Direct Inhibition of RetS Synthesis by RsmA Contributes to Homeostasis of the Gac/Rsm Signaling System.

The Gac/Rsm system is a global regulator of Pseudomonas aeruginosa gene expression. The primary effectors are RsmA and RsmF. Both are RNA-binding proteins that interact with target mRNAs to modulate protein synthesis. RsmA/RsmF recognize GGA sequences presented in the loop portion of stem-loop structures. For repressed targets, the GGA sites usually overlap the ribosome binding site (RBS) and RsmA/RsmF binding inhibits translation initiation. RsmA/RsmF activity is controlled by several small non-coding RNAs (sRNA) that sequester RsmA/RsmF from target mRNAs. The most important sequestering sRNAs are RsmY and RsmZ. Transcription of / is directly controlled by the GacSA two-component regulatory system. GacSA activity is antagonized by RetS, a hybrid sensor kinase. In the absence of , / transcription is derepressed and RsmA/RsmF are sequestered by RsmY/RsmZ. Gac/Rsm system homeostasis is tightly controlled by at least two mechanisms. First, direct binding of RsmA to the and mRNAs inhibits further synthesis of both proteins. Second, RsmA stimulates transcription through an undefined mechanism. In this study we demonstrate that RsmA stimulates transcription by directly inhibiting RetS synthesis. RetS protein levels are elevated 2.5-fold in an mutant. Epistasis experiments demonstrate that the requirement for transcription is entirely suppressed in an double mutant. RsmA directly interacts with the mRNA and requires two distinct GGA sites, one of which overlaps the RBS. We propose a model wherein RsmA inhibits RetS synthesis to promote / transcription and that this acts as a checkpoint to limit RsmA/RsmF availability. The Pseudomonas aeruginosa Gac/Rsm system controls ∼500 genes and governs a critical lifestyle switch by inversely regulating factors that favor acute or chronic colonization. Control of gene expression by the Gac/Rsm system is mediated through RsmA and RsmF, small RNA-binding proteins that interact with target mRNAs to inhibit or promote protein synthesis and/or mRNA stability. RsmA/RsmF activity is governed by two small non-coding RNAs (RsmY and RsmZ) that sequester RsmA/RsmF from target mRNAs. The GacSA two-component regulatory system plays a pivotal role in the Gac/Rsm system by controlling transcription. This study provides insight into the control of homeostasis by demonstrating that RsmA directly targets and inhibits expression of RetS, an orphan sensor kinase critical for transcription.