Regulation of the Extracytoplasmic Stress Factor Sigma E by the Alarmone ppGpp in Escherichia coli
Open Access
- Author:
- Costanzo, Alessandra
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 23, 2006
- Committee Members:
- Sarah Ellen Ades, Committee Chair/Co-Chair
Stephen John Knabel, Committee Member
Katsuhiko Murakami, Committee Member
B Tracy Nixon, Committee Member
Allen T Phillips, Committee Member - Keywords:
- sigma factor competition
Extracytoplasmic stress response
ppGpp - Abstract:
- In bacteria, the ability to sense and respond to environmental stimuli is crucial for adaptation and survival under different conditions. Induction of alternative sigma factors represents an important bacterial strategy to cope with a variety of stresses that compromise cellular homeostasis. Sigma factors are transcription initiation factors and are the subunit of RNA polymerase (RNAP) responsible for binding to promoters allowing RNAP to specifically initiate transcription. In Escherichia coli (E. coli), the alternative sigma factor sigma E is induced upon accumulation of misfolded outer membrane proteins (OMPs), and is responsible for the activation of a stress pathway known as the extracytoplasmic stress response. In the absence of cell envelope stress, sigma E is mostly bound to its main negative regulator, the inner membrane protein RseA. When cell envelope stress is sensed, sigma E is released from RseA and can associate with RNAP. In addition, sigma E is essential for E. coli viability, although its indispensable function is not yet understood. The se regulon includes not only genes that are involved in the stress response, encoding for folding factors and proteases to re-establish a correct folding status of the proteins and to degrade proteins that are irreversibly misfolded, but also a diverse group of genes encoding proteins associated with the cell envelope or required to synthesize components of the cell envelope. These observations suggest that sigma E may be involved in maintaining many aspects of cell envelope integrity, beyond monitoring outer membrane protein folding. In the present study, I reveal a novel function of sigma E in the cell during nutritional stress. I showed that sigma E activity increases upon entry into stationary phase and that the effector molecule guanosine 3'-5'-bispyrophosphate, ppGpp, which binds directly to RNAP, is responsible for such regulation. This signaling pathway is distinct from the one activated by the extracytoplasmic stress response controlled via RseA. I also showed that sigma E can respond to alterations in the levels of ppGpp caused by a variety of methods, starvation for carbon or phosphate, and gratuitious induction of ppGpp, indicating sigma E is likely to have a role in the response to nutritional stress. Once I established the central role of ppGpp in the regulation of sigma E activity, I extended my analysis to determine how ppGpp is modulating such response. ppGpp does not act as a positive regulator of the sigma E synthesis indicating that ppGpp affects the activity of sigma E. ppGpp is best characterized for its role in response to starvation for amino acids. When cells are starved for amino acids, transcription of ribosomal RNA genes decreases and amino acid biosynthetic genes increases. This response reduces ribosome synthesis and boosts amino acid synthesis, allowing cells to adapt and survive when starved for amino acids. ppGpp is required for this response. Mutations that allow cells to bypass the need for ppGpp under starvation conditions have been isolated by other labs in selections based on the observation that a strain lacking ppGpp is unable to grow on a medium lacking amino acids. These suppressor mutations are localized in the rpoB, rpoC and rpoD genes encoding the beta, beta', and sigma 70 subunits of RNAP respectively, suggesting that ppGpp may act directly or indirectly by altering the activity of RNAP, and thus redirecting gene expression. Additional in vitro experiments showed that ppGpp can directly interact with sigma 70-RNAP and, with the help of an accessory protein DksA, directly decrease transcription of rRNA genes and increase transcription of amino acid biosynthetic genes. To determine how ppGpp might regulate the activity of sigma E, I tested whether these mutations could also suppress the defects in the regulation of sigma E in strains lacking ppGpp. The rpoD mutants used in this study are localized in conserved regions of the sigma 70 subunit, which are known to interact with RNAP, and which confer a lower binding affinity to the sigma 70 subunit for core RNAP. The rpoC mutation does not appear to effect sigma/core interactions but has been shown to decrease the stability of open complex. I demonstrated that even though these mutants were isolated based on their effects on genes transcribed by sigma 70, they restore the growth phase-dependent regulation of sigma E activity. In the case of the sigma 70 variant, sigma E activity is not only restored but is nearly constitutive, indicating that decreasing the affinity of sigma 70 for core RNAP allows sigma E to better compete for core RNAP compensating for the absence of ppGpp. In the case of the beta' variant, there are two possible explanations. The mutations may increase the availability of RNAP in the cell by releasing RNAP from the ribosomal RNA genes through the substantial destabilization of open complexes, or these mutations may directly alter transcription initiation by polymerase containing sigma E. These variants restore sigma E activity by either mimicking the action of ppGpp or bypassing the need for ppGpp. ppGpp is known to activate other alternative sigma factors either during entry into stationary phase or during starvation for specific nutrients. The results obtained from my analysis on how ppGpp regulates sigma E not only shed new insights on the role and regulation of sigma E in the cell, but also lead to the idea that ppGpp is a global regulator of alternative sigma factor activity. ppGpp provides a means to coordinately activate multiple stress responses without having to rely on specific signals to independently activate each stress response mediated by a different alternative sigma factor.