Nucleotides Critical for the Interaction of the Streptococcus pyogenes Mga Virulence Regulator with Mga-Regulated Promoter Sequences

• Published in: Journal of Bacteriology 2012-09, Vol.194 (18), p.4904-4919
• Main Authors: Hause, Lara L, McIver, Kevin S
• Format: Article
• Language: English
• Subjects: adenines; Artificial Gene Fusion; Bacterial proteins; Bacterial Proteins - metabolism; Bacteriology; Binding Sites; Biological and medical sciences; complement; deoxyribonuclease I; DNA; DNA Mutational Analysis; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; Enzymes; Fundamental and applied biological sciences. Psychology; gel electrophoresis; Genes, Reporter; luciferase; Luciferases - analysis; Luciferases - genetics; methylation; Microbiology; Miscellaneous; Mutagenesis; nucleotides; Plasmids; promoter regions; Promoter Regions, Genetic; Protein Binding; regulon; Streptococcus pyogenes; Streptococcus pyogenes - metabolism; Streptococcus pyogenes - pathogenicity; Transcription, Genetic; transcriptional activation; Virology; virulence; Virulence Factors - biosynthesis
• ISSN: 0021-9193; 1098-5530; 1067-8832
• DOI: 10.1128/JB.00809-12

The Mga regulator of Streptococcus pyogenes directly activates the transcription of a core regulon that encodes virulence factors such as M protein (emm), C5a peptidase (scpA), and streptococcal inhibitor of complement (sic) by directly binding to a 45-bp binding site as determined by an electrophoretic mobility shift assay (EMSA) and DNase I protection. However, by comparing the nucleotide sequences of all established Mga binding sites, we found that they exhibit only 13.4% identity with no discernible symmetry. To determine the core nucleotides involved in functional Mga-DNA interactions, the M1T1 Pemm1 binding site was altered and screened for nucleotides important for DNA binding in vitro and for transcriptional activation using a plasmid-based luciferase reporter in vivo. Following this analysis, 34 nucleotides within the Pemm1 binding site that had an effect on Mga binding, Mga-dependent transcriptional activation, or both were identified. Of these critical nucleotides, guanines and cytosines within the major groove were disproportionately identified clustered at the 5′ and 3′ ends of the binding site and with runs of nonessential adenines between the critical nucleotides. On the basis of these results, a Pemm1 minimal binding site of 35 bp bound Mga at a level comparable to the level of binding of the larger 45-bp site. Comparison of Pemm with directed mutagenesis performed in the M1T1 Mga-regulated PscpA and Psic promoters, as well as methylation interference analysis of PscpA, establish that Mga binds to DNA in a promoter-specific manner.