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Published on: October 25, 2019
Identification by genetic suppression of Escherichia coli TolB residues important for TolB-Pal interaction
1Unité de Microbiologie et Génétique, UMR 5577, CNRS-INSA-Université Lyon I, F-69622 Villeurbanne, France.
Abstract:
The Tol-Pal system of Escherichia coli is involved in maintaining outer membrane stability. Mutations in tolQ, tolR, tolA, tolB, or pal genes result in sensitivity to bile salts and the leakage of periplasmic proteins. Moreover, some of the tol genes are necessary for the entry of group A colicins and the DNA of filamentous bacteriophages. TolQ, TolR, and TolA are located in the cytoplasmic membrane where they interact with each other via their transmembrane domains. TolB and Pal form a periplasmic complex near the outer membrane. We used suppressor genetics to identify the regions important for the interaction between TolB and Pal. Intragenic suppressor mutations were characterized in a domain of Pal that was shown to be involved in interactions with TolB and peptidoglycan. Extragenic suppressor mutations were located in tolB gene. The C-terminal region of TolB predicted to adopt a beta-propeller structure was shown to be responsible for the interaction of the protein with Pal. Unexpectedly, none of the suppressor mutations was able to restore a correct association between Pal and peptidoglycan, suggesting that interactions between Pal and other components such as TolB may also be important for outer membrane stability.
Insights
The Tol-Pal system maintains E. coli outer membrane stability. Suppressor genetics revealed the TolB C-terminal region interacts with Pal, crucial for membrane integrity.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Cell Structure
Background:
- The Tol-Pal system in Escherichia coli is essential for outer membrane stability.
- Defects in Tol-Pal components lead to bile salt sensitivity and periplasmic protein leakage.
- This system also mediates the entry of colicins and bacteriophage DNA.
Purpose of the Study:
- To identify specific regions involved in the interaction between TolB and Pal using suppressor genetics.
- To elucidate the molecular mechanisms underlying Tol-Pal mediated outer membrane stability.
Main Methods:
- Utilized suppressor genetics to isolate mutations affecting TolB-Pal interactions.
- Characterized intragenic suppressor mutations in Pal and extragenic suppressor mutations in tolB.
- Analyzed the structural and functional roles of specific protein domains in the interaction.
Main Results:
- Identified a domain in Pal involved in interactions with TolB and peptidoglycan.
- Localized extragenic suppressor mutations to the C-terminal region of TolB, which is predicted to form a beta-propeller structure.
- Demonstrated that the C-terminal region of TolB is responsible for Pal interaction.
- Observed that suppressor mutations did not restore Pal-peptidoglycan association, indicating complex interactions.
Conclusions:
- The C-terminal beta-propeller domain of TolB is critical for its interaction with Pal.
- Outer membrane stability relies on intricate interactions involving TolB, Pal, and potentially other components.
- Further investigation is needed to fully understand the Tol-Pal system's role in maintaining bacterial envelope integrity.
