Investigating the genetic regulation of the ECF sigma factor σS in Staphylococcus aureus

Whittney N Burda, Halie K Miller, Christina N Krute, Shane L Leighton, Ronan K Carroll, Lindsey N Shaw

BackgroundWe previously identified an ECF sigma factor, ¿S, that is important in the stress and virulence response of Staphylococcus aureus. Transcriptional profiling of sigS revealed that it is differentially expressed in many laboratory and clinical isolates, suggesting the existence of regulatory networks that modulates its expression.ResultsTo identify regulators of sigS, we performed a pull down assay using S. aureus lysates and the sigS promoter. Through this we identified CymR as a negative effector of sigS expression. Electrophoretic mobility shift assays (EMSAs) revealed that CymR directly binds to the sigS promoter and negatively effects transcription. To more globally explore genetic regulation of sigS, a Tn551 transposon screen was performed, and identified insertions in genes that are involved in amino acid biosynthesis, DNA replication, recombination and repair pathways, and transcriptional regulators. In efforts to identify gain of function mutations, methyl nitro-nitrosoguanidine mutagenesis was performed on a sigS-lacZ reporter fusion strain. From this a number of clones displaying sigS upregulation were subject to whole genome sequencing, leading to the identification of the lactose phosphotransferase repressor, lacR, and the membrane histidine kinase, kdpD, as central regulators of sigS expression. Again using EMSAs we determined that LacR is an indirect regulator of sigS expression, while the response regulator, KdpE, directly binds to the promoter region of sigS.ConclusionsCollectively, our work suggests a complex regulatory network exists in S. aureus that modulates expression of the ECF sigma factor, ¿S.