Pseudomonas aeruginosa lasR mutant fitness in microoxia is supported by an Anr-regulated oxygen-binding hemerythrin

Abstract

Pseudomonas aeruginosa strains with loss-of-function mutations in the transcription factor 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 that was highly induced in the LasR-strains encoded a putative oxygen-binding hemerythrin encoded by PA14_42860 (PA1673) which we named mhr for microoxic hemerythrin. Purified P. aeruginosa Mhr protein contained the predicted di-iron center and binds oxygen with a Kd of 1 {micro}M. Both Anr and Mhr were necessary for fitness in lasR+ and lasR mutant strains in colony biofilms grown in microoxic conditions, and the effects were more striking in the lasR mutant. Among genes in the Anr regulon, mhr was most closely co-regulated with the Anr-controlled high affinity cytochrome c oxidase genes and in the absence of high affinity cytochrome c oxidase activity, deletion of mhr 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 the normoxic biofilm conditions, and metabolomics data indicate that in a lasR mutant, expression of Anr-regulated mhr leads to differences in metabolism in cells grown on LB and artificial sputum medium. Together these data indicate that increased Anr activity in microoxically-grown lasR mutants confers an advantage in part for its regulation of the O2 binding protein Mhr.\n\nSignificancePseudomonas aeruginosa, a versatile bacterium that both lives in environmental habitats and causes life-threatening opportunistic infections, uses quorum sensing to coordinate gene expression with cell density. The lasR gene, which encodes a quorum sensing regulator, is often deleteriously mutated in clinical isolates. Interestingly, LasR-strains have high activity of the oxygen-sensitive transcription factor Anr in microoxic conditions. This report identifies and characterizes an Anr-regulated microoxic hemerythrin that reversibly binds oxygen. We showed both anr and mhr are critical to fitness in microoxia, and these genes uniquely benefit LasR-strains in normoxia. Our findings enrich our understanding of the success of P. aeruginosa as a pulmonary resident through its propensity to lose LasR functionality in the context of low-oxygen infection environments.