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1 Department of Food Science, Food Microbiology, Centre for Advanced Food Studies (LMC), Faculty of Life Sciences, Copenhagen University, Rolighedsvej 30, 1958 Frederiksberg C, Denmark
2 Institute for Hygiene and Toxicology, Federal Research Centre for Nutrition and Food, Karlsruhe, Germany
3 Department of Ecology, Faculty of Life Sciences, Copenhagen University, Copenhagen, Denmark
4 CSIR – Food Research Institute, Accra, Ghana
Correspondence
Dennis S. Nielsen
dn{at}life.ku.dk
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ABSTRACT |
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Maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences showing the phylogenetic position of strains L486, L489T and L499 within the L. casei cluster are available as supplementary figures with the online version of this article.
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MAIN TEXT |
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). During an investigation of the micro-organisms involved in the fermentation of cocoa beans, a number of isolates with unusual properties were isolated from MRS agar and tentatively identified as Lactobacillus species (Nielsen et al., 2007). This study presents the morphological, biochemical and molecular characterization of three of these isolates, designated L486, L489T and L499. Strains L486, L489T, L499 and the reference strains Lactobacillus nagelii DSM 13675T, Lactobacillus satsumensis DSM 16230T and Lactobacillus vini DSM 20605T were grown in MRS broth (Merck) at 30 °C for 2–3 days; for long-term storage, 20 % glycerol was added and the cultures were stored at –80 °C.
The cell morphologies of cultures grown overnight in MRS broth (30 °C) were determined using phase-contrast microscopy and scanning electron microscopy. For the latter, 30 µl culture diluted 10-fold with sterile MilliQ water was filtered through a polycarbonate filter (pore size, 0.2 µm), exposed to osmium vapour for 1 h and coated with gold–palladium. Cells were observed in a FEI Quanta 200 scanning electron microscope at 15 kV. The cells were rod-shaped (0.7–0.8x1.4–2.5 µm), occurring either singly, in pairs or in short chains of three to four cells (Fig. 1). The cells were observed to be highly motile under the phase-contrast microscope. Scanning electron microscopy showed the presence of peritrichous flagella (Fig. 1).
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The Gram reaction and catalase activity were tested using standard methods. Growth at 15 and 45 °C (in MRS broth), in the presence of 6.5 % NaCl and at pH 3.9 and 8.0, gas production from glucose (in MRS broth, with inverted Durham tubes, and determined at 30 °C), the production of ammonia from arginine and the presence of D-meso-diaminopimelic acid in the cell wall were tested by following the protocol of Schillinger & Lücke (1987). The configuration of the lactic acid enantiomer produced was determined enzymically (Boehringer Mannheim) (Schillinger & Lücke, 1987).
The carbohydrate-fermentation patterns of strains L486, L489T and L499 were determined in microtitre plates by following the protocol of Jayne-Williams (1976) and, in addition, using the API 50 CHL identification system (bioMérieux).
For 16S rRNA gene sequencing, DNA was extracted using the method of Björkroth & Korkeala (1996) and the almost-complete 16S rRNA gene was amplified using primers 7f and 1510r (Lane, 1991; Björkroth & Korkeala, 1996). All reactions were carried out in a 50 µl volume containing 1.25 U Taq DNA polymerase (Amersham Biosciences), 5 µl 10x PCR buffer (Amersham Biosciences), 200 µM each dNTP (Amersham Biosciences), 3.0 mM MgCl2 (Amersham Biosciences), 0.1 µM each primer (DNA Technologies), 1 % (v/v) formamide (Merck), 0.1 % (w/v) BSA (New England Biolabs), 20 ng DNA template and sufficient sterile MilliQ water to adjust the volume to 50 µl. The PCR was performed using the following thermocycling program: 5 min initial denaturation at 94 °C, 30 cycles of 94 °C for 90 s, 52 °C for 30 s and 72 °C for 90 s and then a final elongation step at 72 °C for 7 min. Following purification (using the Qiagen PCR purification kit), the PCR products were sequenced in both directions using a CEQ 2000 automated sequencer (Beckman Coulter) and a CEQ 2000 Dye Terminator Cycle Sequencing Quick Start kit (Beckman Coulter) or sent to a commercial sequencing facility (DNA Technology A/S, Aarhus, Denmark). Sequences were corrected manually and aligned using Vector NTI Suite 7 (Informax). The closest phylogenetic relatives were determined by aligning the corrected sequences with 16S rRNA gene sequences in the GenBank database by using the BLAST algorithm (Altschul et al., 1997). Strains L486 and L489T had 100 % identical 16S rRNA gene sequences, whereas L499 differed by one nucleotide from the former two. The 16S rRNA gene sequences of strains L486, L489T, L499 and the sequences of the closest phylogenetic relatives retrieved from the GenBank database were aligned and phylogenetic trees constructed with the neighbour-joining, maximum-likelihood and maximum-parsimony methods using BIONUMERICS, version 3.5 (Applied Maths). Unknown bases were disregarded for the analysis; 1450 nt were included. The statistical reliability of the topology of the phylogenetic trees was evaluated using bootstrap resampling of the data (1000 resamplings) (Fig. 2; also see Supplementary Figs S1 and S2 available in IJSEM Online). In comparisons between the 16S rRNA gene sequence of L489T and those of type strains in the GenBank database, the highest levels of similarity were with L. nagelii DSM 13675T (98.0 %), L. satsumensis DSM 16230T (95.5 %) and L. vini DSM 20605T (93.7 %) (Table 1).
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, as modified by Stackebrandt & Kandler (1978). Strain L486 was not included in the DNA–DNA hybridization studies, as the 16S rRNA gene sequence is 100 % identical to that of strain L489T. The DNA G+C content was determined from the thermal melting temperature (Tm) of the DNA by using a Cary 100 Bio UV-visible spectrophotometer (Varian). DNA–DNA relatedness was determined spectrophotometrically from renaturation rates (De Ley et al., 1970). DNAs from strains L489T and L499 were hybridized with each other and with DNA from L. nagelii DSM 13675T and with DNA from L. satsumensis DSM 16230T. The reassociation value between L489T and L499 was 92.5 %, indicating that the strains are conspecific. Reassociation values of 18–44 % were obtained with respect to L. nagelii DSM 13675T and values of 0–12 % were obtained with respect to L. satsumensis DSM 16230T. All of the DNA–DNA hybridization values obtained for the closest relatives, namely L. nagelii DSM 13675T and L. satsumensis DSM 16230T, were therefore well below the 70 % cut-off value that indicates separate species (Wayne et al., 1987). DNA reassociation values were not determined for L489T and L499 with respect to the phylogenetically closely related species L. vini, as L. vini does not contain D-meso-diaminopimelic acid in the cell wall (Table 1
). The DNA G+C content for strain L489T and for strain L499 was 37.8 mol%, while those for L. nagelii DSM 13675T and L. satsumensis DSM 16230T were 37.7 and 40.2 mol%, respectively (Table 1).
The three strains were also investigated genotypically by using repetitive element palindromic-PCR with the primer GTG5 according to the method of Gevers et al. (2001) for lactic acid bacteria, as described previously (Franz et al., 2006). Strains L486, L489T and L499 clustered closely (r=85 %) and were only distantly related to the type strains of L. nagelii, L. satsumensis and L. vini (Fig. 3).
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). The results obtained in the present study clearly indicate that the strains studied represent a novel species within the genus Lactobacillus, for which the name Lactobacillus ghanensis sp. nov. is proposed. The type strain is L489T (=DSM 18630T=CCUG 53453T).
Description of Lactobacillus ghanensis sp. nov.
Lactobacillus ghanensis (ghan.en'sis. N.L. masc. adj. ghanensis pertaining to Ghana, where the micro-organism was first isolated).
Cells are rod-shaped, 0.7–0.8x1.4–2.5 µm in size and occur singly, in pairs or in short chains comprising three to four cells. Gram-positive, catalase-negative, motile with peritrichous flagella and non-spore-forming. Colonies are 2–3 mm in diameter, white to creamy white, smooth, circular, convex and with entire or slightly uneven edges after 3–4 days of anaerobic growth. Weak growth at 15 °C; good growth at 45 °C. No growth occurs at 6.5 % NaCl. Weak growth at pH 3.9; no growth at pH 8.0. Ammonia is not produced from arginine. No gas is produced from glucose. D(–)- and L(+)-lactic acid are produced as the end products from glucose metabolism. Acid is produced from amygdalin, D-cellobiose, aesculin, D-fructose, D-glucose, D-galactose, D-mannitol, D-mannose, N-acetylglucosamine, L-rhamnose, sucrose, salicin and D-trehalose. Acid is not produced from D-adonitol, starch, D-arabinose, L-arabinose, arbutin, dulcitol, erythritol, D-fucose, L-fucose, gentiobiose, gluconate, glycogen, inositol, inulin, D-lyxose, D-melezitose, D-melibiose, methyl 
-xyloside, D-raffinose, D-ribose, D-sorbitol, D-turanose, xylitol, D-xylose, L-xylose, 2-ketogluconate or 5-ketogluconate. Acid production from glycerol, D-lactose, D-maltose, methyl 
-D-glucoside (delayed reaction), D-sorbitol (delayed reaction), L-sorbose and D-tagatose (delayed reaction) is strain-dependent. Does not produce dextran from sucrose. The cell wall contains peptidoglycan of the D-meso-diaminopimelic acid type. The DNA G+C content is 37.8 mol%.
The type strain, L489T (=DSM 18630T=CCUG 53453T), and all other known strains of the species were isolated from cocoa fermentations in Tafo, Ghana. The description of the type strain corresponds to the description of the species except that no acid is produced from glycerol, maltose, methyl -D-glucoside, D-lactose, sorbitol or tagatose. Strain L499 (=DSM 18631=CCUG 53454) is a reference strain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Torriani, S., Van Reenen, G. A., Klein, G., Reuter, G., Dellaglio, F. & Dicks, L. M. T. (1996). Lactobacillus curvatus subsp. curvatus subsp. nov. and Lactobacillus curvatus subsp. melibiosus subsp. nov. and Lactobacillus sake subsp. sake subsp. nov. and Lactobacillus sake subsp. carnosus subsp. nov., new subspecies of Lactobacillus curvatus Abo-Elnaga and Kandler 1965 and Lactobacillus sake Katagiri, Kitahara, and Fukami 1934 (Klein et al. 1996, emended descriptions), respectively. Int J Syst Evol Microbiol 46, 1158–1163.
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
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