A large-scale whole-genome comparison shows that experimental evolution in response to antibiotics predicts changes in naturally evolved clinical Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections. An increasing number of isolates have mutations that make them antibiotic resistant, making treatment difficult. To identify resistance-associated mutations we experimentally evolved the antibiotic sensitive strain P. aeruginosa PAO1 to become resistant to three widely used anti-pseudomonal antibiotics, ciprofloxacin, meropenem and tobramycin. Mutants could tolerate up to 2048-fold higher concentrations of antibiotic than strain PAO1. Genome sequences were determined for thirteen mutants for each antibiotic. Each mutant had between 2 and 8 mutations. For each antibiotic at least 8 genes were mutated in multiple mutants, demonstrating the genetic complexity of resistance. For all three antibiotics mutations arose in genes known to be associated with resistance, but also in genes not previously associated with resistance. To determine the clinical relevance of mutations uncovered in this study we analysed the corresponding genes in 558 isolates of P. aeruginosa from patients with chronic lung disease and in 172 isolates from the general environment. Many genes identified through experimental evolution had predicted function-altering changes in clinical isolates but not in environmental isolates, showing that mutated genes in experimentally evolved bacteria can predict those that undergo mutation during infection. Additionally, large deletions of up to 479kb arose in experimentally evolved meropenem resistant mutants and large deletions were present in 87 of the clinical isolates. These findings significantly advance understanding of antibiotic resistance in P. aeruginosa and demonstrate the validity of experimental evolution in identifying clinically-relevant resistance-associated mutations.

Expression of Pseudomonas aeruginosa Antibiotic Resistance Genes Varies Greatly during Infections in Cystic Fibrosis Patients.

The lungs of individuals with cystic fibrosis (CF) become chronically infected with Pseudomonas aeruginosa that is difficult to eradicate by antibiotic treatment. Two key P. aeruginosa antibiotic resistance mechanisms are the AmpC ß-lactamase that degrades ß-lactam antibiotics and MexXYOprM, a three-protein efflux pump that expels aminoglycoside antibiotics from the bacterial cells. Levels of antibiotic resistance gene expression are likely to be a key factor in antibiotic resistance but have not been determined during infection. The aims of this research were to investigate the expression of the ampC and mexX genes during infection in patients with CF and in bacteria isolated from the same patients and grown under laboratory conditions. P. aeruginosa isolates from 36 CF patients were grown in laboratory culture and gene expression measured by reverse transcription-quantitative PCR (RT-qPCR). The expression of ampC varied over 20,000-fold and that of mexX over 2,000-fold between isolates. The median expression levels of both genes were increased by the presence of subinhibitory concentrations of antibiotics. To measure P. aeruginosa gene expression during infection, we carried out RT-qPCR using RNA extracted from fresh sputum samples obtained from 31 patients. The expression of ampC varied over 4,000-fold, while mexX expression varied over 100-fold, between patients. Despite these wide variations, median levels of expression of ampC in bacteria in sputum were similar to those in laboratory-grown bacteria. The expression of mexX was higher in sputum than in laboratory-grown bacteria. Overall, our data demonstrate that genes that contribute to antibiotic resistance can be highly expressed in patients, but there is extensive isolate-to-isolate and patient-to-patient variation.

The cysteine dioxygenase homologue from Pseudomonas aeruginosa is a 3-mercaptopropionate dioxygenase.

Thiol dioxygenation is the initial oxidation step that commits a thiol to important catabolic or biosynthetic pathways. The reaction is catalyzed by a family of specific non-heme mononuclear iron proteins each of which is reported to react efficiently with only one substrate. This family of enzymes includes cysteine dioxygenase, cysteamine dioxygenase, mercaptosuccinate dioxygenase, and 3-mercaptopropionate dioxygenase. Using sequence alignment to infer cysteine dioxygenase activity, a cysteine dioxygenase homologue from Pseudomonas aeruginosa (p3MDO) has been identified. Mass spectrometry of P. aeruginosa under standard growth conditions showed that p3MDO is expressed in low levels, suggesting that this metabolic pathway is available to the organism. Purified recombinant p3MDO is able to oxidize both cysteine and 3-mercaptopropionic acid in vitro, with a marked preference for 3-mercaptopropionic acid. We therefore describe this enzyme as a 3-mercaptopropionate dioxygenase. Mössbauer spectroscopy suggests that substrate binding to the ferrous iron is through the thiol but indicates that each substrate could adopt different coordination geometries. Crystallographic comparison with mammalian cysteine dioxygenase shows that the overall active site geometry is conserved but suggests that the different substrate specificity can be related to replacement of an arginine by a glutamine in the active site.

Interactions between an anti-sigma protein and two sigma factors that regulate the pyoverdine signaling pathway in Pseudomonas aeruginosa.

BACKGROUND: Synthesis and uptake of pyoverdine, the primary siderophore of the opportunistic pathogen Pseudomonas aeruginosa, is dependent on two extra-cytoplasmic function (ECF) sigma factors, FpvI and PvdS. FpvI and PvdS are required for expression of the ferri-pyoverdine receptor gene fpvA and of pyoverdine synthesis genes respectively. In the absence of pyoverdine the anti-sigma factor FpvR that spans the cytoplasmic membrane inhibits the activities of both FpvI and PvdS, despite the two sigma factors having low sequence identity. RESULTS: To investigate the interactions of FpvR with FpvI and PvdS, we first used a tandem affinity purification system to demonstrate binding of PvdS by the cytoplasmic region of FpvR in P. aeruginosa at physiological levels. The cytoplasmic region of FpvR bound to and inhibited both FpvI and PvdS when the proteins were co-expressed in Escherichia coli. Each sigma factor was then subjected to error prone PCR and site-directed mutagenesis to identify mutations that increased sigma factor activity in the presence of FpvR. In FpvI, the amino acid changes clustered around conserved region four of the protein and are likely to disrupt interactions with FpvR. Deletion of five amino acids from the C-terminal end of FpvI also disrupted interactions with FpvR. Mutations in PvdS were present in conserved regions two and four. Most of these mutations as well as deletion of thirteen amino acids from the C-terminal end of PvdS increased sigma factor activity independent of whether FpvR was present, suggesting that they increase either the stability of PvdS or its affinity for core RNA polymerase. CONCLUSIONS: These data show that FpvR binds to PvdS in both P. aeruginosa and E. coli, inhibiting its activity. FpvR also binds to and inhibits FpvI and binding of FpvI is likely to involve conserved region four of the sigma factor protein.

Pseudomonas aeruginosa uses multiple pathways to acquire iron during chronic infection in cystic fibrosis lungs.

Pseudomonas aeruginosa chronically infects the lungs of more than 80% of adult patients with cystic fibrosis (CF) and is a major contributor to the progression of disease pathology. P. aeruginosa requires iron for growth and has multiple iron uptake systems that have been studied in bacteria grown in laboratory culture. The purpose of this research was to determine which of these are active during infection in CF. RNA was extracted from 149 sputum samples obtained from 23 CF patients. Reverse transcription-quantitative real-time PCR (RT-qPCR) was used to measure the expression of P. aeruginosa genes encoding transport systems for the siderophores pyoverdine and pyochelin, for heme, and for ferrous ions. Expression of P. aeruginosa genes could be quantified in 89% of the sputum samples. Expression of genes associated with siderophore-mediated iron uptake was detected in most samples but was at low levels in some samples, indicating that other iron uptake mechanisms are active. Expression of genes encoding heme transport systems was also detected in most samples, indicating that heme uptake occurs during infection in CF. feoB expression was detected in all sputum samples, implying an important role for ferrous ion uptake by P. aeruginosa in CF. Our data show that multiple P. aeruginosa iron uptake mechanisms are active in chronic CF infection and that RT-qPCR of RNA extracted from sputum provides a powerful tool for investigating bacterial physiology during infection in CF.

Pseudomonas aeruginosa is oxygen-deprived during infection in cystic fibrosis lungs, reducing the effectiveness of antibiotics.

Pseudomonas aeruginosa infects the lungs of patients with cystic fibrosis. Sputum expectorated from the lungs of patients contains low levels of oxygen, indicating that P. aeruginosa may be oxygen-deprived during infection. During in vitro growth under oxygen-limiting conditions, a P. aeruginosa reference strain increases expression of a cytochrome oxidase with a high affinity for oxygen, and of nitrate and nitrite reductases that enable it to use nitrate instead of oxygen during respiration. Here, we quantified transcription of the genes encoding these three enzymes in sputum samples from 18 infected patients, and in bacteria isolated from the sputum samples and grown in aerobic and anaerobic culture. In culture, expression of all three genes was increased by averages of 20- to 500-fold in anaerobically grown bacteria compared with those grown aerobically, although expression levels varied greatly between isolates. Expression of the same genes in sputum was similar to that of the corresponding bacteria in anaerobic culture. The isolated bacteria were less susceptible to tobramycin and ciprofloxacin, two widely used anti-pseudomonal antibiotics, when grown anaerobically than when grown aerobically. Our findings show that P. aeruginosa experiences oxygen starvation during infection in cystic fibrosis, reducing the effectiveness of antibiotic treatment.

Ceftazidime resistance in Pseudomonas aeruginosa is multigenic and complex.

Pseudomonas aeruginosa causes a wide range of severe infections. Ceftazidime, a cephalosporin, is a key antibiotic for treating infections but a significant proportion of isolates are ceftazidime-resistant. The aim of this research was to identify mutations that contribute to resistance, and to quantify the impacts of individual mutations and mutation combinations. Thirty-five mutants with reduced susceptibility to ceftazidime were evolved from two antibiotic-sensitive P. aeruginosa reference strains PAO1 and PA14. Mutations were identified by whole genome sequencing. The evolved mutants tolerated ceftazidime at concentrations between 4 and 1000 times that of the parental bacteria, with most mutants being ceftazidime resistant (minimum inhibitory concentration [MIC] ≥ 32 mg/L). Many mutants were also resistant to meropenem, a carbapenem antibiotic. Twenty-eight genes were mutated in multiple mutants, with dacB and mpl being the most frequently mutated. Mutations in six key genes were engineered into the genome of strain PAO1 individually and in combinations. A dacB mutation by itself increased the ceftazidime MIC by 16-fold although the mutant bacteria remained ceftazidime sensitive (MIC < 32 mg/L). Mutations in ampC, mexR, nalC or nalD increased the MIC by 2- to 4-fold. The MIC of a dacB mutant was increased when combined with a mutation in ampC, rendering the bacteria resistant, whereas other mutation combinations did not increase the MIC above those of single mutants. To determine the clinical relevance of mutations identified through experimental evolution, 173 ceftazidime-resistant and 166 sensitive clinical isolates were analysed for the presence of sequence variants that likely alter function of resistance-associated genes. dacB and ampC sequence variants occur most frequently in both resistant and sensitive clinical isolates. Our findings quantify the individual and combinatorial effects of mutations in different genes on ceftazidime susceptibility and demonstrate that the genetic basis of ceftazidime resistance is complex and multifactorial.

Differential proteolysis of sigma regulators controls cell-surface signalling in Pseudomonas aeruginosa.

Cell-surface signalling systems are widespread in Gram-negative bacteria. In these systems gene expression occurs following binding of a ligand, commonly a siderophore, to a receptor protein in the outer membrane. The receptor interacts with a sigma regulator protein that extends from the periplasm into the cytoplasm to control the activity of a cognate sigma factor. The mechanisms of signal transduction in cell-surface signalling systems have not been determined. Here we investigate signal transduction in the pyoverdine, ferrichrome and desferrioxamine siderophore systems of Pseudomonas aeruginosa. When pyoverdine is present the sigma regulator FpvR undergoes complete proteolysis resulting in activation of two sigma factors PvdS and FpvI and expression of genes for pyoverdine synthesis and uptake. When pyoverdine is absent subfragments of FpvR inhibit PvdS and FpvI. Similarly, subfragments of the sigma regulators FoxR and FiuR are formed in the absence of desferrioxamine and ferrichrome. These are much less abundant when the siderophores are present and downstream gene expression takes place. In all three systems RseP (MucP/YaeL) is required for complete proteolysis of the sigma regulator and sigma factor activity. These findings indicate that regulated proteolysis is a general mechanism for signal transduction in cell-surface signalling.

Pseudomonas siderophores in the sputum of patients with cystic fibrosis.

The lungs of patients with cystic fibrosis become chronically infected with the bacterium Pseudomonas aeruginosa, which heralds progressive lung damage and a decline in health. Iron is a crucial micronutrient for bacteria and its acquisition is a key factor in infection. P. aeruginosa can acquire this element by secreting pyoverdine and pyochelin, iron-chelating compounds (siderophores) that scavenge iron and deliver it to the bacteria. Siderophore-mediated iron uptake is generally considered a key factor in the ability of P. aeruginosa to cause infection. We have investigated the amounts of pyoverdine in 148 sputum samples from 36 cystic fibrosis patients (30 infected with P. aeruginosa and 6 as negative controls). Pyoverdine was present in 93 samples in concentrations between 0.30 and 51 µM (median 4.6 µM) and there was a strong association between the amount of pyoverdine and the number of P. aeruginosa present. However, pyoverdine was not present, or below the limits of detection (~0.3 µM), in 21 sputum samples that contained P. aeruginosa. Pyochelin was also absent, or below the limits of detection (~1 µM), in samples from P. aeruginosa-infected patients with little or no detectable pyoverdine. Our data show that pyoverdine is an important iron-scavenging molecule for P. aeruginosa in many cystic fibrosis patients, but other P. aeruginosa iron-uptake systems must be active in some patients to satisfy the bacterial need for iron.

Genome evolution drives transcriptomic and phenotypic adaptation in Pseudomonas aeruginosa during 20 years of infection.

The opportunistic pathogen Pseudomonas aeruginosa chronically infects the lungs of patients with cystic fibrosis (CF). During infection the bacteria evolve and adapt to the lung environment. Here we use genomic, transcriptomic and phenotypic approaches to compare multiple isolates of P. aeruginosa collected more than 20 years apart during a chronic infection in a CF patient. Complete genome sequencing of the isolates, using short- and long-read technologies, showed that a genetic bottleneck occurred during infection and was followed by diversification of the bacteria. A 125 kb deletion, an 0.9 Mb inversion and hundreds of smaller mutations occurred during evolution of the bacteria in the lung, with an average rate of 17 mutations per year. Many of the mutated genes are associated with infection or antibiotic resistance. RNA sequencing was used to compare the transcriptomes of an earlier and a later isolate. Substantial reprogramming of the transcriptional network had occurred, affecting multiple genes that contribute to continuing infection. Changes included greatly reduced expression of flagellar machinery and increased expression of genes for nutrient acquisition and biofilm formation, as well as altered expression of a large number of genes of unknown function. Phenotypic studies showed that most later isolates had increased cell adherence and antibiotic resistance, reduced motility, and reduced production of pyoverdine (an iron-scavenging siderophore), consistent with genomic and transcriptomic data. The approach of integrating genomic, transcriptomic and phenotypic analyses reveals, and helps to explain, the plethora of changes that P. aeruginosa undergoes to enable it to adapt to the environment of the CF lung during a chronic infection.

Synthesis of the siderophore pyoverdine in Pseudomonas aeruginosa involves a periplasmic maturation.

Pyoverdines, the main siderophores produced by fluorescent Pseudomonads, comprise a fluorescent dihydroxyquinoline chromophore attached to a strain-specific peptide. These molecules are thought to be synthesized as non-fluorescent precursor peptides that are then modified to give functional pyoverdines. Using the fluorescent properties of PVDI, the pyoverdine produced by Pseudomonas aeruginosa PAO1, we were able to show that PVDI was not present in the cytoplasm of the bacteria, but large amounts of a fluorescent PVDI precursor PVDIp were stored in the periplasm. Like PVDI, PVDIp is able to transport iron into P. aeruginosa cells. Mutation of genes encoding the periplasmic PvdN, PvdO and PvdP proteins prevented accumulation of PVDIp in the periplasm and secretion of PVDI into the growth medium, indicating that these three enzymes are involved in PVDI synthesis. Mutation of the gene encoding PvdQ resulted in the presence of fluorescent PVDI precursor in the periplasm and secretion of a functional fluorescent siderophore that had different isoelectric properties to PVDI, suggesting a role for PvdQ in the periplasmic maturation of PVDI. Mutation of the gene encoding the export ABC transporter PvdE prevented PVDI production and accumulation of PVDIp in the periplasm. These data are consistent with a model in which a PVDI precursor peptide is synthesized in the cytoplasm and exported to the periplasm by PvdE where siderophore maturation, including formation of the chromophore moiety, occurs in a process involving the PvdN, PvdO, PvdP and PvdQ proteins.

Efficient zinc uptake is critical for the ability of Pseudomonas aeruginosa to express virulence traits and colonize the human lung.

We have recently shown that Pseudomonas aeruginosa, an opportunistic pathogen that chronically infects the lungs of patients with cystic fibrosis (CF) and other forms of lung disease, is extremely efficient in recruiting zinc from the environment and that this capability is required for its ability to cause acute lung infections in mice. To verify that P. aeruginosa faces zinc shortage when colonizing the lungs of human patients, we analyzed the expression of three genes that are highly induced under conditions of zinc deficiency (zrmA, dksA2 and rpmE2), in bacteria in the sputum of patients with inflammatory lung disease. All three genes were expressed in all the analyzed sputum samples to a level much higher than that of bacteria grown in zinc-containing laboratory medium, supporting the hypothesis that P. aeruginosa is under zinc starvation during lung infections. We also found that the expression of several virulence traits that play a central role in the ability of P. aeruginosa to colonize the lung is affected by disruption of the most important zinc importing systems. Virulence features dependent on zinc intake include swarming and swimming motility and the ability to form biofilms. Furthermore, alterations in zinc assimilation interfere with the synthesis of the siderophore pyoverdine, suggesting that zinc recruitment could modulate iron uptake and affect siderophore-mediated cell signaling. Our results reveal that zinc uptake is likely to play a key role in the ability of P. aeruginosa to cause chronic lung infections and strongly modulates critical virulence traits of the pathogen. Taking into account the recent discovery that zinc uptake in P. aeruginosa is promoted by the release of a small molecular weight molecule showing high affinity for zinc, our data suggest novel and effective possibilities to control lung infections by these bacteria.

Activation of a Cell Surface Signaling Pathway in Pseudomonas aeruginosa Requires ClpP Protease and New Sigma Factor Synthesis.

Extracytoplasmic function (ECF) sigma factors control expression of large numbers of genes in bacteria. Most ECF sigma factors are inhibited by antisigma proteins, with inhibition being relieved by environmental signals that lead to inactivation of the antisigma protein and consequent sigma factor activity. In cell surface signaling (CSS) systems in Gram negative bacteria antisigma activity is controlled by an outer membrane protein receptor and its ligand. In Pseudomonas aeruginosa one such system controls expression of genes for secretion and uptake of a siderophore, pyoverdine. In this system the activities of two sigma factors σFpvI and σPvdS are inhibited by antisigma protein FpvR20 that binds to the sigma factors, preventing their interaction with core RNA polymerase. Transport of ferripyoverdine by its outer membrane receptor FpvA causes proteolytic degradation of FpvR20, inducing expression of σFpvI- and σPvdS-dependent target genes. Here we show that degradation of FpvR20 and induction of target gene expression was initiated within 1 min of addition of pyoverdine. FpvR20 was only partially degraded in a mutant lacking the intracellular ClpP protease, resulting in an FpvR20 subfragment (FpvR12) that inhibited σFpvI and σPvdS. The translation inhibitor chloramphenicol did not prevent induction of an σFpvI-dependent gene, showing that degradation of FpvR20 released pre-existing σFpvI in an active form. However, chloramphenicol inhibited induction of σPvdS-dependent genes showing that active σPvdS is not released when FpvR20 is degraded and instead, σPvdS must be synthesized in the absence of FpvR20 to be active. These findings show that sigma factor activation occurs rapidly following addition of the inducing signal in a CSS pathway and requires ClpP protease. Induction of gene expression that can arise from release of active sigma from an antisigma protein but can also require new sigma factor synthesis.

An efflux pump is required for siderophore recycling by Pseudomonas aeruginosa.

Pyoverdine (PVDI) is a siderophore produced by Pseudomonas aeruginosa in order to obtain iron. This molecule is composed of a fluorescent chromophore linked to an octapeptide. Following secretion from the bacteria, PVDI chelates iron ions and the resulting Fe-PVDI complexes are taken up by the bacteria through a cell surface receptor protein. The iron is released in the periplasm and the resulting PVDI is recycled, being secreted out of the bacteria by a previously unknown mechanism. Three genes with the potential to encode an efflux system are adjacent to, and coregulated with, genes required for PVDI-mediated iron transport. Mutation of genes encoding this efflux pump (named PvdRT-OpmQ) prevented recycling of PVDI from the periplasm into the extracellular medium. Fluorescence microscopy showed that in the mutant bacteria PVDI accumulated in the periplasm. Gallium (Ga(3+) ), a metal that cannot be removed from PVDI by reduction, is taken up by P. aeruginosa when chelated by PVDI. Recycling did not occur after transport of PVDI-Ga(3+) and fluorescence accumulated in the periplasm even when the PvdRT-OpmQ efflux pump was functional. Cellular fractionation showed that PVDI-synthesizing bacteria lacking PvdRT-OpmQ secreted PVDI but had an approximately 20-fold increase in the amount of PVD present in the periplasm, consistent with an inability to recycle PVDI. Collectively, these data show that PvdRT-OpmQ is involved in recycling of PVDI from the periplasm to the extracellular medium and recycling requires release of the metal ion from PVDI.

An efflux pump is involved in secretion of newly synthesized siderophore by Pseudomonas aeruginosa.

Pseudomonas aeruginosa secretes the fluorescent siderophore, pyoverdine (PVD), to enable iron acquisition. Epifluorescence microscopy and cellular fractionation were used to investigate the role of an efflux pump, PvdRT-OpmQ, in PVD secretion. Bacteria lacking this efflux pump accumulated PVD, or a fluorescent precursor, in the periplasm, due to their inability to efficiently secrete into the media newly synthesized PVD. PvdRT-OpmQ is only the second system identified for secretion of newly synthesized siderophores by Gram negative bacteria.

Characterization of a gene encoding an acetylase required for pyoverdine synthesis in Pseudomonas aeruginosa.

Strains of Pseudomonas aeruginosa secrete one of three pyoverdine siderophores (types I to III). We have characterized a gene, pvdY(II) (for the pvdY gene present in type II P. aeruginosa strains), that is only present in strains that make type II pyoverdine. A mutation in pvdY(II) prevented pyoverdine synthesis. Bioinformatic, genetic, and biochemical approaches indicate that the PvdYII enzyme catalyzes acetylation of hydroxyornithine. Expression of pvdY(II) is repressed by the presence of iron and upregulated by the presence of type II pyoverdine. Characterization of pvdY(II) provides insights into the molecular basis for production of different pyoverdines by different strains of P. aeruginosa.

Mutational analysis of a bifunctional ferrisiderophore receptor and signal-transducing protein from Pseudomonas aeruginosa.

The FpvA protein of Pseudomonas aeruginosa strain PAO1 mediates uptake of a siderophore, ferripyoverdine. It is also a component of a signal transduction pathway that controls production of an exotoxin, a protease, pyoverdine, and FpvA itself. The purpose of the research described here was to dissect these different functions of FpvA. Signaling involves an N-terminal domain of FpvA, and it was shown that this domain is probably located in the periplasm, as expected. Short peptides were inserted at 36 sites within FpvA by linker insertion mutagenesis. The effects of these mutations on the presence of FpvA in the outer membrane, on FpvA-mediated uptake of ferripyoverdine, and on pyoverdine synthesis and gene expression were determined. Five of the mutations resulted in the absence of FpvA from the outer membrane of the bacteria. All of the remaining mutations eliminated either the transport or signaling function of FpvA and most affected both functions. Three mutations prevented transport of ferripyoverdine but had no effect on the signal transduction pathway showing that transport of ferripyoverdine is not required for the trans-membrane signaling process. Conversely, eight mutations affected pyoverdine-mediated signaling but had no effect on transport of ferripyoverdine. These data show that insertions throughout FpvA resulted in loss of function and that signaling and transport are separate and discrete functions of FpvA.

Identification and characterization of novel pyoverdine synthesis genes in Pseudomonas aeruginosa.

Fluorescent pseudomonads secrete yellow-green siderophores named pyoverdines or pseudobactins. These comprise a dihydroxyquinoline derivative joined to a type-specific peptide and, usually, a carboxylic acid or amide. In Pseudomonas aeruginosa strain PAO1, six genes that encode proteins required for pyoverdine synthesis (pvd genes) have been identified previously. Expression of all of these genes requires an alternative sigma factor PvdS. The purpose of this research was to identify other genes that are required for pyoverdine synthesis in P. aeruginosa PAO1. Fourteen candidate genes were identified from the PAO1 genome sequence on the basis of their location in the genome, the functions of homologues in other bacteria, and whether their expression was likely to be PvdS-dependent. The candidate genes were mutated and the effects of the mutations on pyoverdine production were determined. Eight new pvd genes were identified. The presence of homologues of pvd genes in other strains of P. aeruginosa was determined by Southern blotting and in other fluorescent pseudomonads by interrogation of genome sequences. Five pvd genes were restricted to strains of P. aeruginosa that make the same pyoverdine as strain PAO1, suggesting that they direct synthesis of the type-specific peptide. The remaining genes were present in all strains of P. aeruginosa that were examined and homologues were present in other Pseudomonas species. These genes are likely to direct synthesis of the dihydroxyquinoline moiety and the attached carboxylic acid/amide group. It is likely that most if not all of the genes required for pyoverdine synthesis in P. aeruginosa PAO1 have now been identified and this will form the basis for a biochemical description of the pathway of pyoverdine synthesis.

Siderophore-mediated cell signalling in Pseudomonas aeruginosa: divergent pathways regulate virulence factor production and siderophore receptor synthesis.

Under iron-limiting conditions, Pseudomonas aeruginosa produces a siderophore called pyoverdine. Pyoverdine is secreted into the extracellular environment where it chelates iron, and the resulting ferri-pyoverdine complexes are transported back into the bacteria by a cell surface receptor protein FpvA. Pyoverdine also acts as a signalling molecule inducing the production of three secreted virulence factors. Binding of ferri-pyoverdine to FpvA transduces a signal to the periplasmic part of the membrane-spanning antisigma factor FpvR. The signal is transmitted to the cytoplasmic part of FpvR, which controls the activity of an extracytoplasmic family (ECF) sigma factor protein PvdS. This results in the production of the virulence factors pyoverdine, exotoxin A and PrpL endoprotease. Here, we show that a second divergent branch of this signalling pathway regulates the production of the FpvA protein. FpvR negatively regulates the activity of a second ECF sigma factor, FpvI, which is required for the synthesis of FpvA, and the presence of ferri-pyoverdine greatly increases the activity of FpvI so that production of FpvA is induced. To the best of our knowledge, this is the first example of a branched signalling system of this sort and the first example of an antisigma factor protein (FpvR) that directly regulates the activities of two different ECF sigma factor proteins (PvdS and FpvI).