<i>Pseudomonas aeruginosa lasR</i> mutant fitness in microoxia is supported by an Anr-regulated oxygen-binding hemerythrin.

Abstract

<i>Pseudomonas aeruginosa</i> strains with loss-of-function mutations in the transcription factor LasR are frequently encountered in the clinic and the environment. Among the characteristics common to LasR-defective (LasR-) strains is increased activity of the transcription factor Anr, relative to their LasR+ counterparts, in low-oxygen conditions. One of the Anr-regulated genes found to be highly induced in LasR- strains was <i>PA14_42860</i> (<i>PA1673</i>), which we named <i>mhr</i> for microoxic hemerythrin. Purified <i>P. aeruginosa</i> Mhr protein contained the predicted di-iron center and bound molecular oxygen with an apparent <i>K</i>_d of ∼1 µM. Both Anr and Mhr were necessary for fitness in <i>lasR</i>+ and <i>lasR</i> mutant strains in colony biofilms grown in microoxic conditions, and the effects were more striking in the <i>lasR</i> mutant. Among genes in the Anr regulon, <i>mhr</i> was most closely coregulated with the Anr-controlled high-affinity cytochrome <i>c</i> oxidase genes. In the absence of high-affinity cytochrome <i>c</i> oxidases, deletion of <i>mhr</i> no longer caused a fitness disadvantage, suggesting that Mhr works in concert with microoxic respiration. We demonstrate that Anr and Mhr contribute to LasR- strain fitness even in biofilms grown in normoxic conditions. Furthermore, metabolomics data indicate that, in a <i>lasR</i> mutant, expression of Anr-regulated <i>mhr</i> leads to differences in metabolism in cells grown on lysogeny broth or artificial sputum medium. We propose that increased Anr activity leads to higher levels of the oxygen-binding protein Mhr, which confers an advantage to <i>lasR</i> mutants in microoxic conditions.