A Long-Chain Flavodoxin Protects <i>Pseudomonas aeruginosa</i> from Oxidative Stress and Host Bacterial Clearance

<div><p>Long-chain flavodoxins, ubiquitous electron shuttles containing flavin mononucleotide (FMN) as prosthetic group, play an important protective role against reactive oxygen species (ROS) in various microorganisms. <i>Pseudomonas aeruginosa</i> is an opportunistic pathogen which frequently has to face ROS toxicity in the environment as well as within the host. We identified a single ORF, hereafter referred to as <i>fldP</i> (for <b><i>fl</i></b>avo<b><i>d</i></b>oxin from <b><i>P</i></b><i>. aeruginosa</i>), displaying the highest similarity in length, sequence identity and predicted secondary structure with typical long-chain flavodoxins. The gene was cloned and expressed in <i>Escherichia coli</i>. The recombinant product (FldP) could bind FMN and exhibited flavodoxin activity <i>in vitro</i>. Expression of <i>fldP</i> in <i>P. aeruginosa</i> was induced by oxidative stress conditions through an OxyR-independent mechanism, and an <i>fldP</i>-null mutant accumulated higher intracellular ROS levels and exhibited decreased tolerance to H₂O₂ toxicity compared to wild-type siblings. The mutant phenotype could be complemented by expression of a cyanobacterial flavodoxin. Overexpression of FldP in a <i>mutT</i>-deficient <i>P. aeruginosa</i> strain decreased H₂O₂-induced cell death and the hypermutability caused by DNA oxidative damage. FldP contributed to the survival of <i>P. aeruginosa</i> within cultured mammalian macrophages and in infected <i>Drosophila melanogaster</i>, which led in turn to accelerated death of the flies. Interestingly, the <i>fldP</i> gene is present in some but not all <i>P. aeruginosa</i> strains, constituting a component of the <i>P. aeruginosa</i> accessory genome. It is located in a genomic island as part of a self-regulated polycistronic operon containing a suite of stress-associated genes. The collected results indicate that the <i>fldP</i> gene encodes a long-chain flavodoxin, which protects the cell from oxidative stress, thereby expanding the capabilities of <i>P. aeruginosa</i> to thrive in hostile environments.</p>