THE ESSENTIAL FUNCTION OF THE EXTRACYTOPLASMIC FUNCTION SIGMA FACTOR σE IN ESCHERICHIA COLI
Open Access
- Author:
- Hayden, Jennifer Diane
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 16, 2009
- Committee Members:
- Sarah Ellen Ades, Dissertation Advisor/Co-Advisor
Sarah Ellen Ades, Committee Chair/Co-Chair
Jean Elnora Brenchley, Committee Member
Kenneth Charles Keiler, Committee Member
B Tracy Nixon, Committee Member
Edward G Dudley, Committee Member - Keywords:
- sigmaE
bacterial physiology
phosphotransferase system - Abstract:
- All organisms encounter many challenges from their environment. A cell must first sense a stress, transduce information about the stress to other cellular compartments, and mount an appropriate response. Gram-negative bacteria, such as Escherichia coli, have two cellular compartments: the cytoplasm and the cell envelope. The cell envelope is composed of the outer membrane, the inner membrane, and a thin layer of peptidoglycan, which lies in the space between the membranes, called the periplasmic space. Cellular functions must be coordinated between the cytoplasm and the cell envelope, and when stress is sensed in the cell envelope, a signal must be transduced across the inner membrane in order for an appropriate response to be elicited in the cytoplasm. One cell envelope stress response is mediated by the extracytoplasmic function sigma factor, σE. Sigma factors are responsible for binding to and directing RNA polymerase to particular promoters. Various alternative sigma factors are upregulated after a stress is sensed, such that transcription can be redirected and the cell can better respond to the stress. σE is held inactive by its anti-sigma, the inner membrane protein RseA, and is also negatively regulated by the periplasmic protein RseB. Stresses that result in unfolded outer membrane proteins in the cell envelope activate proteolysis of RseA, freeing σE to bind RNA polymerase. σE is known to regulate an array of genes that help the cell respond to stress, such as periplasmic chaperones and foldases. σE also regulates genes involved in outer membrane protein and lipopolysaccharide biosynthesis and assembly. These genes are crucial for rebuilding the cell envelope after a stress is encountered, but must also be properly regulated during non-stress conditions. The gene for σE, rpoE, is essential; although the role of σE in cell envelope stress response is well-understood, it is unclear why σE is required for viability in E. coli. We addressed the question of the requirement for σE by determining the effects of its inhibition. Cells with inhibited σE activity show increased levels of other cell envelope stress responses and morphological defects, called cell envelope blebs; inhibition of σE activity caused eventual cell lysis. Using a genetic selection, we isolated two multicopy suppressors of the need for σE; ptsN and yhbW. We further explored the role of these previously poorly understood genes to determine the mechanism of suppression of the essentiality of σE. Overexpression of ptsN and yhbW lowers basal cell envelope stress responses and suppresses defects caused by disruptions in the flux of outer membrane proteins to the cell envelope. By determining the effects of inhibiting σE activity and isolating multicopy suppressors of lethality due to the lack of σE, we have expanded understanding of the essential function of σE in cell envelope maintenance.