FIGURE 4.

Capsule and hemolysin production is cognately regulated by SP-STK/STP-mediated reversible phosphorylation of CovR. A, the comparison of capsule content based on the biochemical estimation of the hyaluronic acid for the STP mutants and complemented GAS strains with respect to their corresponding wild-type strains. Error bars, S.D. of two independent experiments performed in six wells. B, hemolysin production in the wild-type M1SF370 and M1T1 and their corresponding mutant and complemented strains. Complete lysis of shRBCs achieved upon the addition of 1% Triton X-100 was taken as control (100%). Error bars, S.D. of two independent experiments performed in six wells. C, autoradiogram showing phosphorylation of SP-CovR by SP-STKK and its dephosphorylation by SP-STP in an in vitro kinase assay. Arrows and arrowheads point toward the phosphorylated SP-STKK and SP-CovR, respectively. The parallel Coomassie-stained gel is shown to assess the integrity of the proteins in each case. M, migration of molecular mass standards. D, two-dimensional TLC of phosphorylated SP-CovR revealed phosphorylation at threonine residues in the autoradiogram and the migration of phosphoamino acid standards (tyrosine (pY), serine (pS), and threonine (pT)) by ninhydrin.