Author ORCID Identifier
0000-0003-1407-1451
Date of Graduation
3-2023
Document Type
Dissertation (PhD)
Program Affiliation
Microbiology and Molecular Genetics
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Anna Konovalova, PhD
Committee Member
Mikhail Bogdanov, PhD
Committee Member
Anne-Marie Krachler, PhD
Committee Member
Michael Lorenz, PhD
Committee Member
William Margolin, PhD
Committee Member
Barbara E. Murray, PhD
Abstract
Timely detection and repair of envelope damage are paramount for bacterial survival. The Regulator of Capsule Synthesis (Rcs) stress response is a complex signaling cascade that monitors gram-negative cell envelope integrity and can transduce the stress signals across the multilayered envelope to regulate gene expression in the cytoplasm. The outer membrane (OM) lipoprotein RcsF is the sensory component, but how RcsF functions remains elusive. RcsF interacts with the β-barrel assembly machinery (Bam) complex, which assembles RcsF in complex with OM proteins (OMPs), resulting in RcsF’s partial cell surface exposure. RcsF can also interact with the periplasmic domain of the negative regulator IgaA, derepressing the downstream RcsCDB phosphorelay. Elucidating whether RcsF/Bam or RcsF/OMP interactions are important for its sensing function is challenging because the Bam complex is essential, and partial loss-of-function mutations broadly compromise the OM biogenesis. Using recently isolated rcsF and bamA mutants that impact RcsF/BamA and RcsF/OMP interaction, I was able to test several proposed models for Rcs signaling. The analysis of these mutants showed that Rcs activation in bam mutants results from secondary OM and lipopolysaccharide defects and that RcsF/OMP assembly is required for this activation, supporting an active role of RcsF/OMP complexes in sensing OM stress. I next focused on how the RcsF/IgaA interaction is regulated at the molecular level to activate the signaling in response to stress. Using a site-saturated mutant library of rcsF, we carried out several independent genetic screens to interrogate the mechanism of signal transduction from RcsF to IgaA. We analyzed several distinct classes of rcsF signaling mutants and determined the region of RcsF that is critically important for signal transduction. This region is bifunctional as it is important for RcsF interaction with both IgaA and OMPs. The mutant analysis provides strong evidence for conformational changes in the RcsF/OMP complex mediating signal transduction to IgaA, and the first direct evidence that OMPs play an important regulatory role in Rcs signaling.
Keywords
Rcs phosphorelay, envelope biogenesis, envelope stress response