Direct Inhibition of RetS Synthesis by RsmA Contributes to Homeostasis of the <i>Pseudomonas aeruginosa</i> Gac/Rsm Signaling System

Abstract

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 <i>rsmY</i>/<i>rsmZ</i> is directly controlled by the GacSA two-component regulatory system. GacSA activity is antagonized by RetS, a hybrid sensor kinase. In the absence of <i>retS</i>, <i>rsmY</i>/<i>rsmZ</i> 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 <i>rsmA</i> and <i>rsmF</i> mRNAs inhibits further synthesis of both proteins. Second, RsmA stimulates <i>rsmY/rsmZ</i> transcription through an undefined mechanism. In this study we demonstrate that RsmA stimulates <i>rsmY/rsmZ</i> transcription by directly inhibiting RetS synthesis. RetS protein levels are elevated 2.5-fold in an <i>rsmA</i> mutant. Epistasis experiments demonstrate that the <i>rsmA</i> requirement for <i>rsmY/rsmZ</i> transcription is entirely suppressed in an <i>rsmA, retS</i> double mutant. RsmA directly interacts with the <i>retS</i> mRNA and requires two distinct GGA sites, one of which overlaps the RBS. We propose a model wherein RsmA inhibits RetS synthesis to promote <i>rsmY</i>/<i>rsmZ</i> transcription and that this acts as a checkpoint to limit RsmA/RsmF availability. <b>IMPORTANCE</b> 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 <i>rsmYZ</i> 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 <i>rsmYZ</i> transcription.