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1 BCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
2 Laboratory of Microbiology, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
3 Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing (IMDO), Department of Applied Biological Sciences, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium
Correspondence
Marc Vancanneyt
Marc.Vancanneyt{at}UGent.be
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains LMG 11494 and LMG 11984T are AM158250 and AM158249, respectively.
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Identification of strains of L. brevis using conventional phenotypic methods often leads to ambiguous results (Kandler & Weiss, 1986; Pot et al., 1994). Molecular approaches have proved to be much more reliable (Vogel et al., 1994; Sohier et al., 1999; Guarneri et al., 2001) and have enabled ‘L. brevis-like’ isolates to be assigned to novel taxa such as Lactobacillus acidifarinae, Lactobacillus hammesii, Lactobacillus spicheri and Lactobacillus zymae (Meroth et al., 2004; Valcheva et al., 2005; Vancanneyt et al., 2005). In the present study, reference strains of L. brevis available from the BCCM/LMG Bacteria Collection (http://www.belspo.be/bccm/lmg.htm) were screened genotypically and the results demonstrated that two strains, LMG 11494 and LMG 11984, occupied a distinct position. Further genomic and phenotypic research revealed that these strains represent a single novel species.
Strain LMG 11494 (=NCFB 1058) was isolated from farmhouse red Cheshire cheese and was originally deposited in the NCFB culture collection as L. brevis by A. Hayward in 1957. Strain LMG 11984 (=ATCC 53295) was isolated from wheat and deposited in the ATCC as L. brevis (originally named Sporolactobacillus sp.) by M. Spiller in 1992. It is a patent strain used for the production of leavening barm (Spiller, 1987). Both strains and related reference strains were cultivated and maintained on de Man, Rogosa and Sharpe (MRS) agar medium (pH 6.5; de Man et al., 1960) and incubated at 30 °C for 24–48 h, unless otherwise indicated.
Sequence analysis of the phenylalanyl-tRNA synthase alpha-subunit (pheS) housekeeping gene has been proved to be a robust approach for the identification of enterococci (Naser et al., 2005a). Furthermore, the method is an excellent tool for delineating novel taxa (Naser et al., 2005b; 
vec et al., 2005a, b). In the present study, this methodology was applied to lactobacilli of the Lactobacillus buchneri species group. The primer sequences, amplification conditions and sequencing reactions performed were as described by Naser et al. (2005a). As found previously for enterococci, a species-specific grouping was obtained, as all Lactobacillus species studied formed distinct clusters (data not shown). Only two L. brevis strains, LMG 11494 and LMG 11984, showed an aberrant position. The neighbour-joining tree depicted in Fig. 1 (based upon comparison of partial sequences of 309 bp) revealed the relatedness between strains LMG 11494 and LMG 11984 and type strains of related taxa and showed that the two strains under study constituted a distinct cluster with a gene sequence similarity of 97 %. Nearest neighbours were the type strains of L. acidifarinae, L. hammesii, L. spicheri and L. zymae, with sequence similarities in a significantly lower range of 85–87 %. L. brevis and other taxa of the L. buchneri species group were more distantly related, with sequence similarities below 82 %.
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. Sequence assembly was performed using the AUTOASSEMBLER program (Applied Biosystems). Sequences were aligned with sequences retrieved from GenBank using CLUSTAL_X (Thompson et al., 1997). Phylogenetic analyses and bootstrap analysis (500 replicates) were subsequently performed using the BioNumerics 4.01 software package (Applied Maths). A phylogenetic tree was constructed using the neighbour-joining method (Fig. 2; Saitou & Nei, 1987) and unknown bases were discarded for the analyses. Comparison of the newly determined complete sequences for strains LMG 11494 and LMG 11984 (continuous stretches of 1518 bp) revealed a sequence similarity of 99.9 %. The tree topology obtained with the neighbour-joining method was evaluated and confirmed by maximum parsimony analysis using BioNumerics (data not shown). Comparison with deposited sequences available in the EMBL database classified strains LMG 11494 and LMG 11984 as part of the L. buchneri group (Schleifer & Ludwig, 1995) with the nearest neighbours (>97 % sequence similarity) L. hammesii and L. brevis, showing sequence similarities of 99.2 and 98.1 %, respectively.
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. Densitometric analysis, normalization and interpolation of protein profiles and a numerical analysis were performed by using the GELCOMPAR software package, versions 3.1 and 4.0, respectively (Applied Maths). After comparison with an in-house database containing profiles of nearly all recognized lactic acid bacteria, the two strains could not be distinguished from representatives of L. brevis. Phylogenetically related species, such as L. acidifarinae, L. hammesii, L. spicheri and L. zymae, occupied distinct positions (data not shown). Although this approach is very valuable for species identification of most lactic acid bacteria, some closely related species are not distinguished by this method, as has been shown previously within the Lactobacillus plantarum species group (Torriani et al., 2001) and the Lactobacillus acidophilus species group (Gancheva et al., 1999).
Strains LMG 11494 and LMG 11984 and a representative set of reference strains were investigated by a genotypic screening approach using fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting of whole genomes. FAFLP fingerprinting was performed as described by Thompson et al. (2001) with the following modifications: EcoRI/TaqI was used as the restriction enzyme combination and the 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 using GENESCAN 3.1 software (Applera). Normalized tables of peaks, containing fragments of 50–536 bp, were transferred into the BioNumerics software package, version 3.5, and the computer-generated fingerprints were added to an existing database of FAFLP fingerprints of lactic acid bacteria held at the BCCM/LMG Bacteria Collection. For numerical analysis, the region between the 75 and 500 bp bands of the internal standard were used. Similarity was calculated using the Dice coefficient and clustering was performed using the UPGMA algorithm. The dendrogram obtained from the analysis (Fig. 3) confirmed the distinct taxonomic position of strains LMG 11494 and LMG 11984.
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, using a combination of glass beads and enzymes, but with the following modifications. Volumes were increased tenfold for application on a large scale. SDS-treated cells were vortexed with beads for 30 s. After addition of 16.5 ml buffer (10 mM Tris/HCl, 100 mM EDTA, pH 8.0) and 5 ml 5 M NaCl and gentle mixing, the suspension was incubated at 65 °C for 10 min. Subsequent chloroform/isoamylalcohol extraction, precipitation, spooling of DNA on a glass rod, washing with ethanol and RNase treatment were performed as described by Marmur (1961). For determination of the DNA G+C content, DNA was enzymically degraded into nucleosides as described by Mesbah et al. (1989). The nucleoside mixture was then separated by HPLC using a SymmetryShield RP8 column (Waters) maintained at 37 °C. The solvent was 0.02 M (NH4)H2PO4 (pH 4.0) with 1.5 % acetonitrile. Non-methylated lambda phage DNA (Sigma) was used as the calibration reference. DNA G+C contents of strains LMG 11494 and LMG 11984 were 49 mol%. This value is lower than the value of 52.6 mol% determined for the type strain of L. hammesii and significantly higher than the value of 46 mol% determined for the type strain of L. brevis (Valcheva et al., 2005).
DNA–DNA hybridizations were performed between strains LMG 11494 and LMG 11984 and the type strains of L. brevis and L. hammesii (DNA was prepared as described above). Strain LMG 11984 was further hybridized with the type strains of L. acidifarinae, L. spicheri and L. zymae. The microplate method was used as described by Ezaki et al. (1989) and Goris et al. (1998), using a HTS7000 Bio Assay Reader (Perkin Elmer) for fluorescence measurements. Biotinylated single-stranded DNA (ssDNA) was hybridized with unlabelled ssDNA, which was bound non-covalently to microplate wells. Hybridizations were performed at 40 °C in hybridization mixture (2x SSC, 5x Denhardt's solution, 2.5 % dextran sulphate, 50 % formamide, 100 µg denatured salmon sperm DNA ml–1 and 1.25 µg biotinylated probe DNA ml–1). The DNA–DNA relatedness percentages presented are mean values based on four independent hybridization experiments. Reciprocal reactions (e.g. AxB and BxA) were performed and are also considered as independent hybridization experiments. Strains LMG 11494 and LMG 11984 displayed hybridization values of 35 and 40 %, respectively, with the type strain of L. hammesii and 7 and 8 %, respectively, with the type strain of L. brevis. Strain LMG 11984 yielded values in the range of 5–18 % with the type strains of L. acidifarinae, L. spicheri and L. zymae. The DNA–DNA hybridization value between strains LMG 11494 and LMG 11984 was 90 %, indicating a separate species status for the latter strains.
Growth characteristics and colony morphology were investigated on MRS agar after 24 h incubation at 37 °C under aerobic conditions and are given below in the species description.
Conventional biochemical tests were performed as described by Vancanneyt et al. (2005) and carbohydrate fermentation tests were carried out using API 50 CHL galleries following the manufacturer's instructions (bioMérieux). Metabolites from glucose were lactate, acetate and ethanol, as determined by HPLC (Waters). Isomers of D- and L-lactate were determined enzymically (R-Biopharm) and were in the ratio of 4 : 6 for strains LMG 11494 and LMG 11984. Strains LMG 11494 and LMG 11984 were differentiated from their nearest neighbour L. hammesii by their ability to deaminate arginine and inability to produce acid from aesculin or mannitol (Valcheva et al., 2005). Features that differentiated the two strains from L. brevis were the abilities to produce acid from D-arabitol and methyl 
-xyloside (Vancanneyt et al., 2005). Phenotypic data determined in the present study and by Vancanneyt et al. (2005) also revealed distinct features of the two novel strains which separate them from the more distantly related taxa L. acidifarinae, L. spicheri and L. zymae.
The overall results of the present study enable strains LMG 11494 and LMG 11984 to be assigned as representatives of a novel species, for which the name Lactobacillus parabrevis sp. nov. is proposed.
Description of Lactobacillus parabrevis sp. nov.
Lactobacillus parabrevis (pa.ra.bre'vis. Gr. prep. para like; L. masc. adj. brevis referring to the specific epithet of Lactobacillus brevis; N.L. masc. adj. parabrevis brevis-like, referring to L. brevis).
Cells are rod-shaped, occurring singly or in pairs and in chains, 1.5–5 µm in length and 0.9 µm wide, Gram-positive, catalase-negative, non-spore-forming and non-motile. After 24 h, colonies are beige, circular with a smooth surface and approximately 1 mm in diameter. Growth occurs at 15 °C, but not at 45 °C. Growth occurs at 6 % NaCl. Facultatively anaerobic and produces DL-lactic acid heterofermentatively with acetic acid and ethanol as other metabolites from glucose. Gas is produced from glucose and gluconate. Arginine is deaminated. All strains produce acid from L-arabinose, D-arabitol, gluconate, N-acetylglucosamine, D-glucose, D-fructose, maltose, ribose, D-xylose and methyl -xyloside. All strains test negative for acid production from D-arabinose, L-arabitol, adonitol, amygdalin, arbutin, cellobiose, dulcitol, aesculin, erythritol, D- and L-fucose, -gentiobiose, 2- and 5-ketogluconate, glycerol, glycogen, inositol, inulin, D-lyxose, mannitol, D-mannose, methyl 
-D-mannoside, melezitose, melibiose, D-raffinose, rhamnose, sucrose, salicin, starch, sorbitol, L-sorbose, D-tagatose, trehalose, D-turanose, xylitol and L-xylose. Acid production from galactose, methyl -D-glucoside and lactose is strain-dependent. The G+C content of DNA is 49 mol%.
The type strain, LMG 11984T (=ATCC 53295T), was isolated from wheat. A reference strain, LMG 11494 (=NCFB 1058), has also been isolated from cheese.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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de Man, J. C., Rogosa, M. & Sharpe, M. E. (1960). A medium for the cultivation of lactobacilli. J Appl Bacteriol 23, 130–135.
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