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

Abstract

AbstractPseudomonas aeruginosastrains 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 byPA14_42860(PA1673) which we namedmhrfor microoxic hemerythrin. PurifiedP. aeruginosaMhr protein contained the predicted di-iron center and binds oxygen with aKdof 1 µM. Both Anr and Mhr were necessary for fitness inlasR+ andlasRmutant strains in colony biofilms grown in microoxic conditions, and the effects were more striking in thelasRmutant. Among genes in the Anr regulon,mhrwas most closely co-regulated with the Anr-controlled high affinity cytochromecoxidase genes and in the absence of high affinity cytochromecoxidase activity, deletion ofmhrno 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 alasRmutant, expression of Anr-regulatedmhrleads to differences in metabolism in cells grown on LB and artificial sputum medium. Together these data indicate that increased Anr activity in microoxically-grownlasRmutants confers an advantage in part for its regulation of the O2binding protein Mhr.SignificancePseudomonas 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. ThelasRgene, 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 bothanrandmhrare critical to fitness in microoxia, and these genes uniquely benefit LasR-strains in normoxia. Our findings enrich our understanding of the success ofP. aeruginosaas a pulmonary resident through its propensity to lose LasR functionality in the context of low-oxygen infection environments.