Convergent Evolution of Hyperswarming Leads to Impaired Biofilm Formation in Pathogenic Bacteria

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 toward 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 monoflagellated bacteria in a multiflagellated state and confers a growth rate-independent advantage in swarming. Although 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 trade-off between motility and biofilm formation

Integrated circuit-based electrochemical sensor for spatially resolved detection of redox-active metabolites in biofilms

Despite advances in monitoring spatiotemporal expression patterns of genes and proteins with fluorescent probes, direct detection of metabolites and small molecules remains challenging. A technique for spatially resolved detection of small molecules would benefit the study of redox-active metabolites that are produced by microbial biofilms and can affect their development. Here we present an integrated circuit-based electrochemical sensing platform featuring an array of working electrodes and parallel potentiostat channels. ‘Images’ over a 3.25 0.9 mm2 area can be captured with a diffusion-limited spatial resolution of 750 mm. We demonstrate that square wave voltammetry can be used to detect, identify and quantify (for concentrations as low as 2.6 mM) four distinct redox-active metabolites called phenazines. We characterize phenazine production in both wild-type and mutant Pseudomonas aeruginosa PA14 colony biofilms, and find correlations with fluorescent reporter imaging of phenazine biosynthetic gene expression

Proteinase K goes thermo-labile

Proteinase K, originally from the fungus Tritirachium album, is a highly active serine protease with broad cleavage specificity. This enzyme is widely used to remove proteins/enzymes in nucleic acid samples. However, use of wildtype proteinase K (WTPK) in multi-step enzymatic workflows such as next generation sequencing (NGS) is limited due to its extreme thermostability and ineffective removal by heat treatment. The purpose of this study was to engineer a thermolabile Proteinase K (TLPK) as active as WTPK, which may be fully inactivated at 65°C or below to minimize DNA/RNA damage. Using molecular engineering approaches, we have successfully obtained TLPK. As shown in Figure 1, TLPK is almost as active as WTPK at 37°C using native bovine serum albumin (BSA) as substrate. Importantly, TLPK can be efficiently inactivated within the temperature range of 55°C to 65°C, which is demonstrated by loss of protease activity on bovine serum albumin (BSA) substrate (Figure 2a) and a colorimetric peptide substrate (Figure 2b) after heat treatment. Compared to WTPK, TLPK shows over 20°C more labile to heat inactivation. The melting temperature (Tm) of TLPK is also around 25°C lower than that of WTPK, decreasing from 75.9°C to 50.9°C. TLPK greatly outperforms a broad specificity protease isolated from an arctic marine microbial source, both by specific enzyme activity and thermolability. One of the TLPK applications is it can inactivate heat resistant restriction enzymes such as PvuII and PstI without affecting downstream reactions. The mainstream applications may be its incorporation into multi-step enzymatic workflows such as NGS sample preparation. Unlike WTPK, TLPK can be used to eliminate an enzyme function without contaminating the next enzymatic step in the same reaction vessel. New England Biolabs has tested TLPK and found it to simplify and improve NGS workflows. Please click Additional Files below to see the full abstract

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

Multidrug efflux pumps:structure, function and regulation

Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this Review, we describe recent advances that have increased our understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria. Clinical and laboratory data indicate that efflux pumps function not only in the drug extrusion process but also in virulence and the adaptive responses that contribute to antimicrobial resistance during infection. The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities

Physiology of Pseudomonas Aeruginosa Phenazine Production and Transport

Many bacteria secrete secondary metabolites, whose production is decoupled from active growth in laboratory cultures. Historically, the advantages of secondary metabolite production have mostly been explored in the context of cellular interactions, such as antibiotic effects on competing organisms, damage caused to host tissues during infection, or cell density-dependent signaling. However, recent studies in the opportunistic pathogen Pseudomonas aeruginosa have brought into focus the physiological effects of secondary metabolites on their producer and their implications for multicellular behavior. P. aeruginosa produces antibiotics called phenazines, which can act as mediators to transfer reducing power to an extracellular oxidant and thereby support bacterial survival when oxygen is not accessible. In the crowded environments of biofilms, communities of bacteria surrounded by self-made matrices, this property of phenazines could support energy generation for cells in anoxic subzones. As biofilm formation is a hallmark of P. aeruginosa colonization at various infection sites within the body, I was motivated to investigate the regulation of phenazine production at the level of synthesis and transport, the distribution of phenazines in P. aeruginosa biofilms, and the effects of individual phenazines on P. aeruginosa gene expression and colony biofilm morphogenesis. As part of this work, a novel electrochemical device was developed that enables direct detection of phenazines released from intact colony biofilms. Application of this device and other electrochemical techniques enabled detection of the reactive phenazine intermediate 5-Me-PCA, which was found to be the primary phenazine affecting P. aeruginosa colony morphogenesis. The production of this phenazine was found to be sufficient for activation of the redox-active transcription factor SoxR and full induction of the RND efflux pump MexGHI-OpmD. Finally, results described in this thesis show that 5-Me-PCA is transported by MexGHI-OpmD, constituting a unique demonstration of the self-protective role of an efflux pump in a gram-negative antibiotic-producing bacterium. These findings raise broad questions about the effects of individual phenazines on biofilm cell physiology and have implications for the contributions of individual phenazines to virulence and survival during infection. The technology developed also has potential applications in novel diagnostic and therapeutic approaches. Chapters 1-3 introduce and highlight advances made in understanding secondary metabolite production, with a focus on P. aeruginosa. Chapter 1 provides an introduction to antibiotic production, the concept of self-resistance and other physiological effects of antibiotics in their producers, and infections caused by P. aeruginosa. Chapter 2 reviews recent studies that have brought into focus the physiological effects of secondary metabolites on their producers and their implications for multicellular behavior. Chapter 3 provides an overview of our current understanding of the regulation of phenazine production in pseudomonads and other bacterial species. Chapter 4 describes the development of an integrated circuit-based platform for detection of redox-active metabolites released from multicellular samples, and demonstrates its application to mapping phenazines released from P. aeruginosa biofilms. The study described in Chapter 5 investigates the role of the P. aeruginosa SoxR regulon, which is induced by phenazines, in phenazine transport and shows that the understudied reactive phenazine 5-methylphenazine-1-carboxylic acid (5-Me-PCA) is transported by the RND efflux pump MexGHI-OpmD and is required for wild-type biofilm formation. Chapter 6 describes the development of an assay for 5-Me-PCA production and studies exploring the role of the regulator PsrA in controlling phenazine biosynthesis. Chapter 7 provides an overview of the findings and open questions to be explored in future research. The P. aeruginosa genome contains two nearly identical operons that encode biosynthetic enzymes for the production of phenazine-1-carboxylic acid, the precursor to all of the other phenazines. The study described in Appendix A characterizes the respective contributions of these operons to phenazine production in shaken liquid cultures and biofilms. Appendix B presents evidence that electron acceptor availability influences, and is influenced by, the morphogenesis of P. aeruginosa colony biofilms. Finally, Appendix C describes a screen for commercially available compounds that inhibit production of the phenazine pyocyanin by P. aeruginosa. Together, these findings reveal the unique physiological roles of specific phenazine-related genetic loci and regulatory proteins and of 5-Me-PCA, a phenazine that was previously overlooked due to the technical challenges associated with its detection. They have also uncovered novel aspects of phenazine production in both shaken liquid cultures and biofilms relevant for the development of therapeutics

Microbial Phenazines: Biosynthesis, Agriculture and Health

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

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

Testing biological hypotheses with embodied robots: adaptations, accidents, and by-products in the evolution of vertebrates

Evolutionary robotics allows biologists to test hypotheses about extinct animals. In our case, we modeled some of the first vertebrates, jawless fishes, in order to study the evolution of the trait after which vertebrates are named: vertebrae.

Convergent Evolution of Hyperswarming Leads to Impaired Biofilm Formation in Pathogenic Bacteria

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 toward 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 monoflagellated bacteria in a multiflagellated state and confers a growth rate-independent advantage in swarming. Although 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 trade-off between motility and biofilm formation