Domains Required for Transcriptional Activation Show Conservation in the Mga Family of Virulence Gene Regulators

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Abstract
Mga, or the multigene regulator of the group A streptococcus (GAS) ( Streptococcus pyogenes ), is a transcriptional regulator of virulence genes important for colonization and immune evasion. All serotypes of the GAS possess one of two divergent mga alleles ( mga-1 or mga-2 ), and orthologues of Mga have also been identified in other pathogenic streptococci. To date, the only functional motifs established within Mga are two amino-terminal DNA-binding domains (HTH-3 and HTH-4). To uncover novel domains, a random mutagenesis screen using an M6 Mga ( mga-1 ) was undertaken to find mutations leading to a defect in transcriptional activation of the Mga-regulated emm gene. In addition to mutations in the established DNA-binding domains, the screen also revealed mutations in a region conserved among several Mga orthologues. Alanine scanning helped resolve the boundaries of this c onserved M ga d omain (CMD-1) spanning from residues 10 to 15 of the protein, with the two flanking amino acid residues likely involved in protein stability. Transcriptional reporter analyses demonstrated the importance of CMD-1 for activation of P emm and autoactivation of P mga in the serotype M6 Mga. Mutational analyses showed that both CMD-1 and HTH-4 are also necessary for activation of the promoter target P mrp in a divergent serotype M4 Mga ( mga-2 ), suggesting a conserved functionality. However, in contrast to M6, the M4 Mga mutants did not show a defect in autoregulation. Mutation of similar conserved residues in the Mga-like regulator DmgB from S. dysgalactiae subsp. dysgalactiae showed that CMD-1 and HTH-4 are critical for transcriptional activation in this orthologue, implying that a common mechanism of virulence gene activation may exist for members of the Mga family of regulators.