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.

FIG. 1

Phylogenetic tree showing the relative positions of L. plantarum, L. paraplantarum, and L. pentosus as inferred by the neighbor-joining method with the recA gene from B. subtilis as the outgroup sequence. Bootstrap values for a total of 1,000 replicates are given. Trees generated by the maximum likelihood and parsimony methods have the same topology. The bar indicates 10% sequence divergence. In this tree topology, the phylogenetic distance between organisms is the sum of the horizontal segments.

FIG. 2

Amplification products obtained from the recA multiplex assay. Lane M contained a 1-kb PLUS DNA ladder (Life Technology Inc., Gaithersburg, Md.). Lanes 1 and 2, PCR amplification products from L. paraplantarum LMG 16673^T and NIRD P2, respectively; lanes 3 and 4, PCR amplification products from L. pentosus LMG 10755^T and LMG 18401, respectively; lanes 5 and 6, PCR amplification products from L. plantarum ATCC 14917^T and NCFB 340, respectively; lane 7, L. casei ATCC 334 (negative control).