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A recent workshop titled “Developing Models to Study Polymicrobial Infections,” sponsored by the Dartmouth Cystic Fibrosis Center (DartCF), explored the development of new models to study the polymicrobial infections associated with the airways of persons with cystic fibrosis (CF). The workshop gathered 351 investigators over two virtual sessions. Here, we present the findings of this workshop, summarize some of the challenges involved with developing such models, and suggest three frameworks to tackle this complex problem. The frameworks proposed here, we believe, could be generally useful in developing new model systems for other infectious diseases. Developing and validating new approaches to study the complex polymicrobial communities in the CF airway could open windows to new therapeutics to treat these recalcitrant infections, as well as uncovering organizing principles applicable to chronic polymicrobial infections more generally.

Bacteria commonly live in spatially structured biofilm assemblages, which are encased by an extracellular matrix. Metabolic activity of the cells inside biofilms causes gradients in local environmental conditions, which leads to the emergence of physiologically differentiated subpopulations. Information about the properties and spatial arrangement of such metabolic subpopulations, as well as their interaction strength and interaction length scales are lacking, even for model systems like textitEscherichia coli colony biofilms grown on agar-solidified media. Here, we use an unbiased approach, based on temporal and spatial transcriptome and metabolome data acquired during textitE. coli colony biofilm growth, to study the spatial organization of metabolism. We discovered that alanine displays a unique pattern among amino acids and that alanine metabolism is spatially and temporally heterogeneous. At the anoxic base of the colony, where carbon and nitrogen sources are abundant, cells secrete alanine textitvia the transporter AlaE. In contrast, cells utilize alanine as a carbon and nitrogen source in the oxic nutrient-deprived region at the colony mid-height, textitvia the enzymes DadA and DadX. This spatially structured alanine cross-feeding influences cellular viability and growth in the cross-feeding-dependent region, which shapes the overall colony morphology. More generally, our results on this precisely controllable biofilm model system demonstrate a remarkable spatiotemporal complexity of metabolism in biofilms. A better characterization of the spatiotemporal metabolic heterogeneities and dependencies is essential for understanding the physiology, architecture, and function of biofilms.

Microbiologists have historically been struck by both the beautiful pigmentation of phenazine-producing cultures and the high degree of variability in phenazine production among isolates, conditions, and even repeat experiments. Motivated by an interest in controlling phenazine biosynthesis, they have identified many of the factors that affect the regulation of this process. Phenazine production is controlled by complex regulatory networks. The variability of phenazine production can be explained in part by the effects of environmental conditions on these networks and by strain-specific differences in these networks. In this chapter, we describe the components of a common regulatory cascade that is represented in many phenazine-producing pseudomonads. Membrane sensor proteins and two component sensors control the activity of downstream regulators such as quorum sensing systems and RNA-binding proteins and small RNAs; these cytoplasmic regulators then control the production of phenazine biosynthetic proteins. We highlight examples from specific strains and cases where the mechanistic links may vary among them. We also discuss environmental parameters that have been shown to affect phenazine biosynthesis and compare their effects in different isolates. Ongoing work will further elaborate the details of the environmental sensing and regulatory responses that control production of these dramatically colored compounds. New findings have the potential to support enhanced application of phenazine-producing strains in agriculture, where they promote crop health, and the treatment of infections in which phenazines contribute to bacterial pathogenicity.

The opportunistic pathogen Pseudomonas aeruginosa excretes redox-active small molecules called phenazines. This thesis addresses the possibility that the phenazine pyocyanin acts as an electron acceptor for energy metabolism and exerts beneficial effects on P. aeruginosa physiology. The effects of phenazine production and exposure on P. aeruginosa strain PA14 were examined by comparing the physiological status of the wild type to a mutant defective in phenazine production. Quantification of intracellular NADH and NAD+ pools revealed a more reduced intracellular redox state in the phenazine-null mutant compared to the wild type, consistent with the capacity of P. aeruginosa to reduce pyocyanin. High-performance liquid chromatography of culture metabolites showed that the wild type excreted pyruvate in late stationary phase, indicating that pyocyanin alters flux through central metabolic pathways. We set out to identify mechanisms allowing P. aeruginosa to catalyze pyocyanin redox cycling. Through a genetic screen, we found two loci required for full pyocyanin-dependent ferric citrate reduction activity in P. aeruginosa PA14: (1) the gene gpsA, encoding the soluble glycerol-3-phosphate dehydrogenase (GpsA), and (2) the operon fbcFBC, encoding the respiratory cytochrome bc1 complex. Mutants lacking functional GpsA had oxidized cytoplasms and may be defective in pyocyanin reduction due to a lack of sufficient NADH. In contrast, mutants lacking a functional cytochrome bc1 complex produced ample reducing power for pyocyanin reduction, raising the possibility that the cytochrome bc1 complex directly catalyzes pyocyanin reduction. Pyocyanin has previously been shown to affect the development of P. aeruginosa colonies on agar surfaces: phenazine-null mutants form wrinkled (rugose) colonies, while the wild type forms smooth colonies. Using this colony biofilm assay, we showed that the ΔgpsA mutant forms rugose colonies, consistent with a role for pyocyanin reduction in stimulating smooth colony formation. Modulation of electron acceptor availability through nitrate addition to the medium promoted smooth colony formation in rugose mutants. These results imply that rugosity is an adaptation to electron acceptor limitation. The work in this thesis has provided insight into the physiological relevance of pyocyanin reduction in P. aeruginosa, mechanisms controlling intracellular redox state in bacteria, and mechanisms that may contribute to P. aeruginosa virulence.

Microorganisms exist in the environment as multicellular communities that face the challenge of surviving under nutrientlimited conditions. Chemical communication is an essential part of the way in which these populations coordinate their behavior, and there has been an explosion of understanding in recent years regarding how this is accomplished. Much less, however, is understood about the way these communities sustain their metabolism. Bacteria of the genus Pseudomonas are ubiquitous, and are distinguished by their production of colorful secondary metabolites called phenazines. In this article, we suggest that phenazines, which are produced under conditions of high cell density and nutrient limitation, may be important for the persistence of pseudomonads in the environment.