Negative regulation of the Rcs phosphorelay via IgaA contact with the RcsD phosphotransfer protein

Two-component systems and phosphorelays play central roles in the ability of bacteria to rapidly respond to changing environments. In E. coli and related enterobacteria, the complex Rcs phosphorelay is a critical player in changing bacterial behavior in response to antimicrobial peptides, beta-lactam antibiotics, and other challenges to the cell surface. The Rcs system is unusual in that IgaA, an inner membrane protein, is essential due to its negative regulation of the RcsC/RcsD/RcsB phosphorelay. While it has previously been shown that IgaA transduces signals from the outer membrane lipoprotein RcsF, how it interacts with the phosphorelay was unknown. Here we use in vivo interaction assays and genetic dissection of the critical proteins to demonstrate that IgaA interacts with the phosphorelay protein RcsD, and that this interaction is necessary for regulation. Interactions in periplasmic domains of these two proteins anchor repression of signaling. However, the signaling response depends on a weaker interaction between cytoplasmic loop 1 of IgaA and a truncated PAS domain in RcsD. A point mutation in the PAS domain increases interactions between the two proteins and is sufficient to abolish induction of this phosphorelay. RcsC, the histidine kinase that initiates phosphotransfer through the phosphorelay, appears to be indirectly regulated by IgaA via the contacts with RcsD. Unlike RcsD, and unlike many other histidine kinases, the periplasmic domain of RcsC is not necessary for the response to inducing signals. The multiple contacts between IgaA and RcsD form a poised sensing system, preventing over-activation of this apparently toxic phosphorelay but allowing it to be rapidly and quantitatively responsive to signals. Author Summary The Rcs phosphorelay 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, keeps signaling from this pathway under control when it is not needed, but how it controls the phosphorelay has been unclear. We define a set of critical interactions between IgaA and the phosphotransfer protein RcsD. A periplasmic contact between IgaA and RcsD provides a necessary inhibition of Rcs signaling, modulated further by regulated interactions in the cytoplasmic domains of each protein. This multipartite interaction provides a sensitive regulatory switch.