A phylogenetic tree was constructed to investigate the evolutionary relationships between these proteins. Based on the sequence divergence in amino acid TyrDC sequences (Figure 1), the phylogenetic tree reveals that L. FHPI plantarum TyrDC is closely related to those of L. brevis proteins and made one cluster clearly separated. Similar results were
obtained when phylogenetic tree was constructed with TyrP amino acid sequences (data not shown). These results confirm that the organization of this L. plantarum tdc MEK activation locus is similar to those described for other LAB strains, with contiguous tyrDC and tyrP genes. The phylogenetic tree analysis is consistent with the tdc locus of L. plantarum IR BL0076 strain having been transferred horizontally from L. brevis. Figure 1 Phylogenetic tree comparing 21 TyrDC sequences from various Lactobacillus strains. The amino acid sequences were aligned using the multiple alignment program
CLUSTAL W2. The phylogenetic tree was constructed by using the TreeTop from the GeneBee. Bootstrap values are expressed in percentages and indicated at nodes. The amino acid sequences of TyrDC were obtained from the following accession numbers entries: [GenBank : AF446085] (L. brevis IOEB 9809), [GenBank : YP_796294.1] (L. brevis ATCC 367), [GenBank : ABY71221.1] (L. brevis NS77), [GenBank : ZP_03940842.1] (L. brevis subsp. gravesensis ATCC 27305), [GenBank :AEB91325.1] (Sporolactobacillus sp. P3J), [GenBank
: AAQ73505.1] ICG-001 molecular weight (E. hirae), [GenBank : ZP_05553037] (L. coleohominis 101-4-CHN), [GenBank : ZP_07729457] (L. oris PBo13-T2-3), [GenBank :ZP_06679761] (E. faecium E1071), [GenBank : ZP_06677337] (E. faecium E1162), [GenBank : ZP_00602894.1] (E. faecium DO), [GenBank : ZP_06698865.1] (E. faecium E1679), [GenBank : CAF33980] (E. durans IPLA 655), [GenBank : ZP_05559869] (E. faecalis T8), [GenBank : ZP_07768147] (E. faecalis DAPTO 516), [GenBank : ZP_07771864] (E. faecalis TX0102), [GenBank : ZP_07569615] (E. faecalis TX0109), [GenBank : CBL32775] (Enterococcus sp. 7 L76), [GenBank : ADX79254] (E. faecalis 62) and [GenBank : ZP_04646316] (E. faecalis TUSoD Ef11). Growth of L. plantarum with peptides containing tyrosine Peptides of different sizes were used: Non-specific serine/threonine protein kinase a dipeptide Tyr-Ala containing the tyrosine residue at the N-terminus, a tripeptide Gly-Leu-Tyr with the tyrosine at the C-terminus, and a peptide of four amino acids Gly-Gly-Tyr-Arg, where the tyrosine is in an internal position. The growth was monitored by measuring the OD at 600 nm. L. plantarum IR BL0076 was able to grow in the synthetic medium either with free amino acids (medium 1) or synthetic peptides containing tyrosine (medium 2). The growth curve was the same in the two media (Figure 2), but not in MRS medium (control).