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Universityhttps://www.ebi.ac.uk/ena/browser/view/PRJNA1063516Pseudomonas aeruginosaPseudomonas aeruginosa is an opportunistic human pathogen frequently responsible for multi-drug resistant and virulent infections that urgently need addressing. To find potential targets for enhancing the antibacterial efficacy of the nonredox iron mimetic gallium, we established a doxycycline (Dox)-inducible, genome-wide CRISPRi system enabling comprehensive delineation of responsive target genes influencing P. aeruginosa susceptibility to gallium. This system was demonstrated to be titratable and controllable, exhibiting outstanding efficiency and applicability across different P. aeruginosa strains. We employed CRISPRi-seq screening to identify 618 essential genes in P. aeruginosa PA14, guaranteeing extensive genome coverage and offering robust confirmation for prior Tn-seq studies. Subsequent screening revealed that fprB, coding for ferredoxin NADP+ reductase, plays a role in P. aeruginosa's resistance to gallium treatment. It is worth noting that disturbances in ATP synthesis and metal transport, specifically magnesium and iron, may contribute to the understanding of why fprB inactivation makes P. aeruginosa more vulnerable to gallium. Furthermore, FAD-binding domains in FprB and the transcription levels of fprA have been demonstrated to partially mediate the interaction between P. aeruginosa and gallium. Taken together, our findings indicate that fprA and fprB are key factors in P. aeruginosa's resistance to gallium, making them prime targets for drug development aimed at boosting gallium's therapeutic effectiveness. The CRISPRi-seq screening strategy employed in this research could represent a significant advancement in the genetic analysis of P. aeruginosa and related pathogens, identifying potential therapeutic targets.pathogen:prioritypathogen:bacteriumpathogenENAbacteriatreatment, Therapy, Adrenodoxin, Iron Sulfur Protein Reductase, Iron-Sulfur, Genomes, Peudomonas aeruginosa, Ferredoxin NADP Reductase, Pseudomonas pyocyanea, Iron-Sulfur Protein Reductase, whole genome, Bacterium pyocyaneum, NADPH Ferredoxin Reductase, Treatments, Bacillus pyocyaneus, Bacterium aeruginosum., Ferredoxin:NADP+ oxidoreductase, Pseudomonas polycolor, Micrococcus pyocyaneus, Therapeutic, Iron-Sulfur Protein, NADPH-Ferredoxin Reductase, Adrenodoxin Reductase, NADPH-Ferredoxin, Protein Reductase, disease management, Therapies, probable synonym or variety: "Pseudomonas polycolor" Clara 1930, Treatment, Bacillus aeruginosus, Pseudomonas aeruginosa (Schroeter 1872) Migula 1900 (Approved Lists 1980), Ferredoxin-NADP, ReductasePseudomonas pyocyanea, probable synonym or variety: "Pseudomonas polycolor" Clara 1930, Bacterium aeruginosum., Pseudomonas polycolor, Bacillus aeruginosus, Micrococcus pyocyaneus, Pseudomonas aeruginosa (Schroeter 1872) Migula 1900 (Approved Lists 1980), Bacterium pyocyaneum, Peudomonas aeruginosa, Bacillus pyocyaneusfalsePseudomonas aeruginosaGenome-wide CRISPRi-seq identified ferredoxin-NADP reductase FprB as a synergistic target for gallium therapy in Pseudomonas aeruginosa2025-09-242024-05-11PRJNA1063516287