Regulation of σE by ppGpp in Escherichia coli and the use of famP as a reporter for SigE in Bordetella bronchiseptica

Open Access
Author:
Lee, Jia Yii Airina
Area of Honors:
Biotechnology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Sarah Ellen Ades, Thesis Supervisor
  • Teh Hui Kao, Honors Advisor
  • Wendy Hanna Rose, Faculty Reader
Keywords:
  • Sigma E
  • DksA
  • Bordetella
  • SigE
  • ppGpp
  • membrane stress
  • RseA
Abstract:
When faced with cell envelope stress, the cell employs various sensing mechanisms to activate signal transduction pathways that ultimately lead to changes in its gene expression profile to combat stress. This can be achieved by activating specific transcription factors that allow them to bind to RNA polymerases. One such sigma factor is the alternative sigma factor σE. In E. coli σE is activated upon cell envelope stress such as heat, ethanol stress, and disruptions in the folding of outer membrane porins. Upon activation, the anti-sigma factor RseA is cleaved to release σE, thus allowing it to bind to RNA polymerase. Alternatively, the σE pathway can be triggered by ppGpp with the help of the co-factor DksA. ppGpp functions as a global regulator of gene expression during the stringent response. Previous studies have found that regulation of σE under phosphate starvation conditions is DksA-independent. In this study we found that regulation through ppGpp appears to vary under different starvation stresses. When cells are subjected to carbon starvation, up-regulation of σE activity is fully dependent on DksA. While regulation of σE is well characterized in E. coli, little is known about the SigE pathway in respiratory pathogen B. bronchiseptica. To provide better understanding of the regulation of SigE in Bordetella sp., a reporter has been constructed using the promoter regions of the fam gene in B. bronchiseptica. SigE is able to transcribe the fam promoter in vitro, and the current reporter showed that SigE can similarly transcribe fam in vivo in B. bronchiseptica. The famP::lacZ serves as a tool to shed light on the significance of SigE for the virulence and viability of this respiratory pathogen.