BifA, a cyclic-Di-GMP phosphodiesterase, inversely regulates biofilm formation and swarming motility by Pseudomonas aeruginosa PA14

The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the ΔbifA mutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the ΔbifA mutant but does not contribute to the observed swarming defect. The ΔbifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.     

Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14

We previously reported that SadB, a protein of unknown function, is required for an early step in biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa. Here we report that a mutation in sadB also results in increased swarming compared to the wild-type strain. Our data are consistent with a model in which SadB inversely regulates biofilm formation and swarming motility via its ability both to modulate flagellar reversals in a viscosity-dependent fashion and to influence the production of the Pel exopolysaccharide. We also show that SadB is required to properly modulate flagellar reversal rates via chemotaxis cluster IV (CheIV cluster). Mutational analyses of two components of the CheIV cluster, the methyl-accepting chemotaxis protein PilJ and the PilJ demethylase ChpB, support a model wherein this chemotaxis cluster participates in the inverse regulation of biofilm formation and swarming motility. Epistasis analysis indicates that SadB functions upstream of the CheIV cluster. We propose that P. aeruginosa utilizes a SadB-dependent, chemotaxis-like regulatory pathway to inversely regulate two key surface behaviors, biofilm formation and swarming motility.     

Isolation and characterization of a generalized transducing phage for Pseudomonas aeruginosa strains PAO1 and PA14

A temperate, type IV pilus-dependent, double-stranded DNA bacteriophage named DMS3 was isolated from a clinical strain of Pseudomonas aeruginosa. A clear-plaque variant of this bacteriophage was isolated. DMS3 is capable of mediating generalized transduction within and between P. aeruginosa strains PA14 and PAO1, thus providing a useful tool for the genetic analysis of P. aeruginosa.     

Chemical analysis of cellular and extracellular carbohydrates of a biofilm-forming strain Pseudomonas aeruginosa PA14

Background

Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen, which causes persisting life-threatening infections in cystic fibrosis (CF) patients. Biofilm mode of growth facilitates its survival in a variety of environments. Most P. aeruginosa isolates, including the non-mucoid laboratory strain PA14, are able to form a thick pellicle, which results in a surface-associated biofilm at the air-liquid (A–L) interface in standing liquid cultures. Exopolysaccharides (EPS) are considered as key components in the formation of this biofilm pellicle. In the non-mucoid P. aeruginosa strain PA14, the “scaffolding” polysaccharides of the biofilm matrix, and the molecules responsible for the structural integrity of rigid A–L biofilm have not been identified. Moreover, the role of LPS in this process is unclear, and the chemical structure of the LPS O-antigen of PA14 has not yet been elucidated.

Principal Findings

In the present work we carried out a systematic analysis of cellular and extracellular (EC) carbohydrates of P. aeruginosa PA14. We also elucidated the chemical structure of the LPS O-antigen by chemical methods and 2-D NMR spectroscopy. Our results showed that it is composed of linear trisaccharide repeating units, identical to those described for P. aeruginosa Lanýi type O:2a,c (Lanýi-Bergman O-serogroup 10a, 10c; IATS serotype 19) and having the following structure: -4)-α-L-GalNAcA-(1–3)-α-D-QuiNAc-(1–3)- α-L-Rha-(1-. Furthermore, an EC O-antigen polysaccharide (EC O-PS) and the glycerol-phosphorylated cyclic β-(1,3)-glucans were identified in the culture supernatant of PA14, grown statically in minimal medium. Finally, the extracellular matrix of the thick biofilm formed at the A-L interface contained, in addition to eDNA, important quantities (at least ∼20% of dry weight) of LPS-like material.

Conclusions

We characterized the chemical structure of the LPS O-antigen and showed that the O-antigen polysaccharide is an abundant extracellular carbohydrate of PA14. We present evidence that LPS-like material is found as a component of a biofilm matrix of P. aeruginosa.     

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.     

The Pseudomonas aeruginosa PA14 type III secretion system is expressed but not essential to virulence in the Caenorhabditis elegans-P. aeruginosa pathogenicity model

The Pseudomonas aeruginosa type III secretion system (TTSS), enabling direct injection of toxins into host cells, has been shown to be crucial to virulence in several models of P. aeruginosa pathogenesis. Using the strain PA14 and its isogenic mutant, PA14exsA, we investigated the role of the TTSS during infection of the nematode Caenorhabditis elegans. Although C. elegans N2 was killed by PA14 in an infection like process over 48 to 72 h the same effect was observed following infection with PA14exsA, implying that a functional TTSS was not essential for virulence. This was despite the TTSS being actively expressed during C. elegans infection as demonstrated by the use of green fluorescent reporter constructs and RT-PCR. However, compared to the wild type PA14, PA14exsA did display a reduced rate of killing of C. elegans strain AU1 which harbours a mutation in the sek-1 gene encoding a MAP kinase involved in nematode innate immunity. A fuller understanding of the mechanism of resistance to type III attack in C. elegans may lead to the identification and development of novel therapeutic targets affording protection to TTSS products in man.     

Pseudomonas aeruginosa LD-carboxypeptidase, a serine peptidase with a Ser-His-Glu triad and a nucleophilic elbow

LD-Carboxypeptidases (EC 3.4.17.13) are named for their ability to cleave amide bonds between l- and d-amino acids, which occur naturally in bacterial peptidoglycan. They are specific for the link between meso-diaminopimelic acid and d-alanine and therefore degrade GlcNAc-MurNAc tetrapeptides to the corresponding tripeptides. As only the tripeptides can be reused as peptidoglycan building blocks, ld-carboxypeptidases are thought to play a role in peptidoglycan recycling. Despite the pharmaceutical interest in peptidoglycan biosynthesis, the fold and catalytic type of ld-carboxypeptidases are unknown. Here, we show that a previously uncharacterized open reading frame in Pseudomonas aeruginosa has ld-carboxypeptidase activity and present the crystal structure of this enzyme. The structure shows that the enzyme consists of an N-terminal beta-sheet and a C-terminal beta-barrel domain. At the interface of the two domains, Ser(115) adopts a highly strained conformation in the context of a strand-turn-helix motif that is similar to the "nucleophilic elbow" in alphabeta-hydrolases. Ser(115) is hydrogen-bonded to a histidine residue, which is oriented by a glutamate residue. All three residues, which occur in the order Ser-Glu-His in the amino acid sequence, are strictly conserved in naturally occurring ld-carboxypeptidases and cannot be mutated to alanines without loss of activity. We conclude that ld-carboxypeptidases are serine peptidases with Ser-His-Glu catalytic triads.     

Functional analysis of pyochelin-/enantiopyochelin-related genes from a pathogenicity island of Pseudomonas aeruginosa strain PA14

Genomic islands are foreign DNA blocks inserted in so-called regions of genomic plasticity (RGP). Depending on their gene content, they are classified as pathogenicity, symbiosis, metabolic, fitness or resistance islands, although a detailed functional analysis is often lacking. Here we focused on a 34-kb pathogenicity island of Pseudomonas aeruginosa PA14 (PA14GI-6), which is inserted at RGP5 and carries genes related to those for pyochelin/enantiopyochelin biosynthesis. These enantiomeric siderophores of P. aeruginosa and certain strains of Pseudomonas protegens are assembled by a thiotemplate mechanism from salicylate and two molecules of cysteine. The biochemical function of several proteins encoded by PA14GI-6 was investigated by a series of complementation analyses using mutants affected in potential homologs. We found that PA14_54940 codes for a bifunctional salicylate synthase/salicyl-AMP ligase (for generation and activation of salicylate), that PA14_54930 specifies a dihydroaeruginoic acid (Dha) synthetase (for coupling salicylate with a cysteine-derived thiazoline ring), that PA14_54910 produces a type II thioesterase (for quality control), and that PA14_54880 encodes a serine O-acetyltransferase (for increased cysteine availability). The structure of the PA14GI-6-specified metabolite was determined by mass spectrometry, thin-layer chromatography, and HPLC as (R)-Dha, an iron chelator with antibacterial, antifungal and antitumor activity. The conservation of this genomic island in many clinical and environmental P. aeruginosa isolates of different geographical origin suggests that the ability for Dha production may confer a selective advantage to its host.     

Syndecan-1 shedding is enhanced by LasA, a secreted virulence factor of Pseudomonas aeruginosa

Microbial pathogens frequently take advantage of host systems for their pathogenesis. Shedding of cell surface molecules as soluble extracellular domains (ectodomains) is one of the host responses activated during tissue injury. In this study, we examined whether pathogenic bacteria can modulate shedding of syndecan-1, the predominant syndecan of host epithelia. Our studies found that overnight culture supernatants of Pseudomonas aeruginosa and Staphylococcus aureus enhanced the shedding of syndecan-1 ectodomains, whereas culture supernatants of several other Gram-negative and Gram-positive bacteria had only low levels of activity. Because supernatants from all tested strains of P. aeruginosa (n = 9) enhanced syndecan-1 shedding by more than 4-fold above control levels, we focused our attention on this Gram-negative bacterium. Culture supernatants of P. aeruginosa increased shedding of syndecan-1 in both a concentration- and time-dependent manner, and augmented shedding by various host cells. A 20-kDa shedding enhancer was partially purified from the supernatant through ammonium sulfate precipitation and gel chromatography, and identified by N-terminal sequencing as LasA, a known P. aeruginosa virulence factor. LasA was subsequently determined to be a syndecan-1 shedding enhancer from the findings that (i) immunodepletion of LasA from the partially purified sample resulted in abrogation of its activity to enhance shedding and (ii) purified LasA increased shedding in a concentration-dependent manner. Our results also indicated that LasA enhances syndecan-1 shedding by activation of the host cell's shedding mechanism and not by direct interaction with syndecan-1 ectodomains. Enhanced syndecan-1 shedding may be a means by which pathogenic bacteria take advantage of a host mechanism to promote their pathogenesis.     

The Multifaceted Proteins MvaT and MvaU,Members of the H-NS Family,Control Arginine Metabolism,Pyocyanin Synthesis,and Prophage Activation in Pseudomonas aeruginosa PAO1

The MvaT and MvaU proteins belonging to the H-NS family were identified as DNA-binding proteins that interact with the regulatory region of the aotJQMOP-argR operon for arginine uptake and regulation. Recombinant MvaT and MvaU proteins were purified, and binding of these purified proteins to the aotJ regulatory region was demonstrated using electromobility shift assays. Polyclonal antibodies against purified MvaT and MvaU were prepared and employed in supershift assays to support these observations. Knockout mutations resulting in a single lesion in mvaT or mvaU, as well as knockout mutations resulting in double lesions, were constructed using biparental conjugation, and the absence of MvaT and MvaU in the resulting mutants was confirmed by immunoblot analysis. Using measurements of the β-galactosidase activities from aotJ::lacZ fusions in the mutants and the parental strain, it was found that MvaT and MvaU serve as repressors in control of aotJ expression. The effects of MvaT and MvaU on pyocyanin synthesis and CupA fimbrial expression in these mutants were also analyzed. Pyocyanin synthesis was induced in the single mutants but was completely abolished in the double mutant, suggesting that there is a complicated regulatory scheme in which MvaT and MvaU are essential elements. In comparison, MvaT had a more profound role than MvaU as a repressor of cupA expression; however, a combination of MvaT depletion and MvaU depletion had a strong synergistic effect on cupA. Moreover, prophage Pf4 integrated into the chromosome of Pseudomonas aeruginosa PAO1 was activated in an mvaT mvaU double mutant but not in a single mutant. These results were supported by purification and nucleotide sequencing of replicative-form DNA and by the release of phage particles in plaque assays. In summary, the mvaT mvaU double mutant was viable, and depletion of MvaT and MvaU had serious effects on a variety of physiological functions in P. aeruginosa.The MvaT and MvaU proteins of Pseudomonas aeruginosa belong to the H-NS family of small DNA-binding proteins. MvaT was initially identified in P. mevalonii as a positive regulator for mevalonate catabolism (25). Subsequently, MvaT homologues have been identified in other pseudomonads based on structural and functional similarities; five homologues have been identified in P. putida, three homologues have been identified in P. fluorescens, four homologues have been identified in P. syringae, and two homologues have been identified in P. aeruginosa. In P. putida, the TurA protein represses the Pu promoter of the TOL plasmid in a temperature-dependent manner (24). In P. fluorescens, the MvaT and MvaV proteins regulate the expression of two biocontrol exoproducts, 2,4-diacetyl phloroglucinol and pyoluteorin (1). In P. aeruginosa, MvaT is involved in quorum-sensing responses and biofilm formation. Inactivation of mvaT resulted in increased production of PA-IL lectin and the toxic exoproduct pyocyanin, reduced biofilm formation, increased drug resistance, and reduced swarming motility (5, 33). In addition, the MvaT protein is involved in the phase-variable expression of the fimbrial cup genes involved in biofilm formation. Moreover, DNA microarray analysis has shown that more than 150 genes are influenced by the MvaT protein (27).The MvaU protein shows high levels of similarity to MvaT and can perform some of the MvaT regulatory functions (28). Like other H-NS-related proteins, MvaT and MvaU possess two distinct domains: the N terminus for oligomerization and the C terminus for DNA-binding activity. The MvaT and MvaU proteins can interact through their N-terminal regions and form hetero- and homodimers (28). In general, mvaT mutation seems to have a much more profound effect than mvaU mutation. In a study using chromatin immunoprecipitation coupled with DNA microarrays, the potential MvaT and MvaU binding sites on the genome of P. aeruginosa were identified (3). In the same report, it was concluded that loss of both MvaT and MvaU from the cell cannot be tolerated.In this study, we identified MvaT and MvaU as components of a nucleoprotein complex that controls the aotJQMOP-argR operon for arginine uptake and regulation (21, 22). Using mvaT and mvaU single- and double-mutant constructs, we also demonstrated the consequences of MvaT and MvaU depletion for prophage activation, pyocyanin synthesis, and fimbrial cupA gene expression.     

A Pseudomonas aeruginosa PAO1 acetylcholinesterase is encoded by the PA4921 gene and belongs to the SGNH hydrolase family

Through the use of molecular and biochemical experiments and bioinformatic tools, this work demonstrates that the PA4921 gene of the Pseudomonas aeruginosa PAO1 genome is a gene responsible for cholinesterase (ChoE) activity. Similar to the acetylcholinesterase (AchE) of Zea mays, this ChoE belongs to the SGNH hydrolase family. In mature ChoE, i.e., without a signal peptide, (18)Ser, (78)Gly, (127)N, and (268)H are conserved aminoacyl residues. Acetylthiocholine (ATC) and propionylthiocholine (PTC) are substrates of this enzyme, but butyrylcholine is an inhibitor. The enzyme also catalyzes the hydrolysis of the artificial esters p-nitrophenyl propionate (pNPP) and p-nitrophenyl butyrate (pNPB) but with lower catalytic efficiency with respect to ATC or PTC. The second difference is that pNPP and pNPB did not produce inhibition at high substrate concentrations, as occurred with ATC and PTC. These differences plus preliminary biochemical and kinetic studies with alkylammonium compounds led us to propose that this enzyme is an acetylcholinesterase (AchE) or propionylcholinesterase. Studies performed with the purified recombinant enzyme indicated that the substrate saturation curves and the catalytic mechanism are similar to those properties described for mammalian AchEs. Therefore, the results of this work suggest that the P. aeruginosa ChoE is an AchE that may also be found in Pseudomonas fluorescens.     

Microevolution of the major common Pseudomonas aeruginosa clones C and PA14 in cystic fibrosis lungs

Clones C and PA14 are the worldwide most abundant clonal complexes in the Pseudomonas aeruginosa population. The microevolution of clones C and PA14 was investigated in serial cystic fibrosis (CF) airway isolates collected over 20 years since the onset of colonization. Intraclonal evolution in CF lungs was resolved by genome sequencing of first, intermediate and late isolates and subsequent multimarker SNP genotyping of the whole strain panel. Mapping of sequence reads onto the P. aeruginosa PA14 reference genome unravelled an intraclonal and interclonal sequence diversity of 0.0035% and 0.68% respectively. Clone PA14 diversified into three branches in the patient's lungs, and the PA14 population acquired 15 nucleotide substitutions and a large deletion during the observation period. The clone C genome remained invariant during the first 3 years in CF lungs; however, 15 years later 947 transitions and 12 transversions were detected in a clone C mutL mutant strain. Key mutations occurred in retS, RNA polymerase, multidrug transporter, virulence and denitrification genes. Late clone C and PA14 persistors in the CF lungs were compromised in growth and cytotoxicity, but their mutation frequency was normal even in mutL mutant clades.     

Differential Roles of the Pseudomonas aeruginosa PA14 rpoN Gene in Pathogenicity in Plants, Nematodes, Insects, and Mice

We cloned the rpoN (ntrA, glnF) gene encoding the alternate sigma factor sigma(54) from the opportunistic multihost pathogen Pseudomonas aeruginosa strain PA14. A marker exchange protocol was used to construct the PA14 rpoN insertional mutation rpoN::Gen(r). PA14 rpoN::Gen(r) synthesized reduced levels of pyocyanin and displayed a variety of phenotypes typical of rpoN mutants, including a lack of motility and the failure to grow on nitrate, glutamate, or histidine as the sole nitrogen source. Compared to wild-type PA14, rpoN::Gen(r) was ca. 100-fold less virulent in a mouse thermal injury model and was significantly impaired in its ability to kill the nematode Caenorhabditis elegans. In an Arabidopsis thaliana leaf infectivity assay, although rpoN::Gen(r) exhibited significantly reduced attachment to trichomes, stomata, and the epidermal cell surface, did not attach perpendicularly to or perforate mesophyll cell walls, and proliferated less rapidly in Arabidopsis leaves, it nevertheless elicited similar disease symptoms to wild-type P. aeruginosa PA14 at later stages of infection. rpoN::Gen(r) was not impaired in virulence in a Galleria mellonella (greater wax moth) pathogenicity model. These data indicate that rpoN does not regulate the expression of any genes that encode virulence factors universally required for P. aeruginosa pathogenicity in diverse hosts.