yokota
yokota{at}chem.agr.hokudai.ac.jp
| ABSTRACT |
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Published online ahead of print on 19 July 2002 as DOI 10.1099/ijs.0.02347-0.
The GenBank accession numbers for the 16S rDNA sequences of strains G 12, G 22, G 35T, G 43 and G 44 are AF308146, AF308147, AF317702, AF317703 and AF317704, respectively.
A fuller phylogenetic tree showing the placement of Lactobacillus thermotolerans within the genus Lactobacillus (Fig. I) and a growth curve for strain G 35T (Fig. II) are available as supplementary data in IJSEM Online (http://ijs.sgmjournals.org).
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In the course of studies to isolate thermotolerant micro-organisms for use in the fermentation industry, we have isolated a large number of LAB from various types of natural samples at relatively high temperatures, i.e. 4050 °C. 16S rDNA sequence analysis of these LAB raised the possibility that some of the TLAB isolated from chicken faeces comprised a novel species within the genus Lactobacillus. In the present study, five Lactobacillus strains isolated from chicken faeces that were known to be TLAB were characterized. On the basis of physiological, biochemical and genetic data, it is shown that the five strains represent a novel species within the genus Lactobacillus, for which the name Lactobacillus thermotolerans is proposed.
Strains G 12, G 22, G 35T, G 43 and G 44, isolated as TLAB, were used in this work. For the isolation of TLAB, fresh faecal samples from chickens were collected from the chicken coop of Kasetsart University, Bangkok, Thailand. These faecal samples were inoculated into glucose/peptone/yeast extract (GPY) broth and incubated anaerobically at 40, 45 and 50 °C for 24 h using mixed gases (N2/H2/CO2, 8 : 1 : 1). GPY broth contained (g l-1): glucose, 10; peptone, 5; yeast extract, 10; sodium acetate trihydrate, 2; NaCl, 0·01; Tween 80, 0·5; MgSO4.7H2O, 0·2; MnSO4.4H2O, 0·01; FeSO4.7H2O, 0·035. NaOH was used to adjust the broth to pH 6·8. After appropriate dilutions had been prepared, culture broths were spread onto GPY/BCP agar plates which were prepared by the addition of 5 g CaCO3 l-1, 0·06 g bromocresol purple l-1, 0·05 g cycloheximide l-1 and 20 g agar l-1 to GPY broth. The plates were incubated anaerobically at 40, 45 and 50 °C in anaerobic jars having a H2+CO2 environment generated with a BBL GasPak (Becton Dickinson Microbiology Systems). Colonies that were yellow in colour and formed clear zones were selected as TLAB. In the experiments detailed in this study, cultures were routinely grown anaerobically in MRS broth (Difco) using mixed gases at 42 °C (for strains G 12, G 22, G 35T, G 43 and G 44) and 37 °C (for reference strains). For the preparation of DNA for DNADNA hybridization studies and PCR, cells grown in MRS broth were harvested in the late-exponential phase and DNA was extracted essentially as described by Marmur (1961)
. The 16S rRNA genes of the five novel strains (corresponding to positions 271522 of the Escherichia coli 16S rRNA gene) were amplified by PCR. The six oligonucleotide primers used in the PCR amplification have been described previously (Mori et al., 1997
). The purified PCR products were sequenced directly using a dRhodamine dye terminator cycle sequencing kit (Applied Biosystems) and an automated DNA sequencer (model 377; Applied Biosystems). The resulting sequences were subjected to similarity searches against sequences within the public databases, to determine a possible phylogenetic classification for the novel strains. To determine the closest known relatives of the novel strains, based on 16S rDNA sequences, primary searches were performed in GenBank using the FASTA program (Devereux et al., 1984
). The database sequences representing the best matches to the 16S rDNA sequences of the novel strains were retrieved, and all of the sequences were aligned using the CLUSTAL W software (Thompson et al., 1994
). Sequences in the alignment were corrected manually; approximately 1500 nt, covering the whole range of 16S rDNA sequences, were used in the phylogenetic analysis. A distance matrix was calculated using DNADIST, contained within the PHYLIP package (Felsenstein, 1993
), and the Kimura two-correction parameter, and a phylogenetic tree was constructed using NEIGHBOR, contained within PHYLIP, using the neighbour-joining method (Saitou & Nei, 1987
). The reliability of the individual branches of the tree was assessed by the bootstrap method (1000 replications) using SEQBOOT, DNADIST, NEIGHBOR and CONSENSE (all within the PHYLIP package). The novel strains were further distinguished from their nearest relatives on the basis of physiological and biochemical comparisons. Cells were grown anaerobically on MRS agar or in MRS broth at 42 °C (strains G 12, G 22, G 35T, G 43 and G 44) or 37 °C (reference strains) for 24 h. Catalase and oxidase activity, gas production from glucose, ammonia production from arginine and isomeric type of lactic acid were determined for each strain after they had been cultured in MRS broth. Configuration of the lactic acid was determined by HPLC using an Aminex HPX-87H 300x7·8 mm column (Bio-Rad) by the method described by Otsuka et al. (1994)
. The production of organic acids by each strain was determined by ion-exclusion HPLC using the culture supernatants as samples by the method described by Hoshi et al. (1994)
. Carbohydrate utilization and acid production by the strains were determined using the API 50 CHL system (bioMérieux) with CHL medium, as recommended by the manufacturer. API strips were incubated at 42 °C (novel strains) and 37 °C (reference strains) for up to 48 h. Tests for growth of the strains at 15 and 45 °C were performed in MRS broth; results were recorded after 48 h. To evaluate the thermotolerance of the strains isolated from chicken faeces, optimum temperatures for growth of the novel strains were determined in comparison with reference strains. Each strain was cultured in half-strength MRS broth at temperatures ranging between 15 and 50 °C in waterbaths with standard thermometers. Growth of the strains was determined by measuring the OD660 values of the culture broths with a 20D spectrophotometer (Milton Roy). To determine the optimum temperature for growth of each of the strains, the specific growth rate was calculated from each growth curve for each strain. Detection of diaminopimelic acid in the cell walls of the strains was done with 50100 mg washed cells that had been suspended in 100 µl of potassium phosphate buffer (pH 6·8) containing 0·38 g KH2PO4 l-1, 0·39 g K2HPO4 l-1 and 7·46 g KCl l-1. The cell suspension was treated with 1 ml of 6 M HCl at 100 °C for 18 h. The hydrolysate was applied to a cellulose TLC plate (no. 1.05552; Merck), and developed using methanol/water/6 M HCl/pyridine (80 : 26 : 3 : 10, by vol.) (Komagata & Suzuki, 1987
). The resulting spots were visualized with ninhydrin. For the analysis of the cellular fatty acid composition of the strains, whole-cell fatty acids were converted to methyl esters by treatment with anhydrous methanolic HCl. Methyl esters were extracted with n-hexane (Komagata & Suzuki, 1987
) and analysed by using a GC-MS (Hitachi M7200A GC/3DQMS) equipped with a DB-5ms capillary column coated with 5 % phenylmethylpolysiloxane to a thickness of 250 nm (Hanada et al., 2002
). DNA base content of the strains was determined by the method of Tamaoka & Komagata (1984)
using HPLC following enzymatic digestion of genomic DNA to deoxyribonucleosides. DNADNA hybridization was performed essentially according to the membrane method described by Johnson (1973)
. Tritium-labelled DNA for DNADNA hybridization studies was prepared by using the nick-translation system (Amersham Pharmacia Biotech).
Strains G 12, G 22, G 35T, G 43 and G 44, which were isolated from chicken faeces, were Gram-positive, non-motile, non-spore-forming rods. When grown in MRS broth at 42 °C, cells appeared as short rods of 1x23 µm in size, which occurred singly, in pairs or occasionally in short chains. Good growth of the strains was observed when they were grown under anaerobic conditions in liquid and on solid media. After incubation on MRS agar for 2 days, colonies were white, circular, convex, smooth, opaque and approximately 11·5 mm in diameter. The 16S rDNA sequences of the novel strains were subjected to similarity searches against sequences within GenBank, to infer a possible phylogenetic classification for the strains. The results of these searches revealed that the five novel strains were members of the genus Lactobacillus. This classification was confirmed by a 16S-rDNA-based phylogenetic analysis (Fig. 1
) and by nucleotide sequence similarity values. The phylogenetic tree revealed that the novel strains were included in the Lactobacillus casei/Pediococcus group of the genus Lactobacillus (Collins et al., 1991
). The closest relatives of strain G 35T were found to be Lactobacillus mucosae DSM 13345T (Roos et al., 2000
) and L. fermentum ATCC 14931T, with moderate similarity values of 95·1 and 95·0 %, respectively (Fig. 1
). The branching pattern of the strain cluster with L. fermentum ATCC 14931T was supported by a high bootstrap value (95 %) (Fig. 1
). A fuller phylogenetic tree showing the placement of the five novel strains within the genus Lactobacillus is available as supplementary data in IJSEM Online (Fig. I; http://ijs.sgmjournals.org).
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Description of Lactobacillus thermotolerans sp. nov
Lactobacillus thermotolerans (therm.o.tol.er'ans. Gr. n. thermos heat; L. pres. part. tolerans tolerating; N.L. part. adj. thermotolerans heat-tolerating).
Cells are Gram-positive, non-motile, non-spore-forming, catalase-negative rods of 1x23 µm in size, which occur singly, in pairs or as short chains. After anaerobic growth at 42 °C for 2 days, colonies on MRS agar are 11·5 mm in diameter, white, circular, convex, smooth and opaque. Obligately heterofermentative and produces D- and L-lactic acid. Grows up to 50 °C, but not at 15 °C; optimum temperature for growth is 42 °C. Aesculin is hydrolysed. Arginine is cleaved. Hydrogen sulfide is not produced. Nitrate is not reduced. Gelatin is not liquefied. No dextran is produced from sucrose. Acid is produced from glucose, ribose, L-arabinose, D-xylose and D-fructose. The majority of strains also ferment melibiose and D-raffinose. Glycerol, erythritol, D-arabinose, L-xylose, adonitol, methyl
-xyloside, dulcitol, inositol, methyl
-D-mannoside, arbutin, lactose, inulin, melezitose, starch, glycogen, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate are not fermented. meso-Diaminopimelic acid is not present in the cell wall. DNA G+C content of the type strain is 50·5 mol%. Major cellular fatty acid is a straight chain unsaturated acid, C18 : 1. Isolated from the faeces of chickens in Thailand. The type strain is G 35T (=DSM 14792T =JCM 11425T).
| ACKNOWLEDGEMENTS |
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