The opportunistic pathogen Pseudomonas aeruginosa damages hosts through the production of diverse secreted products, many of which are regulated by quorum sensing (QS). The lasR gene, which encodes a central QS regulator, is frequently mutated in clinical isolates from chronic infections, and loss of LasR function (LasR−) generally impairs the activity of downstream QS regulators RhlR and PqsR. We found that in cocultures containing LasR+ and LasR− strains, LasR− strains hyperproduce the RhlR/RhlI-regulated antagonistic factors pyocyanin and rhamnolipids in diverse models and media and in different strain backgrounds. Diffusible QS autoinducers produced by the wild type were not required for this effect. Using transcriptomics, genetics, and biochemical approaches, we uncovered a reciprocal interaction between wild-type and lasR mutant pairs wherein the iron-scavenging siderophore pyochelin produced by the lasR mutant induced citrate release and cross-feeding from the wild type. Citrate, a metabolite often secreted in low iron environments, stimulated RhlR signaling and RhlI levels in LasR−but not in LasR+ strains. These studies reveal the potential for complex interactions between recently diverged, genetically distinct isolates within populations from single chronic infections. IMPORTANCE Coculture interactions between lasR loss-of-function and LasR+ Pseudomonas aeruginosa strains may explain the worse outcomes associated with the presence of LasR− strains. More broadly, this report illustrates how interactions within a genotypically diverse population, similar to those that frequently develop in natural settings, can promote unpredictably high virulence factor production.

Pseudomonas aeruginosa has a broad metabolic repertoire that facilitates its coexistence with different microbes. Many microbes secrete products that P. aeruginosa can then catabolize, including ethanol, a common fermentation product. Here, we show that under oxygen-limiting conditions P. aeruginosa utilizes AdhA, an NAD-linked alcohol dehydrogenase, as a previously undescribed means for ethanol catabolism. In a rich medium containing ethanol, AdhA, but not the previously described PQQ-linked alcohol dehydrogenase, ExaA, oxidizes ethanol and leads to the accumulation of acetate in culture supernatants. AdhA-dependent acetate accumulation and the accompanying decrease in pH promote P. aeruginosa survival in LB-grown stationary-phase cultures. The transcription of adhA is elevated by hypoxia and under anoxic conditions, and we show that it is regulated by the Anr transcription factor. We have shown that lasR mutants, which lack an important quorum sensing regulator, have higher levels of Anr-regulated transcripts under low-oxygen conditions than their wild-type counterparts. Here, we show that a lasR mutant, when grown with ethanol, has an even larger decrease in pH than the wild type (WT) that is dependent on both anr and adhA. The large increase in AdhA activity is similar to that of a strain expressing a hyperactive Anr-D149A variant. Ethanol catabolism in P. aeruginosa by AdhA supports growth on ethanol as a sole carbon source and electron donor in oxygen-limited settings and in cells growing by denitrification under anoxic conditions. This is the first demonstration of a physiological role for AdhA in ethanol oxidation in P. aeruginosa. IMPORTANCE Ethanol is a common product of microbial fermentation, and the Pseudomonas aeruginosa response to and utilization of ethanol are relevant to our understanding of its role in microbial communities. Here, we report that the putative alcohol dehydrogenase AdhA is responsible for ethanol catabolism and acetate accumulation under low-oxygen conditions and that it is regulated by Anr.

1The opportunistic pathogen Pseudomonas aeruginosa damages hosts through the 2 production of diverse secreted products, many of which are regulated by quorum 3 sensing. The lasR gene, which encodes a central quorum-sensing regulator, is 4 frequently mutated, and loss of LasR function impairs the activity of downstream 5 regulators RhlR and PqsR. We found that in diverse models, the presence of P. 6 aeruginosa wild type causes LasR loss-of-function strains to hyperproduce RhlR/I-7 regulated antagonistic factors, and autoinducer production by the wild type is not 8 required for this effect. We uncovered a reciprocal interaction between isogenic wild 9 type and lasR mutant pairs wherein the iron-scavenging siderophore pyochelin, 10 specifically produced by the lasR mutant, induces citrate release and cross-feeding from 11 wild type. Citrate stimulates RhlR signaling and RhlI levels in LasR-but not in LasR+ 12 strains, and the interactions occur in diverse media. Co-culture interactions between 13 strains that differ by the function of a single transcription factor may explain worse 14 outcomes associated with mixtures of LasR+ and LasR loss-of-function strains. More 15 broadly, this report illustrates how interactions within a genotypically diverse population, 16 similar to those that frequently develop in natural settings, can promote net virulence 17 factor production. (19, 20). Further, some LasR-strains exhibit rewired regulation of quorum sensing 35 (QS)-controlled exoproducts (21) and LasR-strains can activate QS signaling in 36 response to products from other species (22) or in specific culture conditions (23, 24). 37LasR-strains are also frequently found among LasR+ P. aeruginosa strains where 38 exoproducts can be shared or signal cross-feeding can occur (13). 39 P. aeruginosa LasR participates in the regulation of QS in conjunction with two 40 transcription factors: RhlR and PqsR (MvfR). Each of these three regulators has one or 41 more autoinducer ligands: 3-oxo-C12-homoserine lactone (3OC12HSL) for LasR, C4-42 homoserine lactone (C4HSL) for RhlR, and hydroxyalkylquinolones (Pseudomonas 43Quinolone Signal (PQS) and hydroxy-heptyl quinolone (HHQ)) for PqsR (25). In the 44 regulatory networks described in the widely-used P. aeruginosa model strains, LasR is 45 an upstream regulator of RhlR and PqsR signaling, and together these regulators 46 control the expression of a suite of genes associated with virulence including redox-47 active small molecule phenazines (26-28), cyanide (29), proteases (30-33), and 48 rhamnolipid surfactants important for surface motility, biofilm dispersal, and host cell 49 damage (34)(35)(36). 50Other traits that are often heterogeneous across P. aeruginosa isolates relate to 51 strategies for iron acquisition. P. aeruginosa procures iron through the use of 52 siderophores, including pyochelin (37-39) and pyoverdine (40), from heme or through a 53 direct iron uptake system (41-43). It is common to encounter P. aeruginosa strains with 54 loss of function mutations in genes encoding ...