okamoto, a. | Search bioRxiv

bioRxiv2026

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

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.

microbiology↗

bioRxiv2019

Basicity of N5 in semiquinone enhances the rate of respiratory electron outflow in Shewanella oneidensis MR-1

Extracellular electron transport (EET) occurs in environmental iron-reducing bacteria and is mediated by an outer membrane multi-heme cytochrome complex (Cyts). It has critical implications for global mineral cycling and electrochemical microbial catalysis. The rate of EET mediated by multiple heme redox centers significantly increases in the presence of flavins and quinones. Their electron free energy does not entirely account for the fact that differential effects on EET rate enhancement vary significantly by factors [≥]100. Here, we report on whole-cell electrochemical analysis of Shewanella oneidensis MR-1 using six flavin analogs and four quinones. We demonstrated that protonation of the nitrogen atom at position 5 (N5) of the isoalloxazine ring is essential for electron outflow acceleration as a bound non-covalent cofactor of Cyts. EET mediated by Cyts was accelerated at a rate dependent on pKa(N5). The EET rate largely decreased in response to the addition of deuterated water (D2O), while low concentration of D2O (4 %) had little impact on electron free energy difference of the heme and non-covalent bound cofactors, strongly suggesting that the protonation of N5 limits the rate of EET. Our findings directly link EET kinetics to proton transport reaction via N5 and provide a basis for the development of novel strategies for controlling EET-associated biological reactions.\n\nSignificance statementThe potential of various small molecules such as flavins and quinones to enhance the rate of extracellular electron transport (EET) has been exploited to develop environmental energy conversion systems. Flavins and quinones have similar molecular structures but their abilities to enhance EET vary by >100x in Shewanella oneidensis MR-1. These large differences are inconsistent with conventional models, which rely on redox potentials or diffusion constant of shuttling electron mediators. In this study, we demonstrated that the basicity of the nitrogen atom of the isoalloxazine ring (N5) enhances the rate of electron outflow when a flavin or quinone is a non-covalent cofactor of S. oneidensis MR-1 outer membrane c-type cytochromes.

biochemistry↗