Browsing by Subject "Electrophoretic Mobility Shift Assay"
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Item Functional Domains in the Multigene Regulator of the Group A Streptococcus(2006-05-16) Vahling, Cheryl M.; McIver, Kevin S.The group A streptococcus (GAS) is a human pathogen capable of causing a broad range of symptoms from mild erythema to severe tissue destruction. The multigene regulator of the GAS, or Mga, has been shown to bind DNA and activate transcription of virulence genes implicated in colonization and immune evasion in response to environmental conditions. Several different avenues of investigation were undertaken in the present study with an overall goal of identifying and characterizing functional domains within the protein. The first section of this study uses a naturally occurring mutant, previously identified to be deficient in transcriptional activation, as a guide to map functional residues within Mga. Electrophoretic mobility shift assays using purified proteins determined that the defect was not a result of the inability to bind DNA, so gain-of-function mutants were used to restore transcriptional activity to the mutant and pinpoint the residues necessary for full activation. In the second section, a genetic screen was undertaken to identify novel domains within the protein. From this screen, a domain was found within the extreme N-terminus. Sequence homology revealed several proteins in other pathogenic treptococci that shared this motif. As a result, it was named CMD-1 for conserved Mga domain 1, while the group of proteins was subsequently designated as the Mga family of putative virulence gene regulators. Alanine scanning mutagenesis demonstrated the importance of CMD-1 for transcriptional activation in several members of this family. This established that the results yielded from investigations of Mga could be broadened beyond the GAS to provide global insight into possible mechanisms of virulence regulation in other pathogenic streptococci. Finally, an in silico analysis of Mga was performed, revealing several areas with differing degrees of structural similarity to known domains of other bacteria including a receiver domain and a PTS-regulatory domain. Each region was explored to determine if it was functionally active in Mga. Since both domains have been implicated in forming higher order structures, the oligomeric state of Mga was also determined. Overall, characterization of Mga has helped unravel the mechanisms of virulence regulation in pathogenic streptococci.Item Transcriptional Regulation of the MGA Virulence Regulon in Streptococcus Pyogenes(2005-12-19) Almengor, Audry C.; McIver, Kevin S.The group A streptococcus (GAS) is a strict human pathogen that is capable of causing an array of diseases ranging from pharyngitis (strep throat) to necrotizing faciitis (flesh-eating disease). The GAS possesses a transcriptional regulator called Mga, or the multiple gene regulator of the GAS, that responds to growth phase and environmental signals to activate virulence genes necessary for colonization, internalization, and immune evasion of the bacterium. In previous studies, Mga-binding sites were found proximal to the start of transcription in two of the genes that it regulates (emm and scpA), suggesting that Mga activates transcription through stabilization or recruitment of the transcription machinery. Based upon a consensus sequence of the Mga-binding site derived from Pemm and PscpA, two other Mga-regulated genes (sclA and sof/sfbX) were found to have binding sites distal to the start of transcription, suggesting an alternative mechanism of activation. Electrophoretic mobility shift assays (EMSAs) were used to verify the location of Mga binding, and primer extension analysis was used to identify the starts of transcription. A GusA transcriptional reporter with PsclA showed Mga-regulated activity, and the distal Mga-binding site was found to be essential for this activity in vivo. An in vitro transcription assay was adapted for study of this new category of Mga-regulated promoters. In this assay, the sufficiency of Mga to activate transcription at Pemm, PsclA, and Psof/sfbX was assessed and confirmed. Mga is autoregulated and has a complex promoter that consists of two transcriptional start sites and two Mga-binding sites. It is at this promoter that the ability of Mga to respond to environmental cues is thought to take place. An analysis of the mga promoter revealed the presence of a catabolite responsive element (cre site). An EMSA with the catabolite control protein (CcpA) verified its ability to bind specifically to this site. Further, a deletion of the cre site in the context of the whole promoter as well as with respect to its upstream transcriptional start site determined that the cre site is necessary for full transcriptional activity of mga. Consequently, a link between carbohydrate metabolism and virulence gene regulation was established. The studies performed here in conjunction with future experiments on the regulation of mga and Mga-regulated genes should help build our overall knowledge of the molecular mechanisms by which Mga controls virulence in the GAS.