Two DNA‐binding domains of Mga are required for virulence gene activation in the group A streptococcus

Summary Mga is a DNA‐binding protein that activates expression of several important virulence genes in the group A streptococcus (GAS), including those encoding M protein (emm), C5a peptidase (scpA) and Mga (mga). To determine the functionality of four potential helix–turn–helix DNA‐binding motifs (HTH1–HTH4) identified within the amino‐terminus of Mga, alanine substitutions were introduced within each domain in a MBP–Mga fusion allele and purified proteins were assayed for binding to Mga‐specific promoter fragments (Pmga, PscpA and Pemm) in vitro. Although HTH‐1 and HTH‐2 mutations showed wild type DNA‐binding activity, an altered HTH‐3 domain resulted in reduced binding to the three promoters and an HTH‐4 mutant was devoid of detectable binding activity. Plasmid‐encoded expression of the HTH‐3 and HTH‐4 alleles from a constitutive promoter (Pspac) in the mga‐deleted GAS strain JRS519 demonstrated that Mga‐regulated emm expression correlated directly to the DNA‐binding activity observed for each mutant protein in vitro. Single‐copy expression of HTH‐3 and HTH‐4 from their native Pmga resulted in a dramatic reduction in autoregulated mga expression in both mutant strains. Thus, Mga appears to contain two DNA‐binding domains (HTH‐3 and HTH‐4) that are required for direct activation of the Mga virulence regulon in vivo.