An Aerobic Exercise: Defining the Roles of Pseudomonas aeruginosa Terminal Oxidases

The opportunistic pathogen Pseudomonas aeruginosa encodes a large and diverse complement of aerobic terminal oxidases, which is thought to contribute to its ability to thrive in settings with low oxygen availability. In this issue, Arai et al. (J. Bacteriol. 196:4206 – 4215, 2014, doi:http://dx.doi.org/10.1128/JB.02176-14) present a thorough characterization of these five complexes, enabling a more detailed understanding of aerobic respiration in this organism

The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa

Certain members of the fluorescent pseudomonads produce and secrete phenazines. These heterocyclic, redox-active compounds are toxic to competing organisms, and the cause of these antibiotic effects has been the focus of intense research efforts. It is largely unknown, however, how pseudomonads themselves respond to – and survive in the presence of – these compounds. Using Pseudomonas aeruginosa DNA microarrays and quantitative RT-PCR, we demonstrate that the phenazine pyocyanin elicits the upregulation of genes/operons that function in transport [such as the resistance-nodulation-cell division (RND) efflux pump MexGHI-OpmD] and possibly in redox control (such as PA2274, a putative flavin-dependant monooxygenase), and downregulates genes involved in ferric iron acquisition. Strikingly, mexGHI-opmD and PA2274 were previously shown to be regulated by the PA14 quorum sensing network that controls the production of virulence factors (including phenazines). Through mutational analysis, we show that pyocyanin is the physiological signal for the upregulation of these quorum sensing-controlled genes during stationary phase and that the response is mediated by the transcription factor SoxR. Our results implicate phenazines as signalling molecules in both P. aeruginosa PA14 and PAO1

Phenazines affect biofilm formation by Pseudomonas aeruginosa in similar ways at various scales

Some pseudomonads produce phenazines, a group of small, redox-active compounds with diverse physiological functions. In this study, we compared the phenotypes of Pseudomonas aeruginosa strain PA14 and a mutant unable to synthesize phenazines in flow cell and colony biofilms quantitatively. Although phenazine production does not impact the ability of PA14 to attach to surfaces, as has been shown for Pseudomonas chlororaphis Maddula et al., 2006 and Maddula et al., 2008, it influences swarming motility and the surface-to-volume ratio of mature biofilms. These results indicate that phenazines affect biofilm development across a large range of scales, but in unique ways for different Pseudomonas species

An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growth and virulence

Hypoxia is a common challenge faced by bacteria during associations with hosts due in part to the formation of densely packed communities (biofilms). cbb3-type cytochrome c oxidases, which catalyze the terminal step in respiration and have a high affinity for oxygen, have been linked to bacterial pathogenesis. The pseudomonads are unusual in that they often contain multiple full and partial (i.e. ‘orphan’) operons for cbb3-type oxidases and oxidase subunits. Here, we describe a unique role for the orphan catalytic subunit CcoN4 in colony biofilm development and respiration in the opportunistic pathogen Pseudomonas aeruginosa PA14. We also show that CcoN4 contributes to the reduction of phenazines, antibiotics that support redox balancing for cells in biofilms, and to virulence in a Caenorhabditis elegans model of infection. These results highlight the relevance of the colony biofilm model to pathogenicity and underscore the potential of cbb3-type oxidases as therapeutic targets

Redox-Active Antibiotics Control Gene Expression and Community Behavior in Divergent Bacteria

It is thought that bacteria excrete redox-active pigments as antibiotics to inhibit competitors. In Pseudomonas aeruginosa, the endogenous antibiotic pyocyanin activates SoxR, a transcription factor conserved in Proteo- and Actinobacteria. In Escherichia coli, SoxR regulates the superoxide stress response. Bioinformatic analysis coupled with gene expression studies in P. aeruginosa and Streptomyces coelicolor revealed that the majority of SoxR regulons in bacteria lack the genes required for stress responses, despite the fact that many of these organisms still produce redox-active small molecules, which indicates that redox-active pigments play a role independent of oxidative stress. These compounds had profound effects on the structural organization of colony biofilms in both P. aeruginosa and S. coelicolor, which shows that "secondary metabolites" play important conserved roles in gene expression and development

The Pseudomonas aeruginosa Efflux Pump MexGHI-OpmD Transports a Natural Phenazine that Controls Gene Expression and Biofilm Development

Redox-cycling compounds, including endogenously produced phenazine antibiotics, induce expression of the efflux pump MexGHI-OpmD 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 MexGHI-OpmD 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 self-resistance, controlled by a simple circuit, in a Gram-negative pathogen

Spatial alanine metabolism determines local growth dynamics of textitEscherichia 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 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

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 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

Analysis of shared heritability in common disorders of the brain

ience, this issue p. eaap8757 Structured Abstract INTRODUCTION Brain disorders may exhibit shared symptoms and substantial epidemiological comorbidity, inciting debate about their etiologic overlap. However, detailed study of phenotypes with different ages of onset, severity, and presentation poses a considerable challenge. Recently developed heritability methods allow us to accurately measure correlation of genome-wide common variant risk between two phenotypes from pools of different individuals and assess how connected they, or at least their genetic risks, are on the genomic level. We used genome-wide association data for 265,218 patients and 784,643 control participants, as well as 17 phenotypes from a total of 1,191,588 individuals, to quantify the degree of overlap for genetic risk factors of 25 common brain disorders. RATIONALE Over the past century, the classification of brain disorders has evolved to reflect the medical and scientific communities' assessments of the presumed root causes of clinical phenomena such as behavioral change, loss of motor function, or alterations of consciousness. Directly observable phenomena (such as the presence of emboli, protein tangles, or unusual electrical activity patterns) generally define and separate neurological disorders from psychiatric disorders. Understanding the genetic underpinnings and categorical distinctions for brain disorders and related phenotypes may inform the search for their biological mechanisms. RESULTS Common variant risk for psychiatric disorders was shown to correlate significantly, especially among attention deficit hyperactivity disorder (ADHD), bipolar disorder, major depressive disorder (MDD), and schizophrenia. By contrast, neurological disorders appear more distinct from one another and from the psychiatric disorders, except for migraine, which was significantly correlated to ADHD, MDD, and Tourette syndrome. We demonstrate that, in the general population, the personality trait neuroticism is significantly correlated with almost every psychiatric disorder and migraine. We also identify significant genetic sharing between disorders and early life cognitive measures (e.g., years of education and college attainment) in the general population, demonstrating positive correlation with several psychiatric disorders (e.g., anorexia nervosa and bipolar disorder) and negative correlation with several neurological phenotypes (e.g., Alzheimer's disease and ischemic stroke), even though the latter are considered to result from specific processes that occur later in life. Extensive simulations were also performed to inform how statistical power, diagnostic misclassification, and phenotypic heterogeneity influence genetic correlations. CONCLUSION The high degree of genetic correlation among many of the psychiatric disorders adds further evidence that their current clinical boundaries do not reflect distinct underlying pathogenic processes, at least on the genetic level. This suggests a deeply interconnected nature for psychiatric disorders, in contrast to neurological disorders, and underscores the need to refine psychiatric diagnostics. Genetically informed analyses may provide important "scaffolding" to support such restructuring of psychiatric nosology, which likely requires incorporating many levels of information. By contrast, we find limited evidence for widespread common genetic risk sharing among neurological disorders or across neurological and psychiatric disorders. We show that both psychiatric and neurological disorders have robust correlations with cognitive and personality measures. Further study is needed to evaluate whether overlapping genetic contributions to psychiatric pathology may influence treatment choices. Ultimately, such developments may pave the way toward reduced heterogeneity and improved diagnosis and treatment of psychiatric disorders

Physiology and Mechanisms of Pyocyanin Reduction in Pseudomonas aeruginosa

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.</p