Proteomic profiling of the Pseudomonas aeruginosa stringent response highlights global adaptive responses during stationary phase

ABSTRACT Pseudomonas aeruginosa ( P.a ) is a clinically important opportunistic pathogen capable of adapting and surviving a wide range of environments and stress conditions. A central regulator of this adaptability is the stringent response, mediated by the alarmones (p)ppGpp. Activation of the stringent response triggers a global physiological shift away from active metabolism toward stress management and cellular quiescence, a key transition to stationary phase. To date, few studies have investigated the protein-level processes under stringent response control. In this study, we performed shotgun proteomic profiling (LC-MS/MS) of a (p)ppGpp-null relA spoT double knockout mutant (ΔSR) and its isogenic PAO1 parent during late exponential and stationary phase planktonic growth in rich medium. In this first characterization of the stringent response proteome in P.a , we identified 146 differentially abundant proteins (DAP) in late exponential phase ΔSR cells, and 2.3-fold greater DAP (339) in stationary-phase cells. Functional characterization and system-level analysis of the differentially abundant stationary phase proteome highlighted the key role of (p)ppGpp in shutting down a wide range of metabolic processes while inducing adaptive processes such as responses to oxidative stress. Comparative RNA-seq analysis also uncovered incongruent protein and mRNA responses in the ΔSR relative to wild-type cells in several processes, including iron homeostasis and antioxidant defenses. Transcriptomic analysis of the ΔSR also revealed differential expression of numerous sRNAs, including PrrF1/2, raising the possibility that sRNAs may mediate certain stringent-response-dependent post-transcriptional control. Phenotypic validation of the ΔSR proteome confirmed multiple defects in antioxidant defenses and dysregulated pyoverdine biosynthesis. IMPORTANCE Pseudomonas aeruginosa is an important opportunistic human pathogen that causes difficult-to-treat infections and is highly adaptable to stressful environments. A key orchestrator of this adaptability is the stringent response, mediated by the signaling molecule (p)ppGpp. In this study, we characterized the extensive proteome remodeling in the (p)ppGpp-null relA spoT mutant during late exponential and stationary phase and provided the first protein-level analysis of the stringent response in P. aeruginosa . These data highlight the role of (p)ppGpp in shutting down broad metabolic functions and activating adaptive pathways, including stress-resistance responses, particularly during stationary phase. Using comparative proteomics and RNA-seq, we also find that several processes—such as iron regulation and oxidative stress responses—show mismatched RNA and protein changes, suggesting additional layers of post-transcriptional control, potentially involving small regulatory RNAs.