IgaA negatively regulates the Rcs Phosphorelay via contact with the RcsD Phosphotransfer Protein

Author summary The Rcs phosphorelay system plays a central role in allowing enterobacteria to sense and respond to antibiotics, host-produced antimicrobials, and interactions with surfaces. A unique negative regulator, IgaA, attenuates signaling from this pathway when it is not needed, but how IgaA controls the phosphorelay has been unclear. We define a set of critical interactions between IgaA and the phosphotransfer protein RcsD, including a periplasmic contact between IgaA and RcsD that mediates a necessary inhibition of Rcs signaling. Inhibition is further modulated by regulated interactions between the cytoplasmic domains of each protein, providing a sensitive regulatory switch. Two-component systems and phosphorelays play central roles in the ability of bacteria to rapidly respond to changing environments. InE.coliand related enterobacteria, the complex Rcs phosphorelay is a critical player in the bacterial response to antimicrobial peptides, beta-lactam antibiotics, and other disruptions at the cell surface. The Rcs system is unusual in that an inner membrane protein, IgaA, is essential due to its negative regulation of the RcsC/RcsD/RcsB phosphorelay. While it is known that IgaA transduces signals from the outer membrane lipoprotein RcsF, how it interacts with the phosphorelay has remained unknown. Here we performed in vivo interaction assays and genetic dissection of the critical proteins and found that IgaA interacts with the phosphorelay protein RcsD, and that this interaction is necessary for regulation. Interactions between IgaA and RcsD within their respective periplasmic domains of these two proteins anchor repression of signaling. However, the signaling response depends on a second interaction between cytoplasmic loop 1 of IgaA and a truncated Per-Arndt-Sim (PAS-like) domain in RcsD. A single point mutation in the PAS-like domain increased interactions between the two proteins and blocked induction of the phosphorelay. IgaA may regulate RcsC, the histidine kinase that initiates phosphotransfer through the phosphorelay, indirectly, via its contacts with RcsD. Unlike RcsD, and unlike many other histidine kinases, the periplasmic domain of RcsC is dispensable for the response to signals that induce the Rcs phosphorelay system. The multiple contacts between IgaA and RcsD constitute a poised sensing system, preventing potentially toxic over-activation of this phosphorelay while enabling it to rapidly and quantitatively respond to signals.