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).     

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.     

Mutational analysis of an extracytoplasmic-function sigma factor to investigate its interactions with RNA polymerase and DNA

The extracytoplasmic-function (ECF) family of sigma factors comprises a large group of proteins required for synthesis of a wide variety of extracytoplasmic products by bacteria. Residues important for core RNA polymerase (RNAP) binding, DNA melting, and promoter recognition have been identified in conserved regions 2 and 4.2 of primary sigma factors. Seventeen residues in region 2 and eight residues in region 4.2 of an ECF sigma factor, PvdS from Pseudomonas aeruginosa, were selected for alanine-scanning mutagenesis on the basis of sequence alignments with other sigma factors. Fourteen of the mutations in region 2 had a significant effect on protein function in an in vivo assay. Four proteins with alterations in regions 2.1 and 2.2 were purified as His-tagged fusions, and all showed a reduced affinity for core RNAP in vitro, consistent with a role in core binding. Region 2.3 and 2.4 mutant proteins retained the ability to bind core RNAP, but four mutants had reduced or no ability to cause core RNA polymerase to bind promoter DNA in a band-shift assay, identifying residues important for DNA binding. All mutations in region 4.2 reduced the activity of PvdS in vivo. Two of the region 4.2 mutant proteins were purified, and each showed a reduced ability to cause core RNA polymerase to bind to promoter DNA. The results show that some residues in PvdS have functions equivalent to those of corresponding residues in primary sigma factors; however, they also show that several residues not shared with primary sigma factors contribute to protein function.     

Genomics of pyoverdine-mediated iron uptake in pseudomonads

Pyoverdines (PVDs) are complex siderophores produced by members of the fluorescent Pseudomonas. They comprise a dihydroxyquinoline fluorescent chromophore joined to a peptide of remarkably variable length and composition. In Pseudomonas aeruginosa, PVDs also function as signal molecules for the production of virulence factors. Genes responsible for the biosynthesis, excretion, uptake and regulation of these high-affinity siderophores are located either at a single locus or at up to three different loci in the genomes of the four pseudomonads analyzed. The peptide backbone of PVD is assembled by non-ribosomal peptide synthetases (NRPSs) and modified by accessory enzymes in the cytoplasm, and probably the periplasm. Regulation of PVD production and uptake depends on two extracytoplasmic sigma factors (ECF-sigmas), PvdS and FpvI, together with one anti-sigma, FpvR.     

The lipase LipA (PA2862) but not LipC (PA4813) from Pseudomonas aeruginosa influences regulation of pyoverdine production and expression of the sigma factor PvdS

A key element in iron-dependent regulation of iron metabolism and virulence-related functions for Pseudomonas aeruginosa is the sigma factor PvdS. PvdS expression itself is also influenced by iron-independent stimuli. We show that pyoverdine production and pvdS expression depend on one of the two lipases of P. aeruginosa.     

The 'core' and 'accessory' regulons of Pseudomonas-specific extracytoplasmic sigma factors

Pyoverdine is a fluorescent, high-affinity peptide siderophore produced by different Pseudomonas species. The genes for pyoverdine biosynthesis depend on PvdS, an extracytoplasmic sigma factor. In this issue of Molecular Microbiology, Swingle et al. demonstrate that in the plant pathogen Pseudomonas syringae PvdS not only regulates the production of pyoverdine (core regulon), but also controls expression of other genes likely to be involved in the adaptation to the environment (accessory regulon). This accessory regulon is variable, as different sets of genes seem to be recruited according to the Pseudomonas species and its specific ecological niche.     

The pvc Gene Cluster of Pseudomonas aeruginosa: Role in Synthesis of the Pyoverdine Chromophore and Regulation by PtxR and PvdS

A putative operon of four genes implicated in the synthesis of the chromophore moiety of the Pseudomonas aeruginosa siderophore pyoverdine, dubbed pvcABCD (where pvc stands for pyoverdine chromophore), was cloned and sequenced. Mutational inactivation of the pvc genes abrogated pyoverdine biosynthesis, consistent with their involvement in the biosynthesis of this siderophore. pvcABCD expression was negatively regulated by iron and positively regulated by both PvdS, the alternate sigma factor required for pyoverdine biosynthesis, and PtxR, a LysR family activator previously implicated in exotoxin A regulation.     

Role of TonB1 in Pyoverdine-Mediated Signaling in Pseudomonas aeruginosa

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

A novel mechanism of interaction between alpha-synuclein and biological membranes

Conformational abnormalities and aggregation of alpha-synuclein (alpha-syn) have been linked to the pathogenesis of Parkinson's (PD) and related diseases. It has been shown that alpha-syn can stably bind artificial phospholipid vesicles through alpha-helix formation in its N-terminal repeat region. However, little is known about the membrane interaction in cells. In the current study, we determined the membrane-binding properties of alpha-syn to biological membranes by using bi-functional chemical crosslinkers, which allow the detection of transient, but specific, interactions. By utilizing various point mutations and deletions within alpha-syn, we demonstrated that the membrane interaction of alpha-syn in cells is also mediated by alpha-helix formation in the N-terminal repeat region. Moreover, the PD-linked A30P mutation causes reduced membrane binding, which is concordant with the artificial membrane studies. However, contrary to the interaction with artificial membranes, the interaction with biological membranes is rapidly reversible and is not driven by electrostatic attraction. Furthermore, the interaction of alpha-syn with cellular membranes occurs only in the presence of non-protein and non-lipid cytosolic components, which distinguishes it from the spontaneity of the interaction with artificial membranes. More interestingly, addition of the cytosolic preparation to artificial membranes resulted in the transient, charge-independent binding of alpha-syn similar to the interaction with biological membranes. These results suggest that in cells, alpha-syn is engaged in a fundamentally different mode of membrane interaction than the charge-dependent artificial membrane binding, and the mode of interaction is determined by the intrinsic properties of alpha-syn itself and by the cytoplasmic context.     

Genetic evidence for interaction of sigma A with two promoters in Bacillus subtilis.

The specificity of promoter binding by RNA polymerase is governed by the sigma subunit. Recent studies, in which single-amino-acid substitutions in sigma factors have been found to suppress the effects of specific base pair substitutions in promoters, support the model that these sigma factors make sequence-specific contacts with nucleotides at the -10 and -35 regions of promoters. We found that single-amino-acid substitutions in the putative -35 region and -10 region recognition domains of sigma A specifically suppressed the effects of mutations in the -35 and -10 regions, respectively, of two promoters that are expressed in exponentially growing Bacillus subtilis. These mutations change the specificity of sigma A, the primary sigma factor in growing B. subtilis, and demonstrate that this sigma factor interacts with promoters in a manner similar to that of its homolog in Escherichia coli, sigma 70. These mutant derivatives of sigma A also provide a tool that may be useful for determining whether sigma A uses specific promoters in vivo.