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

Abstract

The regulator of capsule synthesis (Rcs) is a complex signaling cascade that monitors gram-negative cell envelope integrity. 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. 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. Our recent discovery that, in the absence of nonessential component BamE, RcsF inhibits function of the central component BamA provided a genetic tool to select mutations that specifically prevent RcsF/BamA interactions. We employed a high-throughput suppressor screen to isolate a collection of such rcsF and bamA mutants and characterized their impact on RcsF/OMP assembly and Rcs signaling. Using these mutants and BamA inhibitors MRL-494L and darobactin, we provide multiple lines of evidence against the model in which RcsF senses Bam complex function. We show 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.

The Wsp system ofPseudomonas aeruginosalinks surface sensing and cell envelope stress

“…E. coli is arguably the best-studied species for cell envelope stress-response systems. Three of the key systems include the CpxAR two-component system ( 32 – 36 ), the Rcs signal transduction system ( 37 – 40 ), and the extracytoplasmic function σ factor, σ E ( 37 , 41 – 44 ). Each system regulates its own set of genes, although there is extensive overlap between the regulons.…”

Bacterial cell widening alters periplasmic size and activates envelope stress responses

“…The Rcs system is activated when BamA fails to bind RcsF and funnel it to OMPs, whereupon newly synthesized RcsF remains facing the periplasm and directly interacts with IgaA to activate the cascade [6, 10]. RcsF can also directly sense lipopolysaccharide (LPS) defects on the OM surface via a set of charged residues [6, 11–13]; activation presumably occurs via RcsF release and/or immobilization facing the periplasm. Interestingly, RcsF signaling is sensitive to periplasmic thickness (i.e.…”

The short chain fatty acid propionic acid activates the Rcs stress response system partially through inhibition of D-alanine racemase

“…The Rcs pathway relies on complex phosphorelay signal transduction through the interplay of multiple inner and outer membrane components including RcsC, RcsD, RcsF, IgaA, RcsB (a response regulator), and RcsB-binding accessory proteins including RcsA (1)(2)(3). RcsF interacts with outer membrane protein OmpA to monitor outer membrane integrity (4).…”

“…Together these components are responsible for transcriptional regulation of genes induced by cell wall and outer membrane stresses, the scope of which is not fully understood. Known Rcs-system activators include mutations, enzymes, peptides, and drugs that affect the outer membrane directly or through inhibition of enzymes necessary for outer membrane biogenesis (4)(5)(6)(7)(8). In addition, mutations, enzymes, or drugs that target peptidoglycan synthesis can activate the Rcs system (7,(9)(10)(11)(12) as well as osmotic and oxidative stress (13,14).…”

“…The Rcs pathway relies on complex phosphorelay signal transduction through the interplay of multiple inner and outer membrane components including RcsC, RcsD, RcsF, IgaA, RcsB (a response regulator), and RcsB-binding accessory proteins including RcsA (1–3). RcsF interacts with outer membrane protein OmpA to monitor outer membrane integrity (4). Together these components are responsible for transcriptional regulation of genes induced by cell wall and outer membrane stresses, the scope of which is not fully understood.…”