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1 Laboratoire de Dynamique, Evolution et Expression de Génomes de Microorganismes, Université Louis Pasteur/CNRS FRE 2326, 28 rue Goethe, 67083 Strasbourg, France
2 Dipartimento Scientifico e Tecnologico, Facoltà di Scienze MM. FF. NN., Università degli Studi di Verona, Strada le Grazie 15, 37134 Verona, Italy
3 Molecular Genetics & Biotechnology unit, National Institute for Medical Research, Edmond Crescent, PMB 2013, Yaba, Lagos, Nigeria
Correspondence
Franco Dellaglio
franco.dellaglio{at}univr.it
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Published online ahead of print on 14 March 2005 as DOI 10.1099/ijs.0.63333-0.
The GenBank/EMBL/DDBJ accession numbers obtained in the present study are: 16S rRNA gene sequence of L. plantarum subsp. argentoratensis DKO 22T, AJ640078; complete recA gene sequences of L. plantarum subsp. plantarum ATCC 14917T, AJ621668; L. plantarum subsp. argentoratensis DKO 22T, AJ640079; L. plantarum subsp. argentoratensis A7, AJ640080; L. pentosus LMG 10755T, AJ621666; L. paraplantarum LMG 16673T, AJ621662; partial cpn60 gene sequences of L. plantarum subsp. argentoratensis DKO 22T, AJ640081; L. plantarum subsp. plantarum ATCC 14917T, AJ640082.
Positions of the primers of species-specific multiplex-PCR assay based on recA gene sequences are shown in a supplementary figure available in IJSEM Online.
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) and in particular to the L. plantarum phylogenetic subgroup (Hammes & Hertel, 2003). This species is characterized by many metabolic activities that allow it to colonize different environments such as dairy products, pickled vegetables, fish products, silage, wine as well as mammal intestinal tracts (Vescovo et al., 1993). The species is highly heterogeneous (Dellaglio et al., 1975). Several studies, which have tried to elucidate intraspecific relationships, led to the description of two novel species: Lactobacillus pentosus and Lactobacillus paraplantarum (Zanoni et al., 1987; Curk et al., 1996). These three closely related species cannot be distinguished by 16S rRNA gene sequence analysis because they show 99 % sequence similarity. They also have very similar fermentation abilities: L. pentosus can be differentiated from L. plantarum only by its capacity to ferment glycerol and xylose (Zanoni et al., 1987). Some exceptions, however, exist: some L. plantarum strains are able to metabolize glycerol as with L. pentosus and not all L. pentosus strains can metabolize xylose (Bringel et al., 1996). Fermentation differences between L. plantarum and L. paraplantarum are even less helpful: the main difference is that strains of the latter species metabolize methyl 
-D-glucopyranoside, whereas 66 % of L. plantarum strains cannot (Bringel et al., 1996).
With regard to molecular techniques, L. plantarum, L. pentosus and L. paraplantarum can be rapidly distinguished using PCR amplification targeting the 16S/23S rRNA gene spacer region (Berthier & Ehrlich, 1998) or the recA gene as described by Torriani et al. (2001). Moreover, a polyphasic approach with a combination of RAPD (randomly amplified polymorphic DNA)-PCR, Southern hybridization and phenotypic traits not only distinguished the three species L. plantarum, L. paraplantarum and L. pentosus, but also revealed the presence of two groups of strains in the species L. plantarum (Bringel et al., 2001): 90 % of these were closely linked to the L. plantarum type strain, ATCC 14917T, and 10 % (14 strains) formed a distinct group. We report here the analyses performed on those 14 ‘atypical’ strains and, on the basis of collected data, a novel subspecies of L. plantarum is proposed.
The studied bacterial strains listed in Table 1 were grown in MRS at 37 °C. After genomic DNA extraction (Marmur, 1961), the recA-nested multiplex-PCR assay was performed as described by Torriani et al. (2001). This multiplex-PCR assay uses a reverse primer (pRev) in combination with three species-specific forward primers (planF, pentF and paraF) to distinguish the closely related species of the L. plantarum group. Three species-specific amplification products of different length were obtained: 318 bp for L. plantarum, 218 bp for L. pentosus and 107 bp for L. paraplantarum. The 14 atypical strains, isolated from vegetable sources, displayed an unexpected pattern characterized by two amplification bands, i.e. the 318 bp band typical of L. plantarum, and an additional 120 bp band (see Fig. 1). To evaluate the origin and the specificity of this new band, further PCR amplifications were performed on the 14 strains and on L. plantarum ATCC 14917T. Each of the three species-targeted forward primers was assayed in combination with the reverse primer pRev in separate PCR assays. As expected, a unique 318 bp band was obtained when the primer specific for L. plantarum species was used. By contrast, in the presence of the primer pentF, specific for L. pentosus, a single amplification product of 120 bp was obtained from all 14 strains (data not shown), but no amplification was detected with the type strain of L. plantarum. Sequencing confirmed that both amplicons are recA gene fragments. Moreover, the recA gene sequences of the 14 strains were more similar to the recA gene of L. plantarum ATCC 14917T, confirming that they belong to the species L. plantarum. The positions of the multiplex-PCR primers on the recA gene sequence of L. plantarum ATCC 14917T (GenBank accession no. AJ621668), L. plantarum DKO 22T (AJ640079), L. pentosus LMG 10755T (AJ621666) and L. paraplantarum LMG 16673T (AJ621662) are given in Supplementary Fig. S1 available in IJSEM Online.
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lists the fermentation abilities of the 14 strains evaluated using the API-50 CHL detection kit as described by the manufacturer (bioMérieux). All the L. plantarum strains and the type strains of L. pentosus and L. paraplantarum fermented galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetylglucosamine, amygdalin, aesculin, salicin, cellobiose, maltose, sucrose and 
-gentiobiose. None of the strains was able to ferment L-sorbitol, inulin, D-lyxose, erythritol, D-arabinose, L-xylose, adonitol, methyl -xyloside, inositol, D-tagatose, DL-fucose, L-arabitol, 2-ketogluconate or 5-ketogluconate. By contrast, different fermentation trends were observed between the two groups of L. plantarum strains for five carbohydrates (Table 2). All strains were negative for catalase activity (Freney et al., 1999). Growth at 15 °C (positive) and 45 °C (negative) was tested in MRS broth. Hydrolysis of arginine determined according to the method described by Sharpe (1979) was not positive for any strain. Lactic acid isomers were assayed enzymically using the DL-lactate test kit (Boehringer) after growth in MRS. The 14 atypical L. plantarum strains formed both isomers, with the D isomer accounting for 65–70 % of the total lactic acid produced.
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). Phylogenetic analysis was performed with the MEGA version 2.1 program (Kumar et al., 2001): the distance matrix was obtained with Tamura's three-parameter model and tree reconstruction was obtained with minimum evolution clustering. An analysis of the robustness of the tree was obtained by using a bootstrap approach with 1000 replicates. A maximum-parsimony method was also applied with default options. Maximum-likelihood analysis was performed with the DNAML program in the PHYLIP software package. Very low divergence of sequences (similarity of 99 %) in the phylogenetic trees confirmed strongly that strain DKO 22T belongs to the L. plantarum group (data not shown). Sequence analysis of the 16S rRNA gene did not allow us to distinguish very closely related species whereas protein-encoding genes did (Torriani et al., 2001). Therefore, the complete recA gene sequences of selected L. plantarum strains (ATCC 14917T, DKO 22T, A7) and of the L. pentosus and L. paraplantarum type strains were obtained. The corresponding recA genes were PCR-amplified using primers annealing the flanking gene sequences, which were conserved in the sequenced genomes (http://www.tigr.org) of bacteria belonging to the phylum Firmicutes (data not shown). The upstream cinA gene (accession no. NT01LP2031 in L. plantarum WCFS1, according to TIGR annotation) encodes a putative competence-damage protein. The downstream gene (accession no. NT01LP2029 in L. plantarum WCFS1, according to TIGR annotation) encodes a conserved protein with unknown function. The recA-flanking gene sequences were used to design degenerate primers to amplify two overlapping fragments that included recA. A 1·4 kb fragment corresponding to the 5'-region was amplified with degenerate primers cinA-f (5'-CGAGTTTTGCGGTTTTGTGGNATHGGNGA-3') and AN-r (5'-CRATTTTTTCACGRATYTG-3'). The reaction mixture contained 300 ng DNA template, 2 mM MgCl2, 0·2 mM dNTPs, 1 µM each primer and 2·5 U Taq DNA polymerase (Sigma) in a standard reaction buffer. After an initial denaturation of 5 min at 94 °C, 30 cycles of 1 min at 94 °C, 1·5 min at 44 °C, 1·5 min at 72 °C and a final extension at 72 °C for 5 min were performed. The 1·1 kb 3'-end fragment was amplified using AN-f (5'-AAATYGAWGGNGARATGGG-3') and HDIG-r (5'-TGTTCACGAAAAATTTGATTTTCTTCYTTNGCYTC-3'). Reaction mixtures contained 300 ng DNA template, 2 mM MgCl2, 0·1 mM dNTPs, 0·5 µM each primer and 1·5 U Taq DNA polymerase (Sigma) in a standard reaction buffer. Reaction conditions were the same as those described above, except primer annealing was performed at 42 °C rather than 44 °C. The amplified fragments from L. plantarum DKO 22T, A7 and ATCC 14917T, L. pentosus LMG 10755T and L. paraplantarum LMG 16673T were sequenced. The recA gene sequence of L. plantarum WCFS1, available in the EMBL database (GenBank/EMBL accession no. AL935258), was retrieved from its complete genome sequence (Kleerebezem et al., 2003). The sequenced strain WCFS1 is isogenic to strain NCIMB 8826, which was included in the present study (Table 1
) and gave a single 318 bp multiplex-PCR assay amplification product as obtained with the L. plantarum type strain (see Fig. 1). The recA gene was longer in L. plantarum (1143 bp) than in L. pentosus and in L. paraplantarum (both 1137 bp). Alignments of the L. plantarum recA nucleotide sequences revealed 100 % similarity between strains DKO 22T and A7, 99·7 % similarity between strains ATCC 14917T and WCFS1, but only 93 % similarity between the two L. plantarum recA gene clusters (strains DKO 22T/A7 versus ATCC 14917T/WCFS1). L. plantarum strains shared about 81–82 % sequence similarity with L. pentosus and L. paraplantarum when similarity values were calculated on matching positions in L. pentosus and L. paraplantarum 1134 bp recA genes (without the stop codon). Thus, recA gene sequence analysis not only clearly distinguished between L. plantarum, L. paraplantarum and L. pentosus, but also identified two separate groups within the L. plantarum species. A phylogenetic tree based on the complete recA gene sequences was obtained as described above and is shown in Fig. 2. The two L. plantarum clusters deduced from the complete sequence analysis of recA genes in four strains was validated in the other L. plantarum strains by comparing a 750 bp internal recA sequence obtained as described by Dellaglio et al. (2004). About 20 ng PCR product was restricted with endonucleases AfaI, BamHI and SalI (Takara; 5 U, 1 h). The same restriction patterns found in L. plantarum DKO 22T were found in 12 of the 14 atypical strains (data not shown). The two exceptions, strains DK 19 and SF2A35B, showed the same restriction pattern as for L. plantarum ATCC 14917T, suggesting sequence divergence in their restriction enzyme recognition sites.
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). In DKO 22T, a C
T point mutation in the recA sequence (nucleotide 505 in GenBank accession number AJ640079) was linked to the specific annealing of primer pentF. The same point mutation was also found in strain A7 but not in L. plantarum ATCC 14917T and WCFS1 (see Supplementary Fig. S1). Thus, in the recA-nested multiplex-PCR test, the two L. plantarum groups can be discriminated on the basis of a CT sequence divergence. Comparative sequence analysis on a second protein-encoding gene, cpn60, was also performed. Part of the cpn60 gene was amplified using the degenerate primers cpn-f (5'-CTTGGGCCCAAAAGGCMGNAAYGTNGT-3') and cpn-r (5'-CCAACCGTTCTTGCAATTTTTCNCKRTCRAA-3'). The reaction mixture (20 µl) contained 100 ng template DNA, 2 mM MgCl2, 0·1 mM dNTPs, 1 µM of each primer and 1·5 U Taq polymerase in a standard reaction buffer. After an initial denaturation of 5 min at 94 °C, 30 cycles of 1 min at 94 °C, 1 min at 55 °C, 1·5 min at 72 °C and a final extension at 72 °C for 7 min were performed. Amplicons of the expected length of about 1·0 kb were obtained for all the strains. Amplification products of about 450 bp were sequenced for both L. plantarum DKO 22T and ATCC 14917T. The two groups of L. plantarum could not be distinguished with cpn60 partial sequence analysis as their sequences were highly similar (99·8 %).
In conclusion, the 14 atypical strains belonged to L. plantarum as previously determined with Southern blot analysis with species-specific probes and RAPD-PCR (Bringel et al., 2001), with DNA–DNA hybridization of LP85-2 with the type strain of L. plantarum (Bringel et al., 1996) and with multiplex-PCR assay. However, for five carbohydrates, these 14 strains had different fermentation trends from those of the other L. plantarum strains tested (Table 2). Moreover, the 14 strains were easily distinguished based on highly standardized, reproducible and independent molecular tests, such as Southern analysis (Bringel et al., 1996), RAPD-PCR analysis (Bringel et al., 2001), recA multiplex-PCR assay (Torriani et al., 2001) and recA gene sequence analysis. Moreover, comparative analysis of 20 strains of L. plantarum using microarrays genotyped L. plantarum strains into two clearly distinguishable groups in agreement with the results of this study (unpublished data; personal communication of D. Molenaar, F. Bringel, F. Schuren, W. de Vos, R. Siezen and M. Kleerebezem). These results justify a novel subspecies designation for the 14 atypical strains in the species L. plantarum, for which the name Lactobacillus plantarum subsp. argentoratensis subsp. nov. is proposed. According to Rule 40b of the Bacteriological Code (Lapage et al., 1992), the description of a new subspecies automatically creates the subspecies L. plantarum subsp. plantarum, which contains the type strain (ATCC 14917T).
Description of Lactobacillus plantarum subsp. plantarum subsp. nov.
Lactobacillus plantarum subsp. plantarum (plan.ta'rum. L. fem. n. planta a sprout; M.L. n. planta a plant; M.L. gen. pl. n. plantarum of plants).
Gram-positive, non-motile, non-spore-forming straight rods with rounded ends, usually 0·9–1·2 µm by 3–8 µm, that occur singly, in pairs or in short chains. Facultatively anaerobic; growth occurs at 15 °C but not at 45 °C. Both isomers of lactic acid are produced. Gas from glucose is not produced. Catalase is not produced, but some strains may exhibit pseudocatalase activity especially if grown under glucose limitation. Amygdalin, cellobiose, aesculin, D-fructose, galactose, -gentiobiose, N-acetylglucosamine, D-glucose, lactose, maltose, mannitol, D-mannose, melibiose, trehalose, salicin and sucrose are fermented. Adonitol, D-arabinose, L-arabitol, erythritol, DL-fucose, 5-ketogluconate, methyl -xylose, 2-ketogluconate, inositol, inulin, D-lyxose, L-sorbitol, D-tagatose and DL-xylose are not fermented. Arginine is not deaminated. meso-Diaminopimelic acid is present in the cell wall and ribitol or glycerol have been described to be present as teichoic acids. G+C content of the DNA is 44–46 mol%. Considering phylogenetic placement based on 16S rRNA gene sequence analysis, L. plantarum belongs to the L. plantarum phylogenetic subgroup (Hammes & Hertel, 2003), very closely related to L. paraplantarum and L. pentosus. The three species are easily distinguishable by means of molecular techniques: Southern hybridization with a pyr probe on BglI digestion of chromosomal DNA (Bringel et al., 1996, 2001), multiplex species-specific PCR analysis (Torriani et al., 2001) and recA gene sequencing. Such techniques also allow the differentiation of the two subspecies of L. plantarum, L. plantarum subsp. plantarum and L. plantarum subsp. argentoratensis. When analysed by a specific multiplex-PCR approach (Torriani et al., 2001), DNA of strains belonging to L. plantarum subsp. plantarum produce a single amplification product of 318 bp. A rough separation of the two subspecies of L. plantarum may also be obtained based on fermentation profiles of dulcitol, melezitose, methyl -D-mannoside, L-arabinose, D-turanose (Table 2): usually, L. plantarum subsp. plantarum strains do not ferment dulcitol but do metabolize melezitose, methyl -D-mannoside, L-arabinose and D-turanose.
The type strain is ATCC 14917T (=NCDO 1752T=DSM 20174T).
Description of Lactobacillus plantarum subsp. argentoratensis subsp. nov.
Lactobacillus plantarum subsp. argentoratensis (ar.gen.to.ra.ten'sis. L. masc. adj. argentoratensis of or pertaining to Argentor
tus, the Roman name of the city of Strasbourg in Alsace, France, where the 14 strains were first collected and analysed).
Morphological, physiological and biochemical characteristics are the same as those of L. plantarum subsp. plantarum, with only a few exceptions: strains belonging to L. plantarum subsp. argentoratensis are not able to metabolize either melezitose or methyl -D-mannoside. Strains of this subspecies may be reliably differentiated from strains of L. plantarum subsp. plantarum by Southern hybridization with a pyr probe on BglI digestion of chromosomal DNA (Bringel et al., 1996, 2001), by multiplex species-specific PCR analysis (Torriani et al., 2001) and by recA gene sequencing, as reported in the present paper. When analysed by a specific multiplex-PCR approach (Torriani et al., 2001), DNA of strains belonging to this subspecies produce two amplification products, of 318 bp (specific for the species L. plantarum) and 120 bp (specific for the L. plantarum subsp. argentoratensis subspecies).
The type strain is DKO 22T (=CIP 108320T=DSM 16365T).
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