A molecular mechanism that stabilizes cooperative secretions in Pseudomonas aeruginosa.
ABSTRACT: Bacterial populations frequently act as a collective by secreting a wide range of compounds necessary for cell-cell communication, host colonization and virulence. How such behaviours avoid exploitation by spontaneous 'cheater' mutants that use but do not contribute to secretions remains unclear. We investigate this question using Pseudomonas aeruginosa swarming, a collective surface motility requiring massive secretions of rhamnolipid biosurfactants. We first show that swarming is immune to the evolution of rhlA(-) 'cheaters'. We then demonstrate that P. aeruginosa resists cheating through metabolic prudence: wild-type cells secrete biosurfactants only when the cost of their production and impact on individual fitness is low, therefore preventing non-secreting strains from gaining an evolutionary advantage. Metabolic prudence works because the carbon-rich biosurfactants are only produced when growth is limited by another growth limiting nutrient, the nitrogen source. By genetically manipulating a strain to produce the biosurfactants constitutively we show that swarming becomes cheatable: a non-producing strain rapidly outcompetes and replaces this obligate cooperator. We argue that metabolic prudence, which may first evolve as a direct response to cheating or simply to optimize growth, can explain the maintenance of massive secretions in many bacteria. More generally, prudent regulation is a mechanism to stabilize cooperation.
Project description:Many unicellular organisms live in multicellular communities that rely on cooperation between cells. However, cooperative traits are vulnerable to exploitation by non-cooperators (cheaters). We expand our understanding of the molecular mechanisms that allow multicellular systems to remain robust in the face of cheating by dissecting the dynamic regulation of cooperative rhamnolipids required for swarming in Pseudomonas aeruginosa. We combine mathematical modeling and experiments to quantitatively characterize the integration of metabolic and population density signals (quorum sensing) governing expression of the rhamnolipid synthesis operon rhlAB. The combined computational/experimental analysis reveals that when nutrients are abundant, rhlAB promoter activity increases gradually in a density dependent way. When growth slows down due to nutrient limitation, rhlAB promoter activity can stop abruptly, decrease gradually or even increase depending on whether the growth-limiting nutrient is the carbon source, nitrogen source or iron. Starvation by specific nutrients drives growth on intracellular nutrient pools as well as the qualitative rhlAB promoter response, which itself is modulated by quorum sensing. Our quantitative analysis suggests a supply-driven activation that integrates metabolic prudence with quorum sensing in a non-digital manner and allows P. aeruginosa cells to invest in cooperation only when the population size is large enough (quorum sensing) and individual cells have enough metabolic resources to do so (metabolic prudence). Thus, the quantitative description of rhlAB regulatory dynamics brings a greater understating to the regulation required to make swarming cooperation stable.
Project description:How does metabolism influence social behavior? This fundamental question at the interface of molecular biology and social evolution is hard to address with experiments in animals, and therefore, we turned to a simple microbial system: swarming in the bacterium Pseudomonas aeruginosa. Using genetic engineering, we excised a locus encoding a key metabolic regulator and disrupted P. aeruginosa's metabolic prudence, the regulatory mechanism that controls expression of swarming public goods and protects this social behavior from exploitation by cheaters. Then, using experimental evolution, we followed the joint evolution of the genome, the metabolome and the social behavior as swarming re-evolved. New variants emerged spontaneously with mutations that reorganized the metabolome and compensated in distinct ways for the disrupted metabolic prudence. These experiments with a unicellular organism provide a detailed view of how metabolism-currency of all physiological processes-can determine the costs and benefits of a social behavior and ultimately influence how an organism behaves towards other organisms of the same species.
Project description:Cooperation has fascinated biologists since Darwin. How did cooperative behaviors evolve despite the fitness cost to the cooperator? Bacteria have cooperative behaviors that make excellent models to take on this age-old problem from both proximate (molecular) and ultimate (evolutionary) angles. We delve into Pseudomonas aeruginosa swarming, a phenomenon where billions of bacteria move cooperatively across distances of centimeters in a matter of a few hours. Experiments with swarming have unveiled a strategy called metabolic prudence that stabilizes cooperation, have showed the importance of spatial structure, and have revealed a regulatory network that integrates environmental stimuli and direct cooperative behavior, similar to a machine learning algorithm. The study of swarming elucidates more than proximate mechanisms: It exposes ultimate mechanisms valid to all scales, from cells in cancerous tumors to animals in large communities.
Project description:Pseudomonas aeruginosa is an opportunistic pathogen that can cause a wide range of infections and inflammations in a variety of hosts, such as chronic biofilm associated lung infections in Cystic Fibrosis patients. Phosphate, an essential nutrient, has been recognized as an important signal that affects virulence in P. aeruginosa. In the current study we examined the connection between phosphate regulation and surface motility in P. aeruginosa. We focused on two important genes, pstS, which is involved in phosphate uptake, and phoB, a central regulator that responds to phosphate starvation. We found that a mutant lacking pstS is constantly starved for phosphate and has a hyper swarming phenotype. Phosphate starvation also induced swarming in the wild type. The phoB mutant, on the other hand, did not express phosphate starvation even when phosphate was limited and showed no swarming. A double mutant lacking both genes (pstS and phoB) showed a similar phenotype to the phoB mutant (i.e. no swarming). This highlights the role of phoB in controlling swarming motility under phosphate-depleted conditions. Finally, we were able to demonstrate that PhoB controls swarming by up-regulating the Rhl quorum sensing system in P. aeruginosa, which resulted in hyper production of rhamonlipids: biosurfactants that are known to induce swarming motility.
Project description:Swarming is a surface-associated motile behavior that plays an important role in the rapid spread, colonization, and subsequent establishment of bacterial communities. In Pseudomonas aeruginosa, swarming is dependent upon a functional flagella and aided by the production of biosurfactants. AmrZ, a conserved transcription factor across pseudomonads, has been shown to be a global regulator of multiple genes important for virulence and ecological fitness. In this study, we expand this concept of global control to swarming motility by showing that deletion of amrZ results in a severe defect in swarming, while multicopy expression of this gene stimulates swarming of P. aeruginosa. Mechanistic studies showed that the swarming defect of an amrZ mutant does not involve changes of biosurfactant production but is associated with flagellar malfunction. The ?amrZ mutant exhibits increased levels of the second messenger cyclic di-GMP (c-di-GMP) compared to the wild-type strain, under swarming conditions. We found that the diguanylate cyclase GcbA was the main contributor to the increased accumulation of c-di-GMP observed in the ?amrZ mutant and was a strong inhibitor of flagellar-dependent motility. Our results revealed that the GcbA-dependent inhibition of motility required the presence of two c-di-GMP receptors containing a PilZ domain: FlgZ and PA14_56180. Furthermore, the ?amrZ mutant exhibits enhanced production of Pel polysaccharide. Epistasis analysis revealed that GcbA and the Pel polysaccharide act independently to limit swarming in ?amrZ. Our results support a role for AmrZ in controlling swarming motility, yet another social behavior besides biofilm formation that is crucial for the ability of P. aeruginosa to colonize a variety of surfaces. The central role of AmrZ in controlling these behaviors makes it a good target for the development of treatments directed to combat P. aeruginosa infections.
Project description:Pseudomonas aeruginosa is a ubiquitous bacterium that survives in many environments, including as an acute and chronic pathogen in humans. Substantial evidence shows that P. aeruginosa behavior is affected by its motility, and appendages known as flagella and type IV pili (TFP) are known to confer such motility. The role these appendages play when not facilitating motility or attachment, however, is unclear. Here we discern a passive intercellular role of TFP during flagellar-mediated swarming of P. aeruginosa that does not require TFP extension or retraction. We studied swarming at the cellular level using a combination of laboratory experiments and computational simulations to explain the resultant patterns of cells imaged from in vitro swarms. Namely, we used a computational model to simulate swarming and to probe for individual cell behavior that cannot currently be otherwise measured. Our simulations showed that TFP of swarming P. aeruginosa should be distributed all over the cell and that TFP-TFP interactions between cells should be a dominant mechanism that promotes cell-cell interaction, limits lone cell movement, and slows swarm expansion. This predicted physical mechanism involving TFP was confirmed in vitro using pairwise mixtures of strains with and without TFP where cells without TFP separate from cells with TFP. While TFP slow swarm expansion, we show in vitro that TFP help alter collective motion to avoid toxic compounds such as the antibiotic carbenicillin. Thus, TFP physically affect P. aeruginosa swarming by actively promoting cell-cell association and directional collective motion within motile groups to aid their survival.
Project description:<i>Pseudomonas aeruginosa</i> is among the many bacteria that swarm, where groups of cells coordinate to move over surfaces. It has been challenging to determine the behavior of single cells within these high-cell-density swarms. To track individual cells within <i>P. aeruginosa</i> swarms, we imaged a fluorescently labeled subset of the larger population. Single cells at the advancing swarm edge varied in their motility dynamics as a function of time. From these data, we delineated four phases of early swarming prior to the formation of the tendril fractals characteristic of <i>P. aeruginosa</i> swarming by collectively considering both micro- and macroscale data. We determined that the period of greatest single-cell motility does not coincide with the period of greatest collective swarm expansion. We also noted that flagellar, rhamnolipid, and type IV pilus motility mutants exhibit substantially less single-cell motility than the wild type.<b>IMPORTANCE</b> Numerous bacteria exhibit coordinated swarming motion over surfaces. It is often challenging to assess the behavior of single cells within swarming communities due to the limitations of identifying, tracking, and analyzing the traits of swarming cells over time. Here, we show that the behavior of <i>Pseudomonas aeruginosa</i> swarming cells can vary substantially in the earliest phases of swarming. This is important to establish that dynamic behaviors should not be assumed to be constant over long periods when predicting and simulating the actions of swarming bacteria.
Project description: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.
Project description:Cooperative behaviors provide a collective benefit, but are considered costly for the individual. Here, we report that these costs vary dramatically in different contexts and have opposing effects on the selection for non-cooperating cheaters. We investigate a prominent example of bacterial cooperation, the secretion of the peptide siderophore pyoverdine by Pseudomonas aeruginosa, under different nutrient-limiting conditions. Using metabolic modeling, we show that pyoverdine incurs a fitness cost only when its building blocks carbon or nitrogen are growth-limiting and are diverted from cellular biomass production. We confirm this result experimentally with a continuous-culture approach. We show that pyoverdine non-producers (cheaters) enjoy a large fitness advantage in co-culture with producers (cooperators) and spread to high frequency when limited by carbon, but not when limited by phosphorus. The principle of nutrient-dependent fitness costs has implications for the stability of cooperation in pathogenic and non-pathogenic environments, in biotechnological applications, and beyond the microbial realm.Cooperative behaviour among individuals provides a collective benefit, but is considered costly. Using Pseudomonas aeruginosa as a model system, the authors show that secretion of the siderophore pyoverdine only incurs a fitness cost and favours cheating when its building blocks carbon or nitrogen are growth-limiting.
Project description:We investigate the effect of bacteriophage infection and antibiotic treatment on the coordination of swarming, a collective form of flagellum- and pilus-mediated motility in bacteria. We show that phage infection of the opportunistic bacterial pathogen <i>Pseudomonas aeruginosa</i> abolishes swarming motility in the infected subpopulation and induces the release of the <i>Pseudomonas</i> quinolone signaling molecule PQS, which repulses uninfected subpopulations from approaching the infected area. These mechanisms have the overall effect of limiting the infection to a subpopulation, which promotes the survival of the overall population. Antibiotic treatment of <i>P. aeruginosa</i> elicits the same response, abolishing swarming motility and repulsing approaching swarms away from the antibiotic-treated area through a PQS-dependent mechanism. Swarms are entirely repelled from the zone of antibiotic-treated <i>P. aeruginosa</i>, consistent with a form of antibiotic evasion, and are not repelled by antibiotics alone. PQS has multiple functions, including serving as a quorum-sensing molecule, activating an oxidative stress response, and regulating the release of virulence and host-modifying factors. We show that PQS serves additionally as a stress warning signal that causes the greater population to physically avoid cell stress. The stress response at the collective level observed here in <i>P. aeruginosa</i> is consistent with a mechanism that promotes the survival of bacterial populations.<b>IMPORTANCE</b> We uncover a phage- and antibiotic-induced stress response in the clinically important opportunistic pathogen <i>Pseudomonas aeruginosa</i> Phage-infected <i>P. aeruginosa</i> subpopulations are isolated from uninfected subpopulations by the production of a stress-induced signal. Activation of the stress response by antibiotics causes <i>P. aeruginosa</i> to physically be repelled from the area containing antibiotics altogether, consistent with a mechanism of antibiotic evasion. The stress response observed here could increase <i>P. aeruginosa</i> resilience against antibiotic treatment and phage therapy in health care settings, as well as provide a simple evolutionary strategy to avoid areas containing stress.