FIG. 1.

Random PCR mutagenesis screen used to identify domains involved in Mga-dependent transcriptional activation. Random mutations were generated in mga using the GeneMorph PCR mutagenesis kit (Stratagene). Mutated M6 mga alleles were placed on a plasmid under the constitutive promoter Pspac in an mga-deleted strain containing a promoter fusion of the Mga-regulated Pemm to a promoterless gusA reporter gene in the chromosome of M6 GAS. Resulting strains were plated onto THY plates containing X-Gluc to reveal clones deficient in transcriptional activation (light blue or white). Whole-cell lysates from clones showing a defect in activation were extracted, and Mga protein levels were determined using Western blot analysis. Plasmid DNA from clones producing wild-type levels of protein was isolated, sequenced, and aligned to the wild-type M6 mga gene to identify mutations.