Physiological Concentrations of Calcium Interact with Alginate and Extracellular DNA in the Matrices of Pseudomonas aeruginosa Biofilms to Impede Phagocytosis by Neutrophils.

Biofilms are communities of interacting microbes embedded in a matrix of polymer, protein, and other materials. Biofilms develop distinct mechanical characteristics that depend on their predominant matrix components. These matrix components may be produced by microbes themselves or, for infections in vivo, incorporated from the host environment. Pseudomonas aeruginosa (P. aeruginosa) is a human pathogen that forms robust biofilms that extensively tolerate antibiotics and effectively evade clearance by the immune system. Two of the important bacterial-produced polymers in the matrices of P. aeruginosa biofilms are alginate and extracellular DNA (eDNA), both of which are anionic and therefore have the potential to interact electrostatically with cations. Many physiological sites of infection contain significant concentrations of the calcium ion (Ca2+). In this study, we investigate the structural and mechanical impacts of Ca2+ supplementation in alginate-dominated biofilms grown in vitro, and we evaluate the impact of targeted enzyme treatments on clearance by immune cells. We use multiple-particle tracking microrheology to evaluate the changes in biofilm viscoelasticity caused by treatment with alginate lyase or DNase I. For biofilms grown without Ca2+, we correlate a decrease in relative elasticity with increased phagocytic success. However, we find that growth with Ca2+ supplementation disrupts this correlation except in the case where both enzymes are applied. This suggests that the calcium cation may be impacting the microstructure of the biofilm in nontrivial ways. Indeed, confocal laser scanning fluorescence microscopy and scanning electron microscopy reveal unique Ca2+-dependent eDNA and alginate microstructures. Our results suggest that the presence of Ca2+ drives the formation of structurally and compositionally discrete microdomains within the biofilm through electrostatic interactions with the anionic matrix components eDNA and alginate. Further, we observe that these structures serve a protective function as the dissolution of both components is required to render biofilm bacteria vulnerable to phagocytosis by neutrophils.

Physiological concentrations of calcium interact with alginate and extracellular DNA in the matrices of Pseudomonas aeruginosa biofilms to impede phagocytosis by neutrophils.

Biofilms are communities of interacting microbes embedded in a matrix of polymer, protein, and other materials. Biofilms develop distinct mechanical characteristics that depend on their predominant matrix components. These matrix components may be produced by microbes themselves or, for infections in vivo, incorporated from the host environment. Pseudomonas aeruginosa is a human pathogen that forms robust biofilms that extensively tolerate antibiotics and effectively evade clearance by the immune system. Two of the important bacterial-produced polymers in the matrices of P. aeruginosa biofilms are alginate and extracellular DNA (eDNA), both of which are anionic and therefore have the potential to interact electrostatically with cations. Many physiological sites of infection contain significant concentrations of the calcium ion (Ca2+). In this study we investigate the structural and mechanical impacts of Ca2+ supplementation in alginate-dominated biofilms grown in vitro and we evaluate the impact of targeted enzyme treatments on clearance by immune cells. We use multiple particle tracking microrheology to evaluate the changes in biofilm viscoelasticity caused by treatment with alginate lyase and/or DNAse I. For biofilms grown without Ca2+, we correlate a decrease in relative elasticity with increased phagocytic success. However, we find that growth with Ca2+ supplementation disrupts this correlation except in the case where both enzymes are applied. This suggests that the calcium cation may be impacting the microstructure of the biofilm in non-trivial ways. Indeed, confocal laser scanning fluorescence microscopy and scanning electron microscopy reveal unique Ca2+-dependent eDNA and alginate microstructures. Our results suggest that the presence of Ca2+ drives the formation of structurally and compositionally discrete microdomains within the biofilm through electrostatic interactions with the anionic matrix components eDNA and alginate. Further, we observe that these structures serve a protective function as the dissolution of both components is required to render biofilm bacteria vulnerable to phagocytosis by neutrophils.

Incorporation of collagen into Pseudomonas aeruginosa and Staphylococcus aureus biofilms impedes phagocytosis by neutrophils.

Biofilms are communities of microbes embedded in a matrix of extracellular polymeric substances (EPS). Matrix components can be produced by biofilm organisms and can also originate from the environment and then be incorporated into the biofilm. For example, we have recently shown that collagen, a host-produced protein that is abundant in many different infection sites, can be taken up into the biofilm matrix, altering biofilm mechanics. The biofilm matrix protects bacteria from clearance by the immune system, and some of that protection likely arises from the mechanical properties of the biofilm. Pseudomonas aeruginosa and Staphylococcus aureus are common human pathogens notable for forming biofilm infections in anatomical sites rich in collagen. Here, we show that the incorporation of Type I collagen into P. aeruginosa and S. aureus biofilms significantly hinders phagocytosis of biofilm bacteria by human neutrophils. However, enzymatic treatment with collagenase, which breaks down collagen, can partly or entirely negate the protective effect of collagen and restore the ability of neutrophils to engulf biofilm bacteria. From these findings, we suggest that enzymatic degradation of host materials may be a potential way to compromise biofilm infections and enhance the efficacy of the host immune response without promoting antibiotic resistance. Such an approach might be beneficial both in cases where the infecting species is known and also in cases wherein biofilm components are not readily known, such as multispecies infections or infections by unknown species.

The accumulation and growth of Pseudomonas aeruginosa on surfaces is modulated by surface mechanics via cyclic-di-GMP signaling.

Attachment of bacteria onto a surface, consequent signaling, and accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms. While recent reports have hinted that surface mechanics may affect the accumulation of bacteria on that surface, the processes that underlie bacterial perception of surface mechanics and modulation of accumulation in response to surface mechanics remain largely unknown. We use thin and thick hydrogels coated on glass to create composite materials with different mechanics (higher elasticity for thin composites; lower elasticity for thick composites) but with the same surface adhesivity and chemistry. The mechanical cue stemming from surface mechanics is elucidated using experiments with the opportunistic human pathogen Pseudomonas aeruginosa combined with finite-element modeling. Adhesion to thin composites results in greater changes in mechanical stress and strain in the bacterial envelope than does adhesion to thick composites with identical surface chemistry. Using quantitative microscopy, we find that adhesion to thin composites also results in higher cyclic-di-GMP levels, which in turn result in lower motility and less detachment, and thus greater accumulation of bacteria on the surface than does adhesion to thick composites. Mechanics-dependent c-di-GMP production is mediated by the cell-surface-exposed protein PilY1. The biofilm lag phase, which is longer for bacterial populations on thin composites than on thick composites, is also mediated by PilY1. This study shows clear evidence that bacteria actively regulate differential accumulation on surfaces of different stiffnesses via perceiving varied mechanical stress and strain upon surface engagement.

Bacterial mechanosensing of surface stiffness promotes signaling and growth leading to biofilm formation by Pseudomonas aeruginosa.

The attachment of bacteria onto a surface, consequent signaling, and the accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms. While recent reports have hinted that the stiffness of a surface may affect the accumulation of bacteria on that surface, the processes that underlie bacterial perception of and response to surface stiffness are unknown. Furthermore, whether, and how, the surface stiffness impacts biofilm development, after initial accumulation, is not known. We use thin and thick hydrogels to create stiff and soft composite materials, respectively, with the same surface chemistry. Using quantitative microscopy, we find that the accumulation, motility, and growth of the opportunistic human pathogen Pseudomonas aeruginosa respond to surface stiffness, and that these are linked through cyclic-di-GMP signaling that depends on surface stiffness. The mechanical cue stemming from surface stiffness is elucidated using finite-element modeling combined with experiments - adhesion to stiffer surfaces results in greater changes in mechanical stress and strain in the bacterial envelope than does adhesion to softer surfaces with identical surface chemistry. The cell-surface-exposed protein PilY1 acts as a mechanosensor, that upon surface engagement, results in higher cyclic-di-GMP levels, lower motility, and greater accumulation on stiffer surfaces. PilY1 impacts the biofilm lag phase, which is extended for bacteria attaching to stiffer surfaces. This study shows clear evidence that bacteria actively respond to different stiffness of surfaces where they adhere via perceiving varied mechanical stress and strain upon surface engagement.

Microrheology of Pseudomonas aeruginosa biofilms grown in wound beds.

A new technique was used to measure the viscoelasticity of in vivo Pseudomonas aeruginosa biofilms. This was done through ex vivo microrheology measurements of in vivo biofilms excised from mouse wound beds. To our knowledge, this is the first time that the mechanics of in vivo biofilms have been measured. In vivo results are then compared to typical in vitro measurements. Biofilms grown in vivo are more relatively elastic than those grown in a wound-like medium in vitro but exhibited similar compliance. Using various genetically mutated P. aeruginosa strains, it is observed that the contributions of the exopolysaccharides Pel, Psl, and alginate to biofilm viscoelasticity were different for the biofilms grown in vitro and in vivo. In vitro experiments with collagen containing medium suggest this likely arises from the incorporation of host material, most notably collagen, into the matrix of the biofilm when it is grown in vivo. Taken together with earlier studies that examined the in vitro effects of collagen on mechanical properties, we conclude that collagen may, in some cases, be the dominant contributor to biofilm viscoelasticity in vivo.

High-throughput assays show the timescale for phagocytic success depends on the target toughness.

Phagocytic immune cells can clear pathogens from the body by engulfing them. Bacterial biofilms are communities of bacteria that are bound together in a matrix that gives biofilms viscoelastic mechanical properties that do not exist for free-swimming bacteria. Since a neutrophil is too small to engulf an entire biofilm, it must be able to detach and engulf a few bacteria at a time if it is to use phagocytosis to clear the infection. We recently found a negative correlation between the target elasticity and phagocytic success. That earlier work used time-consuming, manual analysis of micrographs of neutrophils and fluorescent beads. Here, we introduce and validate flow cytometry as a fast and high-throughput technique that increases the number of neutrophils analyzed per experiment by two orders of magnitude, while also reducing the time required to do so from hours to minutes. We also introduce the use of polyacrylamide gels in our assay for engulfment success. The tunability of polyacrylamide gels expands the mechanical parameter space we can study, and we find that high toughness and yield strain, even with low elasticity, also impact the phagocytic success as well as the timescale thereof. For stiff gels with low-yield strain, and consequent low toughness, phagocytic success is nearly four times greater when neutrophils are incubated with gels for 6 h than after only 1 h of incubation. In contrast, for soft gels with high-yield strain and consequent high toughness, successful engulfment is much less time-sensitive, increasing by less than a factor of two from 1 to 6 h incubation.

Effect of collagen and EPS components on the viscoelasticity of Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa is an opportunistic pathogen that causes thousands of deaths every year in part due to its ability to form biofilms composed of bacteria embedded in a matrix of self-secreted extracellular polysaccharides (EPS), e-DNA, and proteins. In chronic wounds, biofilms are exposed to the host extracellular matrix, of which collagen is a major component. How bacterial EPS interacts with host collagen and whether this interaction affects biofilm viscoelasticity is not well understood. Since physical disruption of biofilms is often used in their removal, knowledge of collagen's effects on biofilm viscoelasticity may enable new treatment strategies that are better tuned to biofilms growing in host environments. In this work, biofilms are grown in the presence of different concentrations of collagen that mimic in vivo conditions. In order to explore collagen's interaction with EPS, nine strains of P. aeruginosa with different patterns of EPS production were used to grow biofilms. Particle tracking microrheology was used to characterize the mechanical development of biofilms over two days. Collagen is found to decrease biofilm compliance and increase relative elasticity regardless of the EPS present in the system. However, this effect is minimized when biofilms overproduce EPS. Collagen appears to become a de facto component of the EPS, through binding to bacteria or physical entanglement.

Quantitative confocal microscopy and calibration for measuring differences in cyclic-di-GMP signalling by bacteria on biomedical hydrogels.

The growth of bacterial biofilms on implanted medical devices causes harmful infections and device failure. Biofilm development initiates when bacteria attach to and sense a surface. For the common nosocomial pathogen Pseudomonas aeruginosa and many others, the transition to the biofilm phenotype is controlled by the intracellular signal and second messenger cyclic-di-GMP (c-di-GMP). It is not known how biomedical materials might be adjusted to impede c-di-GMP signalling, and there are few extant methods for conducting such studies. Here, we develop such a method. We allowed P. aeruginosa to attach to the surfaces of poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These bacteria contained a plasmid for a green fluorescent protein (GFP) reporter for c-di-GMP. We used laser-scanning confocal microscopy to measure the dynamics of the GFP reporter for 3 h, beginning 1 h after introducing bacteria to the hydrogel. We controlled for the effects of changes in bacterial metabolism using a promoterless plasmid for GFP, and for the effects of light passing through different hydrogels being differently attenuated by using fluorescent plastic beads as 'standard candles' for calibration. We demonstrate that this method can measure statistically significant differences in c-di-GMP signalling associated with different PEGDA gel types and with the surface-exposed protein PilY1.

Suspended multiwalled, acid-functionalized carbon nanotubes promote aggregation of the opportunistic pathogen Pseudomonas aeruginosa.

The increasing prevalence of carbon nanotubes (CNTs) as components of new functional materials has the unintended consequence of causing increases in CNT concentrations in aqueous environments. Aqueous systems are reservoirs for bacteria, including human and animal pathogens, that can form biofilms. At high concentrations, CNTs have been shown to display biocidal effects; however, at low concentrations, the interaction between CNTs and bacteria is more complicated, and antimicrobial action is highly dependent upon the properties of the CNTs in suspension. Here, impact of low concentrations of multiwalled CNTs (MWCNTs) on the biofilm-forming opportunistic human pathogen Pseudomonas aeruginosa is studied. Using phase contrast and confocal microscopy, flow cytometry, and antibiotic tolerance assays, it is found that sub-lethal concentrations (2 mg/L) of MWCNTs promote aggregation of P. aeruginosa into multicellular clusters. However, the antibiotic tolerance of these "young" bacterial-CNT aggregates is similar to that of CNT-free cultures. Overall, our results indicate that the co-occurrence of MWCNTs and P. aeruginosa in aqueous systems, which promotes the increased number and size of bacterial aggregates, could increase the dose to which humans or animals are exposed.

Principles and Applications of Biological Membrane Organization.

Many critical biological events, including biochemical signaling, membrane traffic, and cell motility, originate at membrane surfaces. Each such event requires that members of a specific group of proteins and lipids rapidly assemble together at a specific site on the membrane surface. Understanding the biophysical mechanisms that stabilize these assemblies is critical to decoding and controlling cellular functions. In this article, we review progress toward a quantitative biophysical understanding of the mechanisms that drive membrane heterogeneity and organization. We begin from a physical perspective, reviewing the fundamental principles and key experimental evidence behind each proposed mechanism. We then shift to a biological perspective, presenting key examples of the role of heterogeneity in biology and asking which physical mechanisms may be responsible. We close with an applied perspective, noting that membrane heterogeneity provides a novel therapeutic target that is being exploited by a growing number of studies at the interface of biology, physics, and engineering.

Assaying How Phagocytic Success Depends on the Elasticity of a Large Target Structure.

Biofilm infections can consist of bacterial aggregates that are an order of magnitude larger than neutrophils, phagocytic immune cells that densely surround aggregates but do not enter them. Because a neutrophil is too small to engulf the entire aggregate, it must be able to detach and engulf a few bacteria at a time if it is to use phagocytosis to clear the infection. Current research techniques do not provide a method for determining how the success of phagocytosis, here defined as the complete engulfment of a piece of foreign material, depends on the mechanical properties of a larger object from which the piece must be removed before being engulfed. This article presents a step toward such a method. By varying polymer concentration or cross-linking density, the elastic moduli of centimeter-sized gels are varied over the range that was previously measured for Pseudomonas aeruginosa biofilms grown from clinical bacterial isolates. Human neutrophils are isolated from blood freshly drawn from healthy adult volunteers, exposed to gel containing embedded beads for 1 h, and removed from the gel. The percentage of collected neutrophils that contain beads that had previously been within the gels is used to measure successful phagocytic engulfment. Both increased polymer concentration in agarose gels and increased cross-linking density in alginate gels are associated with a decreased success of phagocytic engulfment. Upon plotting the percentage of neutrophils showing successful engulfment as a function of the elastic modulus of the gel to which they were applied, it is found that data from both alginate and agarose gels collapse onto the same curve. This suggests that gel mechanics may be impacting the success of phagocytosis and demonstrates that this experiment is a step toward realizing methods for measuring how the mechanics of a large target, or a large structure in which smaller targets are embedded, impact the success of phagocytic engulfment.

Bacterial mechanosensing: the force will be with you, always.

Whether bacteria are in the planktonic state, free-swimming or free-floating in liquid, or in the biofilm state, sessile on surfaces, they are always subject to mechanical forces. The long, successful evolutionary history of bacteria implies that they are capable of adapting to varied mechanical forces, and probably even actively respond to mechanical cues in their changing environments. However, the sensing of mechanical cues by bacteria, or bacterial mechanosensing, has been under-investigated. This leaves the mechanisms underlying how bacteria perceive and respond to mechanical cues largely unknown. In this Review, we first examine the surface-associated behavior of bacteria, outline the clear evidence for bacterial mechanosensing and summarize the role of flagella, type-IV pili, and envelope proteins as potential mechanosensors, before presenting indirect evidence for mechanosensing in bacteria. The general themes underlying bacterial mechanosensing that we highlight here may provide a framework for future research.

Antimicrobial Activity of Ibuprofen against Cystic Fibrosis-Associated Gram-Negative Pathogens.

Clinical trials have demonstrated the benefits of ibuprofen therapy in cystic fibrosis (CF) patients, an effect that is currently attributed to ibuprofen's anti-inflammatory properties. Yet, a few previous reports demonstrated an antimicrobial activity of ibuprofen as well, although none investigated its direct effects on the pathogens found in the CF lung, which is the focus of this work. Determination of ibuprofen's in vitro antimicrobial activity against Pseudomonas aeruginosa and Burkholderia species strains through measurements of the endpoint number of CFU and growth kinetics showed that ibuprofen reduced the growth rate and bacterial burden of the tested strains in a dose-dependent fashion. In an in vitroPseudomonas biofilm model, a reduction in the rate of biomass accumulation over 8 h of growth with ibuprofen treatment was observed. Next, an acute Pseudomonas pneumonia model was used to test this antimicrobial activity after the oral delivery of ibuprofen. Following intranasal inoculation, ibuprofen-treated mice exhibited lower CFU counts and improved survival compared with the control animals. Preliminary biodistribution studies performed after the delivery of ibuprofen to mice by aerosol demonstrated a rapid accumulation of ibuprofen in serum and minimum retention in lung tissue and bronchoalveolar lavage fluid. Therefore, ibuprofen-encapsulated polymeric nanoparticles (Ibu-NPs) were formulated to improve the pharmacokinetic profile. Ibu-NPs formulated for aerosol delivery inhibited the growth of P. aeruginosa in vitro and may provide a convenient dosing method. These results provide an additional explanation for the previously observed therapeutic effects of ibuprofen in CF patients and further strengthen the argument for its use by these patients.

Dynamic Fingering in Adhered Lipid Membranes.

Artificial lipid membranes incorporating proteins have frequently been used as models for the dynamic organization of biological structures in living cells as well as in the development of biology-inspired technologies. We report here on the experimental demonstration and characterization of a pattern-forming process that occurs in a lipid bilayer membrane adhered via biotin-avidin binding to a second lipid membrane that is supported by a solid substrate. Adhesion regions are roughly circular with a diameter of about 25 µm. Using confocal fluorescence microscopy, we record time series of dynamic fingering patterns that grow in the upper lipid membrane and intermembrane biotin-avidin bonds. The fingers are micrometer-scale elongated pores that grow from the edge of an already-stabilized hole. Finger growth is saltatory on the scale of tens of seconds. We find that as the fingers grow and the density of adhesion proteins increases, the rate of finger growth decreases exponentially and the width of newly formed fingers decreases linearly. We show that these findings are consistent with a thermodynamic description of dynamic pore formation and stabilization.

Evolutionary adaptations of biofilms infecting cystic fibrosis lungs promote mechanical toughness by adjusting polysaccharide production.

Biofilms are communities of microbes embedded in a matrix of extracellular polymeric substances, largely polysaccharides. Multiple types of extracellular polymeric substances can be produced by a single bacterial strain. The distinct polymer components of biofilms are known to provide chemical protection, but little is known about how distinct extracellular polysaccharides may also protect biofilms against mechanical stresses such as shear or phagocytic engulfment. Decades-long infections of Pseudomonas. aeruginosa biofilms in the lungs of cystic fibrosis patients are natural models for studies of biofilm fitness under pressure from antibiotics and the immune system. In cystic fibrosis infections, production of the extracellular polysaccharide alginate has long been known to increase with time and to chemically protect biofilms. More recently, it is being recognized that chronic cystic fibrosis infections also evolve to increase production of another extracellular polysaccharide, Psl; much less is known about Psl's protective benefits to biofilms. We use oscillatory bulk rheology, on biofilms grown from longitudinal clinical isolates and from genetically-manipulated lab strains, to show that increased Psl stiffens biofilms and increases biofilm toughness, which is the energy cost to cause the biofilm to yield mechanically. Further, atomic force microscopy measurements reveal greater intercellular cohesion for higher Psl expression. Of the three types of extracellular polysaccharides produced by P. aeruginosa, only Psl increases the stiffness. Stiffening by Psl requires CdrA, a protein that binds to mannose groups on Psl and is a likely cross-linker for the Psl components of the biofilm matrix. We compare the elastic moduli of biofilms to the estimated stresses exerted by neutrophils during phagocytosis, and infer that increased Psl could confer a mechanical protection against phagocytic clearance.

The Pseudomonas aeruginosa PSL Polysaccharide Is a Social but Noncheatable Trait in Biofilms.

Extracellular polysaccharides are compounds secreted by microorganisms into the surrounding environment, and they are important for surface attachment and maintaining structural integrity within biofilms. The social nature of many extracellular polysaccharides remains unclear, and it has been suggested that they could function as either cooperative public goods or as traits that provide a competitive advantage. Here, we empirically tested the cooperative nature of the PSL polysaccharide, which is crucial for the formation of biofilms in Pseudomonas aeruginosa We show that (i) PSL is not metabolically costly to produce; (ii) PSL provides population-level benefits in biofilms, for both growth and antibiotic tolerance; (iii) the benefits of PSL production are social and are shared with other cells; (iv) the benefits of PSL production appear to be preferentially directed toward cells which produce PSL; (v) cells which do not produce PSL are unable to successfully exploit cells which produce PSL. Taken together, this suggests that PSL is a social but relatively nonexploitable trait and that growth within biofilms selects for PSL-producing strains, even when multiple strains are on a patch (low relatedness at the patch level).IMPORTANCE Many studies have shown that bacterial traits, such as siderophores and quorum sensing, are social in nature. This has led to an impression that secreted traits act as public goods, which are costly to produce but benefit both the producing cell and its surrounding neighbors. Theories and subsequent experiments have shown that such traits are exploitable by asocial cheats, but we show here that this does not always hold true. We demonstrate that the Pseudomonas aeruginosa exopolysaccharide PSL provides social benefits to populations but that it is nonexploitable, because most of the fitness benefits accrue to PSL-producing cells. Our work builds on an increasing body of work showing that secreted traits can have both private and public benefits to cells.

Mechanosensing of shear by Pseudomonas aeruginosa leads to increased levels of the cyclic-di-GMP signal initiating biofilm development.

Biofilms are communities of sessile microbes that are phenotypically distinct from their genetically identical, free-swimming counterparts. Biofilms initiate when bacteria attach to a solid surface. Attachment triggers intracellular signaling to change gene expression from the planktonic to the biofilm phenotype. For Pseudomonas aeruginosa, it has long been known that intracellular levels of the signal cyclic-di-GMP increase upon surface adhesion and that this is required to begin biofilm development. However, what cue is sensed to notify bacteria that they are attached to the surface has not been known. Here, we show that mechanical shear acts as a cue for surface adhesion and activates cyclic-di-GMP signaling. The magnitude of the shear force, and thereby the corresponding activation of cyclic-di-GMP signaling, can be adjusted both by varying the strength of the adhesion that binds bacteria to the surface and by varying the rate of fluid flow over surface-bound bacteria. We show that the envelope protein PilY1 and functional type IV pili are required mechanosensory elements. An analytic model that accounts for the feedback between mechanosensors, cyclic-di-GMP signaling, and production of adhesive polysaccharides describes our data well.

Singly Flagellated Pseudomonas aeruginosa Chemotaxes Efficiently by Unbiased Motor Regulation.

UNLABELLED: Pseudomonas aeruginosais an opportunistic human pathogen that has long been known to chemotax. More recently, it has been established that chemotaxis is an important factor in the ability ofP. aeruginosato make biofilms. Genes that allowP. aeruginosato chemotax are homologous with genes in the paradigmatic model organism for chemotaxis,Escherichia coli However,P. aeruginosais singly flagellated andE. colihas multiple flagella. Therefore, the regulation of counterclockwise/clockwise flagellar motor bias that allowsE. colito efficiently chemotax by runs and tumbles would lead to inefficient chemotaxis byP. aeruginosa, as half of a randomly oriented population would respond to a chemoattractant gradient in the wrong sense. HowP. aeruginosaregulates flagellar rotation to achieve chemotaxis is not known. Here, we analyze the swimming trajectories of single cells in microfluidic channels and the rotations of cells tethered by their flagella to the surface of a variable-environment flow cell. We show thatP. aeruginosachemotaxes by symmetrically increasing the durations of both counterclockwise and clockwise flagellar rotations when swimming up the chemoattractant gradient and symmetrically decreasing rotation durations when swimming down the chemoattractant gradient. Unlike the case forE. coli, the counterclockwise/clockwise bias stays constant forP. aeruginosa We describeP. aeruginosa's chemotaxis using an analytical model for symmetric motor regulation. We use this model to do simulations that show that, givenP. aeruginosa's physiological constraints on motility, its distinct, symmetric regulation of motor switching optimizes chemotaxis. IMPORTANCE: Chemotaxis has long been known to strongly affect biofilm formation by the opportunistic human pathogenP. aeruginosa, whose essential chemotaxis genes have homologues inE. coli, which achieves chemotaxis by biasing the relative probability of counterclockwise and clockwise flagellar rotation. However, the physiological difference between multiflagellatedE. coliand singly flagellatedP. aeruginosaimplies that biased motor regulation should preventP. aeruginosapopulations from chemotaxing efficiently. Here, we used experiments, analytical modeling, and simulations to demonstrate thatP. aeruginosauses unbiased, symmetric regulation of the flagellar motor to maximize its chemotaxis efficiency. This mode of chemotaxis was not previously known and demonstrates a new variant of a paradigmatic signaling system in an important human pathogen.

Shaping the Growth Behaviour of Biofilms Initiated from Bacterial Aggregates.

Bacterial biofilms are usually assumed to originate from individual cells deposited on a surface. However, many biofilm-forming bacteria tend to aggregate in the planktonic phase so that it is possible that many natural and infectious biofilms originate wholly or partially from pre-formed cell aggregates. Here, we use agent-based computer simulations to investigate the role of pre-formed aggregates in biofilm development. Focusing on the initial shape the aggregate forms on the surface, we find that the degree of spreading of an aggregate on a surface can play an important role in determining its eventual fate during biofilm development. Specifically, initially spread aggregates perform better when competition with surrounding unaggregated bacterial cells is low, while initially rounded aggregates perform better when competition with surrounding unaggregated cells is high. These contrasting outcomes are governed by a trade-off between aggregate surface area and height. Our results provide new insight into biofilm formation and development, and reveal new factors that may be at play in the social evolution of biofilm communities.