Phenazines Regulate Nap-Dependent Denitrification in Pseudomonas aeruginosa Biofilms.

Microbes in biofilms face the challenge of substrate limitation. In particular, oxygen often becomes limited for cells in Pseudomonas aeruginosa biofilms growing in the laboratory or during host colonization. Previously we found that phenazines, antibiotics produced by P. aeruginosa, balance the intracellular redox state of cells in biofilms. Here, we show that genes involved in denitrification are induced in phenazine-null (Δphz) mutant biofilms grown under an aerobic atmosphere, even in the absence of nitrate. This finding suggests that resident cells employ a bet-hedging strategy to anticipate the potential availability of nitrate and counterbalance their highly reduced redox state. Consistent with our previous characterization of aerobically grown colonies supplemented with nitrate, we found that the pathway that is induced in Δphz mutant colonies combines the nitrate reductase activity of the periplasmic enzyme Nap with the downstream reduction of nitrite to nitrogen gas catalyzed by the enzymes Nir, Nor, and Nos. This regulatory relationship differs from the denitrification pathway that functions under anaerobic growth, with nitrate as the terminal electron acceptor, which depends on the membrane-associated nitrate reductase Nar. We identified the sequences in the promoter regions of the nap and nir operons that are required for the effects of phenazines on expression. We also show that specific phenazines have differential effects on nap gene expression. Finally, we provide evidence that individual steps of the denitrification pathway are catalyzed at different depths within aerobically grown biofilms, suggesting metabolic cross-feeding between community subpopulations.IMPORTANCE An understanding of the unique physiology of cells in biofilms is critical to our ability to treat fungal and bacterial infections. Colony biofilms of the opportunistic pathogen Pseudomonas aeruginosa grown under an aerobic atmosphere but without nitrate express a denitrification pathway that differs from that used for anaerobic growth. We report that the components of this pathway are induced by electron acceptor limitation and that they are differentially expressed over the biofilm depth. These observations suggest that (i) P. aeruginosa exhibits "bet hedging," in that it expends energy and resources to prepare for nitrate availability when other electron acceptors are absent, and (ii) cells in distinct biofilm microniches may be able to exchange substrates to catalyze full denitrification.

Bow-tie signaling in c-di-GMP: Machine learning in a simple biochemical network.

Bacteria of many species rely on a simple molecule, the intracellular secondary messenger c-di-GMP (Bis-(3'-5')-cyclic dimeric guanosine monophosphate), to make a vital choice: whether to stay in one place and form a biofilm, or to leave it in search of better conditions. The c-di-GMP network has a bow-tie shaped architecture that integrates many signals from the outside world-the input stimuli-into intracellular c-di-GMP levels that then regulate genes for biofilm formation or for swarming motility-the output phenotypes. How does the 'uninformed' process of evolution produce a network with the right input/output association and enable bacteria to make the right choice? Inspired by new data from 28 clinical isolates of Pseudomonas aeruginosa and strains evolved in laboratory experiments we propose a mathematical model where the c-di-GMP network is analogous to a machine learning classifier. The analogy immediately suggests a mechanism for learning through evolution: adaptation though incremental changes in c-di-GMP network proteins acquires knowledge from past experiences and enables bacteria to use it to direct future behaviors. Our model clarifies the elusive function of the ubiquitous c-di-GMP network, a key regulator of bacterial social traits associated with virulence. More broadly, the link between evolution and machine learning can help explain how natural selection across fluctuating environments produces networks that enable living organisms to make sophisticated decisions.

Electron-shuttling antibiotics structure bacterial communities by modulating cellular levels of c-di-GMP.

Diverse organisms secrete redox-active antibiotics, which can be used as extracellular electron shuttles by resistant microbes. Shuttle-mediated metabolism can support survival when substrates are available not locally but rather at a distance. Such conditions arise in multicellular communities, where the formation of chemical gradients leads to resource limitation for cells at depth. In the pathogenic bacterium Pseudomonas aeruginosa PA14, antibiotics called phenazines act as oxidants to balance the intracellular redox state of cells in anoxic biofilm subzones. PA14 colony biofilms show a profound morphogenic response to phenazines resulting from electron acceptor-dependent inhibition of ECM production. This effect is reminiscent of the developmental responses of some eukaryotic systems to redox control, but for bacterial systems its mechanistic basis has not been well defined. Here, we identify the regulatory protein RmcA and show that it links redox conditions to PA14 colony morphogenesis by modulating levels of bis-(3',5')-cyclic-dimeric-guanosine (c-di-GMP), a second messenger that stimulates matrix production, in response to phenazine availability. RmcA contains four Per-Arnt-Sim (PAS) domains and domains with the potential to catalyze the synthesis and degradation of c-di-GMP. Our results suggest that phenazine production modulates RmcA activity such that the protein degrades c-di-GMP and thereby inhibits matrix production during oxidizing conditions. RmcA thus forms a mechanistic link between cellular redox sensing and community morphogenesis analogous to the functions performed by PAS-domain-containing regulatory proteins found in complex eukaryotes.

Candida albicans ethanol stimulates Pseudomonas aeruginosa WspR-controlled biofilm formation as part of a cyclic relationship involving phenazines.

In chronic infections, pathogens are often in the presence of other microbial species. For example, Pseudomonas aeruginosa is a common and detrimental lung pathogen in individuals with cystic fibrosis (CF) and co-infections with Candida albicans are common. Here, we show that P. aeruginosa biofilm formation and phenazine production were strongly influenced by ethanol produced by the fungus C. albicans. Ethanol stimulated phenotypes that are indicative of increased levels of cyclic-di-GMP (c-di-GMP), and levels of c-di-GMP were 2-fold higher in the presence of ethanol. Through a genetic screen, we found that the diguanylate cyclase WspR was required for ethanol stimulation of c-di-GMP. Multiple lines of evidence indicate that ethanol stimulates WspR signaling through its cognate sensor WspA, and promotes WspR-dependent activation of Pel exopolysaccharide production, which contributes to biofilm maturation. We also found that ethanol stimulation of WspR promoted P. aeruginosa colonization of CF airway epithelial cells. P. aeruginosa production of phenazines occurs both in the CF lung and in culture, and phenazines enhance ethanol production by C. albicans. Using a C. albicans adh1/adh1 mutant with decreased ethanol production, we found that fungal ethanol strongly altered the spectrum of P. aeruginosa phenazines in favor of those that are most effective against fungi. Thus, a feedback cycle comprised of ethanol and phenazines drives this polymicrobial interaction, and these relationships may provide insight into why co-infection with both P. aeruginosa and C. albicans has been associated with worse outcomes in cystic fibrosis.

Redox-driven regulation of microbial community morphogenesis.

During growth on surfaces, diverse microbial communities display topographies with captivating patterns. The quality and quantity of matrix excreted by resident cells play major roles in determining community architecture. Two current publications indicate that the cellular redox state and respiratory activity are important parameters affecting matrix output in the divergent bacteria Pseudomonas aeruginosa and Bacillus subtilis. These and related studies have identified regulatory proteins with the potential to respond to changes in redox state and respiratory electron transport and modulate the activity of the signal transduction pathways that control matrix production. These developments hint at the critical mechanistic links between environmental sensing and community behavior, and provide an exciting new context within which to interpret the molecular details of biofilm structure determination.

Morphological optimization for access to dual oxidants in biofilms.

A major theme driving research in biology is the relationship between form and function. In particular, a longstanding goal has been to understand how the evolution of multicellularity conferred fitness advantages. Here we show that biofilms of the bacterium Pseudomonas aeruginosa produce structures that maximize cellular reproduction. Specifically, we develop a mathematical model of resource availability and metabolic response within colony features. This analysis accurately predicts the measured distribution of two types of electron acceptors: oxygen, which is available from the atmosphere, and phenazines, redox-active antibiotics produced by the bacterium. Using this model, we demonstrate that the geometry of colony structures is optimal with respect to growth efficiency. Because our model is based on resource dynamics, we also can anticipate shifts in feature geometry based on changes to the availability of electron acceptors, including variations in the external availability of oxygen and genetic manipulation that renders the cells incapable of phenazine production.

Control of Candida albicans metabolism and biofilm formation by Pseudomonas aeruginosa phenazines.

Candida albicans has developmental programs that govern transitions between yeast and filamentous morphologies and between unattached and biofilm lifestyles. Here, we report that filamentation, intercellular adherence, and biofilm development were inhibited during interactions between Candida albicans and Pseudomonas aeruginosa through the action of P. aeruginosa-produced phenazines. While phenazines are toxic to C. albicans at millimolar concentrations, we found that lower concentrations of any of three different phenazines (pyocyanin, phenazine methosulfate, and phenazine-1-carboxylate) allowed growth but affected the development of C. albicans wrinkled colony biofilms and inhibited the fungal yeast-to-filament transition. Phenazines impaired C. albicans growth on nonfermentable carbon sources and led to increased production of fermentation products (ethanol, glycerol, and acetate) in glucose-containing medium, leading us to propose that phenazines specifically inhibited respiration. Methylene blue, another inhibitor of respiration, also prevented the formation of structured colony biofilms. The inhibition of filamentation and colony wrinkling was not solely due to lowered extracellular pH induced by fermentation. Compared to smooth, unstructured colonies, wrinkled colony biofilms had higher oxygen concentrations within the colony, and wrinkled regions of these colonies had higher levels of respiration. Together, our data suggest that the structure of the fungal biofilm promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by bacterial molecules such as phenazines or compounds with similar activities disrupts these pathways. These findings may suggest new ways to limit fungal biofilms in the context of disease. IMPORTANCE Many of the infections caused by Candida albicans, a major human opportunistic fungal pathogen, involve both morphological transitions and the formation of surface-associated biofilms. Through the study of C. albicans interactions with the bacterium Pseudomonas aeruginosa, which often coinfects with C. albicans, we have found that P. aeruginosa-produced phenazines modulate C. albicans metabolism and, through these metabolic effects, impact cellular morphology, cell-cell interactions, and biofilm formation. We suggest that the structure of C. albicans biofilms promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by phenazines inhibits biofilm development. Our findings not only provide insight into interactions between these species but also provide valuable insights into novel pathways that could lead to the development of new therapies to treat C. albicans infections.

Bacterial community morphogenesis is intimately linked to the intracellular redox state.

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.

The carbon monoxide releasing molecule CORM-2 attenuates Pseudomonas aeruginosa biofilm formation.

Chronic infections resulting from biofilm formation are difficult to eradicate with current antimicrobial agents and consequently new therapies are needed. This work demonstrates that the carbon monoxide-releasing molecule CORM-2, previously shown to kill planktonic bacteria, also attenuates surface-associated growth of the gram-negative pathogen Pseudomonas aeruginosa by both preventing biofilm maturation and killing bacteria within the established biofilm. CORM-2 treatment has an additive effect when combined with tobramycin, a drug commonly used to treat P. aeruginosa lung infections. CORM-2 inhibited biofilm formation and planktonic growth of the majority of clinical P. aeruginosa isolates tested, for both mucoid and non-mucoid strains. While CORM-2 treatment increased the production of reactive oxygen species by P. aeruginosa biofilms, this increase did not correlate with bacterial death. These data demonstrate that CO-RMs possess potential novel therapeutic properties against a subset of P. aeruginosa biofilm related infections.

Redox eustress: roles for redox-active metabolites in bacterial signaling and behavior.

SIGNIFICANCE: Plant biologists and microbiologists have long discussed and debated the physiological roles of so-called "redox-active metabolites." These are natural products with unusually high redox activity that are not directly required for active growth. Generally, the biological roles of these compounds have been ascribed to interspecies competition and virulence, and they have been considered important sources of distress. RECENT ADVANCES: In this review, we discuss two examples of redox-active metabolites: nitric oxide and phenazines. Both are known for their toxic effects in some organisms and conditions but have recently been shown to provide benefits for some organisms under other conditions. CRITICAL ISSUES: Biologists are identifying new roles for redox-active metabolites that are not directly related to their toxicity. These roles prompt us to suggest a dismissal of the paradigm that all biological stress is negative (i.e., distress). FUTURE DIRECTIONS: A more accurate view of redox couples requires characterization of their specific biological effects in a condition-dependent manner. The responses to these compounds can be termed "distress" or "eustress," depending on whether they inhibit survival, provide protection from a compound that would otherwise inhibit survival, or promote survival.