13Two-component systems and phosphorelays play central roles in the ability of bacteria 14 to rapidly respond to changing environments. In E. coli and related enterobacteria, the 15 complex Rcs phosphorelay is a critical player in changing bacterial behavior in response to 16 antimicrobial peptides, beta-lactam antibiotics, and other challenges to the cell surface. The
17Rcs system is unusual in that IgaA, an inner membrane protein, is essential due to its negative 18 regulation of the RcsC/RcsD/RcsB phosphorelay. While it has previously been shown that IgaA 19 transduces signals from the outer membrane lipoprotein RcsF, how it interacts with the 20 phosphorelay was unknown. Here we use in vivo interaction assays and genetic dissection of 21 the critical proteins to demonstrate that IgaA interacts with the phosphorelay protein RcsD, and 22 Author Summary
35The Rcs phosphorelay plays a central role in allowing enterobacteria to sense and respond to 36 antibiotics, host-produced antimicrobials, and interactions with surfaces. A unique negative regulator, 37IgaA, keeps signaling from this pathway under control when it is not needed, but how it controls the 38 phosphorelay has been unclear. We define a set of critical interactions between IgaA and the 39 phosphotransfer protein RcsD. A periplasmic contact between IgaA and RcsD provides a necessary 40 inhibition of Rcs signaling, modulated further by regulated interactions in the cytoplasmic domains of 41 each protein. This multipartite interaction provides a sensitive regulatory switch. 42IgaA negatively regulates the Rcs Phosphorelay via contact with RcsD 4 43 101 cause the loss of ability to de-phosphorylate RcsB that has acquired phosphate from other 102 sources [14][15][16]. In our assay conditions, the rcsC deletion strain produces a signal that is 3-4 103 fold above WT. 104 The rcsD deletion shown, rcsD541, increased PrprA-mCherry expression in a manner 105 similar to the rcsC deletion. rcsD is encoded upstream of rcsB, with the major promoters for 106 rcsB inside the rcsD coding region [17]. This affects the way rcsD deletion alleles can be 107 constructed. In addition, in both Salmonella and E. coli, some transcripts from the rcsD 108 IgaA negatively regulates the Rcs Phosphorelay via contact with RcsD 7 promoter may continue through to rcsB, though apparently at a much lower level [17, 18]. 109 Four different rcsD alleles were examined, each designed to leave the rcsB promoters intact 110 (S1D Fig). These include rcsD carrying an H842A mutation in the phosphotransfer domain 111 active site, as well as rcsD containing stop codons after codon 841 (rcsD841*). The overall 112 amount of PrprA-mCherry expression seems to differ modestly between rcsD541 and rcsD543, 113 even though the deleted regions in both alleles share the same boundaries. rcsD541 also gave 114 higher PrprA-mCherry signal than the point mutants (S1D Fig). When checked by western blot 115 with a polyclonal RcsD antibody, it is evident that rcsD541, rcsD543 and rcsD841* are all dev...