Evolutionary analysis in Enterobacterales of the Rcs-repressor protein IgaA unveils two cytoplasmic small β-barrel domains central for function

The Rcs sensor system, comprised by the proteins RcsB/RcsC/RcsD and RcsF, is used by bacteria of the order Enterobacterales to withstand envelope damage. Under non-stress conditions, the system is repressed by the membrane protein IgaA. How IgaA has evolved within Enterobacterales in concert with the Rcs system has not been explored. Here, we report phylogenetic data supporting co-evolution of IgaA with the inner membrane proteins RcsC and RcsD. Functional assays showed that IgaA from representative genera as Shigella and Dickeya, but not those from Yersinia or the endosymbionts Photorhabdus and Sodalis, repress the Rcs system when expressed in a heterogenous host like Salmonella enterica serovar Typhimurium. IgaA structural features have therefore diverged among Enterobacterales. Modelling of IgaA structure unveiled one periplasmic and two cytoplasmic {beta}-rich architectures forming partially-closed small {beta}-barrel (SBB) domains related to OB (oligonucleotide/oligosaccharide binding motif) fold domains. Interactions among conserved residues were mapped in a connector linking SBB-1 domain of cytoplasmic region cyt1 to SBB-2 domain of region cyt2 (residues E180-R265); the C-terminus of cyt1 facing cyt2 (R188-E194-D309 and T191-H326); and, between cyt2-cyt3 regions (H293-E328-R686). These interactions identify a previously unnoticed "hybrid" SBB-2 domain. We also identified interactions absent in the IgaA variants not functional in S. Typhimurium, including H192-P249, which links cyt1 to cyt2, R255-D313 and D287-R314. A short -helix (6) located in the SSB-1 domain is also missing in the non-complementing IgaA tested. Taken together, our data support a central role of the two cytoplasmic SBB domains in IgaA function and evolution. SIGNIFICANCEThe "intracellular growth attenuator A" protein (IgaA) was first reported as repressor of the Rcs system in S. enterica serovar Typhimurium. IgaA orthologs were later studied in other genera and families of the Enterobacterales order, mainly in Escherichia coli. Despite intense investigation about the mechanism by which IgaA controls the Rcs system, the extent at which IgaA evolved within families of the Enterobacterales order has not been investigated. Using a combination of functional assays and in silico structural analyses, our work provides a detail map of conserved and divergent residues in IgaA representing interactions occurring in all Enterobacterales and others that may have diverged concomitantly to interacting proteins, probably for responding to specific environments. Future studies involving mutagenesis of these residues in IgaA of Enterobacterales families and genera of interest will certainly provide valuable insights into the regulation acting in the IgaA-Rcs axis.