| Amino acid residues that are important for metal binding and catalysis in Gram-positive phosphotyrosine phosphatases were identified in Streptococcus thermophilus Wzh/EpsB proteins. The Wzh protein from S. thermophilus MR-1C was purified after heterologous expression and tested for phosphatase activity against synthetic phosphotyrosine and phosphoserine/threonine peptides. The purified Wzh protein was able to remove phosphate from both phosphotyrosine peptides tested and the phosphatase activity of Wzh was dramatically reduced by the presence of the phosphotyrosine phosphatase inhibitor sodium vanadate at concentrations of 1, 5, and 10 mM. Purified Wzh had no activity against the synthetic phosphoserine/threonine peptide. These results established that Wzh functions as a phosphotyrosine phosphatase. By using the yeast two-hybrid system, strong intraspecific protein interactions were detected in S. thermophilus MR-1C, Streptococcus iniae 9066, and Lactococcus lactis subsp. cremoris JRF1 between the putative transmembrane activation protein (Wzd, CpsC, and EpsA, respectively) and the putative protein tyrosine kinase (Wze, CpsD, and EpsB, respectively). Weaker protein interactions take place forming a dimer between two identical protein tyrosine kinases and between the protein tyrosine kinase and phosphotyrosine phosphatase (Wzh, CpsB, and EpsC, respectively) in these species. Protein-protein interactions involving a S. thermophilus MR-1C Wzd/Wze fusion protein and Wzd and Wze indicated that these proteins may form multi-protein complexes. All combinations of the S. thermophilus Wzh, Wzd, Wze, Wzg (regulation), CpsE (glycosyl-1-phosphate transferase), CpsS (polymerization), CpsL (unknown), CpsW (regulation), and CpsU (membrane translocation) proteins were analyzed for protein-protein interactions but no additional interactions were discovered. For each of the intraspecific interactions detected, interspecific interactions were also detected when one protein was from S. iniae and the other was from S. thermophilus. Interactions were also observed between two protein tyrosine kinases when one protein was from either of the Streptococcus species and the other from L. lactis subsp. cremoris. These results and sequence comparisons performed in this study support the conclusion that interactions among the components of the tyrosine kinase/phosphatase regulatory system are conserved in the family Streptococcaceae. Interspecific protein-protein interactions suggest that functional regulatory complexes can be formed in naturally occurring and genetically engineered recombinant strains. |
