Many microbial species form multicellular structures comprising elaborate wrinkles and concentric rings, yet the rules governing their architecture are poorly understood. The opportunistic pathogen Pseudomonas aeruginosa produces phenazines, small molecules that act as alternate electron acceptors to oxygen and nitrate to oxidize the intracellular redox state and that influence biofilm morphogenesis. Here, we show that the depth occupied by cells within colony biofilms correlates well with electron acceptor availability. Perturbations in the environmental provision, endogenous production, and utilization of electron acceptors affect colony development in a manner consistent with redox control. Intracellular NADH levels peak before the induction of colony wrinkling. These results suggest that redox imbalance is a major factor driving the morphogenesis of P. aeruginosa biofilms and that wrinkling itself is an adaptation that maximizes oxygen accessibility and thereby supports metabolic homeostasis. This type of redox-driven morphological change is reminiscent of developmental processes that occur in metazoans.

Evolutionary biologists have postulated that several fitness advantages may be conferred by the maintenance of duplicate genes, including environmental adaptation resulting from differential regulation. We examined the expression and physiological contributions of two redundant operons in the adaptable bacterium Pseudomonas aeruginosa PA14. These operons, phzA1-G1 (phz1) and phzA2-G2 (phz2), encode nearly identical sets of proteins that catalyze the synthesis of phenazine-1-carboxylic acid, the precursor for several phenazine derivatives. Phenazines perform diverse roles in P. aeruginosa physiology and act as virulence factors during opportunistic infections of plant and animal hosts. Although reports have indicated that phz1 is regulated by the Pseudomonas quinolone signal, factors controlling phz2 expression have not been identified, and the relative contributions of these redundant operons to phenazine biosynthesis have not been evaluated. We found that in liquid cultures, phz1 was expressed at higher levels than phz2, although phz2 showed a greater contribution to phenazine production. In colony biofilms, phz2 was expressed at high levels, whereas phz1 expression was not detectable, and phz2 was responsible for virtually all phenazine production. Analysis of mutants defective in quinolone signal synthesis revealed a critical role for 4-hydroxy-2-heptylquinoline in phz2 induction. Finally, deletion of phz2, but not of phz1, decreased lung colonization in a murine model of infection. These results suggest that differential regulation of the redundant phz operons allows P. aeruginosa to adapt to diverse environments. G ene duplications give rise to genetic redundancy, an unstable condition that would not be expected to persist over time (1). This property is exhibited by diverse genomes (2-4), however, and evolutionary theorists have proposed several mechanisms whereby duplicate genes might provide selective advantages (5); for example, redundancy may be favored when spatial or temporal differences in expression enable tissue-specific variation or survival under varying environmental conditions (6, 7). Products of duplicated genes are involved in crucial cellular processes, including signal transduction, development, and metabolism (8).The genome of the bacterium Pseudomonas aeruginosa, an opportunistic pathogen that thrives in both soil and host environments, contains two redundant seven-gene operons termed phzA1-G1 (phz1) and phzA2-G2 (phz2). These operons are nearly identical (∼98% similarity at the DNA level), and each encodes the biosynthetic enzymes for phenazine-1-carboxylic acid (PCA) (9). Downstream modifications derivatize this precursor, generating other phenazines (SI Appendix, Fig. S1A). Pseudomonad phenazines are toxic to many other organisms and cell types due to their inherent redox activity (10, 11). Studies conducted in various plant and animal models of infection have implicated phenazines in colonization and pathogenicity (12, 13). In addition, recent studies have elucidated beneficial roles...

Most bacteria in nature live in surface-associated communities rather than planktonic populations. Nonetheless, how surface-associated environments shape bacterial evolutionary adaptation remains poorly understood. Here we show that subjecting Pseudomonas aeruginosa to repeated rounds of swarming, a collective form of surface migration, drives remarkable parallel evolution towards a hyperswarmer phenotype. In all independently evolved hyperswarmers, the reproducible hyperswarming phenotype is caused by parallel point mutations in a flagellar synthesis regulator, FleN, which locks the naturally mono-flagellated bacteria in a multi-flagellated state and confers a growth-rate independent advantage in swarming. Even though hyperswarmers outcompete the ancestral strain in swarming competitions, they are strongly outcompeted in biofilm formation, which is an essential trait for P. aeruginosa in environmental and clinical settings. The finding that evolution in swarming colonies reliably produces evolution of poor biofilm formers supports the existence of an evolutionary tradeoff between motility and biofilm formation.

Redox-cycling compounds, including endogenously produced phenazine antibiotics, induce expression of the efflux pump MexGHIOpmD in the opportunistic pathogen Pseudomonas aeruginosa. Previous studies of P. aeruginosa virulence, physiology, and biofilm development have focused on the blue phenazine pyocyanin and the yellow phenazine-1-carboxylic acid (PCA). In P. aeruginosa phenazine biosynthesis, conversion of PCA to pyocyanin is presumed to proceed through the intermediate 5-methylphenazine-1-carboxylate (5-Me-PCA), a reactive compound that has eluded detection in most laboratory samples. Here, we apply electrochemical methods to directly detect 5-Me-PCA and find that it is transported by MexGHIOpmD in P. aeruginosa strain PA14 planktonic and biofilm cells. We also show that 5-Me-PCA is sufficient to fully induce MexGHI-OpmD expression and that it is required for wild-type colony biofilm morphogenesis. These physiological effects are consistent with the high redox potential of 5-Me-PCA, which distinguishes it from other well-studied P. aeruginosa phenazines. Our observations highlight the importance of this compound, which was previously overlooked due to the challenges associated with its detection, in the context of P. aeruginosa gene expression and multicellular behavior. This study constitutes a unique demonstration of efflux-based selfresistance, controlled by a simple circuit, in a Gram-negative pathogen.phenazine | RND efflux | MexGHI-OpmD | biofilm | antibiotic