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Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers.

ABSTRACT: In this study, we succeeded in differentiating Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum by means of recA gene sequence comparison. Short homologous regions of about 360 bp were amplified by PCR with degenerate consensus primers, sequenced, and analyzed, and 322 bp were considered for the inference of phylogenetic trees. Phylograms, obtained by parsimony, maximum likelihood, and analysis of data matrices with the neighbor-joining model, were coherent and clearly separated the three species. The validity of the recA gene and RecA protein as phylogenetic markers is discussed. Based on the same sequences, species-specific primers were designed, and a multiplex PCR protocol for the simultaneous distinction of these bacteria was optimized. The sizes of the amplicons were 318 bp for L. plantarum, 218 bp for L. pentosus, and 107 bp for L. paraplantarum. This strategy permitted the unambiguous identification of strains belonging to L. plantarum, L. pentosus, and L. paraplantarum in a single reaction, indicating its applicability to the speciation of isolates of the L. plantarum group.

SUBMITTER: Torriani S

PROVIDER: S-EPMC93042 | biostudies-literature | 2001 Aug

REPOSITORIES: biostudies-literature

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Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers.

Torriani S S Felis G E GE Dellaglio F F

Applied and environmental microbiology 20010801 8

In this study, we succeeded in differentiating Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum by means of recA gene sequence comparison. Short homologous regions of about 360 bp were amplified by PCR with degenerate consensus primers, sequenced, and analyzed, and 322 bp were considered for the inference of phylogenetic trees. Phylograms, obtained by parsimony, maximum likelihood, and analysis of data matrices with the neighbor-joining model, were coherent and cl ...[more]

PMID: 11472918

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Project description:BACKGROUND: Tannase (tannin acyl hydrolase, EC 3.1.1.20) specifically catalyzes the hydrolysis of the galloyl ester bonds in hydrolyzable tannins to release gallic acid. The enzyme was found not only in fungal species but also many bacterial species including Lactobacillus plantarum, L. paraplantarum, and L. pentosus. Recently, we identified and expressed a tannase gene of L. plantarum, tanLpl, to show remarkable differences to characterized fungal tannases. However, little is known about genes responsible for tannase activities of L. paraplantarum and L. pentosus. We here identify the tannase genes (i.e. tanLpa and tanLpe) of the above lactobacilli species, and describe their molecular diversity among the strains as well as enzymological difference between species inclusive of L. plantarum. RESULTS: The genes encoding tannase, designated tanLpa and tanLpe, were cloned from Lactobacillus paraplantarum NSO120 and Lactobacillus pentosus 21A-3, which shared 88% and 72% amino acid identity with TanLpl, cloned from Lactobacillus plantarum ATCC 14917(T), respectively. These three enzymes could comprise a novel tannase subfamily of independent lineage, because no other tannases in the databases share significant sequence similarity with them. Each of tanLpl, tanLpa, and tanLpe was expressed in Bacillus subtilis RIK 1285 and recombinant enzymes were secreted and purified. The K(m) values of the enzymes on each galloyl ester were comparable; however, the k(cat)/K(m) values of TanLpa for EGCg, ECg, Cg, and GCg were markedly higher than those for TanLpl and TanLpe. Their enzymological properties were compared to reveal differences at least in substrate specificity. CONCLUSION: Two tannase genes responsible for tannase activities of L. paraplantarum and L. pentosus were identified and characterized. TanLpl, TanLpa and TanLpe forming a phylogenetic cluster in the known bacterial tannase genes and had a limited diversity in each other. Their enzymological properties were compared to reveal differences at least in substrate specificity. This is the first comparative study of closely related bacterial tannases.

Dataset Information

Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers.

Publications

Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers.

Similar Datasets

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