FIG 7.

The SpeB^A−/SsE^A+ phenotype of in vivo-selected MGAS2221 variants is caused by covRS mutations. (A) SsE PAF acetylhydrolase activity in the culture supernatant of SpeB^A− variants with covS mutations and SpeB^A+ isolates from MGAS2221-infected mice. (B) Western blots for SsE and Spy0469 (control) in culture supernatants showing positive detection of SsE in SpeB^A− variants (strains 1 through 11) but not in SpeB^A+ isolates (strains 12 and 13) and MGAS2221 (strain 14). (C) Complementation of SpeB^A− variants with pGRV-CovRS (strains 6 and 7) and pCovSC (the other strains) converted the SpeB^A− phenotype of the mutants into the SpeB^A+ phenotype, and pDCBB controls for pCovSC did not change the status of SpeB production. Strain numbers correspond to those in Table 1. (D) SsE PAF-AH activity in the culture supernatant of pDCBB (control)- and pCovSC-transformed SpeB^A− variants with covS mutations listed in Table 1. (E) SsE PAF-AH activity in culture supernatants of the two CovR mutants and their complemented strains with integrated pGRV-CovRS.