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Related Experiment Video

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High-throughput suppressor screen demonstrates that RcsF monitors outer membrane integrity and not Bam complex

Muralidhar Tata1, Santosh Kumar1, Sarah R Lach1,2

  • 1Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030.

Proceedings of the National Academy of Sciences of the United States of America
|August 5, 2021
PubMed
Summary

The regulator of capsule synthesis (Rcs) pathway senses outer membrane stress. RcsF protein, interacting with outer membrane proteins, is crucial for this sensing, not by directly monitoring the essential Bam complex function.

Keywords:
Rcs phosphorelayenvelope biogenesisenvelope stress responsesurface-exposed lipoproteins

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Cell Envelope Biogenesis

Background:

  • The regulator of capsule synthesis (Rcs) pathway monitors gram-negative bacterial cell envelope integrity.
  • Outer membrane (OM) lipoprotein RcsF is the sensory component, but its precise function is unclear.
  • RcsF interacts with the essential β-barrel assembly machinery (Bam) complex.

Purpose of the Study:

  • To investigate the role of RcsF/Bam complex interactions in Rcs signaling.
  • To determine if RcsF senses Bam complex function or outer membrane stress.
  • To elucidate the mechanism of Rcs pathway activation.

Main Methods:

  • Utilized a genetic tool based on BamE absence to select for mutations affecting RcsF/BamA interactions.
  • Employed a high-throughput suppressor screen to isolate rcsF and bamA mutants.
  • Characterized mutant impacts on RcsF/OMP assembly and Rcs signaling using inhibitors like MRL-494L and darobactin.

Main Results:

  • Provided evidence against RcsF sensing Bam complex function directly.
  • Demonstrated that Rcs activation in bam mutants arises from secondary OM and lipopolysaccharide defects.
  • Showed RcsF/OMP assembly is necessary for Rcs activation, indicating a role in sensing OM stress.

Conclusions:

  • RcsF does not directly sense Bam complex function.
  • RcsF/OMP complexes play an active role in sensing outer membrane stress.
  • Rcs pathway activation is linked to OM integrity and lipopolysaccharide defects.