Genetic Analysis of the ts-Lethal Mutant Δpa0665 / pTS-pa0665 Reveals Its Role in Cell Morphology and Oxidative Phosphorylation in Pseudomonas aeruginosa .

Pa0665 in Pseudomonas aeruginosa shares homologous sequences with that of the essential A-type iron-sulfur (Fe-S) cluster insertion protein ErpA in Escherichia coli . However, its essentiality in P. aeruginosa and its complementation with E. coli erpA has not been experimentally examined. To fulfill this task, we constructed plasmid-based ts -mutant Δpa0665 / pTS-pa0665 using a three-step protocol. The mutant displayed growth defects at 42 °C, which were complemented by expressing ec.erpA . Microscopic observations indicated a petite cell phenotype for Δpa0665 / pTS-pa0665 at 42 °C, correlated with the downregulation of the oprG gene. RNA sequencing revealed significant transcriptional changes in genes associated with the oxidative phosphorylation (OXPHOS) system, aligning with reduced ATP levels in Δpa0665 / pTS-pa0665 under 42 °C. Additionally, the ts -mutant showed heightened sensitivity to H 2 O 2 at 42 °C. Overall, our study demonstrates the essential role of pa0665 for OXPHOS function and is complemented by ec.erpA . We propose that the plasmid-based ts -allele is useful for genetic analysis of essential genes of interest in P. aeruginosa .