Purification of Global Regulator, Spx, and RNA Polymerase from Staphylococcus aureus for Use in In Vitro Transcription of Redox Genes

Spx is a global regulator discovered in Bacillus subtilis to suppress basal growth and development processes and activate transcription of genes involved in thiol homeostasis when a cell encounters oxidative stress. Its activity relies on reversible thiol-disulfide bond formation and binding RNA polymerase rather than DNA. The discovery that Staphylococcus aureus global virulence regulator, SarA, is more active upon cysteine reduction suggests that redox response could mediate virulence in this important human pathogen. We describe the cloning of spx from S. aureus strain RN6390, overexpression in Escherichia coli, and purification of native protein. Antibodies against Spx were raised for western analysis. Spx from S. aureus was highly active in a B. subtilis in vitro transcription system, stimulating expression of trxB, the gene encoding thioredoxin reductase, without reducing agents. RNA polymerase was partially purified from S. aureus, and the enzyme was active, catalyzing transcription of rpsD, but not trxB.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Biological Sciences

Year

2006

Author dc:creator

• McBride, Jane

Contributors dc:contributor

• Clancy, Mary
• Timpte, Candace
• Hurlburt, Barry

Repository record dc:identifier

https://scholarworks.uno.edu/td/495

OAI identifier oai:identifier

oai:scholarworks.uno.edu:td-1495