SummaryUnder iron-limiting conditions, Pseudomonas aeruginosa produces a siderophore called pyoverdine. Pyoverdine is secreted into the extracellular environment where it chelates iron, and the resulting ferripyoverdine 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).

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.

Background: Thiol dioxygenation is catalyzed by enzymes specific for each substrate. Results: Kinetic, structural, and spectroscopic data describe an enzyme from P. aeruginosa that is a 3-mercaptopropionate dioxygenase with secondary cysteine dioxygenase activity. Conclusion: An arginine to glutamine switch and the absence of a cis-peptide bond correlate with substrate preference. Significance: Characterization of this enzyme deepens our understanding of substrate specificity in thiol dioxygenases.

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.

dPseudomonas 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.

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.

SummaryCell-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.

a b s t r a c tPseudomonas 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.