Cyclic-di-GMP-Mediated Repression of Swarming Motility by Pseudomonas aeruginosa: the pilY1 Gene and Its Impact on Surface-Associated Behaviors

The intracellular signaling molecule cyclic-di-GMP (c-di-GMP) has been shown to influence surface-associated behaviors of Pseudomonas aeruginosa, including biofilm formation and swarming motility. Previously, we reported a role for the bifA gene in the inverse regulation of biofilm formation and swarming motility. The bifA gene encodes a c-di-GMP-degrading phosphodiesterase (PDE), and the ΔbifA mutant exhibits increased cellular pools of c-di-GMP, forms hyperbiofilms, and is unable to swarm. In this study, we isolated suppressors of the ΔbifA swarming defect. Strains with mutations in the pilY1 gene, but not in the pilin subunit pilA gene, show robust suppression of the swarming defect of the ΔbifA mutant, as well as its hyperbiofilm phenotype. Despite the ability of the pilY1 mutation to suppress all the c-di-GMP-related phenotypes, the global pools of c-di-GMP are not detectably altered in the ΔbifA ΔpilY1 mutant relative to the ΔbifA single mutant. We also show that enhanced expression of the pilY1 gene inhibits swarming motility, and we identify residues in the putative VWA domain of PilY1 that are important for this phenotype. Furthermore, swarming repression by PilY1 specifically requires the diguanylate cyclase (DGC) SadC, and epistasis analysis indicates that PilY1 functions upstream of SadC. Our data indicate that PilY1 participates in multiple surface behaviors of P. aeruginosa, and we propose that PilY1 may act via regulation of SadC DGC activity but independently of altering global c-di-GMP levels.Pseudomonas aeruginosa forms surface-attached communities known as biofilms, and this microbe is also capable of surface-associated motility, including twitching and swarming. The mechanism by which cells regulate and coordinate these various surface-associated behaviors, or how these microbes transition from one surface behavior to another, has yet to be elucidated. Given that P. aeruginosa is capable of such diverse surface-associated lifestyles, this Gram-negative organism serves as a useful model to address questions regarding the regulation of surface-associated behaviors.Recent studies indicate that biofilm formation and swarming motility by P. aeruginosa are inversely regulated via a common pathway (12, 27, 37). Important factors that influence early biofilm formation by P. aeruginosa strain PA14 include control of flagellar motility and the robust production of the Pel exopolysaccharide (EPS). Swarming occurs when cells move across a hydrated, viscous semisolid surface, and like biofilm formation, flagellar function is important for this surface-associated motility. Additionally, swarming requires production of rhamnolipid surfactant acting as a surface-wetting agent (25, 58). In contrast to biofilm formation, swarming motility is enhanced in strains which are defective for the production of Pel EPS (12).The inverse regulation of biofilm formation and swarming motility is reminiscent of the regulation of sessile and motile behaviors that occurs in a wide range of bacterial species via the intracellular signaling molecule cyclic-di-GMP (c-di-GMP) (17, 24, 50, 51, 56). High levels of this signaling molecule promote sessile behaviors and inhibit motility, whereas low levels of c-di-GMP favor motile behaviors (8, 9, 22, 56). Recently, we reported that the BifA phosphodiesterase, which catalyzes the breakdown of c-di-GMP, inversely regulates biofilm formation and swarming motility (27). In addition, Merritt et al. reported that SadC, a diguanylate cyclase (DGC) which synthesizes c-di-GMP, participates with BifA to modulate cellular c-di-GMP levels and thus regulate biofilm formation and swarming motility (37).Consistent with a role for BifA as a c-di-GMP phosphodiesterase, ΔbifA mutants exhibit increased cellular pools of c-di-GMP relative to the wild type (WT) (27). Phenotypically, ΔbifA mutants form hyperbiofilms and are unable to swarm. The hyperbiofilm phenotype of the ΔbifA mutant results largely from increased synthesis of the pel-derived polysaccharide; that is, the ΔbifAΔpel double mutant shows a marked decrease in biofilm formation compared to the ΔbifA mutant (27). Interestingly, elevated Pel polysaccharide production alone is not sufficient to explain the swarming defect of the ΔbifA mutant, as the ΔbifAΔpel double mutant recovers only minimal swarming ability (27). These data indicate that high levels of c-di-GMP inhibit swarming motility in a largely Pel-independent manner.To better understand how elevated c-di-GMP levels in the cell inhibit swarming motility, we exploited the swarming defect of the ΔbifA mutant, and using a genetic screen, we identified suppressors in the ΔbifA background that restored the ability to swarm. Here we report a role for the PilY1 protein in repression of swarming motility in the ΔbifA mutant background. Our data are consistent with a model in which PilY1 functions upstream of the c-di-GMP diguanylate cyclase SadC to regulate swarming motility by P. aeruginosa.     

Organoselenium Coating on Cellulose Inhibits the Formation of Biofilms by Pseudomonas aeruginosa and Staphylococcus aureus

Among the most difficult bacterial infections encountered in treating patients are wound infections, which may occur in burn victims, patients with traumatic wounds, necrotic lesions in people with diabetes, and patients with surgical wounds. Within a wound, infecting bacteria frequently develop biofilms. Many current wound dressings are impregnated with antimicrobial agents, such as silver or antibiotics. Diffusion of the agent(s) from the dressing may damage or destroy nearby healthy tissue as well as compromise the effectiveness of the dressing. In contrast, the antimicrobial agent selenium can be covalently attached to the surfaces of a dressing, prolonging its effectiveness. We examined the effectiveness of an organoselenium coating on cellulose discs in inhibiting Pseudomonas aeruginosa and Staphylococcus aureus biofilm formation. Colony biofilm assays revealed that cellulose discs coated with organoselenium completely inhibited P. aeruginosa and S. aureus biofilm formation. Scanning electron microscopy of the cellulose discs confirmed these results. Additionally, the coating on the cellulose discs was stable and effective after a week of incubation in phosphate-buffered saline. These results demonstrate that 0.2% selenium in a coating on cellulose discs effectively inhibits bacterial attachment and biofilm formation and that, unlike other antimicrobial agents, longer periods of exposure to an aqueous environment do not compromise the effectiveness of the coating.Among the most difficult bacterial infections encountered in treating patients are wound infections, which may occur in burn victims (10), patients with traumatic wounds (33), people with diabetes (27), and patients with surgical wounds (29, 31). Two of the more common causative agents of wound infections are Staphylococcus aureus and Pseudomonas aeruginosa (10, 27, 29, 31, 33). Such infections often lead to fatality; the mortality rate among patients infected with P. aeruginosa ranges from 26% to 55% (9, 49), while mortality from S. aureus infection ranges from 19% to 38% (28, 46, 50). As opportunistic pathogens, S. aureus and P. aeruginosa cause few infections in healthy individuals but readily cause infection once host defenses are compromised, such as with the removal of skin from burns (10). S. aureus infection originates from the normal flora of either the patient or health care workers (48), while P. aeruginosa is acquired from the environment surrounding the patient (41). Once established on the skin, S. aureus and P. aeruginosa are then able to colonize the wound. Infection results if the organisms proliferate in the wound environment.Both P. aeruginosa and S. aureus often exist within burn wounds as biofilms (43, 47). A biofilm is presently defined as a sessile microbial community characterized by cells that are irreversibly attached either to a substratum or to each other (16). Biofilms, which can attain over 100 μm in thickness, are made up of multiple layers of bacteria in an exopolysaccharide matrix (12, 16, 42). Sauer et al. showed that P. aeruginosa biofilms form in distinct developmental stages: reversible attachment, irreversible attachment, two stages of maturation, and a dispersion phase (42). Clinically, biofilms present serious medical management problems through their association with different chronic infections (37). During vascular catheter-related infections and sepsis, biofilms serve as a reservoir of bacteria from which planktonic cells detach and spread throughout the tissue and/or enter the circulatory system, resulting in bacteremia or septicemia (15). Factors specific to the bacterium may influence the formation of bacterial biofilms at different infection sites or surfaces. For example, during the initial attachment stage the flagellum, lipopolysaccharide, and possibly outer membrane proteins play a major role in bringing P. aeruginosa into proximity with the surface as well as mediating the interaction with the substratum (12). Using the murine model of thermal injury, we recently showed that P. aeruginosa forms a biofilm within the thermally injured tissues (43). Clinically, the surgeons debride the infected or dead tissues; however, a few microorganisms may remain on the tissue surface and reinitiate biofilm formation.Antibiotics, silver, or chitosan, attached to or embedded in gauze, have been shown to be efficacious in preventing wound infection (21, 24, 26, 36). However, the resistance of P. aeruginosa and S. aureus to available antibiotics severely limits the choices for antibiotic treatment (13, 40). Additionally, silver compounds, such as silver nitrate and silver sulfadiazine, leaching from dressings are toxic to human fibroblasts even at low concentrations (20, 25). Thus, effective alternative antimicrobial agents that contact the thermally injured/infected tissues and prevent the development of bacterial biofilms are required. Previous studies have shown that selenium (Se) can be covalently bound to a solid matrix and retain its ability to catalyze the formation of superoxide radicals (O₂^·−) (30). These superoxide radicals inhibit bacterial attachment to the solid surface (30). In this study, we examined the ability of a newly synthesized organoselenium-methacrylate polymer (Se-MAP) to block biofilm formation by both S. aureus and P. aeruginosa. These bacteria were chosen since they cause a major share of wound infections and because drug-resistant forms of these bacteria have become a serious problem in the treatment and management of these wound infections (6, 13, 17, 18, 38). Results of the study show that 0.2% (wt/wt) Se in Se-MAP covalently attached to cellulose discs inhibited P. aeruginosa and S. aureus biofilm formation. This could lead to the development of a selenium-based antimicrobial coating for cotton materials that will prevent the bacterial attachment and colonization that can ultimately lead to bacterial biofilm formation during chronic infections.     

Activation of ExoU Phospholipase Activity Requires Specific C-Terminal Regions

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that utilizes a type III secretion system to subvert host innate immunity. Of the 4 known effector proteins injected into eukaryotic cells, ExoS and ExoU are cytotoxic. The cytotoxic phenotype of ExoU depends on the enzymatic activity of the patatin-like phospholipase A₂ domain localized to the N-terminal half of the protein. Amino acid residues located within the C-terminal region of ExoU are postulated to be required for trafficking or localization to the plasma membrane of eukaryotic cells. This report describes the characterization of a transposon-based linker insertion library in ExoU. Utilizing an unbiased screening approach and sensitive methods for measuring enzymatic activity, we identified regions of ExoU that are critical for activation of the phospholipase activity by the only known cofactor, SOD1. Insertions at D572 and L618 reduced the rate of substrate cleavage. Enzymatic activity could be restored to almost parental levels when SOD1 concentrations were increased, suggesting that the linker insertion disrupted the interaction between ExoU and SOD1. An enzyme-linked immunosorbent assay (ELISA)-based binding test was developed to measure ExoU-SOD1 binding. These experiments suggest that ExoU activation by SOD1 is hampered by linker insertion. ExoU derivatives harboring minimal phospholipase activity retained biological activity in tissue culture assays. These proteins affected primarily cellular architecture in a manner similar to that of ExoT. Our studies suggest that conformational changes in ExoU are facilitated by SOD1. Importantly, the level of phospholipase activity influences the biological outcome of ExoU intoxication.Pseudomonas aeruginosa is a Gram-negative bacterium responsible for severe and potentially fatal opportunistic infections. As a contributor to nosocomial infections, P. aeruginosa is a leading cause of hospital-acquired and ventilator-associated pneumonias (40). Furthermore, P. aeruginosa is responsible for ulcerative keratitis and ocular disease found in conjunction with the use of soft contact lenses (2, 10, 54). Infections with this pathogen are of critical concern for individuals admitted with severe burns, due to the bacterium''s ability to colonize and persist in damaged tissues (35). Patients suffering from cystic fibrosis often succumb to severe lung infections and inflammation due to colonization with antibi otic-resistant, mucoid strains of P. aeruginosa (3). The expression of multiple efflux pumps and the ability to inactivate and modify antibiotics make P. aeruginosa dangerous and difficult to treat (27). Several investigators are exploring ways, as adjuncts or alternatives to antibiotic treatment, to neutralize virulence factors that contribute to the ability of P. aeruginosa to suppress host innate and adaptive immune responses (17, 21, 22, 52).Many Gram-negative bacteria, including P. aeruginosa, encode one or more type III secretion systems (T3SS), which are thought to aid in pathogenesis and increase disease severity (19, 32, 39). Four effectors are translocated by the T3SS of P. aeruginosa and include ExoS, ExoT, ExoU, and ExoY (8, 23, 56, 57). The activity of each effector is dependent upon interaction with a cofactor present in eukaryotic but not prokaryotic cells. ExoS and ExoT are bifunctional enzymes that possess both Rho GTPase-activating protein and ADP-ribosyltransferase activities (23, 25, 51). The ADP ribosylation of eukaryotic proteins by ExoS and ExoT requires activation by members of the 14-3-3 family of scaffolding proteins (13). ExoY is an adenylyl cyclase that causes the accumulation of cyclic AMP (cAMP) in intoxicated cells. The eukaryotic cofactor required for ExoY activity has not been identified (57). ExoU, a potent A₂ phospholipase responsible for membrane disruption and cellular lysis, requires superoxide dismutase 1 (SOD1) for the detection of enzymatic activity (43, 46).ExoU is an important virulence factor of P. aeruginosa, as it causes rapid cell death during in vitro infections and is associated with poor clinical outcomes (19, 39, 44). Several studies have used truncation analyses, linker mutagenesis, and site-specific amino acid substitutions to define regions of ExoU important for various functions (7, 36). ExoU is a 74-kDa, hydrophilic, and slightly acidic protein with a pI of 5.9 (8). The first 52 amino acids are required for interaction with the chaperone SpcU and may be important for translocation through the T3SS (7, 9). Enzymatic activity is attributed to the patatin-like phospholipase domain located between residues 107 and 357 (34, 46). Two catalytic residues, S142 and D344, and a sequence encoding an oxyanion hole (₁₁₂GGAK₁₁₅) are located within this domain (34, 46). The oxyanion hole is thought to stabilize the negative charge of the intermediate structure during substrate cleavage (5). C-terminal residues of ExoU, specifically the last 137 amino acids, have been implicated in membrane localization after translocation into mammalian cells (37). The domain or region(s) required for the activation of ExoU by SOD1 have not been identified.In this study, linker-scanning mutagenesis (the insertion of 15 nucleotides randomly throughout the coding sequence) was used to identify regions of exoU that impair activation of phospholipase activity by SOD1. Our data support the model that SOD1 may be facilitating the activation of ExoU by altering the conformational properties of the enzyme. Understanding the molecular mechanisms mediating SOD1 and ExoU interaction may contribute to the design of therapeutics for the treatment of acute P. aeruginosa infections.     

A Putative ABC Transporter,HatABCDE, Is among Molecular Determinants of Pyomelanin Production in Pseudomonas aeruginosa

Pyomelanin overproduction is a common phenotype among Pseudomonas aeruginosa isolates recovered from cystic fibrosis and urinary tract infections. Its prevalence suggests that it contributes to the persistence of the producing microbial community, yet little is known about the mechanisms of its production. Using transposon mutagenesis, we identified factors that contribute to melanogenesis in a clinical isolate of P. aeruginosa. In addition to two enzymes already known to be involved in its biosynthesis (homogentisate dioxygenase and hydroxyphenylpyruvate dioxygenase), we identified 26 genes that encode regulatory, metabolic, transport, and hypothetical proteins that contribute to the production of homogentisic acid (HGA), the monomeric precursor of pyomelanin. One of these, PA14_57880, was independently identified four times and is predicted to encode the ATP-binding cassette of an ABC transporter homologous to proteins in Pseudomonas putida responsible for the extrusion of organic solvents from the cytosol. Quantification of HGA production by P. aeruginosa PA14 strains missing the predicted subcomponents of this transporter confirmed its role in HGA production: mutants unable to produce the ATP-binding cassette (PA14_57880) or the permease (PA14_57870) produced substantially less extracellular HGA after growth for 20 h than the parental strain. In these mutants, concurrent accumulation of intracellular HGA was observed. In addition, quantitative real-time PCR revealed that intracellular accumulation of HGA elicits upregulation of these transport genes. Based on their involvement in homogentisic acid transport, we rename the genes of this operon hatABCDE.Pseudomonas aeruginosa is a metabolically versatile, opportunistic pathogen that is a major cause of life-threatening infections in patients with burn wounds, compromised immunity, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) (23, 41). A major contributor to P. aeruginosa''s pathogenicity is its remarkable genomic plasticity, which often results is a wide range of phenotypic variation among isolates obtained from both acute and chronic infections. These phenotypes include small colony variant formation (24), alginate overproduction (36), hyperpigmentation (22), autoaggregation (13), and autolysis (64). Many of these phenotypes evolve as infections progress, and most have been ascribed to “loss-of-function” genome diversification that promotes long-term survival in the host environment (54). In this regard, recent studies have stimulated interest in another example of a loss-of-function phenotype, the mutation or deletion of hmgA, which encodes the homogentisate 1,2-dioxygenase enzyme. The absence of this protein leads to the accumulation and subsequent export of homogentisic acid (HGA), which ultimately aggregates into the pyomelanin polymer that manifests as a reddish brown pigmentation of P. aeruginosa colonies and their surrounding milieu (Fig. ​(Fig.1A)1A) (5, 49).Open in a separate windowFIG. 1.Pyomelanin production by the PA14 ΔhmgA and DKN343 strains. (A) Homogentisate pathway for the catabolism of chorismate and aromatic amino acids. Enzyme names are shown above the arrows for each step. A mutation or deletion of the hmgA gene (encoding homogentisate 1,2-dioxygenase) leads to the accumulation of pyomelanin. (B) Pyomelanin overproduction by the PA14 ΔhmgA mutant is abolished when complemented with an intact hmgA gene. Complementation of a melanogenic clinical P. aeruginosa isolate, DKN343, with hmgA results in no phenotypic change, indicating that other factors contribute to its pigmentation.Production of pyomelanin (and other forms of melanin) has been described to occur in a wide range of bacterial species, including Aeromonas (4), Azotobacter (51), Azospirillum (50), Bacillus (3), Legionella (8), Marinomonas (33), Micrococcus (40), Mycobacterium (45), Proteus (1), Rhizobium (12), Shewanella (61), Sinorhizobium (38), Streptomyces (67), and Vibrio (63) species. Notably, isolates of other bacterial species associated with chronic infections of the CF lung, Burkholderia cenocepacia and Stenotrophomonas maltophilia, can also be melanogenic (28, 58), suggesting a possible role for this pigment in the establishment and/or persistence of infection. Some genera produce melanin under normal conditions via polyphenol oxidases or laccases, while others synthesize the pigment only in response to specific environmental conditions (17, 35). Many species, however, including P. aeruginosa, overproduce pyomelanin as a result of a point mutation in hmgA or large chromosomal deletions of loci containing the homogentisate operon (2, 19). While these genetic variations have been frequently reported, there is little understanding of the competitive advantage, if any, that this pigment confers to the producing bacterium.Proposed roles for pyomelanin include the enhancement of bacterial surface attachment (20), extracellular electron transfer (61), iron reduction/acquisition (8), induction of virulence factor expression (63), heavy metal binding (21), and protection from environmental stress (11, 28, 32, 44, 53, 65). A protective role has also been proposed to occur in P. aeruginosa PA14, where pyomelanin was shown to contribute to the persistence of an overproducing strain in a chronic CF infection model in mice (49). However, given that melanogenic isolates have been recovered from laboratory-grown communities of P. aeruginosa PAO1 (5, 56), it is probable that pyomelanin plays other roles in addition to protection against host defense mechanisms.As a first step toward gaining a better understanding of pyomelanin function, we sought to identify the molecular determinants of its production in P. aeruginosa. By screening a library of pTnTet/minimariner transposon mutants of a pyomelanin-overproducing clinical isolate for alterations in pigmentation, we identified several genes whose products are involved in tyrosine catabolism, central metabolic pathways, nucleotide biosynthesis, regulation, and membrane transport, in addition to hypothetical proteins of unknown function. We chose to further characterize the gene identified most frequently in our screen, one annotated as encoding a putative ATP-binding cassette of an ABC-type transporter. Here, we demonstrate that this transporter is involved in HGA transport and the subsequent extracellular formation of pyomelanin.     

Increase in Rhamnolipid Synthesis under Iron-Limiting Conditions Influences Surface Motility and Biofilm Formation in Pseudomonas aeruginosa

Iron is an essential element for life but also serves as an environmental signal for biofilm development in the opportunistic human pathogen Pseudomonas aeruginosa. Under iron-limiting conditions, P. aeruginosa displays enhanced twitching motility and forms flat unstructured biofilms. In this study, we present evidence suggesting that iron-regulated production of the biosurfactant rhamnolipid is important to facilitate the formation of flat unstructured biofilms. We show that under iron limitation the timing of rhamnolipid expression is shifted to the initial stages of biofilm formation (versus later in biofilm development under iron-replete conditions) and results in increased bacterial surface motility. In support of this observation, an rhlAB mutant defective in biosurfactant production showed less surface motility under iron-restricted conditions and developed structured biofilms similar to those developed by the wild type under iron-replete conditions. These results highlight the importance of biosurfactant production in determining the mature structure of P. aeruginosa biofilms under iron-limiting conditions.The biofilm mode of bacterial growth is a surface-attached state in which cells are closely packed and encased in an extracellular polymeric matrix (10, 27). Biofilms are abundant in nature and are of clinical, environmental, and industrial importance. Biofilm development is known to follow a series of complex but discrete and tightly regulated steps (18, 27), including (i) microbial attachment to the surface, (ii) growth and aggregation of cells into microcolonies, (iii) maturation, and (iv) dissemination of progeny cells that can colonize new niches. Over the last decade, several key processes important for biofilm formation have been identified, including quorum sensing (12) and surface motility (28).One of the best-studied model organisms for biofilm development is the bacterium Pseudomonas aeruginosa (10), a notorious opportunistic pathogen which causes many types of infections, including biofilm-associated chronic lung infections in individuals with cystic fibrosis (10, 24, 41). Like most organisms, P. aeruginosa requires iron for growth, as iron serves as a cofactor for enzymes that are involved in many basic cellular functions and metabolic pathways. Recent work has shown that at iron concentrations that are not limiting for growth, this metal serves as a signal for biofilm development (40). Iron limitation imposed, for example, by the mammalian iron chelator lactoferrin blocks the ability of P. aeruginosa biofilms to mature from thin layers of cells attached to a surface into large multicellular mushroom-like biofilm structures (40). By chelating iron, lactoferrin induces twitching motility (a specialized form of surface motility), which causes the cells to move across the surface instead of settling down to form structured communities (39, 40). In a recent paper, Berlutti et al. (5) provided further support for the role of iron in cell aggregation and biofilm formation. They reported that in the liquid phase, iron limitation induced motility and transition to the free-living (i.e., planktonic) mode of growth, while increased iron concentrations facilitated cell aggregation and biofilm formation. We recently demonstrated that iron limitation-induced twitching motility is regulated by quorum sensing (31). Quorum sensing allows bacteria to sense and respond to their population density via the production of small diffusible signal molecules. In P. aeruginosa and many other Gram-negative bacteria, these signal molecules are N-acyl homoserine lactones (acyl-HSLs), which have specific receptors (R proteins) (16, 30). P. aeruginosa possesses two acyl-HSL quorum-sensing systems, one for production of and response to N-3-oxo-dodecanoyl homoserine lactone (3OC₁₂-HSL) (LasR-LasI) and the other for production of and response to N-butanoyl homoserine lactone (C₄-HSL) (RhlR-RhlI) (35, 37). We have reported that an rhlI mutant unable to synthesize the C₄-HSL signal was impaired in iron limitation-induced twitching motility and formed structured biofilms under iron-limiting conditions (31).The correlation between twitching motility, the RhlR-RhlI quorum-sensing system, and iron-regulated biofilm formation led us to hypothesize that rhamnolipids are involved in mediating this process. Rhamnolipids are surface-active amphipathic molecules composed of a hydrophobic lipid and a hydrophilic sugar moiety and compose the main constituents of the biosurfactant produced by P. aeruginosa (reviewed in reference 42). The biosurfactant is required for a form of surface motility called swarming, where it functions as a wetting agent and reduces surface tension (8, 14). Furthermore, elements constituting the biosurfactant were recently shown to modulate the swarming behavior by acting as chemotactic-like stimuli (43). Rhamnolipids are also important in maintaining biofilm structure and inducing biofilm dispersion (6, 11, 29). Their synthesis requires the expression of the rhlAB operon, which is regulated by the RhlR-RhlI quorum-sensing system (14, 25, 32) and is also induced under iron-limiting conditions (14).In this study, we test this hypothesis and demonstrate that rhamnolipid production is induced under iron-limiting conditions and that this promotes twitching motility. We found that increased expression of rhamnolipid synthesis genes during early biofilm development under iron-limiting conditions induces surface motility and results in formation of a thin flat biofilm. Furthermore, a mutant that is incapable of synthesizing rhamnolipids does not display twitching motility under iron-limiting conditions and thus forms structured biofilms under these conditions. These results highlight the importance of biosurfactant production in determining the architecture of mature P. aeruginosa biofilms under iron-limiting conditions.     

Role of TonB1 in Pyoverdine-Mediated Signaling in Pseudomonas aeruginosa

Pyoverdines are siderophores secreted by Pseudomonas aeruginosa. Uptake of ferripyoverdine in P. aeruginosa PAO1 occurs via the FpvA receptor protein and requires the energy-transducing protein TonB1. Interaction of (ferri)pyoverdine with FpvA activates pyoverdine gene expression in a signaling process involving the cytoplasmic-membrane-spanning anti-sigma factor FpvR and the sigma factor PvdS. Here, we show that mutation of a region of FpvA that interacts with TonB1 (the TonB box) prevents this signaling process, as well as inhibiting bacterial growth in the presence of the iron-chelating compound ethylenediamine-di(o-hydroxy-phenylacetic acid). Signaling via wild-type FpvA was also eliminated in strains lacking TonB1 but was unaffected in strains lacking either (or both) of two other TonB proteins in P. aeruginosa, TonB2 and TonB3. An absence of pyoverdine-mediated signaling corresponded with proteolysis of PvdS. These data show that interactions between FpvA and TonB1 are required for (ferri)pyoverdine signal transduction, as well as for ferripyoverdine transport, consistent with a mechanistic link between the signaling and transport functions of FpvA.Pseudomonas aeruginosa is an opportunistic pathogen that is able to cause severe infections in patients with cystic fibrosis and in immunocompromised individuals, such as burn victims. Under conditions of iron limitation, P. aeruginosa secretes an iron-scavenging compound (siderophore) called pyoverdine. Ferripyoverdine is transported back into the bacteria by an outer membrane (OM) receptor protein, FpvA. The transport of ferripyoverdine via FpvA requires energy provided by a TonB complex (36, 42, 50). TonB is an energy-transducing protein that couples the energy of the cytoplasmic membrane (CM) to a variety of OM receptors required for the import of ferrisiderophores and other molecules. TonB acts in a complex with two CM-associated proteins, ExbB and ExbD, both of which are required for full TonB function (5, 37). The TonB-ExbB-ExbD complex has been identified in many gram-negative bacterial species and is thought to be a conserved mechanism for energy transduction to OM receptor proteins (31). TonB-dependent receptors contain a conserved protein motif known as the TonB box (5). Direct interaction between TonB and the TonB box has been demonstrated for several TonB-dependent receptors (8, 26, 33, 35, 47). Mutations of the TonB box, particularly mutations that are likely to affect the secondary structure, can result in a TonB-uncoupled phenotype characterized by loss of TonB-dependent functions (ferrisiderophore transport) with no loss of TonB-independent functions, such as internalization of bacteriophage (37).The P. aeruginosa PAO1 genome contains three tonB genes, tonB1 (PA5531) (36), tonB2 (PA0197) (55), and tonB3 (PA0406) (20), encoding proteins of 342, 270, and 319 amino acids (aa), respectively. The TonB1 and TonB2 amino acid sequences display 31% identity over a section of 187 aa, but otherwise, the three PAO1 TonB proteins show similarity (30 to 40% aa identity) to each other only over short (<70-aa) regions. TonB1 is considered to be the primary TonB protein involved in iron transport in P. aeruginosa. tonB1 mutants are impaired for growth in iron-limited medium and are defective for siderophore-mediated iron transport and heme utilization (36, 50, 55). Moreover, direct interaction between TonB1 and the ferripyoverdine receptor FpvA has been demonstrated in vitro (1). The tonB2 gene is not required for growth in iron-limited medium (55). However, tonB1 tonB2 double mutants grow even less well under iron limitation than tonB1 mutants, indicating that TonB2 may be able to partially complement TonB1 in its role in iron acquisition (55). The tonB3 gene is required for twitching motility and assembly of extracellular pili (20), but it is not known whether TonB3 has a role in iron acquisition. Genes encoding ExbB and ExbD proteins are located directly downstream of tonB2 (55) but are not found in association with tonB1 or tonB3.Besides its role in ferripyoverdine transport, FpvA is part of a signal transduction pathway and thus belongs to a subset of TonB-dependent receptors known as TonB-dependent transducers (reviewed in references 23 and 51). Mutational analysis has shown that the ferripyoverdine transport and signaling roles of FpvA are separate and discrete functions (21, 46). Besides FpvA, the signal transduction pathway involves a CM-spanning anti-sigma factor protein, FpvR, and (ferri)pyoverdine. (It was previously thought that both ferri- and apopyoverdine could bind FpvA (43). However, it was recently reported that only ferripyoverdine is able to form a high-affinity interaction with FpvA (13). The designation (ferri)pyoverdine will be used here to represent the active signaling molecule. FpvA and (ferri)pyoverdine regulate the activity of FpvR, which in turn regulates the activities of two extracytoplasmic function family sigma factors, PvdS and FpvI (3, 25). Upon binding of (ferri)pyoverdine to FpvA, a signal is transmitted to FpvR, resulting in activation of PvdS and FpvI. Activation of PvdS is required for maximal synthesis of pyoverdine itself, as well as two secreted proteins (25). Activation of FpvI leads to increased expression of fpvA (3, 39). In the absence of pyoverdine-mediated signaling, caused by the lack of FpvA or pyoverdine or overexpression of FpvR, suppression of PvdS- and FpvI-dependent gene expression occurs (3, 25), and this is associated with proteolysis of PvdS (49).Analogous siderophore transport and signaling systems involving an OM TonB-dependent transducer, a CM-bound anti-sigma factor, and an extracytoplasmic function family sigma factor have been described in other bacteria, including the ferric citrate (Fec) system in Escherichia coli and the pseudobactin (Pup) system in Pseudomonas putida (reviewed in reference 6). The TonB protein is required for signaling in both the Fec (14, 33) and Pup (24) systems. Similarly, a TonB system is required for hemophore transport and signaling in Serratia marcescens (4). The aim of this study was to investigate whether TonB was required for pyoverdine-mediated signaling in P. aeruginosa, and if so, to identify which of the three TonB proteins was involved.     

The Ferrichrome Uptake Pathway in Pseudomonas aeruginosa Involves an Iron Release Mechanism with Acylation of the Siderophore and Recycling of the Modified Desferrichrome

The uptake of iron into Pseudomonas aeruginosa is mediated by two major siderophores produced by the bacterium, pyoverdine and pyochelin. The bacterium is also able of utilize several heterologous siderophores of bacterial or fungal origin. In this work, we have investigated the iron uptake in P. aeruginosa PAO1 by the heterologous ferrichrome siderophore. ⁵⁵Fe uptake assays showed that ferrichrome is transported across the outer membrane primarily (80%) by the FiuA receptor and to a lesser extent (20%) by a secondary transporter. Moreover, we demonstrate that like in the uptake of ferripyoverdine and ferripyochelin, the energy required for both pathways of ferrichrome uptake is provided by the inner membrane protein TonB1. Desferrichrome-⁵⁵Fe uptake in P. aeruginosa was also dependent on the expression of the permease FiuB, suggesting that this protein is the inner membrane transporter of the ferrisiderophore. A biomimetic fluorescent analogue of ferrichrome, RL1194, was used in vivo to monitor the kinetics of iron release from ferrichrome in P. aeruginosa in real time. This dissociation involves acylation of ferrichrome and its biomimetic analogue RL1194 and recycling of both modified siderophores into the extracellular medium. FiuC, an N-acetyltransferase, is certainly involved in this mechanism of iron release, since its mutation abolished desferrichrome-⁵⁵Fe uptake. The acetylated derivative reacts with iron in the extracellular medium and is able to be taken up again by the cells. All these observations are discussed in light of the current knowledge concerning ferrichrome uptake in P. aeruginosa and in Escherichia coli.Iron is essential for life for practically all living organisms and plays a number of key roles in biology. DNA and RNA synthesis, glycolysis, energy generation by electron transport, nitrogen fixation, and photosynthesis are examples of processes in which iron-containing enzymes play vital roles. However, under physiological conditions iron forms highly insoluble ferric hydroxide complexes, which severely limits its bioavailability. To overcome the problem of iron inaccessibility, bacteria excrete high-affinity iron chelators termed siderophores, which are able to solubilize iron and deliver it into the cells (3, 64).Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is capable of infecting a wide variety of animal, insects, and plants. As a human pathogen, P. aeruginosa is the leading source of Gram-negative nosocomial infections (59) and causes chronic lung infections in approximately 90% of individuals suffering from cystic fibrosis (40). Under iron-limited conditions, P. aeruginosa produces two major siderophores, pyoverdine (PVD) (62) and pyochelin (PCH) (15). P. aeruginosa is also capable of utilizing numerous siderophores secreted by other microorganisms: pyoverdins from other pseudomonas, enterobactin (49), cepabactin (45), mycobactin and carboxymycobactin (38), fungal siderophores (ferrichrome [39]; deferrioxamines [39, 60]; and desferrichrysin, desferricrocin, and coprogen [44]), and natural occurring chelators such as citrate (14, 23) (for a review, see reference 47).In Gram-negative bacteria, the uptake of ferrisiderophores always involves a specific transporter at the level of the outer membrane (4). The energy required for this process is provided by the proton motive force (PMF) of the inner membrane by means of an inner membrane complex comprising TonB, ExbB, and ExbD (21, 51, 63). In silico analysis of the P. aeruginosa genome (http://www.pseudomonas.com) revealed 32 genes encoding putative TonB-dependent transporters (13), of which only 12 are involved in metal (mostly iron) uptake (38). FpvA and FpvB are the outer membrane transporters involved in the uptake of PVD-Fe (19, 48), and FptA transports PCH-Fe (25). Concerning the heterologous siderophores, there are two transporters, FoxA and FiuA, involved in the transport of ferrioxamine B and ferrichrome (39). The mechanism involved in the translocation of ferrisiderophores across the outer membrane by the TonB-dependent transporters has been studied mostly in E. coli (for a review, see reference 5) and in the case of P. aeruginosa has been studied only for the FpvA/PVD and the FptA/PCH systems. The structures of FpvA (8, 11, 65) and FptA (12) have been solved and their interactions with PVD and PCH investigated at the molecular level (26, 27, 45, 53). Three tonB genes, encoding the energy coupler TonB, have been found in the P. aeruginosa genome, i.e., tonB1, tonB2, and tonB3. Disruption of tonB1 abrogates PVD- and PCH-mediated iron uptake (50, 58) and heme uptake (67). Inactivation of tonB2 has no adverse effect on iron or heme acquisition, but tonB1 tonB2 double mutants are more compromised with respect to growth in iron-restricted medium than is a single tonB1 knockout mutant (67). Mutation of tonB3 appears to result in defective twitching motility (28), and the gene product is most likely not involved in iron uptake.In P. aeruginosa, many ferrisiderophore outer membrane transporters are also involved in a signaling cascade regulating the expression of genes involved in iron uptake. This is the case for FpvA (PVD uptake), FoxA (ferrioxamine), and FiuA (ferrichrome) (38, 39, 43, 61). Such a signaling cascade involves an extracytoplasmic function (ECF) sigma factor and an inner membrane anti-sigma factor. Equivalent cell surface signaling is present in Escherichia coli for ferricitrate uptake by FecA but not for ferrichrome, ferrioxamine, and enterobactin uptake by FhuA, FhuE, and FepA, respectively.Little is known about the translocation of ferrisiderophores across the inner membrane in P. aeruginosa. In E. coli, this step involves a specific ABC transporter for almost every siderophore used by this bacterium: FhuBCD for the uptake of ferrichrome and ferrioxamine (33-36), FecBCD for the uptake of ferricitrate (6, 56) and, FepBCDG for the uptake of ferrienterobactin (9). In P. aeruginosa, the only characterized inner membrane siderophore transport protein is FptX, a proton motive force-dependent permease, which functions in PCH-Fe utilization (16). The inner membrane FoxB is involved in the utilization of ferrichrome and ferrioxamine B, but it remains to be determined whether this protein functions in the transport of ferrisiderophore or in the release of iron from ferrichrome or ferrioxamine (17). The genome clearly shows a fepBCDG homologue for the transport of ferrienterobactin. For the other iron uptake pathways present in P. aeruginosa, the transporters involved at the level of the inner membrane have not been identified. An import ABC transporter is present in the pvd locus (PA2407 to PA2410; http://www.pseudomonas.com), but its mutation does not affect PVD-Fe uptake (46).In P. aeruginosa, the mechanism of ferrisiderophore dissociation has been investigated only for the PVD pathway. This step occurs in the periplasm by a mechanism involving no chemical siderophore modification but involving a reduction of iron and a recycling of the siderophore into the extracellular medium by the PvdRT-OpmQ efflux pump (20, 54, 66). In E. coli, the mechanism of ferrisiderophore dissociation has been investigated for the ferrichrome and ferrienterobactin pathways. Iron release from ferrichrome occurs in the cytoplasm and probably involves iron reduction (41) followed by acetylation of the siderophore and its recycling into the growth medium (24). For the ferrienterobactin pathway, a cytoplasmic esterase hydrolyzes the siderophore (7).In the present work, we have investigated the ferrichrome pathway in P. aeruginosa using both ferrichrome and a fluorescently labeled biomimetic ferrichrome analogue. We evaluated the siderophore properties of the fluorescent analogue and identified the different transporters involved in the uptake across the outer and inner membranes. Furthermore, we demonstrated that following ferrichrome uptake, iron is released from the siderophore by a mechanism involving an acetylation of the chelator and the modified desferrichrome is secreted back into the growth medium.     

The Glycerol-3-Phosphate Permease GlpT Is the Only Fosfomycin Transporter in Pseudomonas aeruginosa

Fosfomycin is transported into Escherichia coli via both glycerol-3-phosphate (GlpT) and a hexose phosphate transporter (UhpT). Consequently, the inactivation of either glpT or uhpT confers increased fosfomycin resistance in this species. The inactivation of other genes, including ptsI and cyaA, also confers significant fosfomycin resistance. It has been assumed that identical mechanisms are responsible for fosfomycin transport into Pseudomonas aeruginosa cells. The study of an ordered library of insertion mutants in P. aeruginosa PA14 demonstrated that only insertions in glpT confer significant resistance. To explore the uniqueness of this resistance target in P. aeruginosa, the linkage between fosfomycin resistance and the use of glycerol-3-phosphate was tested. Fosfomycin-resistant (Fos-R) mutants were obtained in LB and minimal medium containing glycerol as the sole carbon source at a frequency of 10⁻⁶. However, no Fos-R mutants grew on plates containing fosfomycin and glycerol-3-phosphate instead of glycerol (mutant frequency, ≤5 × 10⁻¹¹). In addition, 10 out of 10 independent spontaneous Fos-R mutants, obtained on LB-fosfomycin, harbored mutations in glpT, and in all cases the sensitivity to fosfomycin was recovered upon complementation with the wild-type glpT gene. The analysis of these mutants provides additional insights into the structure-function relationship of glycerol-3-phosphate the transporter in P. aeruginosa. Studies with glucose-6-phosphate and different mutant derivatives strongly suggest that P. aeruginosa lacks a specific transport system for this sugar. Thus, glpT seems to be the only fosfomycin resistance mutational target in P. aeruginosa. The high frequency of Fos-R mutations and their apparent lack of fitness cost suggest that Fos-R variants will be obtained easily in vivo upon the fosfomycin treatment of P. aeruginosa infections.Pseudomonas aeruginosa is an opportunistic, life-threatening bacterial pathogen that especially affects critically ill patients in intensive care units or those suffering from chronic respiratory diseases such as cystic fibrosis (19, 40). Its 6.3-Mb genome supports its enormous metabolic versatility and, consequently, its adaptability to almost any challenging environment. One of the consequences of this versatility is the rapid adaptation to stressful environmental conditions, including starvation, desiccation, and antibiotic treatments (14, 40). Mutants resistant to one or several antibiotics will evolve during sufficiently prolonged treatments, this being a process facilitated by the presence of hypermutable alleles (31, 32). After years of treating cystic fibrosis patients with antibiotics, P. aeruginosa became unavoidably resistant to many or all of them (5). Multidrug-resistant strains of P. aeruginosa are an important problem for the treatment of nosocomial outbreaks and cystic fibrosis patients (27, 37). Currently, the treatment of multidrug-resistant P. aeruginosa requires the combination of various antimicrobial agents. Fosfomycin (Fos) has been reported to be effective in combination with other antipseudomonal agents (6, 29, 42, 44). The proportion of Fos-resistant (Fos-R) strains in clinical isolates of P. aeruginosa currently is not well known, and even the mechanisms that support Fos resistance in P. aeruginosa are not clear. Thus, the knowledge of the molecular bases involved in the development of spontaneous Fos resistance in P. aeruginosa is of particular interest.Fos is a unique broad-spectrum bactericidal antibiotic that is chemically unrelated to any other known antimicrobial agent used to treat urinary tract and gastrointestinal infections in humans (9, 35). It binds UDP-GlcNAc enol-pyruvyltransferase (MurA), acting as a phosphoenolpyruvate analogue and avoiding the formation of UDP-N-acetylglucosamine-3-O-enolpyruvate from UDP-N-acetylglucosamine and phosphoenolpyruvate (12, 33). Fos is taken up actively into bacterial cells via transport systems. In Escherichia coli, Fos is imported through two nutrient transport systems, the glycerol-3-phosphate (glycerol-3-P) transporter (GlpT) and glucose-6-phosphate (glucose-6-P) transporter (UhpT), to achieve its target and inhibits the initial step in cell wall synthesis (12, 17). The expression of these transport systems is induced by their substrates (glycerol-3-P and glucose-6P) and requires the presence of the cyclic AMP receptor protein (cAMP-CRP) complex (23, 30). Additionally, the high-level expression of UhpT requires the regulatory genes uhpA, uhpB, and uhpC (12, 30). Therefore, Fos-R strains isolated in E. coli contain mutations that prevent Fos transport using GlpT or UhpT (23, 30). Plasmid-encoded resistance also has been described previously (4, 41).In this paper, we describe the screening and analysis of Fos-R clones in a P. aeruginosa PA14 ordered insertional library (18). In addition, we studied the mutations responsible for the spontaneous resistance to Fos in P. aeruginosa PA14, the effect of these mutations on the in vitro growth rate, and the uniqueness of the mutational target.