The L-lactate dehydrogenases of Pseudomonas aeruginosa are conditionally regulated but both contribute to survival during macrophage infection.

Pseudomonas aeruginosa is an opportunistic pathogen that thrives in environments associated with human activity, including soil and water altered by agriculture or pollution. Because L-lactate is a significant product of plant and animal metabolism, it is available to serve as a carbon source for P. aeruginosa in the diverse settings it inhabits. Here, we evaluate P. aeruginosa's production and use of its redundant L-lactate dehydrogenases, termed LldD and LldA. We confirm that the protein LldR represses lldD and identify a new transcription factor, called LldS, that activates lldA; these distinct regulators and the genomic contexts of lldD and lldA contribute to their differential expression. We demonstrate that the lldD and lldA genes are conditionally controlled in response to lactate isomers as well as to glycolate and - hydroxybutyrate, which, like lactate, are -hydroxycarboxylates. We also show that lldA is induced when iron availability is low. Our examination of lldD and lldA expression across depth in biofilms indicates a complex pattern that is consistent with the effects of glycolate production, iron availability, and cross-regulation on enzyme preference. Finally, macrophage infection assays revealed that both lldD and lldA contribute to persistence within host cells, underscoring the potential role of L-lactate as a carbon source during P. aeruginosa-eukaryote interactions. Together, these findings help us understand the metabolism of a key resource that may promote P. aeruginosa's success as a resident of contaminated environments and animal hosts.

Cellular arrangement impacts metabolic activity and antibiotic tolerance in Pseudomonas aeruginosa biofilms.

Cells must access resources to survive, and the anatomy of multicellular structures influences this access. In diverse multicellular eukaryotes, resources are provided by internal conduits that allow substances to travel more readily through tissue than they would via diffusion. Microbes growing in multicellular structures, called biofilms, are also affected by differential access to resources and we hypothesized that this is influenced by the physical arrangement of the cells. In this study, we examined the microanatomy of biofilms formed by the pathogenic bacterium Pseudomonas aeruginosa and discovered that clonal cells form striations that are packed lengthwise across most of a mature biofilm's depth. We identified mutants, including those defective in pilus function and in O-antigen attachment, that show alterations to this lengthwise packing phenotype. Consistent with the notion that cellular arrangement affects access to resources within the biofilm, we found that while the wild type shows even distribution of tested substrates across depth, the mutants show accumulation of substrates at the biofilm boundaries. Furthermore, we found that altered cellular arrangement within biofilms affects the localization of metabolic activity, the survival of resident cells, and the susceptibility of subpopulations to antibiotic treatment. Our observations provide insight into cellular features that determine biofilm microanatomy, with consequences for physiological differentiation and drug sensitivity.

MpaR-driven expression of an orphan terminal oxidase subunit supports Pseudomonas aeruginosa biofilm respiration and development during cyanogenesis.

IMPORTANCE: Cyanide is an inhibitor of heme-copper oxidases, which are required for aerobic respiration in all eukaryotes and many prokaryotes. This fast-acting poison can arise from diverse sources, but mechanisms by which bacteria sense it are poorly understood. We investigated the regulatory response to cyanide in the pathogenic bacterium Pseudomonas aeruginosa, which produces cyanide as a virulence factor. Although P. aeruginosa has the capacity to produce a cyanide-resistant oxidase, it relies primarily on heme-copper oxidases and even makes additional heme-copper oxidase proteins specifically under cyanide-producing conditions. We found that the protein MpaR controls expression of cyanide-inducible genes in P. aeruginosa and elucidated the molecular details of this regulation. MpaR contains a DNA-binding domain and a domain predicted to bind pyridoxal phosphate (vitamin B6), a compound that is known to react spontaneously with cyanide. These observations provide insight into the understudied phenomenon of cyanide-dependent regulation of gene expression in bacteria.

Spatial heterogeneity in biofilm metabolism elicited by local control of phenazine methylation.

Within biofilms, gradients of electron acceptors such as oxygen stimulate the formation of physiological subpopulations. This heterogeneity can enable cross-feeding and promote drug resilience, features of the multicellular lifestyle that make biofilm-based infections difficult to treat. The pathogenic bacterium Pseudomonas aeruginosa produces pigments called phenazines that can support metabolic activity in hypoxic/anoxic biofilm subzones, but these compounds also include methylated derivatives that are toxic to their producer under some conditions. In this study, we uncover roles for the global regulators RpoS and Hfq/Crc in controlling the beneficial and detrimental effects of methylated phenazines in biofilms. Our results indicate that RpoS controls phenazine methylation by modulating activity of the carbon catabolite repression pathway, in which the Hfq/Crc complex inhibits translation of the phenazine methyltransferase PhzM. We find that RpoS indirectly inhibits expression of CrcZ, a small RNA that binds to and sequesters Hfq/Crc, specifically in the oxic subzone of P. aeruginosa biofilms. Deletion of rpoS or crc therefore leads to overproduction of methylated phenazines, which we show leads to increased metabolic activity-an apparent beneficial effect-in hypoxic/anoxic subpopulations within biofilms. However, we also find that under specific conditions, biofilms lacking RpoS and/or Crc show increased sensitivity to phenazines indicating that the increased metabolic activity in these mutants comes at a cost. Together, these results suggest that complex regulation of PhzM allows P. aeruginosa to simultaneously exploit the benefits and limit the toxic effects of methylated phenazines.

Molecular insights into RmcA-mediated c-di-GMP consumption: Linking redox potential to biofilm morphogenesis in Pseudomonas aeruginosa.

The ability of many bacteria to form biofilms contributes to their resilience and makes infections more difficult to treat. Biofilm growth leads to the formation of internal oxygen gradients, creating hypoxic subzones where cellular reducing power accumulates, and metabolic activities can be limited. The pathogen Pseudomonas aeruginosa counteracts the redox imbalance in the hypoxic biofilm subzones by producing redox-active electron shuttles (phenazines) and by secreting extracellular matrix, leading to an increased surface area-to-volume ratio, which favors gas exchange. Matrix production is regulated by the second messenger bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) in response to different environmental cues. RmcA (Redox modulator of c-di-GMP) from P. aeruginosa is a multidomain phosphodiesterase (PDE) that modulates c-di-GMP levels in response to phenazine availability. RmcA can also sense the fermentable carbon source arginine via a periplasmic domain, which is linked via a transmembrane domain to four cytoplasmic Per-Arnt-Sim (PAS) domains followed by a diguanylate cyclase (DGC) and a PDE domain. The biochemical characterization of the cytoplasmic portion of RmcA reported in this work shows that the PAS domain adjacent to the catalytic domain tunes RmcA PDE activity in a redox-dependent manner, by differentially controlling protein conformation in response to FAD or FADH2. This redox-dependent mechanism likely links the redox state of phenazines (via FAD/FADH2 ratio) to matrix production as indicated by a hyperwrinkling phenotype in a macrocolony biofilm assay. This study provides insights into the role of RmcA in transducing cellular redox information into a structural response of the biofilm at the population level. Conditions of resource (i.e. oxygen and nutrient) limitation arise during chronic infection, affecting the cellular redox state and promoting antibiotic tolerance. An understanding of the molecular linkages between condition sensing and biofilm structure is therefore of crucial importance from both biological and engineering standpoints.

Cell arrangement impacts metabolic activity and antibiotic tolerance in Pseudomonas aeruginosa biofilms.

Cells must access resources to survive, and the anatomy of multicellular structures influences this access. In diverse multicellular eukaryotes, resources are provided by internal conduits that allow substances to travel more readily through tissue than they would via diffusion. Microbes growing in multicellular structures, called biofilms, are also affected by differential access to resources and we hypothesized that this is influenced by the physical arrangement of the cells. In this study, we examined the microanatomy of biofilms formed by the pathogenic bacterium Pseudomonas aeruginosa and discovered that clonal cells form striations that are packed lengthwise across most of a mature biofilm's depth. We identified mutants, including those defective in pilus function and in O-antigen attachment, that show alterations to this lengthwise packing phenotype. Consistent with the notion that cellular arrangement affects access to resources within the biofilm, we found that while the wild type shows even distribution of tested substrates across depth, the mutants show accumulation of substrates at the biofilm boundaries. Furthermore, we found that altered cellular arrangement within biofilms affects the localization of metabolic activity, the survival of resident cells, and the susceptibility of subpopulations to antibiotic treatment. Our observations provide insight into cellular features that determine biofilm microanatomy, with consequences for physiological differentiation and drug sensitivity.

Cyanide-dependent control of terminal oxidase hybridization by Pseudomonas aeruginosa MpaR.

Pseudomonas aeruginosa is a common, biofilm-forming pathogen that exhibits complex pathways of redox metabolism. It produces four different types of terminal oxidases for aerobic respiration, and for one of these-the cbb3-type terminal oxidases-it has the capacity to produce at least 16 isoforms encoded by partially redundant operons. It also produces small-molecule virulence factors that interact with the respiratory chain, including the poison cyanide. Previous studies had indicated a role for cyanide in activating expression of an "orphan" terminal oxidase subunit gene called ccoN4 and that the product contributes to P. aeruginosa cyanide resistance, fitness in biofilms, and virulence-but the mechanisms underlying this process had not been elucidated. Here, we show that the regulatory protein MpaR, which is predicted to be a pyridoxal phosphate-binding transcription factor and is encoded just upstream of ccoN4, controls ccoN4 expression in response to endogenous cyanide. Paradoxically, we find that cyanide production is required to support CcoN4's contribution to respiration in biofilms. We identify a palindromic motif required for cyanide- and MpaR-dependent expression of ccoN4 and co-expressed, adjacent loci. We also characterize the regulatory logic of this region of the chromosome. Finally, we identify residues in the putative cofactor-binding pocket of MpaR that are required for ccoN4 expression. Together, our findings illustrate a novel scenario in which the respiratory toxin cyanide acts as a signal to control gene expression in a bacterium that produces the compound endogenously.

Spatial heterogeneity in biofilm metabolism elicited by local control of phenazine methylation.

Within biofilms, gradients of electron acceptors such as oxygen stimulate the formation of physiological subpopulations. This heterogeneity can enable cross-feeding and promote drug resilience, features of the multicellular lifestyle that make biofilm-based infections difficult to treat. The pathogenic bacterium Pseudomonas aeruginosa produces pigments called phenazines that can support metabolic activity in hypoxic/anoxic biofilm subzones, but these compounds also include methylated derivatives that are toxic to their producer under some conditions. Here, we uncover roles for the global regulators RpoS and Hfq/Crc in controlling the beneficial and detrimental effects of methylated phenazines in biofilms. Our results indicate that RpoS controls phenazine methylation by modulating activity of the carbon catabolite repression pathway, in which the Hfq/Crc complex inhibits translation of the phenazine methyltransferase PhzM. We find that RpoS indirectly inhibits expression of CrcZ, a small RNA that binds to and sequesters Hfq/Crc, specifically in the oxic subzone of P. aeruginosa biofilms. Deletion of rpoS or crc therefore leads to overproduction of methylated phenazines, which we show leads to increased metabolic activity-an apparent beneficial effect-in hypoxic/anoxic subpopulations within biofilms. However, we also find that biofilms lacking Crc show increased sensitivity to an exogenously added methylated phenazine, indicating that the increased metabolic activity in this mutant comes at a cost. Together, these results suggest that complex regulation of PhzM allows P. aeruginosa to simultaneously exploit the benefits and limit the toxic effects of methylated phenazines.

Light/Dark and Temperature Cycling Modulate Metabolic Electron Flow in Pseudomonas aeruginosa Biofilms.

Sunlight drives phototrophic metabolism, which affects redox conditions and produces substrates for nonphototrophs. These environmental parameters fluctuate daily due to Earth's rotation, and nonphototrophic organisms can therefore benefit from the ability to respond to, or even anticipate, such changes. Circadian rhythms, such as daily changes in body temperature, in host organisms can also affect local conditions for colonizing bacteria. Here, we investigated the effects of light/dark and temperature cycling on biofilms of the opportunistic pathogen Pseudomonas aeruginosa PA14. We grew biofilms in the presence of a respiratory indicator dye and found that enhanced dye reduction occurred in biofilm zones that formed during dark intervals and at lower temperatures. This pattern formation occurred with cycling of blue, red, or far-red light, and a screen of mutants representing potential sensory proteins identified two with defects in pattern formation, specifically under red light cycling. We also found that the physiological states of biofilm subzones formed under specific light and temperature conditions were retained during subsequent condition cycling. Light/dark and temperature cycling affected expression of genes involved in primary metabolic pathways and redox homeostasis, including those encoding electron transport chain components. Consistent with this, we found that cbb3-type oxidases contribute to dye reduction under light/dark cycling conditions. Together, our results indicate that cyclic changes in light exposure and temperature have lasting effects on redox metabolism in biofilms formed by a nonphototrophic, pathogenic bacterium. IMPORTANCE Organisms that do not obtain energy from light can nevertheless be affected by daily changes in light exposure. Many aspects of animal and fungal physiology fluctuate in response to these changes, including body temperature and the activities of antioxidant and other redox enzymes that play roles in metabolism. Whether redox metabolism is affected by light/dark and temperature cycling in bacteria that colonize such circadian organisms has not been studied in detail. Here, we show that growth under light/dark and temperature cycling lead to rhythmic changes in redox metabolism in Pseudomonas aeruginosa and identify proteins involved in this response. P. aeruginosa is a major cause of health care-associated infections and is designated a serious threat by the CDC due to its recalcitrance during treatments. Our findings have the potential to inform therapeutic strategies that incorporate controlled light exposure or consider P. aeruginosa's responses to conditions in the host.

Gradients and consequences of heterogeneity in biofilms.

Historically, appreciation for the roles of resource gradients in biology has fluctuated inversely to the popularity of genetic mechanisms. Nevertheless, in microbiology specifically, widespread recognition of the multicellular lifestyle has recently brought new emphasis to the importance of resource gradients. Most microorganisms grow in assemblages such as biofilms or spatially constrained communities with gradients that influence, and are influenced by, metabolism. In this Review, we discuss examples of gradient formation and physiological differentiation in microbial assemblages growing in diverse settings. We highlight consequences of physiological heterogeneity in microbial assemblages, including division of labour and increased resistance to stress. Our impressions of microbial behaviour in various ecosystems are not complete without complementary maps of the chemical and physical geographies that influence cellular activities. A holistic view, incorporating these geographies and the genetically encoded functions that operate within them, will be essential for understanding microbial assemblages in their many roles and potential applications.

Sensory Perception in Bacterial Cyclic Diguanylate Signal Transduction.

Cyclic diguanylate (c-di-GMP) signal transduction systems provide bacteria with the ability to sense changing cell status or environmental conditions and then execute suitable physiological and social behaviors in response. In this review, we provide a comprehensive census of the stimuli and receptors that are linked to the modulation of intracellular c-di-GMP. Emerging evidence indicates that c-di-GMP networks sense light, surfaces, energy, redox potential, respiratory electron acceptors, temperature, and structurally diverse biotic and abiotic chemicals. Bioinformatic analysis of sensory domains in diguanylate cyclases and c-di-GMP-specific phosphodiesterases as well as the receptor complexes associated with them reveals that these functions are linked to a diverse repertoire of protein domain families. We describe the principles of stimulus perception learned from studying these modular sensory devices, illustrate how they are assembled in varied combinations with output domains, and summarize a system for classifying these sensor proteins based on their complexity. Biological information processing via c-di-GMP signal transduction not only is fundamental to bacterial survival in dynamic environments but also is being used to engineer gene expression circuitry and synthetic proteins with à la carte biochemical functionalities.

Spatial alanine metabolism determines local growth dynamics of Escherichia coli colonies.

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 Escherichia 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 E. 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 via 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, via 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.

Model Systems to Study the Chronic, Polymicrobial Infections in Cystic Fibrosis: Current Approaches and Exploring Future Directions.

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 35+ 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.

Pseudomonas aeruginosa PA14 produces R-bodies, extendable protein polymers with roles in host colonization and virulence.

R-bodies are long, extendable protein polymers formed in the cytoplasm of some bacteria; they are best known for their role in killing of paramecia by bacterial endosymbionts. Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes (referred to as the reb cluster) with potential to confer production of R-bodies and that have been implicated in virulence. Here, we show that products of the PA14 reb cluster associate with R-bodies and control stochastic expression of R-body structural genes. PA14 expresses reb genes during colonization of plant and nematode hosts, and R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that P. aeruginosa R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. Our observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection.

Metabolic Heterogeneity and Cross-Feeding in Bacterial Multicellular Systems.

Cells in assemblages differentiate and perform distinct roles. Though many pathways of differentiation are understood at the molecular level in multicellular eukaryotes, the elucidation of similar processes in bacterial assemblages is recent and ongoing. Here, we discuss examples of bacterial differentiation, focusing on cases in which distinct metabolisms coexist and those that exhibit cross-feeding, with one subpopulation producing substrates that are metabolized by a second subpopulation. We describe several studies of single-species systems, then segue to studies of multispecies metabolic heterogeneity and cross-feeding in the clinical setting. Many of the studies described exemplify the application of new techniques and modeling approaches that provide insights into metabolic interactions relevant for bacterial growth outside the laboratory.

Light-Mediated Decreases in Cyclic di-GMP Levels Inhibit Structure Formation in Pseudomonas aeruginosa Biofilms.

Light is known to trigger regulatory responses in diverse organisms, including slime molds, animals, plants, and phototrophic bacteria. However, light-dependent processes in nonphototrophic bacteria, and those of pathogens in particular, have received comparatively little research attention. In this study, we examined the impact of light on multicellular development in Pseudomonas aeruginosa, a leading cause of biofilm-based bacterial infections. We grew P. aeruginosa strain PA14 in a colony morphology assay and found that growth under prolonged exposure to low-intensity blue light inhibited biofilm matrix production and thereby the formation of vertical biofilm structures (i.e., "wrinkles"). Light-dependent inhibition of biofilm wrinkling was correlated with low levels of cyclic di-GMP (c-di-GMP), consistent with the role of this signal in stimulating matrix production. A screen of enzymes with the potential to catalyze c-di-GMP synthesis or degradation identified c-di-GMP phosphodiesterases that contribute to light-dependent inhibition of biofilm wrinkling. One of these, RmcA, was previously characterized by our group for its role in mediating the effect of redox-active P. aeruginosa metabolites called phenazines on biofilm wrinkle formation. Our results suggest that an RmcA sensory domain that is predicted to bind a flavin cofactor is involved in light-dependent inhibition of wrinkling. Together, these findings indicate that P. aeruginosa integrates information about light exposure and redox state in its regulation of biofilm development.IMPORTANCE Light exposure tunes circadian rhythms, which modulate the immune response and affect susceptibility to infection in plants and animals. Though molecular responses to light are defined for model plant and animal hosts, analogous pathways that function in bacterial pathogens are understudied. We examined the response to light exposure in biofilms (matrix-encased multicellular assemblages) of the nonphotosynthetic bacterium Pseudomonas aeruginosa We found that light at intensities that are not harmful to human cells inhibited biofilm maturation via effects on cellular signals. Because biofilm formation is a critical factor in many types of P. aeruginosa infections, including burn wound infections that may be exposed to light, these effects could be relevant for pathogenicity.

Interdependency of Respiratory Metabolism and Phenazine-Associated Physiology in Pseudomonas aeruginosa PA14.

Extracellular electron transfer (EET), the reduction of compounds that shuttle electrons to distal oxidants, can support bacterial survival when preferred oxidants are not directly accessible. EET has been shown to contribute to virulence in some pathogenic organisms and is required for current generation in mediator-based fuel cells. In several species, components of the electron transport chain (ETC) have been implicated in electron shuttle reduction, raising the question of how shuttling-based metabolism is integrated with primary routes of metabolic electron flow. The clinically relevant bacterium Pseudomonas aeruginosa can utilize carbon sources (i.e., electron donors) covering a broad range of reducing potentials and possesses a branched ETC that can be modulated to optimize respiratory efficiency. It also produces electron shuttles called phenazines that facilitate intracellular redox balancing, increasing the complexity of its metabolic potential. In this study, we investigated the reciprocal influence of respiratory metabolism and phenazine-associated physiology in P. aeruginosa PA14. We found that phenazine production affects respiratory activity and terminal oxidase gene expression and that carbon source identity influences the mechanisms enabling phenazine reduction. Furthermore, we found that growth in biofilms, a condition for which phenazine metabolism is critical to normal development and redox balancing, affects the composition of the P. aeruginosa phenazine pool. Together, these findings can aid interpretation of P. aeruginosa behavior during host infection and provide inroads to understanding the cross talk between primary metabolism and shuttling-based physiology in the diverse bacteria that carry out EET.IMPORTANCE The clinically relevant pathogen Pseudomonas aeruginosa uses diverse organic compounds as electron donors and possesses multiple enzymes that transfer electrons from central metabolism to O2 These pathways support a balanced intracellular redox state and produce cellular energy. P. aeruginosa also reduces secondary metabolites called phenazines to promote redox homeostasis and virulence. In this study, we examined the reciprocal relationship between these primary and secondary routes of electron flow. We found that phenazines affect respiratory function and that the complement of phenazines produced is strongly affected by growth in assemblages called biofilms. These results provide a more nuanced understanding of P. aeruginosa redox metabolism and may inform strategies for treating persistent infections caused by this bacterium.

Phenazine production promotes antibiotic tolerance and metabolic heterogeneity in Pseudomonas aeruginosa biofilms.

Antibiotic efficacy can be antagonized by bioactive metabolites and other drugs present at infection sites. Pseudomonas aeruginosa, a common cause of biofilm-based infections, releases metabolites called phenazines that accept electrons to support cellular redox balancing. Here, we find that phenazines promote tolerance to clinically relevant antibiotics, such as ciprofloxacin, in P. aeruginosa biofilms and that this effect depends on the carbon source provided for growth. We couple stable isotope labeling with stimulated Raman scattering microscopy to visualize biofilm metabolic activity in situ. This approach shows that phenazines promote metabolism in microaerobic biofilm regions and influence metabolic responses to ciprofloxacin treatment. Consistent with roles of specific respiratory complexes in supporting phenazine utilization in biofilms, phenazine-dependent survival on ciprofloxacin is diminished in mutants lacking these enzymes. Our work introduces a technique for the chemical imaging of biosynthetic activity in biofilms and highlights complex interactions between bacterial products, their effects on biofilm metabolism, and the antibiotics we use to treat infections.

The Pseudomonas aeruginosa Complement of Lactate Dehydrogenases Enables Use of d- and l-Lactate and Metabolic Cross-Feeding.

Pseudomonas aeruginosa is the most common cause of chronic, biofilm-based lung infections in patients with cystic fibrosis (CF). Sputum from patients with CF has been shown to contain oxic and hypoxic subzones as well as millimolar concentrations of lactate. Here, we describe the physiological roles and expression patterns of P. aeruginosa lactate dehydrogenases in the contexts of different growth regimes. P. aeruginosa produces four enzymes annotated as lactate dehydrogenases, three of which are known to contribute to anaerobic or aerobic metabolism in liquid cultures. These three are LdhA, which reduces pyruvate to d-lactate during anaerobic survival, and LldE and LldD, which oxidize d-lactate and l-lactate, respectively, during aerobic growth. We demonstrate that the fourth enzyme, LldA, performs redundant l-lactate oxidation during growth in aerobic cultures in both a defined MOPS (morpholinepropanesulfonic acid)-based medium and synthetic CF sputum media. However, LldA differs from LldD in that its expression is induced specifically by the l-enantiomer of lactate. We also show that the P. aeruginosa lactate dehydrogenases perform functions in colony biofilms that are similar to their functions in liquid cultures. Finally, we provide evidence that the enzymes LdhA and LldE have the potential to support metabolic cross-feeding in biofilms, where LdhA can catalyze the production of d-lactate in the anaerobic zone, which is then used as a substrate in the aerobic zone. Together, these observations further our understanding of the metabolic pathways that can contribute to P. aeruginosa growth and survival during CF lung infection.IMPORTANCE Lactate is thought to serve as a carbon and energy source during chronic infections. Sites of bacterial colonization can contain two enantiomers of lactate: the l-form, generally produced by the host, and the d-form, which is usually produced by bacteria, including the pulmonary pathogen Pseudomonas aeruginosa Here, we characterize P. aeruginosa's set of four enzymes that it can use to interconvert pyruvate and lactate, the functions of which depend on the availability of oxygen and specific enantiomers of lactate. We also show that anaerobic pyruvate fermentation triggers production of the aerobic d-lactate dehydrogenase in both liquid cultures and biofilms, thereby enabling metabolic cross-feeding of lactate over time and space between subpopulations of cells. These metabolic pathways might contribute to P. aeruginosa growth and survival in the lung.

Pseudomonas aeruginosa PumA acts on an endogenous phenazine to promote self-resistance.

The activities of critical metabolic and regulatory proteins can be altered by exposure to natural or synthetic redox-cycling compounds. Many bacteria, therefore, possess mechanisms to transport or transform these small molecules. The opportunistic pathogen Pseudomonas aeruginosa PA14 synthesizes phenazines, redox-active antibiotics that are toxic to other organisms but have beneficial effects for their producer. Phenazines activate the redox-sensing transcription factor SoxR and thereby induce the transcription of a small regulon, including the operon mexGHI-opmD, which encodes an efflux pump that transports phenazines, and PA14_35160 (pumA), which encodes a putative monooxygenase. Here, we provide evidence that PumA contributes to phenazine resistance and normal biofilm development, particularly during exposure to or production of strongly oxidizing N-methylated phenazines. We show that phenazine resistance depends on the presence of residues that are conserved in the active sites of other putative and characterized monooxygenases found in the antibiotic producer Streptomyces coelicolor. We also show that during biofilm growth, PumA is required for the conversion of phenazine methosulfate to unique phenazine metabolites. Finally, we compare ∆mexGHI-opmD and ∆pumA strains in assays for colony biofilm morphogenesis and SoxR activation, and find that these deletions have opposing phenotypic effects. Our results suggest that, while MexGHI-OpmD-mediated efflux has the effect of making the cellular phenazine pool more reducing, PumA acts on cellular phenazines to make the pool more oxidizing. We present a model in which these two SoxR targets function simultaneously to control the biological activity of the P. aeruginosa phenazine pool.