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1 Laboratory of Microbiology, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium
2 Federal Research Centre for Nutrition and Food, Institute of Hygiene and Toxicology, Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany
3 BCCM/LMG Bacteria Collection, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium
4 Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Department of Applied Biological Sciences and Engineering, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
Correspondence
Katrien De Bruyne
Katrien.DeBruyne{at}UGent.be
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of LMG 23990T is AM600682. The GenBank/EMBL/DDBJ accession numbers for the pheS, rpoA and atpA gene sequences reported in this paper are AM711136–AM711355, as indicated in Supplementary Figs S1–S6.
Supplementary figures are available with the online version of this paper.
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TOPABSTRACTMAIN TEXTREFERENCES |
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, 2003), Leuconostoc gasicomitatum from marinated broiler meat strips (Susiluoto et al., 2003), Leuconostoc durionis from tempoyak, fermented durian (Leisner et al., 2005), and Leuconostoc ficulneum and Leuconostoc pseudoficulneum from ripe figs (Antunes et al., 2002; Chambel et al., 2006).
Strain LMG 23990T was isolated during an investigation of the microbial populations associated with the fermentation of coffee in Ethiopia. A combined action of lactic acid bacteria and/or endogenous coffee enzymes has been reported to play a role in the mucilage degradation (Vaughn et al., 1958; Arunga, 1973). In this respect Leuconostoc mesenteroides was found to solubilize pectic substances (Juven et al., 1985). Whereas the majority of Leuconostoc isolates from coffee were identified to the species level by rep-PCR (Caroline, 2005; Böhringer, 2006), this strain did not cluster with any known Leuconostoc reference or type species. Subsequent 16S rRNA gene sequence analysis indicated that it might represent a novel Leuconostoc species. In the present study additional analyses were performed on reference strains of other Leuconostoc species obtained from the BCCM/LMG Bacteria Collection, Ghent, Belgium and DSMZ, Braunschweig, Germany (Table 1). All strains except Leuconostoc gelidum strains were cultivated on MRS agar (de Man et al., 1960) at 30 °C for 24 h. L. gelidum strains were cultivated at 22 °C for 48 h.
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, as modified for Gram-positive bacteria, as described by Björkroth & Korkeala (1996). PCR products were purified and commercially sequenced at GATC Biotech as described previously (Kostinek et al., 2005). Using FASTA analysis at the EMBL database, the closest related bacteria were identified as members of the genus Leuconostoc. Evolutionary distances were calculated using the Jukes & Cantor (1969) evolutionary model and a phylogenetic tree (Fig. 1) was constructed using the neighbour-joining method (Saitou & Nei, 1987) with the BioNumerics software package, version 4.61 (Applied Maths). To evaluate the reliability of the topology of the neighbour-joining tree 500 bootstrap resamplings of the data were performed (Fig. 1). The novel strain belonged to a cluster of species together with Leuconostoc lactis and Leuconostoc citreum. The 16S rRNA gene sequence similarity values between strain LMG 23990T and its closest relatives L. citreum ATCC 49370T and L. lactis JCM 6123T were 99.6 and 99.0 %, respectively.
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. Densitometric analysis, normalization, interpolation of protein profiles and numerical analysis were performed by using the BioNumerics software package, version 4.61 (Applied Maths). The whole-cell protein profile of strain LMG 23990T was different from those of strains belonging to its two closest phylogenetic neighbours, L. lactis and L. citreum (Fig. 2).
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with the following modifications: EcoRI/TaqI was used as the restriction enzyme combination, and primer combination E01/T01 (both having an adenosine extension at the 3'-end) was applied for selective PCR. The resulting electrophoretic patterns were tracked and normalized with the GENESCAN 3.1 software package (Applera). Normalized tables of peaks were transferred into BioNumerics software package, version 4.61 (Applied Maths). The FAFLP fingerprint of strain LMG 23990T was different from those of representatives of its closest phylogenetic neighbours and of other type strains of all established Leuconostoc species (Fig. 3).
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). The total genomic DNA used for these hybridization experiments and for the determination of the DNA base composition was extracted and purified according to the method of Marmur (1961), as modified by Stackebrandt & Kandler (1979). The DNA–DNA relatedness level was determined using the spectrophotometric method of De Ley et al. (1970). Hybridization levels of 24 and 53 % were found between strain LMG 23990T and L. citreum DSM 5577T, and L. lactis DSM 20202T DSM 8581T, respectively. These DNA reassociation values were well below the 70 % threshold value recommended for species delineation (Wayne et al., 1987), indicating that strain LMG 23990T represents a distinct species.
The DNA base composition of strain LMG 23990T was determined as described by Mesbah et al. (1989) using a Waters Breeze HPLC system and XBridge Shield RP18 column. The solvent used was 0.02 M NH4H2PO4 (pH 4.0) and 1.5 % (v/v) acetonitrile. Non-methylated lambda phage (Sigma) and E. coli LMG 2093 DNAs were used as calibration reference and control, respectively. The DNA G+C content of strain LMG 23990T was 43.5 mol%, which is within the expected mol% G+C range of the genus Leuconostoc (38–44 %).
Recently, novel techniques have been developed to provide a rapid identification of Leuconostoc species. Lee et al. (2000) and Macian et al. (2004) reported identification using multiplex PCR targeting the 16S rRNA genes. Chenoll et al. (2003) evaluated the use of rDNA-based techniques such as intergenic spacer region restriction analysis and amplified rDNA restriction analysis. Accurate species identification, however, often requires a polyphasic approach, including 16S rRNA gene sequencing, DNA–DNA hybridizations, SDS-PAGE of whole-cell proteins and FAFLP of genomic DNA. Unfortunately the use of 16S rRNA is not discriminatory enough to differentiate closely related species within the genus Leuconostoc and the use of fingerprint patterns is restricted due to difficult inter-laboratory reproducibility. A solution to this problem is offered by the sequencing of housekeeping genes, which is expected to bring a new dimension into the study of genomic relationships at the inter- and intraspecies level (Stackebrandt et al., 2002). Recently, the phylogenetic structure of the Leuconostoc–Oenococcus–Weissella clade was evaluated by comparison of 16S rRNA, dnaA, gyrB, rpoC and dnaK gene sequence analysis of a restricted set of species within these genera. While some clades were well defined in every gene tree, many other clades were shown to be gene specific (Chelo et al., 2007). In general, the phylogenies obtained with the different genes showed a consistency with the 16S rRNA phylogeny.
Sequence analysis of genes encoding the phenylalanyl-tRNA synthase alpha subunit (pheS), RNA polymerase alpha subunit (rpoA) and the alpha subunit of ATP synthase (atpA) has been successfully applied for the accurate identification of Enterococcus (Naser et al., 2005a, b) and Lactobacillus species (S. Naser and others, unpublished data). In the present study, we used the primers listed in Table 2 for the amplification and sequencing of the same set of genes. The primer combinations pheS-21-F/pheS-23-R, rpoA-21-F/rpoA-23-R and atpA-20-F/atpA-26-R amplified the target genes of most strains. Where necessary, an alternative primer combination for rpoA (rpoA-20-F/rpoA-22-R) and atpA (atpA-20-F/atpA-26-R) was used. Amplification conditions and sequencing reactions were performed as described by Naser et al. (2005a, b). To assess inter- and intraspecies variation, we included multiple strains per species where possible. Bacterial strains, depositors and their sources are listed in Table 1
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). For the genes pheS, rpoA and atpA this observation is not supported, which is in agreement with other sequence analyses (Chelo et al., 2007). According to Chelo et al. (2007), who evaluated the evolutionary rates of the 16S rRNA gene, dnaA, gyrB, rpoC and dnaK for the Leuconostoc–Oenococcus–Weissella clade, this is due to the greater evolutionary rate of the 16S rRNA gene compared to other genes. Philippe et al. (2005) demonstrated that in cases of low rates of evolution, there is a tendency for the slow-evolving taxa to resemble outgroup sequences. In particular the difference in branch evolutionary rates between the genes could explain the position of L. fallax in 16S rRNA gene phylogeny, as the result of a relatively low rate of evolution in comparison to other Leuconostoc species. In all gene phylogenies the clade of L. ficulneum, L. pseudoficulneum, L. durionis and L. fructosum resulted in the most peripheral group. The same group was also observed as the most divergent line in the comparative phylogenies obtained by Chelo et al. (2007). For each of the genes studied, species were clearly delineated above 93, 98 and 98 % pheS, rpoA and atpA gene sequence similarity, respectively. For most pairs of species, the intraspecies diversity was consistently smaller than the interspecies similarity towards their nearest neighbours for each of the three genes studied and a clear separation of the species was obtained. The only exception was the differentiation between L. inhae and L. gelidum in the rpoA-based phylogenetic tree (Supplementary Fig. S2). Nevertheless, the separation between the two species was supported by a bootstrap value of 100. In general, the differences between intraspecies diversity and interspecies similarity were smaller for the rpoA and atpA genes compared with the pheS gene. None of the loci examined, nor the concatenated sequences allowed discrimination between the subspecies within L. mesenteroides. On the basis of present data, the strains classified as L. mesenteroides subsp. mesenteroides LMG 8159 and Leuconostoc pseudomesenteroides LMG 11499 form a peculiar clade in the three gene phylogenies. These strains should probably be classified as L. mesenteroides; however, to formally clarify their taxonomic position DNA–DNA hybridizations should be performed. According to Konstantinidis et al. (2006), the minimum number of genes needed for multilocus differentiation between species is three because of potential events of horizontal gene transfer or recombination. In the present study, the concatenated gene sequences indeed proved valuable for the differentiation of all Leuconostoc species (Fig. 4).
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. The API 50CHL identification system (bioMérieux) was used to determine the carbohydrate fermentation profile. Results of this characterization are given in the species description. Table 3 gives an overview of the physiological differences between the novel species and the most closely related species. It is evident from these summarized physiological data that strain LMG 23990T can be distinguished from related Leuconostoc species by a combination of acid production tests from sugars.
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Description of Leuconostoc holzapfelii sp. nov.
Leuconostoc holzapfelii (hol.za.pfel'i.i. N.L. gen. masc. n. holzapfelii, of Holzapfel, in honour of Professor Dr W. H. Holzapfel, in recognition of his outstanding work in the area of lactic acid bacterium taxonomy and physiology).
Cells are Gram-positive, non-motile, ovoid or short rods, approximately 0.8–1.0x2.0–3.0 µm in size, and mostly occur in pairs or short chains. Colonies grown on MRS agar at 30 °C for 2 days are approximately 1 mm in diameter, beige, smooth and circular. Facultatively anaerobic, catalase-negative. The D-(-) isomer of lactic acid is produced from glucose with gas formation. Growth occurs at 10 and 37 °C, but not at 4 or 40 °C. Does not grow in the presence of 6.5 % NaCl. Grows at pH 3.9. Ammonia is not produced from arginine. Slime is produced from sucrose. Acid is produced from L-arabinose, galactose, glucose, fructose, mannose, methyl 
-D-glucoside, N-acetylglucosamine, maltose, melibiose, trehalose, raffinose, D-turanose and gluconate. Acid is not produced from glycerol, erythritol, D-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl β-D-xyloside, sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl -D-mannoside, amygdalin, arbutin, aesculin, salicin, cellobiose, lactose, sucrose, inulin, melezitose, amygdalin, glycogen, xylitol, gentiobiose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-ketogluonate or 5-ketogluconate. The DNA G+C content was 43.5 mol%.
The type strain, LMG 23990T (=CCUG 54536T), was isolated from coffee fermentation in Ethiopia.
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ACKNOWLEDGEMENTS |
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