RcsF-independent mechanisms of signaling within the Rcs Phosphorelay

Abstract The Rcs (regulator of capsule synthesis) phosphorelay is a conserved cell envelope stress response mechanism in enterobacteria. It responds to perturbations at the cell surface and the peptidoglycan layer from a variety of sources, including antimicrobial peptides, beta-lactams, and changes in osmolarity. RcsF, an outer membrane lipoprotein, is the sensor for this pathway and activates the phosphorelay by interacting with an inner membrane protein IgaA. IgaA is essential; it negatively regulates the signaling by interacting with the phosphotransferase RcsD. We previously showed that RcsF-dependent signaling does not require the periplasmic domain of the histidine kinase RcsC and identified a dominant negative mutant of RcsD that can block signaling via increased interactions with IgaA. However, how the inducting signals are sensed and how signal is transduced to activate the transcription of the Rcs regulon remains unclear. In this study, we investigated how the Rcs cascade functions without its only known sensor, RcsF and characterized the underlying regulatory mechanisms for three distinct RcsF-independent inducers. Previous reports showed that Rcs signaling can be induced in the absence of RcsF by a loss of function mutation in the periplasmic oxidoreductase DsbA or by overexpression of the DnaK cochaperone DjlA. We identified an inner membrane protein, DrpB, as a multicopy RcsF-independent Rcs activator in E. coli . The loss of the periplasmic oxidoreductase DsbA and the overexpression of the DnaK cochaperone DjlA each trigger the Rcs cascade in the absence of RcsF by weakening IgaA-RcsD interactions in different ways. In contrast, the cell-division associated protein DrpB uniquely requires the RcsC periplasmic domain for signaling; this domain is not needed for RcsF-dependent signaling. This suggests the possibility that RcsC acts as a sensor for some Rcs signals. Overall, the results add new understanding to how this complex phosphorelay can be activated by diverse mechanisms. Author summary The Rcs phosphorelay signaling cascade regulates the expression of genes related to capsule synthesis, biofilm formation, virulence, and cell division in Enterobacteria and is critical for cell membrane integrity and response to beta-lactam antibiotics and antimicrobial peptides. RcsF is the sole known sensor, but other proteins have been reported to activate this pathway in the absence of RcsF. We have discovered a novel RcsF-independent Rcs activator and found that each of three RcsF-independent proteins activate the system differently. Most significantly, we find that the histidine kinase RcsC can be involved in signal sensing independently of RcsF. Our study sheds light into the complex mechanisms of Rcs activation and adds to our knowledge of non-orthodox signaling systems across organisms.