Abstract
Escherichia coli RseP, a member of the site-2 protease family of intramembrane proteases, is involved in the activation of the σE extracytoplasmic stress response and elimination of signal peptides from the cytoplasmic membrane. However, whether RseP has additional cellular functions is unclear. In this study, we used mass spectrometry–based quantitative proteomic analysis to search for new substrates that might reveal unknown physiological roles for RseP. Our data showed that the levels of several Fec system proteins encoded by the fecABCDE operon (fec operon) were significantly decreased in an RseP-deficient strain. The Fec system is responsible for the uptake of ferric citrate, and the transcription of the fec operon is controlled by FecI, an alternative sigma factor, and its regulator FecR, a single-pass transmembrane protein. Assays with a fec operon expression reporter demonstrated that the proteolytic activity of RseP is essential for the ferric citrate–dependent upregulation of the fec operon. Analysis using the FecR protein and FecR-derived model proteins showed that FecR undergoes sequential processing at the membrane and that RseP participates in the last step of this sequential processing to generate the N-terminal cytoplasmic fragment of FecR that participates in the transcription of the fec operon with FecI. A shortened FecR construct was not dependent on RseP for activation, confirming this cleavage step is the essential and sufficient role of RseP. Our study unveiled that E. coli RseP performs the intramembrane proteolysis of FecR, a novel physiological role that is essential for regulating iron uptake by the ferric citrate transport system.
Highlights
While bacterial cellular membranes act as a barrier to protect a cell from extrinsic damages caused by various xenobiotics and hazardous changes in environmental conditions, they must mediate selective import of nutrients and other small molecules and transduction of signals from the external milieu to adapt to environmental changes
We performed proteomic analysis to identify the substrates of RseP, the site-2 protease (S2P) family intramembrane protease (IMP) of E. coli, to explore novel functions of this protease and unveiled that RseP is involved in the regulation of the Fec system (Ferric citrate uptake system) genes through the intramembrane cleavage of a novel physiological substrate, FecR
Our proteomic analysis of the membrane proteins identified multiple membrane components of the Fec system (FecA, FecD, and FecE) encoded by the fec operon as proteins whose levels were significantly decreased in the RseP-deficient strain, suggesting that RseP is required for the expression of the fec operon
Summary
While bacterial cellular membranes act as a barrier to protect a cell from extrinsic damages caused by various xenobiotics and hazardous changes in environmental conditions, they must mediate selective import of nutrients and other small molecules and transduction of signals from the external milieu to adapt to environmental changes. We introduced the reporter plasmid into the ΔompA ΔompC ΔrseP cells (rseP can be deleted in a strain lacking the two outer membrane proteins OmpA and OmpC [61]) and examined the effect of rseP disruption on the transcriptional activation of the fec operon in response to ferric citrate.
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