Genome-wide fitness profiling reveals flagellar rotation as an energetic liability during anaerobic maintenance in Pseudomonas aeruginosa
- 1California Institute of Technology
Abstract
Bacteria in nature and disease frequently spend time in non-growing states, yet the genes that allow cells to survive without growing remain poorly understood. Using Pseudomonas aeruginosa strain PA14 as a model system to study non-growth powered by anaerobic phenazine cycling, we employed randomly barcoded transposon-insertion sequencing (RB-TnSeq) to identify the genes required for this maintenance state using a platform that sustains anaerobic survival via continuous phenazine reoxidation. We found 167 genes whose disruption altered survival, including genes involved in transcription, translation, protein quality control, and cell envelope maintenance. Comparing our results to published TnSeq data from other forms of energy-limited growth arrest (conditions where electron acceptor or carbon availability constrains energy conservation below that required for cell growth/division) in PA14 revealed that while a subset of genes are fitness determinants across distinct growth-arrested states, most are condition-specific. Notably, genes involved in flagellar regulation and assembly were broadly detrimental to survival under this maintenance condition. Leveraging a high-throughput electrochemical system that allows for quantitative and mechanistic dissection of the phenazine cycling-dependent maintenance state, we found that flagellar abundance influences cells survival, metabolic rate, and ATP levels. Moreover, removing the flagellar stator proteins MotAC, which are required for flagellar rotation but not assembly, reversed these defects, indicating that the energetic cost of flagella comes from their rotation rather than construction under these conditions. These results show that in a low-powered maintenance state, limiting energy-dissipating processes, such as proton-motive force loss through flagellar rotation, supports cell survival.
Related
- Comprehensive interaction profiling and machine learning prediction of bacteriophage infectivity across clinically diverse Pseudomonas aeruginosa
- Proteomic profiling of the Pseudomonas aeruginosa stringent response highlights global adaptive responses during stationary phase
- 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
@misc{demirer2026genomewide,
title = {{Genome-wide fitness profiling reveals flagellar rotation as an energetic liability during anaerobic maintenance in Pseudomonas aeruginosa}},
author = {K. Demirer and R. A. Melnyk and H. K. Carlson and a. okamoto and A. M. Deutschbauer and D. K. Newman},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.04.749331},
url = {https://www.biorxiv.org/content/10.64898/2026.09.04.749331v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Genome-wide fitness profiling reveals flagellar rotation as an energetic liability during anaerobic maintenance in Pseudomonas aeruginosa
AU - K. Demirer
AU - R. A. Melnyk
AU - H. K. Carlson
AU - a. okamoto
AU - A. M. Deutschbauer
AU - D. K. Newman
PY - 2026
DA - 2026/09/04
PB - bioRxiv
DO - 10.64898/2026.09.04.749331
UR - https://www.biorxiv.org/content/10.64898/2026.09.04.749331v1
AB - Bacteria in nature and disease frequently spend time in non-growing states, yet the genes that allow cells to survive without growing remain poorly understood. Using Pseudomonas aeruginosa strain PA14 as a model system to study non-growth powered by anaerobic phenazine cycling, we employed randomly barcoded transposon-insertion sequencing (RB-TnSeq) to identify the genes required for this maintenance state using a platform that sustains anaerobic survival via continuous phenazine reoxidation. We found 167 genes whose disruption altered survival, including genes involved in transcription, translation, protein quality control, and cell envelope maintenance. Comparing our results to published TnSeq data from other forms of energy-limited growth arrest (conditions where electron acceptor or carbon availability constrains energy conservation below that required for cell growth/division) in PA14 revealed that while a subset of genes are fitness determinants across distinct growth-arrested states, most are condition-specific. Notably, genes involved in flagellar regulation and assembly were broadly detrimental to survival under this maintenance condition. Leveraging a high-throughput electrochemical system that allows for quantitative and mechanistic dissection of the phenazine cycling-dependent maintenance state, we found that flagellar abundance influences cells survival, metabolic rate, and ATP levels. Moreover, removing the flagellar stator proteins MotAC, which are required for flagellar rotation but not assembly, reversed these defects, indicating that the energetic cost of flagella comes from their rotation rather than construction under these conditions. These results show that in a low-powered maintenance state, limiting energy-dissipating processes, such as proton-motive force loss through flagellar rotation, supports cell survival.
ER -