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bioRxiv · 10.64898/2026.09.04.749331

Genome-wide fitness profiling reveals flagellar rotation as an energetic liability during anaerobic maintenance in Pseudomonas aeruginosa

Demirer, K.·Melnyk, R. A.·Carlson, H. K.·okamoto, a.·Deutschbauer, A. M.·Newman, D. K.
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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.

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Demirer, K., Melnyk, R. A., Carlson, H. K., okamoto, a., Deutschbauer, A. M., Newman, D. K.. 2026-09-07. Genome-wide fitness profiling reveals flagellar rotation as an energetic liability during anaerobic maintenance in Pseudomonas aeruginosa. https://doi.org/10.64898/2026.09.04.749331

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