Conformational rearrangements in the sensory RcsF/OMP complex mediate signal transduction across the bacterial cell envelope

Abstract

Timely detection and repair of envelope damage are paramount for bacterial survival. The Regulator of Capsule Synthesis (Rcs) stress response can transduce the stress signals across the multilayered gram-negative cell envelope to regulate gene expression in the cytoplasm. Previous studies defined the overall pathway, which begins with the sensory lipoprotein RcsF interacting with several outer membrane proteins (OMPs). RcsF can also interact with the periplasmic domain of the negative regulator IgaA, derepressing the downstream RcsCDB phosphorelay. However, how the RcsF/IgaA interaction is regulated at the molecular level to activate the signaling in response to stress remains poorly understood. In this study, we used a site-saturated mutant library of rcsF to carry 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.

Evolutionary analysis and structure modelling of the Rcs-repressor IgaA unveil a functional role of two cytoplasmic small β-barrel (SBB) domains

“…The most accepted model of communication between IgaA and the Rcs system is a control at the periplasmic side exerted by an IgaA-RcsF interaction [ 10 , 12 ], which is relieved upon stress, an alteration that is supposed to be translated to cytoplasmic regions of IgaA. This model, however, contrasts at some extent with our functional studies with IgaA from different Enterobacterales families and the structural in silico analyses presented here.…”

“…Various studies have shown that IgaA interacts directly with outer membrane lipoprotein RcsF [ 10 , 12 ] and the inner membrane protein RcsD [ 6 , 7 ]. These interactions represent the activated and resting states of the Rcs phosphorelay, respectively.…”

“…An interaction between the outer membrane protein RcsF and the periplasmic region of IgaA was proposed to relieve its repression over RcsC/RcsD [ 10 ]. More recent studies have reported interactions between outer membrane proteins and RcsF, modulating the RcsF-IgaA interaction [ 11 , 12 ], and, between RcsD and IgaA [ 6 , 7 ], this latter involving periplasmic and cytoplasmic domains [ 7 ].…”

BamE directly interacts with BamA and BamD coordinating their functions

“…Because the Bam complex stability does not always correlate with function (McCabe et al, 2017), we used RcsF/OMP assembly as a direct functional readout. In the absence of BamE cells fail to assemble RcsF complex with partner OMPs (Lach et al, 2023), but we use OmpA as a convenient readout in the formaldehyde crosslinking experiments (Tata et al, 2021; Tata & Konovalova, 2019). RcsF/OmpA complex is significantly reduced in the absence of BamE, and instead, RcsF accumulated on BamA (Figure 3c).…”

“…Our previous research provided the first direct evidence of the specialized functions of Bam lipoproteins by demonstrating that BamE is uniquely required for the assembly of the OM complex between the lipoprotein RcsF and its OMP partners (Konovalova et al, 2016; Tata & Konovalova, 2019). The RcsF/OMP complexes function in the Regulator of Capsule Synthesis (Rcs) stress response, allowing RcsF to monitor the integrity of the OM (Lach et al, 2023; Tata et al, 2021). Identification of the RcsF/OMP complexes as the first substrate of the Bam complex that requires BamE activity presented an opportunity to study a molecular function of BamE.…”