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International Centre for Brewing and Distilling, School of Life Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK
Correspondence
Fergus G. Priest
f.g.priest{at}hw.ac.uk
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SAT is AY445815.
RAPD profiles and a neighbour-joining tree based on partial tuf gene sequences are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; van Beek & Priest, 2002). In well-maintained distilleries their numbers are low at the beginning of the fermentation, but, once the yeast has completed the alcoholic fermentation, they grow prolifically during the ‘late lactic fermentation’ and are considered to confer positive flavour notes to the spirit (Takatani & Ikemoto, 2004). The most common species encountered are Lactobacillus brevis, Lactobacillus fermentum and Lactobacillus paracasei, but numerous other Lactobacillus species, lactococci, leuconostocs and weissellas have been detected, particularly in the early stages of the fermentation, when the alcohol concentration is relatively low (van Beek & Priest, 2003).
Analysis of Scotch whisky fermentations using denaturing gradient gel electrophoresis revealed an uncultured bacterium in the late stages of fermentation that was closely related to a bacterium isolated from a Japanese malt whisky fermentation and referred to as strain Y10. Phylogenetic studies based on the 16S rRNA gene sequence of strain Y10 suggested that this bacterium was a member of the Lactobacillus acidophilus group (van Beek & Priest, 2002). This group comprises L. acidophilus sensu stricto, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus and Lactobacillus johnsonii (Johnson et al., 1980; Fujisawa et al., 1992; Gancheva et al., 1999), with three relatively recent additions: Lactobacillus amylolyticus, isolated from malt and beer wort (Bohak et al., 1998), Lactobacillus iners, associated with human sources (Falsen et al., 1999) and Lactobacillus kitasatonis, from chicken intestines (Mukai et al., 2003). In this study, we have investigated the phylogenetic relationships between the Japanese and Scottish isolates from whisky fermentations and shown that they constitute a distinct taxon within the L. acidophilus lineage, for which we propose the name Lactobacillus suntoryeus sp. nov.
Eight strains of lactic acid bacteria were isolated from four different distilleries in Japan and Scotland. The Japanese strains were isolated on three occasions: strains Y10, H8 and W6 in 1989, strains YD, HD and PD in 1993 and strain SAT in 1999. Strains Y10, YD and SAT were isolated from the Yamazaki distillery, strains H8, W6 and HD from the Hakushu distillery and PD from the Suntory pilot plant. Strain M4 was isolated from the Glenkinchie distillery in southern Scotland in 2002. For isolation of strain M4, a fermentation sample was diluted in MRS broth, plated onto MRS agar (Oxoid) supplemented with maltose (10 g l–1), brain heart infusion broth (Oxoid; 10 g l–1) and cycloheximide (100 µg ml–1) and incubated anaerobically for 48 h at 37 °C. Isolated colonies were purified on the same medium by streak plating three times and a representative isolate was stored at –70 °C in MRS broth containing 20 % (v/v) glycerol as strain M4. Distillery isolates were routinely cultured on supplemented MRS agar or broth (without cycloheximide) at 37 °C anaerobically. The main reason why these organisms had not been recovered in earlier studies (e.g. Makanjuola & Springham, 1984; Simpson et al., 2001) was probably due to their growth requirements; they grow poorly, if at all, unless MRS-based media are supplemented with brain heart infusion or similar. Reference strains L. gallinarum DSM 10532T and L. helveticus NCIMB 11971T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and the National Collection of Industrial and Marine Bacteria (NCIMB), respectively, and cultured in MRS agar or broth at 37 °C.
Chromosomal DNA was isolated using the PUREGENE DNA Isolation kit (Gentra Systems) from cultures grown in supplemented MRS broth for 24 h at 37 °C. The manufacturer's protocol for DNA isolation from Gram-positive bacteria was modified by the addition of 140 U mutanolysin (Sigma) ml–1 and 50 µg lysozyme (Sigma) ml–1 to the lytic enzyme solution and the suspension was incubated at 37 °C for 45 min to effect lysis. Larger amounts of DNA were prepared by phenol extraction as described previously (Simpson et al., 2001). DNA concentration was determined by the diphenylamine method (Burton, 1968). Randomly amplified polymorphic DNA (RAPD) patterns were generated as described previously (Simpson et al., 2001).
Fragments of genes for sequence determination were amplified by PCR using the following reaction mixture (50 µl): 5 µl DyNAzyme EXT buffer (Finnzymes), 1 µl 10 mM dNTPs, 2 µl 50 mM MgCl2, 10 pmol each primer, 100 ng DNA and 1 U DyNAzyme EXT polymerase. Amplifications were performed with the following thermal cycle: 95 °C for 5 min, 30 cycles of 95 °C for 10 s, an annealing step of 1 min (temperature specified below) and elongation at 72 °C for 2 min, with a final elongation step at 72 °C for 10 min. 16S rRNA genes were amplified using universal primers 27f (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492r (5'-TACGGYTACCTTGTTACGACTT-3'), with an annealing temperature of 56 °C. tuf genes were amplified using primers tuf-01-f (5'-GTTGTTGCTGCAACTGATGG-3') and tuf-02-r (5'-CCATTTCAGTACCTTCTGGC-3'), with an annealing temperature of 56 °C. These were designed from an alignment of tuf gene sequences from L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum and L. helveticus and enabled amplification of a region from nt 19 to 760 (numbering according to the L. acidophilus ATCC 4356T gene sequence). S-layer genes were amplified using various primers with corresponding annealing temperatures. The initial primers S01-f (5'-AGAATYGTTAGCGYTGCTGC-3') and S02-r (5'-CGTTGTGCTTCAAAGTACGC-3') at 50 °C enabled partial sequencing of the 5' end of the PCR products from all four strains (three distillery isolates and L. gallinarum DSM 10532T). New primers were then prepared for strains SAT and Y10, S03-f (5'-GACCACATGAAGAAAAATTT-3') and Jap3-S04-r (5'-CAGCACCATCAACAGCAATA-3') at 50 °C; for strain M4, S01-f and M4-S04-r (5'-TCGTCAGCAGTAGTAGATGG-3') at 50 °C; and for L. gallinarum DSM 10532T, S01-f and galli-S04-r (5'-GTTCATAGCCTTAAGAGCTG-3') at 56 °C, as conserved regions suitable for all strains were not available. This resulted in fragments of 848 bp (nt 49–896) for strains Y10 and SAT, 711 bp (nt 70–780) for strain M4 and 808 bp (nt 70–877) for L. gallinarum DSM 10532T all based on L. acidophilus ATCC 4356T gene sequence numbering. PCR products were purified by PEG8000 precipitation and sequenced using the ABI Prism Big Dye Terminator cycle sequencing ready reaction kit and the ABI 310 capillary sequencing system (Applied Biosystems). Due to the presence of multiple copies of the S-layer gene in L. gallinarum DSM 10532T, PCR products were cloned in Escherichia coli with the TOPO TA Cloning kit (Invitrogen) and sequenced after PCR amplification with primers S01-f and galli-S04-r as described above. Alignments were prepared with CLUSTAL X and phylogenetic trees were constructed using PAUP 4 (http://paup.csit.fsu.edu/).
For DNA–DNA hybridizations, probe DNAs were labelled with digoxigenin using random-priming and the DIG High Prime DNA Labelling and Detection Starter kit II (Roche). DNA (1 µg) was immobilized on nylon membranes, hybridized at 42 °C and washed at high stringency (65 °C) as described previously (Simpson et al., 2001). Labelled probe was detected by chemiluminescence and quantified with ImageMaster TotalLab software (Amersham Pharmacia Biotech).
Partial 16S rRNA gene sequences (around 300 bp from the 5' end) of the eight strains were identical. RAPD analysis provided further evidence that the distillery isolates were closely related. RAPD patterns are highly discriminatory for members of the L. acidophilus group and can be used to distinguish strains of the various species, as well as to provide discrimination within species (Du Plessis & Dicks, 1995; Gancheva et al., 1999; Ventura & Zink, 2002). The identical patterns generated from strains SAT and Y10 and the very similar pattern from strain M4 were consistent with the isolates belonging to a single species (see Supplementary Fig. A available in IJSEM Online).
The virtually complete 16S rRNA gene sequences of strains SAT, Y10 and M4 and of L. helveticus NCIMB 11971T were determined and a phylogenetic tree was constructed using 16S rRNA gene sequences retrieved from GenBank for other members of the L. acidophilus group and L. brevis. The three distillery isolates had identical 16S rRNA gene sequences and the tree topology confirmed that they were members of the L. acidophilus group (Fig. 1). The 16S rRNA genes of these strains shared 99·3 and 98·1 % sequence similarity with L. helveticus and L. gallinarum 16S rRNA genes, respectively.
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) and slp, which encodes a surface-layer protein. A neighbour-joining tree based on partial sequences of 681 bp of the tuf genes fully supported the tree topology obtained with 16S rRNA gene sequences. The three distillery isolates had identical tuf alleles and were again most closely related to L. helveticus and L. gallinarum (see Supplementary Fig. B available in IJSEM Online), confirming the value of this gene as a reliable indicator of phylogenetic relationships within this group of bacteria (Chavagnat et al., 2002).
Lactobacilli of the L. acidophilus group, apart from L. johnsonii and L. gasseri, possess a highly variable surface-layer protein (Boot et al., 1996) that has been used as a taxonomic tool for identification of L. crispatus (Horie et al., 2002). We amplified and sequenced partial slp genes from the three distillery isolates and L. gallinarum DSM 10532T. The topology of the neighbour-joining tree constructed from these sequences and homologous sequences from GenBank (Fig. 2) differed from that based on 16S rRNA and tuf genes, and the distillery isolates did not form a monophyletic group. Indeed, the allele of strain M4 shared very low sequence similarity (approx. 54 %) with those of the Japanese strains. Surface-protein genes are subject to greater selective pressure and change more rapidly than housekeeping genes such as tuf, particularly in pathogenic bacteria exposed to immune responses (van Loo et al., 2002). The inconsistencies between the slp tree and those derived from 16S rRNA and tuf genes indicated that the slp gene may not provide an accurate picture of the phylogenetic relationships of these organisms and may be subject to lateral transfer.
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The three distillery strains SAT, Y10 and M4 were all Gram-positive, catalase-negative homofermentative rods. After anaerobic incubation in modified MRS broth for 24 h, SAT cells were rods approximately 10 µm long (ranging from 2 to 30 µm), M4 cells were smaller (from 2 to 15 µm) and Y10 cells formed long filaments (from 20 to 200 µm). The carbohydrate fermentation patterns of the three distillery strains and L. gallinarum DSM 10532T were determined using the API 50 CHL system (bioMérieux). The three distillery strains had similar profiles except for the production of acid from galactose and starch, which was positive for strain M4 and negative for strains SAT and Y10 (Table 1). The phylogenetic analyses, DNA hybridization results and distinguishing phenotypic characteristics (Table 1) confirmed these bacteria as members of a novel species, for which we propose the name Lactobacillus suntoryeus sp. nov. in recognition of the first isolation of these bacteria.
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Gram-positive rods varying in length from 2 µm to long filaments. Non-motile, non-sporulating and catalase negative. After 48 h incubation on MRS agar (supplemented with maltose and brain heart infusion broth), colonies are circular, shiny, creamy white in colour and 2–5 mm in diameter. Growth is poor in unsupplemented MRS medium. Growth occurs at 37 and 45 °C, but not at 15 °C. Strictly homofermentative. Acid is produced from N-acetyl glucosamine, amygdalin, arbutin, cellobiose, aesculin, fructose, gentiobiose, glucose, maltose, mannose, salicin, sucrose and in some cases from galactose and starch (type strain negative). Adonitol, D-arabinose, L-arabinose, D- and L-arabitol, dulcitol, erythritol, D- and L-fucose, gluconate, glycerol, inositol, inulin, 2-ketogluconate, 5-ketogluconate, D-lyxose, mannitol, melezitose, methyl 
-D-glucoside, methyl -D-mannoside, methyl 
-D-xyloside, rhamnose, ribose, sorbitol, sorbose, D-tagatose, D-turanose, xylitol and D- and L-xylose are not fermented. Strains have been isolated from whisky fermentations.
The type strain is strain SAT (=LMG 22464T=NCIMB 14005T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Boot, H. J., Kolen, C. P., Pot, B., Kersters, K. & Pouwels, P. H. (1996). The presence of two S-layer-protein-encoding genes is conserved among species related to Lactobacillus acidophilus. Microbiology 142, 2375–2384.
Burton, K. (1968). Determination of DNA concentration with diphenylamine. Methods Enzymol 12, 163–166.[CrossRef]
Chavagnat, F., Haueter, M., Jimeno, J. & Casey, M. G. (2002). Comparison of partial tuf gene sequences for the identification of lactobacilli. FEMS Microbiol Lett 217, 177–183.[CrossRef][Medline]
Du Plessis, E. M. & Dicks, L. M. (1995). Evaluation of random amplified polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii. Curr Microbiol 31, 114–118.[CrossRef][Medline]
Falsen, E., Pascual, C., Sjödén, B., Ohlén, M. & Collins, M. D. (1999). Phenotypic and phylogenetic characterization of a novel Lactobacillus species from human sources: description of Lactobacillus iners sp. nov. Int J Syst Bacteriol 49, 217–221.
Fujisawa, T., Benno, Y., Yaeshima, T. & Mitsuoka, T. (1992). Taxonomic study of the Lactobacillus acidophilus group, with recognition of Lactobacillus gallinarum sp. nov. and Lactobacillus johnsonii sp. nov. and synonymy of Lactobacillus acidophilus group A3 (Johnson et al. 1980) with the type strain of Lactobacillus amylovorus (Nakamura 1981). Int J Syst Bacteriol 42, 487–491.
Gancheva, A., Pot, B., Vanhonacker, K., Hoste, B. & Kersters, K. (1999). A polyphasic approach towards the identification of strains belonging to Lactobacillus acidophilus and related species. Syst Appl Microbiol 22, 573–585.[Medline]
Horie, M., Kajikawa, H. S. & Toba, T. (2002). Identification of Lactobacillus crispatus by polymerase chain reaction targeting S-layer protein gene. Lett Appl Microbiol 35, 57–61.[Medline]
Johnson, J. L., Phelps, C. F., Cummins, C. S., London, J. & Gasser, F. (1980). Taxonomy of the Lactobacillus acidophilus group. Int J Syst Bacteriol 30, 53–68.
Makanjuola, D. B. & Springham, D. G. (1984). Identification of lactic acid bacteria isolated from different stages of the Scotch whisky fermentation. J Inst Brew 90, 13–19.
Mukai, T., Arihara, K., Ikeda, A., Nomura, K., Suzuki, F. & Ohori, H. (2003). Lactobacillus kitasatonis sp. nov., from chicken intestine. Int J Syst Evol Microbiol 53, 2055–2059.
Simpson, K. L., Pettersson, B. & Priest, F. G. (2001). Characterization of lactobacilli from Scotch malt whisky distilleries and description of Lactobacillus ferintoshensis sp. nov., a new species isolated from malt whisky fermentations. Microbiology 147, 1007–1016.
Takatani, T. & Ikemoto, H. (2004). Contribution of bacterial microflora in malt whisky quality. In Distilled Spirits: Tradition and Innovation, pp. 197–207. Edited by J. H. Bryce & G. G. Stewart. Nottingham: Nottingham University Press.
van Beek, S. & Priest, F. G. (2002). Evolution of the lactic acid bacterial community during malt whisky fermentation: a polyphasic study. Appl Environ Microbiol 68, 297–305.
van Beek, S. & Priest, F. G. (2003). Bacterial diversity in Scotch whisky fermentations as revealed by denaturing gradient gel electrophoresis. J Am Soc Brew Chem 61, 10–14.
van Loo, I. H. M., Heuvelman, K. J., King, A. J. & Mooi, F. R. (2002). Multilocus sequence typing of Bordetella pertussis based on surface protein genes. J Clin Microbiol 40, 1994–2001.
Ventura, M. & Zink, R. (2002). Specific identification and molecular typing analysis of Lactobacillus johnsonii by using PCR-based methods and pulsed-field gel electrophoresis. FEMS Microbiol Lett 217, 141–154.[CrossRef][Medline]
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