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

Abstract

1 Pseudomonas aeruginosa strains with loss-of-function mutations in the transcription factor 2 are frequently encountered in the clinic and the environment. Among the characteristics common 3 to LasR-defective (LasR-) strains is increased activity of the transcription factor Anr, relative to 4 their LasR+ counterparts, in low oxygen conditions. One of the Anr-regulated genes that was 5 highly induced in the LasR-strains encoded a putative oxygen-binding hemerythrin encoded by 6 PA14_42860 (PA1673) which we named mhr for microoxic hemerythrin. Purified P. aeruginosa 7 Mhr protein contained the predicted di-iron center and binds oxygen with a Kd of 1 µM. Both Anr 8 and Mhr were necessary for fitness in lasR+ and lasR mutant strains in colony biofilms grown in 9 microoxic conditions, and the effects were more striking in the lasR mutant. Among genes in the 10 Anr regulon, mhr was most closely co-regulated with the Anr-controlled high affinity cytochrome 11 c oxidase genes and in the absence of high affinity cytochrome c oxidase activity, deletion of mhr 12 no longer caused a fitness disadvantage suggesting that Mhr works in concert with microoxic 13 respiration. We demonstrate that Anr and Mhr contribute to LasR-strain fitness even in the 14 normoxic biofilm conditions, and metabolomics data indicate that in a lasR mutant, expression of 15 Anr-regulated mhr leads to differences in metabolism in cells grown on LB and artificial sputum 16 medium. Together these data indicate that increased Anr activity in microoxically-grown lasR 17 mutants confers an advantage in part for its regulation of the O2 binding protein Mhr. 18 Significance 19Pseudomonas aeruginosa, a versatile bacterium that both lives in environmental habitats 20 and causes life-threatening opportunistic infections, uses quorum sensing to coordinate gene 21 expression with cell density. The lasR gene, which encodes a quorum sensing regulator, is often 22 deleteriously mutated in clinical isolates. Interestingly, LasR-strains have high activity of the 23 oxygen-sensitive transcription factor Anr in microoxic conditions. This report identifies and 24 characterizes an Anr-regulated microoxic hemerythrin that reversibly binds oxygen. We showed 25 both anr and mhr are critical to fitness in microoxia, and these genes uniquely benefit LasR-26 strains in normoxia. Our findings enrich our understanding of the success of P. aeruginosa as a 27 pulmonary resident through its propensity to lose LasR functionality in the context of low-oxygen 28 infection environments. 30 31Pseudomonas aeruginosa is a devastating pathogen for healthcare systems worldwide 32 and causes opportunistic infections at multiple body sites that are extremely difficult to treat. P. 33aeruginosa is especially damaging within the context of the genetic disease cystic fibrosis (CF), 34where it establishes chronic infections of the airway and is a major predictor of morbidity and 35 mortality (1, 2). The success of P. aeruginosa in disease is due to a confluence of factors, 36including intrinsic an...

Intraspecies Signaling between Common Variants of Pseudomonas aeruginosa Increases Production of Quorum-Sensing-Controlled Virulence Factors

“…1E ). We have previously shown that Anr activity is higher in ∆ lasR strains and contributes to the competitive fitness of the ∆ lasR strain against WT P. aeruginosa in colony biofilms ( 27 , 45 ), but Anr was not required for coculture pyocyanin production ( Fig. S2 ).…”

“…Planktonic cultures were grown on roller drums at 37°C. Artificial sputum medium (ASM) was made as described previously ( 27 ).…”

“…LasR− clinical isolates (CIs) are frequently found among strains with functional LasR (LasR+) where exoproducts can be shared or signal cross-feeding can occur ( 14 ), and some LasR− clinical isolates exhibit rewired QS regulation ( 25 ). Loss of LasR function also confers some fitness advantages, including altered catabolic profiles ( 26 ) and enhanced growth in low oxygen ( 27 , 28 ), which may contribute to bacterial burden. Further, LasR− strains can activate QS in response to specific fungal products ( 29 ) or culture conditions ( 30 , 31 ).…”

Intra-species signaling betweenPseudomonas aeruginosagenotypes increases production of quorum sensing controlled virulence factors

“…1D ). We have previously shown that Anr activity is higher in Δ lasR strains and contributes to Δ lasR competitive fitness against WT P. aeruginosa in colony biofilms (53, 54), but Anr was not required for co-culture pyocyanin production by Δ lasR ( Fig. S2 ).…”

“…There are several possible explanations for this apparent contradiction. Loss of lasR function confers some fitness advantages including altered catabolic profiles (18) and enhanced growth in low oxygen (19, 20). Further, some LasR-strains exhibit rewired regulation of quorum sensing (QS)-controlled exoproducts (21) and LasR-strains can activate QS signaling in response to products from other species (22) or in specific culture conditions (23, 24).…”