Transcriptional Activation of sclA by Mga Requires a Distal Binding Site in Streptococcus pyogenes

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Abstract
Streptococcus pyogenes (the group A streptococcus [GAS]) is a medically significant pathogen of humans, causing a range of diseases from pharyngitis to necrotizing fasciitis. Several important GAS virulence genes are under the control of a pleiotropic regulator called Mga, or the multiple gene regulator of GAS, including the gene encoding the streptococcal collagen-like protein, or sclA . Analysis of the genome sequence upstream of sclA revealed two potential Mga-binding sites with homology to the published Mga-binding element, which were called P sclA -I (distal) and P sclA -II (proximal) based on their location relative to a predicted start of transcription. Primer extension was used to confirm that the Mga-dependent transcriptional start site for sclA was located adjacent to the proximal P sclA -II binding site. By using overlapping P sclA promoter probes and purified Mga-His fusion protein, it was shown by electrophoretic mobility shift assays that, unlike other Mga-regulated promoters, Mga binds only to a distal DNA-binding site (P sclA -I). Binding of Mga to P sclA -I could be competed with cold probes corresponding to known Mga-regulated promoters (P emm , P scpA , and P mga ) but not with a nonspecific probe or the proximal P sclA -II fragment. With the use of a plasmid-based green fluorescent protein transcriptional reporter system, the full-length P sclA was not sufficient to reproduce normal Mga-regulated activation. However, studies using a single-copy gusA transcriptional reporter system integrated at the native sclA chromosomal locus clearly demonstrated that the distal P sclA -I binding site is required for Mga regulation. Therefore, P sclA represents a new class of Mga-regulated promoters that requires a single distal binding site for activation.