Surface hardness impairment of quorum sensing and swarming for Pseudomonas aeruginosa

The importance of rhamnolipid to swarming of the bacterium Pseudomonas aeruginosa is well established. It is frequently, but not exclusively, observed that P. aeruginosa swarms in tendril patterns--formation of these tendrils requires rhamnolipid. We were interested to explain the impact of surface changes on P. aeruginosa swarm tendril development. Here we report that P. aeruginosa quorum sensing and rhamnolipid production is impaired when growing on harder semi-solid surfaces. P. aeruginosa wild-type swarms showed huge variation in tendril formation with small deviations to the "standard" swarm agar concentration of 0.5%. These macroscopic differences correlated with microscopic investigation of cells close to the advancing swarm edge using fluorescent gene reporters. Tendril swarms showed significant rhlA-gfp reporter expression right up to the advancing edge of swarming cells while swarms without tendrils (grown on harder agar) showed no rhlA-gfp reporter expression near the advancing edge. This difference in rhamnolipid gene expression can be explained by the necessity of quorum sensing for rhamnolipid production. We provide evidence that harder surfaces seem to limit induction of quorum sensing genes near the advancing swarm edge and these localized effects were sufficient to explain the lack of tendril formation on hard agar. We were unable to artificially stimulate rhamnolipid tendril formation with added acyl-homoserine lactone signals or increasing the carbon nutrients. This suggests that quorum sensing on surfaces is controlled in a manner that is not solely population dependent.     

Rhamnolipids modulate swarming motility patterns of Pseudomonas aeruginosa

Pseudomonas aeruginosa is capable of twitching, swimming, and swarming motility. The latter form of translocation occurs on semisolid surfaces, requires functional flagella and biosurfactant production, and results in complex motility patterns. From the point of inoculation, bacteria migrate as defined groups, referred to as tendrils, moving in a coordinated manner capable of sensing and responding to other groups of cells. We were able to show that P. aeruginosa produces extracellular factors capable of modulating tendril movement, and genetic analysis revealed that modulation of these movements was dependent on rhamnolipid biosynthesis. An rhlB mutant (deficient in mono- and dirhamnolipid production) and an rhlC mutant (deficient in dirhamnolipid production) exhibited altered swarming patterns characterized by irregularly shaped tendrils. In addition, agar supplemented with rhamnolipid-containing spent supernatant inhibited wild-type (WT) swarming, whereas agar supplemented with spent supernatant from mutants that do not make rhamnolipids had no effect on WT P. aeruginosa swarming. Addition of purified rhamnolipids to swarming medium also inhibited swarming motility of the WT strain. We also show that a sadB mutant does not sense and/or respond to other groups of swarming cells and this mutant was capable of swarming on media supplemented with rhamnolipid-containing spent supernatant or purified rhamnolipids. The abilities to produce and respond to rhamnolipids in the context of group behavior are discussed.     

Flow directs surface-attached bacteria to twitch upstream

Bacteria inhabit a wide variety of environments in which fluid flow is present, including healthcare and food processing settings and the vasculature of animals and plants. The motility of bacteria on surfaces in the presence of flow has not been well characterized. Here we focus on Pseudomonas aeruginosa, an opportunistic human pathogen that thrives in flow conditions such as in catheters and respiratory tracts. We investigate the effects of flow on P. aeruginosa cells and describe a mechanism in which surface shear stress orients surface-attached P. aeruginosa cells along the flow direction, causing cells to migrate against the flow direction while pivoting in a zig-zag motion. This upstream movement is due to the retraction of type IV pili by the ATPase motors PilT and PilU and results from the effects of flow on the polar localization of type IV pili. This directed upstream motility could be beneficial in environments where flow is present, allowing bacteria to colonize environments that cannot be reached by other surface-attached bacteria.     

Optimized plant compound with potent anti-biofilm activity across gram-negative species

Many human diseases, including cystic fibrosis lung infections, are caused or exacerbated by bacterial biofilms. Specialized modes of motility, including swarming and twitching, allow gram-negative bacteria to spread across surfaces and form biofilms. Compounds that inhibit these motilities could slow the spread of biofilms, thereby allowing antibiotics to work better. We previously demonstrated that a set of plant-derived triterpenes, including oleanolic acid and ursolic acid, inhibit formation of Escherichia coli and Pseudomonas aeruginosa biofilms, and alter expression of genes involved in chemotaxis and motility. In the present study, we have prepared a series of analogs of oleanolic acid. The analogs were evaluated against clinical isolates of E. coli and P. aeruginosa in biofilm formation assays and swarming assays. From these analogs, compound 9 was selected as a lead compound for further development. Compound 9 inhibits E. coli biofilm formation at 4 µg/mL; it also inhibits swarming at ≤1 µg/mL across multiple clinical isolates of P. aeruginosa, E. coli, Burkholderia cepacia, and Salmonella enterica, and at <0.5 µg/mL against multiple agricultural strains. Compound 9 also potentiates the activity of the antibiotics tobramycin and colistin against swarming P. aeruginosa; this is notable, as tobramycin and colistin are inhaled antibiotics commonly used to treat P. aeruginosa lung infections in people with cystic fibrosis. qPCR experiments suggested that 9 alters expression of genes involved in regulating Type IV pili; western blots confirmed that expression of Type IV pili components PilA and PilY1 decreases in P. aeruginosa in the presence of 9.     

Nanoscale Pulling of Type IV Pili Reveals Their Flexibility and Adhesion to Surfaces over Extended Lengths of the Pili

Type IV pili (T4P) are very thin protein filaments that extend from and retract into bacterial cells, allowing them to interact with and colonize a broad array of chemically diverse surfaces. The physical aspects that allow T4P to mediate adherence to many different surfaces remain unclear. Atomic force microscopy (AFM) nanoscale pulling experiments were used to measure the mechanical properties of T4P of a mutant strain of Pseudomonas aeruginosa PAO1 unable to retract its T4P. After adhering bacteria to the end of an AFM cantilever and approaching surfaces of mica, gold, or polystyrene, we observed adhesion of the T4P to all of the surfaces. Pulling of single and multiple T4P on retraction of the cantilever from the surfaces could be described using the worm-like chain (WLC) model. Distinct peaks in the measured distributions of the best-fit values of the persistence length L_p on two different surfaces provide strong evidence for close-packed bundling of very flexible T4P. In addition, we observed force plateaus indicating that adhesion of the T4P to both hydrophilic and hydrophobic surfaces occurs along extended lengths of the T4P. These data shed new light, to our knowledge, on T4P flexibility and support a low-affinity, high-avidity adhesion mechanism that mediates bacteria-surface interactions.     

Sink to Source Transition in Tendrils of a Semileafless Mutant, Pisum sativum cv Curly

Sink to source transition parallels loss of thigmotropic capacity in tendrils of a semileafless mutant, Pisum sativum cv Curly. Macroscopic tendril development is subdivided based on thigmotropic capacity. Stage I is the elongation stage and, although the rate of photosynthesis is similar to that of stage II and III tendrils, dark respiration rates are higher in stage I. During stage II, tendrils are thigmotropic and act as a sink. Even though stage II tendrils have CO₂ exchange characteristics similar to those of stage III tendrils, which are coiled, our fluorescein, ¹⁴C-partitioning, and ¹¹C-translocation experiments suggest that stage I and II tendrils do not export carbon. Only stage III tendrils act as sources of newly fixed carbon. Export from them is blocked by cold, heat girdling of the petiole, or anoxia treatment of the tendrils. A late stage II tendril complex, in which coiling is occurring, may be exporting photoassimilates; however, this phenomenon can be attributed to the fact that the pea leaf is a compound structure and there may be one or more stage III tendrils, no longer thigmotropic, within the tendril complex. Photosynthetic maturity in pea tendrils occurs at stage III and is characterized by the ability of these tendrils to export photoassimilates.     

Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa

Bacteriophage infection has profound effects on bacterial biology. Clustered regular interspaced short palindromic repeats (CRISPRs) and cas (CRISPR-associated) genes are found in most archaea and many bacteria and have been reported to play a role in resistance to bacteriophage infection. We observed that lysogenic infection of Pseudomonas aeruginosa PA14 with bacteriophage DMS3 inhibits biofilm formation and swarming motility, both important bacterial group behaviors. This inhibition requires the CRISPR region in the host. Mutation or deletion of five of the six cas genes and one of the two CRISPRs in this region restored biofilm formation and swarming to DMS3 lysogenized strains. Our observations suggest a role for CRISPR regions in modifying the effects of lysogeny on P. aeruginosa.     

Evolutionary,genetic, environmental and hormonal-induced plasticity in the fate of organs arising from axillary meristems in Passiflora spp.

Tendrils can be found in different plant species. In legumes such as pea, tendrils are modified leaves produced by the vegetative meristem but in the grape vine, a same meristem is used to either form a tendril or an inflorescence. Passiflora species originated in ecosystems in which there is dense vegetation and competition for light. Thus climbing on other plants in order to reach regions with higher light using tendrils is an adaptive advantage. In Passiflora species, after a juvenile phase, every leaf has a subtending vegetative meristem, and a separate meristem that forms both flowers and a tendril. Thus, flowers are formed once a tendril is formed yet whether or not this flower will reach bloom depends on the environment. For example, in Passiflora edulis flowers do not develop under shaded conditions, so that tendrils are needed to bring the plant to positions were flowers can develop. This separate meristem generally forms a single tendril in different Passiflora species yet the number and position of flowers formed from the same meristem diverges among species. Here we display the variation among species as well as variation within a single species, P. edulis. We also show that the number of flowers within a specific genotype can be modulated by applying Cytokinins. Finally, this separate meristem is capable of transforming into a leaf-producing meristem under specific environmental conditions. Thus, behind what appears to be a species-specific rigid program regarding the fate of this meristem, our study helps to reveal a plasticity normally restrained by genetic, hormonal and environmental constraints.     

Influence of Pseudomonas aeruginosa pvdQ Gene on Altering Antibiotic Susceptibility Under Swarming Conditions

In Pseudomonas aeruginosa PAO1, the pvdQ gene has been shown to have at least two functions. It encodes the acylase enzyme and hydrolyzes 3-oxo-C12-HSL, the key signaling molecule of quorum sensing system. In addition, pvdQ is involved in swarming motility. It is required and up-regulated during swarming motility, which is triggered by high cell densities. As high density bacterial populations also display elevated antibiotics resistance, studies have demonstrated swarm-cell differentiation in P. aeruginosa promotes increased resistance to various antibiotics. PvdQ acts as a signal during swarm-cell differentiation, and thus may play a role in P. aeruginosa antibiotic resistance. The aim of this study was to examine whether pvdQ was involved in modifying antibiotic susceptibility during swarming conditions and to investigate the mechanism by which this occurred. We constructed the PAO1pMEpvdQ strain, which overproduces PvdQ. PAO1pMEpvdQ promotes swarming motility, while PAO1ΔpvdQ abolishes swarming motility. In addition, both PAO1 and PAO1pMEpvdQ acquired resistance to ceftazidime, ciprofloxacin, meropenem, polymyxin B, and gentamicin, though PAO1pMEpvdQ exhibited a twofold to eightfold increase in antibiotic resistance compared to PAO1. These results indicate that pvdQ plays an important role in elevating antibiotic resistance via swarm-cell differentiation and possibly other mechanisms as well. We analyzed outer membrane permeability. Our data also suggest that pvdQ decreases P. aeruginosa outer membrane permeability, thereby elevating antibiotic resistance under swarming conditions. Our results suggest new approaches for reducing P. aeruginosa resistance.     

Self-produced extracellular stimuli modulate the Pseudomonas aeruginosa swarming motility behaviour

Pseudomonas aeruginosa presents three types of motilities: swimming, twitching and swarming. The latter is characterized by rapid and coordinated group movement over a semisolid surface resulting from morphological differentiation and intercellular interactions. A striking feature of P. aeruginosa swarming motility is the formation of migrating tendrils producing colonies with complex fractal-like patterns. Previous studies have shown that normal swarming motility is intimately related to the production of extracellular surface-active molecules: rhamnolipids (RLs), composed of monorhamnolipids (mono-RLs) and dirhamnolipids (di-RLs), and 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs). Here, we report that (i) di-RLs attract active swarming cells while HAAs behave as strong repellents, (ii) di-RLs promote and HAAs inhibit tendril formation and migration, (iii) di-RLs and HAAs display different diffusion kinetics on a surface as di-RLs spread faster than HAAs in agar, (iv) di-RLs and HAAs have no effect on swimming cells, suggesting that swarming cells are different from swimming cells not only in morphology but also at the regulatory level and (v) mono-RLs act as wetting agents. We propose a model explaining how HAAs and di-RLs together modulate the behaviour of swarming migrating cells by acting as self-produced negative and positive chemotactic-like stimuli.     

Chelator-Induced Dispersal and Killing of Pseudomonas aeruginosa Cells in a Biofilm

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as Staphylococcus aureus. EDTA can also kill planktonic cells of Proteobacteria like Pseudomonas aeruginosa. In this study we demonstrate that EDTA is a potent P. aeruginosa biofilm disrupter. In Tris buffer, EDTA treatment of P. aeruginosa biofilms results in 1,000-fold greater killing than treatment with the P. aeruginosa antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells. P. aeruginosa biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of P. aeruginosa cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect P. aeruginosa biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.     

Cyclic Di-GMP-Mediated Repression of Swarming Motility by Pseudomonas aeruginosa PA14 Requires the MotAB Stator

The second messenger cyclic diguanylate (c-di-GMP) plays a critical role in the regulation of motility. In Pseudomonas aeruginosa PA14, c-di-GMP inversely controls biofilm formation and surface swarming motility, with high levels of this dinucleotide signal stimulating biofilm formation and repressing swarming. P. aeruginosa encodes two stator complexes, MotAB and MotCD, that participate in the function of its single polar flagellum. Here we show that the repression of swarming motility requires a functional MotAB stator complex. Mutating the motAB genes restores swarming motility to a strain with artificially elevated levels of c-di-GMP as well as stimulates swarming in the wild-type strain, while overexpression of MotA from a plasmid represses swarming motility. Using point mutations in MotA and the FliG rotor protein of the motor supports the conclusion that MotA-FliG interactions are critical for c-di-GMP-mediated swarming inhibition. Finally, we show that high c-di-GMP levels affect the localization of a green fluorescent protein (GFP)-MotD fusion, indicating a mechanism whereby this second messenger has an impact on MotCD function. We propose that when c-di-GMP level is high, the MotAB stator can displace MotCD from the motor, thereby affecting motor function. Our data suggest a newly identified means of c-di-GMP-mediated control of surface motility, perhaps conserved among Pseudomonas, Xanthomonas, and other organisms that encode two stator systems.     

SadC reciprocally influences biofilm formation and swarming motility via modulation of exopolysaccharide production and flagellar function

Pseudomonas aeruginosa has served as an important organism in the study of biofilm formation; however, we still lack an understanding of the mechanisms by which this microbe transitions to a surface lifestyle. A recent study of the early stages of biofilm formation implicated the control of flagellar reversals and production of an exopolysaccharide (EPS) as factors in the establishment of a stable association with the substratum and swarming motility. Here we present evidence that SadC (PA4332), an inner membrane-localized diguanylate cyclase, plays a role in controlling these cellular functions. Deletion of the sadC gene results in a strain that is defective in biofilm formation and a hyperswarmer, while multicopy expression of this gene promotes sessility. A ΔsadC mutant was additionally found to be deficient in EPS production and display altered reversal behavior while swimming in high-viscosity medium, two behaviors proposed to influence biofilm formation and swarming motility. Epistasis analysis suggests that the sadC gene is part of a genetic pathway that allows for the concomitant regulation of these aspects of P. aeruginosa surface behavior. We propose that SadC and the phosphodiesterase BifA (S. L. Kuchma et al., J. Bacteriol. 189:8165-8178, 2007), via modulating levels of the signaling molecule cyclic-di-GMP, coregulate swarming motility and biofilm formation as P. aeruginosa transitions from a planktonic to a surface-associated lifestyle.     

Subinhibitory Concentration of Kanamycin Induces the Pseudomonas aeruginosa type VI Secretion System

Pseudomonas aeruginosa is a Gram-negative bacterium found in natural environments including plants, soils and warm moist surfaces. This organism is also in the top ten of nosocomial pathogens, and prevalent in cystic fibrosis (CF) lung infections. The ability of P. aeruginosa to colonize a wide variety of environments in a lasting manner is associated with the formation of a resistant biofilm and the capacity to efficiently outcompete other microorganisms. Here we demonstrate that sub-inhibitory concentration of kanamycin not only induces biofilm formation but also induces expression of the type VI secretion genes in the H1-T6SS cluster. The H1-T6SS is known for its role in toxin production and bacterial competition. We show that the antibiotic induction of the H1-T6SS only occurs when a functional Gac/Rsm pathway is present. These observations may contribute to understand how P. aeruginosa responds to antibiotic producing competitors. It also suggests that improper antibiotic therapy may enhance P. aeruginosa colonization, including in the airways of CF patients.     

Periodic Reversals in Paenibacillus dendritiformis Swarming

Bacterial swarming is a type of motility characterized by a rapid and collective migration of bacteria on surfaces. Most swarming species form densely packed dynamic clusters in the form of whirls and jets, in which hundreds of rod-shaped rigid cells move in circular and straight patterns, respectively. Recent studies have suggested that short-range steric interactions may dominate hydrodynamic interactions and that geometrical factors, such as a cell''s aspect ratio, play an important role in bacterial swarming. Typically, the aspect ratio for most swarming species is only up to 5, and a detailed understanding of the role of much larger aspect ratios remains an open challenge. Here we study the dynamics of Paenibacillus dendritiformis C morphotype, a very long, hyperflagellated, straight (rigid), rod-shaped bacterium with an aspect ratio of ∼20. We find that instead of swarming in whirls and jets as observed in most species, including the shorter T morphotype of P. dendritiformis, the C morphotype moves in densely packed straight but thin long lines. Within these lines, all bacteria show periodic reversals, with a typical reversal time of 20 s, which is independent of their neighbors, the initial nutrient level, agar rigidity, surfactant addition, humidity level, temperature, nutrient chemotaxis, oxygen level, illumination intensity or gradient, and cell length. The evolutionary advantage of this unique back-and-forth surface translocation remains unclear.