Gene_locus Report for: pseae-PA2934

Recurrent Pseudomonas aeruginosa infections coupled with robust, damaging neutrophilic inflammation characterize the chronic lung disease cystic fibrosis (CF). The proresolving lipid mediator, 15-epi lipoxin A4 (15-epi LXA4), plays a critical role in limiting neutrophil activation and tissue inflammation, thus promoting the return to tissue homeostasis. Here, we show that a secreted P. aeruginosa epoxide hydrolase, cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), can disrupt 15-epi LXA4 transcellular biosynthesis and function. In the airway, 15-epi LXA4 production is stimulated by the epithelial-derived eicosanoid 14,15-epoxyeicosatrienoic acid (14,15-EET). Cif sabotages the production of 15-epi LXA4 by rapidly hydrolyzing 14,15-EET into its cognate diol, eliminating a proresolving signal that potently suppresses IL-8-driven neutrophil transepithelial migration in vitro. Retrospective analyses of samples from patients with CF supported the translational relevance of these preclinical findings. Elevated levels of Cif in bronchoalveolar lavage fluid were correlated with lower levels of 15-epi LXA4, increased IL-8 concentrations, and impaired lung function. Together, these findings provide structural, biochemical, and immunological evidence that the bacterial epoxide hydrolase Cif disrupts resolution pathways during bacterial lung infections. The data also suggest that Cif contributes to sustained pulmonary inflammation and associated loss of lung function in patients with CF.

The CFTR inhibitory factor (Cif) is an epoxide hydrolase (EH) virulence factor secreted by the bacterium Pseudomonas aeruginosa. Sequence alignments reveal a pattern of Cif-like substitutions that proved to be characteristic of a new subfamily of bacterial EHs. At the same time, crystallographic and mutagenetic data suggest that EH activity is required for virulence and that Cif's active site remains generally compatible with a canonical two-step EH mechanism. A hallmark of this mechanism is the formation of a covalent hydroxyalkyl-enzyme intermediate by nucleophilic attack. In several well-studied EHs, this intermediate has been captured at near stoichiometric levels, presumably reflecting rate-limiting hydrolysis. Here we show by mass spectrometry that only minimal levels of the expected intermediate can be trapped with WT Cif. In contrast, substantial amounts of intermediate are recovered from an active-site mutant (Cif-E153Q) that selectively targets the second, hydrolytic release step. Utilizing Cif-E153Q and a previously reported nucleophile mutant (Cif-D129S), we then captured Cif in the substrate-bound, hydroxyalkyl-intermediate, and product-bound states for 1,2-epoxyhexane, yielding the first crystallographic snapshots of an EH at these key stages along the reaction coordinate. Taken together, our data illuminate the proposed two-step hydrolytic mechanism of a new class of bacterial virulence factor. They also suggest that the failure of WT Cif to accumulate a covalent hydroxyalkyl-enzyme intermediate reflects an active-site chemistry in which hydrolysis is no longer the rate-limiting step, a noncanonical kinetic regime that may explain similar observations with a number of other EHs.

Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.

Recurrent Pseudomonas aeruginosa infections coupled with robust, damaging neutrophilic inflammation characterize the chronic lung disease cystic fibrosis (CF). The proresolving lipid mediator, 15-epi lipoxin A4 (15-epi LXA4), plays a critical role in limiting neutrophil activation and tissue inflammation, thus promoting the return to tissue homeostasis. Here, we show that a secreted P. aeruginosa epoxide hydrolase, cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), can disrupt 15-epi LXA4 transcellular biosynthesis and function. In the airway, 15-epi LXA4 production is stimulated by the epithelial-derived eicosanoid 14,15-epoxyeicosatrienoic acid (14,15-EET). Cif sabotages the production of 15-epi LXA4 by rapidly hydrolyzing 14,15-EET into its cognate diol, eliminating a proresolving signal that potently suppresses IL-8-driven neutrophil transepithelial migration in vitro. Retrospective analyses of samples from patients with CF supported the translational relevance of these preclinical findings. Elevated levels of Cif in bronchoalveolar lavage fluid were correlated with lower levels of 15-epi LXA4, increased IL-8 concentrations, and impaired lung function. Together, these findings provide structural, biochemical, and immunological evidence that the bacterial epoxide hydrolase Cif disrupts resolution pathways during bacterial lung infections. The data also suggest that Cif contributes to sustained pulmonary inflammation and associated loss of lung function in patients with CF.

Pseudomonas aeruginosa secretes an epoxide hydrolase with catalytic activity that triggers degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and perturbs other host defense networks. Targets of this CFTR inhibitory factor (Cif) are largely unknown, but include an epoxy-fatty acid. In this class of signaling molecules, chirality can be an important determinant of physiological output and potency. Here we explore the active-site chemistry of this two-step alpha/beta-hydrolase and its implications for an emerging class of virulence enzymes. In combination with hydrolysis data, crystal structures of 15 trapped hydroxyalkyl-enzyme intermediates reveal the stereochemical basis of Cif's substrate specificity, as well as its regioisomeric and enantiomeric preferences. The structures also reveal distinct sets of conformational changes that enable the active site to expand dramatically in two directions, accommodating a surprising array of potential physiological epoxide targets. These new substrates may contribute to Cif's diverse effects in vivo, and thus to the success of P. aeruginosa and other pathogens during infection.

The CFTR inhibitory factor (Cif) is an epoxide hydrolase (EH) virulence factor secreted by the bacterium Pseudomonas aeruginosa. Sequence alignments reveal a pattern of Cif-like substitutions that proved to be characteristic of a new subfamily of bacterial EHs. At the same time, crystallographic and mutagenetic data suggest that EH activity is required for virulence and that Cif's active site remains generally compatible with a canonical two-step EH mechanism. A hallmark of this mechanism is the formation of a covalent hydroxyalkyl-enzyme intermediate by nucleophilic attack. In several well-studied EHs, this intermediate has been captured at near stoichiometric levels, presumably reflecting rate-limiting hydrolysis. Here we show by mass spectrometry that only minimal levels of the expected intermediate can be trapped with WT Cif. In contrast, substantial amounts of intermediate are recovered from an active-site mutant (Cif-E153Q) that selectively targets the second, hydrolytic release step. Utilizing Cif-E153Q and a previously reported nucleophile mutant (Cif-D129S), we then captured Cif in the substrate-bound, hydroxyalkyl-intermediate, and product-bound states for 1,2-epoxyhexane, yielding the first crystallographic snapshots of an EH at these key stages along the reaction coordinate. Taken together, our data illuminate the proposed two-step hydrolytic mechanism of a new class of bacterial virulence factor. They also suggest that the failure of WT Cif to accumulate a covalent hydroxyalkyl-enzyme intermediate reflects an active-site chemistry in which hydrolysis is no longer the rate-limiting step, a noncanonical kinetic regime that may explain similar observations with a number of other EHs.

The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. On the basis of these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif and have the potential for clinical application.

Opportunistic pathogens exploit diverse strategies to sabotage host defenses. Pseudomonas aeruginosa secretes the CFTR inhibitory factor Cif and thus triggers loss of CFTR, an ion channel required for airway mucociliary defense. However, the mechanism of action of Cif has remained unclear. It catalyzes epoxide hydrolysis, but there is no known role for natural epoxides in CFTR regulation. It was demonstrated that the hydrolase activity of Cif is strictly required for its effects on CFTR. A small-molecule inhibitor that protects this key component of the mucociliary defense system was also uncovered. These results provide a basis for targeting the distinctive virulence chemistry of Cif and suggest an unanticipated role of physiological epoxides in intracellular protein trafficking.

Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.

Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8(+) T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that secretes a multitude of virulence factors during the course of infection. Among these is Cif, an epoxide hydrolase (EH) that reduces the functional localization of the cystic fibrosis transmembrane conductance regulator in epithelial cells. In addition to being the first reported EH virulence factor, Cif possesses unique sequence deviations from canonical EH motifs. Foremost among these is the substitution of a histidine for the first epoxide ring-opening tyrosine in the active site. To test the functional equivalence of Tyr and His side chains at this position, we have generated the mutant Cif-H177Y. Structural analysis confirms that both the WT His and mutant Tyr side chains can be accommodated without large-scale conformational changes. However, the Tyr mutant is functionally inactive. Based on a detailed analysis of the structure of the Tyr mutant, it appears that Cif's main-chain conformation imposes a functional requirement for a His at this position. Comparison with canonical EH structures reveals additional conformational differences, which are coupled to divergent sequence characteristics. When used to probe the genomes of other opportunistic pathogens, these sequence-structure criteria uncover candidate sequences that appear to form a distinct subfamily of Cif-like epoxide hydrolases characterized by a conserved His/Tyr ring-opening pair.

Pseudomonas aeruginosa secretes an epoxide hydrolase virulence factor that reduces the apical membrane expression of ABC transporters such as the cystic fibrosis transmembrane conductance regulator (CFTR). This virulence factor, named CFTR inhibitory factor (Cif), is regulated by a TetR-family, epoxide-responsive repressor known as CifR via direct binding and repression. We identified two sites of CifR binding in the intergenic space between cifR and morB, the first gene in the operon containing the cif gene. We have mapped these binding sites and found they are 27 bp in length, and they overlap the -10 and +1 sites of both the cifR and morB regulatory region and the start of transcription, respectively. In addition, we found that CifR binds to each repression site with differing affinity. Mutagenesis of these binding sites resulted in a loss of DNA binding in vitro, and mutation of one of these sites in vivo resulted in an increase in transcription of both the cif and cifR genes. We characterized cif and cifR gene expression in sputum and found that, whereas cif gene expression varied relative to an in vitro coculture control, cifR gene expression was consistently higher. Analysis of a longitudinal sample of CF isolates from nine patients revealed that Cif protein was expressed over time, although variably, and these changes could not be linked to mutations in the cifR gene or the promoters of these genes. Finally, we tested CifR responsiveness to other epoxides and showed that CifR can respond to multiple epoxides to various degrees.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD), pneumonia, cystic fibrosis (CF), and bronchiectasis. Cif (PA2934), a bacterial toxin secreted in outer membrane vesicles (OMV) by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens.

Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and is also compatible with other alpha/beta hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8 A resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of alpha/beta hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. Reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression may require hydrolysis of an extended endogenous epoxide substrate.

The opportunistic pathogen Pseudomonas aeruginosa secretes a protein that triggers the accelerated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells. This protein, which is known as the CFTR inhibitory factor (Cif), acts as a virulence factor and may facilitate airway colonization by P. aeruginosa. Based on sequence similarity Cif appears to be an epoxide hydrolase (EH), but it lacks several of the conserved features found in the active sites of canonical members of the EH family. Here, the crystallization of purified recombinant Cif by vapor diffusion is reported. The crystals formed in space group C2, with unit-cell parameters a = 167.4, b = 83.6, c = 88.3 A, beta = 100.6 degrees . The crystals diffracted to 2.39 A resolution on a rotating-anode source. Based on the calculated Matthews coefficient (2.2 A(3) Da(-1)), it appears that the asymmetric unit contains four molecules.

Pseudomonas aeruginosa isolates have a highly conserved core genome representing up to 90% of the total genomic sequence with additional variable accessory genes, many of which are found in genomic islands or islets. The identification of the Liverpool Epidemic Strain (LES) in a children's cystic fibrosis (CF) unit in 1996 and its subsequent observation in several centers in the United Kingdom challenged the previous widespread assumption that CF patients acquire only unique strains of P. aeruginosa from the environment. To learn about the forces that shaped the development of this important epidemic strain, the genome of the earliest archived LES isolate, LESB58, was sequenced. The sequence revealed the presence of many large genomic islands, including five prophage clusters, one defective (pyocin) prophage cluster, and five non-phage islands. To determine the role of these clusters, an unbiased signature tagged mutagenesis study was performed, followed by selection in the chronic rat lung infection model. Forty-seven mutants were identified by sequencing, including mutants in several genes known to be involved in Pseudomonas infection. Furthermore, genes from four prophage clusters and one genomic island were identified and in direct competition studies with the parent isolate; four were demonstrated to strongly impact on competitiveness in the chronic rat lung infection model. This strongly indicates that enhanced in vivo competitiveness is a major driver for maintenance and diversifying selection of these genomic prophage genes.

We previously reported that the novel Pseudomonas aeruginosa toxin Cif is capable of decreasing apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR). We further demonstrated that Cif is capable of degrading the synthetic epoxide hydrolase (EH) substrate S-NEPC [(2S,3S)-trans-3-phenyl-2-oxiranylmethyl 4-nitrophenol carbonate], suggesting that Cif may be reducing apical membrane expression of CFTR via its EH activity. Here we report that Cif is capable of degrading the xenobiotic epoxide epibromohydrin (EBH) to its vicinal diol 3-bromo-1,2-propanediol. We also demonstrate that this epoxide is a potent inducer of cif gene expression. We show that the predicted TetR family transcriptional repressor encoded by the PA2931 gene, which is immediately adjacent to and divergently transcribed from the cif-containing, three-gene operon, negatively regulates cif gene expression by binding to the promoter region immediately upstream of the cif-containing operon. Furthermore, this protein-DNA interaction is disrupted by the epoxide EBH in vitro, suggesting that the binding of EBH by the PA2931 protein product drives the disassociation from its DNA-binding site. Given its role as a repressor of cif gene expression, we have renamed PA2931 as CifR. Finally, we demonstrate that P. aeruginosa strains isolated from cystic fibrosis patient sputum with increased cif gene expression are impaired for the expression of the cifR gene.

One of the hallmarks of the Gram-negative bacterium Pseudomonas aeruginosa is its ability to thrive in diverse environments that includes humans with a variety of debilitating diseases or immune deficiencies. Here we report the complete sequence and comparative analysis of the genomes of two representative P. aeruginosa strains isolated from cystic fibrosis (CF) patients whose genetic disorder predisposes them to infections by this pathogen. The comparison of the genomes of the two CF strains with those of other P. aeruginosa presents a picture of a mosaic genome, consisting of a conserved core component, interrupted in each strain by combinations of specific blocks of genes. These strain-specific segments of the genome are found in limited chromosomal locations, referred to as regions of genomic plasticity. The ability of P. aeruginosa to shape its genomic composition to favor survival in the widest range of environmental reservoirs, with corresponding enhancement of its metabolic capacity is supported by the identification of a genomic island in one of the sequenced CF isolates, encoding enzymes capable of degrading terpenoids produced by trees. This work suggests that niche adaptation is a major evolutionary force influencing the composition of bacterial genomes. Unlike genome reduction seen in host-adapted bacterial pathogens, the genetic capacity of P. aeruginosa is determined by the ability of individual strains to acquire or discard genomic segments, giving rise to strains with customized genomic repertoires. Consequently, this organism can survive in a wide range of environmental reservoirs that can serve as sources of the infecting organisms.

P-glycoprotein (Pgp), a member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily, is a major drug efflux pump expressed in normal tissues, and is overexpressed in many human cancers. Overexpression of Pgp results in reduced intracellular drug concentration and cytotoxicity of chemotherapeutic drugs and is thought to contribute to multidrug resistance of cancer cells. The involvement of Pgp in clinical drug resistance has led to a search for molecules that block Pgp transporter activity to improve the efficacy and pharmacokinetics of therapeutic agents. We have recently identified and characterized a secreted toxin from Pseudomonas aeruginosa, designated cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif). Cif reduces the apical membrane abundance of CFTR, also an ABC transporter, and inhibits the CFTR-mediated chloride ion secretion by human airway and kidney epithelial cells. We report presently that Cif also inhibits the apical membrane abundance of Pgp in kidney, airway, and intestinal epithelial cells but has no effect on plasma membrane abundance of multidrug resistance protein 1 or 2. Cif increased the drug sensitivity to doxorubicin in kidney cells expressing Pgp by 10-fold and increased the cellular accumulation of daunorubicin by 2-fold. Thus our studies show that Cif increases the sensitivity of Pgp-overexpressing cells to doxorubicin, consistent with the hypothesis that Cif affects Pgp functional expression. These results suggest that Cif may be useful to develop a new class of specific inhibitors of Pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs, and at improving the bioavailability of Pgp transport substrates.

We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2934 [corrected], and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl(-) ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in alpha/beta hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe.

BACKGROUND: Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important opportunistic human pathogen. Generally, the acquisition of genes in the form of pathogenicity islands distinguishes pathogenic isolates from nonpathogens. We therefore sequenced a highly virulent strain of P. aeruginosa, PA14, and compared it with a previously sequenced (and less pathogenic) strain, PAO1, to identify novel virulence genes. RESULTS: The PA14 and PAO1 genomes are remarkably similar, although PA14 has a slightly larger genome (6.5 megabses [Mb]) than does PAO1 (6.3 Mb). We identified 58 PA14 gene clusters that are absent in PAO1 to determine which of these genes, if any, contribute to its enhanced virulence in a Caenorhabditis elegans pathogenicity model. First, we tested 18 additional diverse strains in the C. elegans model and observed a wide range of pathogenic potential; however, genotyping these strains using a custom microarray showed that the presence of PA14 genes that are absent in PAO1 did not correlate with the virulence of these strains. Second, we utilized a full-genome nonredundant mutant library of PA14 to identify five genes (absent in PAO1) required for C. elegans killing. Surprisingly, although these five genes are present in many other P. aeruginosa strains, they do not correlate with virulence in C. elegans. CONCLUSION: Genes required for pathogenicity in one strain of P. aeruginosa are neither required for nor predictive of virulence in other strains. We therefore propose that virulence in this organism is both multifactorial and combinatorial, the result of a pool of pathogenicity-related genes that interact in various combinations in different genetic backgrounds.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections. A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants. Here we report the complete sequence of P. aeruginosa strain PAO1. At 6.3 million base pairs, this is the largest bacterial genome sequenced, and the sequence provides insights into the basis of the versatility and intrinsic drug resistance of P. aeruginosa. Consistent with its larger genome size and environmental adaptability, P. aeruginosa contains the highest proportion of regulatory genes observed for a bacterial genome and a large number of genes involved in the catabolism, transport and efflux of organic compounds as well as four potential chemotaxis systems. We propose that the size and complexity of the P. aeruginosa genome reflect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.