The <i>Pseudomonas aeruginosa</i> DedA protein PA4029 is an undecaprenyl phosphate flippase important for polymyxin resistance

Undecaprenyl phosphate (C55-P) is a critical lipid carrier required for the transport of cell envelope precursors across the cytoplasmic membrane in bacteria. Recent studies have identified proteins of the DedA family and DUF368 domain family as C55-P flippases in both Gram-positive and Gram-negative organisms. However, their roles remain undefined in many clinically relevant pathogens. Here, we screened for DedA and DUF368 proteins in <i>Pseudomonas aeruginosa</i> and assessed their functional importance. We show that PA4029, a DedA family membrane protein, is involved in C55-P recycling. Deletion of PA4029 sensitizes cells to fosmidomycin and limits the emergence of spontaneous colistin-resistant mutants. Using native mass spectrometry, we demonstrate that PA4029 binds C55-P with high affinity and selectivity over membrane phospholipids, and that this interaction is disrupted by the C55-P targeting antibiotic amphomycin. We also show that a DUF368 protein, found in some <i>Pseudomonas</i> species lacking PA4029 orthologs, can functionally substitute for PA4029 in <i>P. aeruginosa</i>, suggesting divergent strategies for C55-P recycling in this genus. Together, these findings position PA4029 within the conserved DedA-mediated lipid carrier pathway and highlight its importance for cell envelope homeostasis and antibiotic resistance in <i>P. aeruginosa</i>.IMPORTANCEBacteria use lipid carrier undecaprenyl phosphate (C55-P) to build and maintain their cell envelope, which is necessary for survival and is the target of many antibiotics. Recent studies have implicated DedA family proteins in C55-P transport, but how these proteins function in important pathogens like <i>Pseudomonas aeruginosa</i> remains uncharacterized. In this work, we uncover a specific DedA protein, PA4029, and show its involvement in C55-P recycling and importance for bacteria's ability to develop resistance to the last-resort antibiotic colistin. These findings extend the relevance of DedA-mediated lipid transport to one of the most dreaded human pathogens.