Proteomic profiling of the Pseudomonas aeruginosa stringent response highlights global adaptive responses during stationary phase
6 affiliations
- 1Department of Microbiology and Immunology, McGill University, Montreal, Canada
- 2Department of Microbiology and Immunology, Faculty of Biomedical Sciences, University of Otago, Dunedin, New Zealand
- 3Division of Geriatric Medicine, Department of Medicine, McGill University, Montreal, QC, Canada
- 4Department of Medicine, McGill University, Montreal, Canada
- 5Meakins-Christie Laboratories, Research Institute of the McGill University Health Centre, Montreal, Canada
- 6Proteomics and Molecular Analysis Platform, Research Institute of the McGill University Health Centre, Montreal, Canada
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.
Related
- Genome-wide fitness profiling reveals flagellar rotation as an energetic liability during anaerobic maintenance in Pseudomonas aeruginosa
- Comprehensive interaction profiling and machine learning prediction of bacteriophage infectivity across clinically diverse Pseudomonas aeruginosa
- Host immune stress reveals a role for potassium homeostasis in Pseudomonas aeruginosa aggregate stability
- LytM family endopeptidase LmdC is controlled by regulated auto processing and proteolytic destruction in Pseudomonas aeruginosa
- Mining the Pseudomonas aeruginosa genome for regulators of type III secretion system gene expression using FT-Tn-seq
BibTeX
@article{rich2026proteomic,
title = {{Proteomic profiling of the Pseudomonas aeruginosa stringent response highlights global adaptive responses during stationary phase}},
author = {Jacquelyn Rich and Samuel J. T. Wardell and Dorival Martins and Malika Khakhimova and Gowthami Sampathkumar and Tevy Chan and Thi Tuyet Mai Nguyen and Geoffrey McKay and Lorne Taylor and Daniel Pletzer and Dao Nguyen},
journal = {mSystems},
year = {2026},
month = sep,
doi = {10.1128/msystems.01742-25},
url = {https://doi.org/10.1128/msystems.01742-25},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Proteomic profiling of the Pseudomonas aeruginosa stringent response highlights global adaptive responses during stationary phase
AU - Jacquelyn Rich
AU - Samuel J. T. Wardell
AU - Dorival Martins
AU - Malika Khakhimova
AU - Gowthami Sampathkumar
AU - Tevy Chan
AU - Thi Tuyet Mai Nguyen
AU - Geoffrey McKay
AU - Lorne Taylor
AU - Daniel Pletzer
AU - Dao Nguyen
PY - 2026
DA - 2026/09/17
JO - mSystems
DO - 10.1128/msystems.01742-25
UR - https://doi.org/10.1128/msystems.01742-25
AB - 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.
SN - 2379-5077
ER -