Iron-regulated transcription of the pvdA gene in Pseudomonas aeruginosa: effect of Fur and PvdS on promoter activity.
ABSTRACT: The pvdA gene, encoding the enzyme L-ornithine N5-oxygenase, catalyzes a key step of the pyoverdin biosynthetic pathway in Pseudomonas aeruginosa. Expression studies with a promoter probe vector made it possible to identify three tightly iron-regulated promoter regions in the 5.9-kb DNA fragment upstream of pvdA. The promoter governing pvdA expression was located within the 154-bp sequence upstream of the pvdA translation start site. RNA analysis showed that expression of PvdA is iron regulated at the transcriptional level. Primer extension and S1 mapping experiments revealed two 5'termini of the pvdA transcript, 68 bp (T1) and 43 bp (T2) 5' of the PvdA initiation. The pvdA transcripts were monocystronic, with T1 accounting for 90% of the pvdA mRNA. Fur box-like sequences were apparently absent in the regions 5' of pvdA transcription start sites. A sequence motif resembling the -10 hexamer of AlgU-dependent promoters and the iron starvation box of pyoverdin genes controlled by the sigmaE -like factor PvdS were identified 5' of the T1 start site. The minimum DNA region required for iron-regulated promoter activity was mapped from bp -41 to -154 relative to the ATG translation start site of pvdA. We used pvdA'::lacZ transcriptional fusions and Northern (RNA) analyses to study the involvement of Fur and PvdS in the iron-regulated expression of pvdA. Two fur mutants of P. aeruginosa were much less responsive than wild-type PAO1 to the iron-dependent regulation of pvdA expression. Transcription from the pvdA promoter did not occur in a heterologous host unless in the presence of the pvdS gene in trans and was abrogated in a pvdS mutant of P. aeruginosa. Interaction of the Fur repressor with a 150-bp fragment encompassing the pvdS promoter was demonstrated in vivo by the Fur titration assay and confirmed in vitro by gel retardation experiments with a partially purified Fur preparation. Conversely, the promoter region of pvdA did not interact with Fur. Our results support the hypothesis that the P. aeruginosa Fur repressor indirectly controls pvdA transcription through the intermediary sigma factor PvdS; in the presence of sufficient iron, Fur blocks the pvdS promoter, thus preventing PvdS expression and consequently transcription of pvdA and other pyoverdin biosynthesis genes.
Project description:In Pseudomonas aeruginosa, iron modulates gene expression through a cascade of negative and positive regulatory proteins. The master regulator Fur is involved in iron-dependent repression of several genes. One of these genes, pvdS, was predicted to encode a putative sigma factor responsible for the transcription of a subset of genes of the Fur regulon. PvdS appears to belong to a structurally and functionally distinct subgroup of the extracytoplasmic function family of alternative sigma factors. Members of this subgroup, also including PbrA from Pseudomonas fluorescens, PfrI and PupI from Pseudomonas putida, and FecI from Escherichia coli, are controlled by the Fur repressor, and they activate transcription of genes for the biosynthesis or the uptake of siderophores. Evidence is provided that the PvdS protein of P. aeruginosa is endowed with biochemical properties of eubacterial sigma factors, as it spontaneously forms 1:1 complexes with the core fraction of RNA polymerase (RNAP, alpha(2)betabeta' subunits), thereby promoting in vitro binding of the PvdS-RNAP holoenzyme to the promoter region of the pvdA gene. These functional features of PvdS are consistent with the presence of structural domains predicted to be involved in core RNAP binding, promoter recognition, and open complex formation. The activity of pyoverdin biosynthetic (pvd) promoters was significantly lower in E. coli overexpressing the multicopy pvdS gene than in wild-type P. aeruginosa PAO1 carrying the single gene copy, and pvd::lacZ transcriptional fusions were silent in both pfrI (the pvdS homologue) and pfrA (a positive regulator of pseudobactin biosynthetic genes) mutants of P. putida WCS358, while they are expressed at PAO1 levels in wild-type WCS358. Moreover, the PvdS-RNAP holoenzyme purified from E. coli lacked the ability to generate in vitro transcripts from the pvdA promoter. These observations suggest that at least one additional positive regulator could be required for full activity of the PvdS-dependent transcription complex both in vivo and in vitro. This is consistent with the presence of a putative activator binding site (the iron starvation box) at variable distance from the transcription initiation sites of promoters controlled by the iron starvation sigma factors PvdS, PfrI, and PbrA of fluorescent pseudomonads.
Project description:We previously identified a novel regulator of the exotoxin A gene (toxA) in Pseudomonas aeruginosa, PtxR, that belongs to the LysR family of prokaryotic regulatory proteins. Preliminary data also suggest that PtxR affects the expression of siderophores in P. aeruginosa. Because toxA expression and siderophore production in this organism are coordinately regulated by the ferric uptake regulator (Fur) and the Fur-regulated alternative sigma factor PvdS, regulation of ptxR itself in the context of these regulators was examined. RNase protection analyses of ptxR transcription revealed that there are two independent transcription initiation sites (T1 and T2). While transcription from the promoter of T1 is constitutive throughout the growth cycle of PAO1, transcription from the second promoter (P2) is negatively affected by iron. Transcription from the P2 promoter is constitutive in a fur mutant under microaerobic conditions but still iron regulated during aerobic growth. High concentrations (>100 nM) of the ferric uptake regulatory protein (Fur) failed to bind to either of the promoter regions of ptxR in either gel mobility shift assays or DNase I footprint experiments. These results indicate that Fur indirectly regulates the iron-dependent expression of ptxR. Iron-regulated transcription of ptxR from the P2 promoter, but not constitutive expression from the P1 promoter, was dependent on the Fur-regulated alternative sigma factor gene pvdS, even under aerobic conditions. Consequently, there are two levels of iron-regulated expression of ptxR. The iron-regulated expression of ptxR under microaerobic conditions from the P2 promoter of ptxR is mediated indirectly by Fur through the iron-regulated expression of pvdS. In contrast, pvdS-mediated iron regulation of ptxR under aerobic conditions is Fur independent.
Project description:The enzyme L-ornithine N5-oxygenase catalyzes the hydroxylation of L-ornithine (L-Orn), which represents an early step in the biosynthesis of the peptidic moiety of the fluorescent siderophore pyoverdin in Pseudomonas aeruginosa. A gene bank of DNA from P. aeruginosa PAO1 (ATCC 15692) was constructed in the broad-host-range cosmid pLAFR3 and mobilized into the L-Orn N5-oxygenase-defective (pvdA) P. aeruginosa mutant PALS124. Screening for fluorescent transconjugants made it possible to identify the trans-complementing cosmid pPV4, which was able to restore pyoverdin synthesis and L-Orn N5-oxygenase activity in the pvdA mutant PALS124. The 17-kb PAO1 DNA insert of pPV4 contained at least two genetic determinants involved in pyoverdin synthesis, i.e., pvdA and pvdC4, as shown by complementation analysis of a set of mutants blocked in different steps of the pyoverdin biosynthetic pathway. Deletion analysis, subcloning, and transposon mutagenesis enabled us to locate the pvdA gene in a minimum DNA fragment of 1.7 kb flanked by two SphI restriction sites. Complementation of the pvdA mutation was under stringent iron control; both pyoverdin synthesis and L-Orn N5-oxygenase activity were undetectable in cells of the trans-complemented mutant which had been grown in the presence of 100 microM FeCl3. The entire nucleotide sequence of the pvdA gene, from which the primary structure of the encoded polypeptide was deduced, was determined. The pvdA structural gene is 1,278 bp; the cloned DNA fragment contains at the 5' end of the gene a putative ribosome-binding site but apparently lacks known promoterlike sequences. The P. aeruginosa L-Orn N5-oxygenase gene codes for a 426-amino-acid peptide with a predicted molecular mass of 47.7 kDa and an isoelectric point of 8.1. The enzyme shows approximately 50% homology with functional analogs, i.e., L-lysine N6-hydroxylase of aerobactin-producing Escherichia coli and L-Orn N5-oxygenase of ferrichrome-producing Ustilago maydis. The pvdA gene was expressed in P. aeruginosa under the control of the T7 promoter. Induction of the T7 RNA polymerase system resulted in parallel increases of the L-Orn N5-oxygenase activity and of the amount of a 47.7-kDa polypeptide. We also constructed a site-specific pvdA mutant by insertion of a tetracycline-resistance cassette in the chromosomal pvdA gene of P. aeruginosa PAO1. Similarly to strain PALS124, the pvdA mutant obtained by gene disruption also disclosed no pyoverdin synthesis, lacked L-Orn N5-oxygenase activity, was complemented by the cloned pvdA gene, and produced pyoverdin at wild-type levels when fed with the biosynthetic precursor L-N5-OH-Orn. Southern blot analysis indicated that genes homologous to pvdA could be located within a 1.7-kb DNA fragment from SphI-digested genomic DNA of different hydroxamate-producing Pseudomonas spp. Our results suggest that omega-amino acid oxygenases have been conserved over a wide evolutionary range and probably evolved from a common ancestor.
Project description:Burkholderia cenocepacia mutants that fail to produce the siderophore ornibactin were obtained following mutagenesis with mini-Tn5Tp. These mutants were shown to be growth restricted under conditions of iron depletion. In eight of the mutants, the transposon had integrated into one of two genes, orbI and orbJ, encoding nonribosomal peptide synthetases. In the other mutant, the transposon had inserted into an open reading frame, orbS, located upstream from orbI. The polypeptide product of orbS exhibits a high degree of similarity to the Pseudomonas aeruginosa extracytoplasmic function (ECF) sigma factor PvdS but possesses an N-terminal extension of approximately 29 amino acids that is not present in PvdS. Three predicted OrbS-dependent promoters were identified within the ornibactin gene cluster, based on their similarity to PvdS-dependent promoters. The iron-regulated activity of these promoters was shown to require OrbS. Transcription of the orbS gene was found to be under the control of an iron-regulated sigma(70)-dependent promoter. This promoter, but not the OrbS-dependent promoters, was shown to be a target for repression by the global regulator Fur. Our results demonstrate that production of ornibactin by B. cenocepacia in response to iron starvation requires transcription of an operon that is dependent on the Fur-regulated ECF sigma factor gene orbS. A mechanism is also proposed for the biosynthesis of ornibactin.
Project description:Pseudomonas aeruginosa, which causes serious infections in immunocompromised patients, produces numerous virulence factors, including exotoxin A and the siderophore pyoverdine. As production of these virulence factors is influenced by the host environment, we examined the effect serum has on global transcription within P. aeruginosa strain PAO1 at different phases of growth in an iron-deficient medium. At early exponential phase, serum significantly enhanced expression of 138 genes, most of which are repressed by iron, including pvdS, regA and the pyoverdine synthesis genes. However, serum did not interfere with the repression of these genes by iron. Serum enhanced regA expression in a fur mutant of PAO1 but not in a pvdS mutant. The serum iron-binding protein apotransferrin, but not ferritin, enhanced regA and pvdS expression. However, in PAO1 grown in a chemically defined medium that contains no iron, serum but not apotransferrin enhanced pvdS and regA expression. While complement inactivation failed to eliminate this effect, albumin absorption reduced the effect of serum on pvdS and regA expression in the iron-deficient medium chelexed tryptic soy broth dialysate. Additionally, albumin absorption eliminated the effect of serum on pvdS and regA expression in the chemically defined medium. These results suggest that serum enhances the expression of P. aeruginosa iron-controlled genes by two mechanisms: one through apotransferrin and another one through albumin.
Project description:The ornithine hydroxylase from Pseudomonas aeruginosa (PvdA) catalyzes the FAD-dependent hydroxylation of the side chain amine of ornithine, which is subsequently formylated to generate the iron-chelating hydroxamates of the siderophore pyoverdin. PvdA belongs to the class B flavoprotein monooxygenases, which catalyze the oxidation of substrates using NADPH as the electron donor and molecular oxygen. Class B enzymes include the well studied flavin-containing monooxygenases and Baeyer-Villiger monooxygenases. The first two structures of a class B N-hydroxylating monooxygenase were determined with FAD in oxidized (1.9 ? resolution) and reduced (3.03 ? resolution) states. PvdA has the two expected Rossmann-like dinucleotide-binding domains for FAD and NADPH and also a substrate-binding domain, with the active site at the interface between the three domains. The structures have NADP(H) and (hydroxy)ornithine bound in a solvent-exposed active site, providing structural evidence for substrate and co-substrate specificity and the inability of PvdA to bind FAD tightly. Structural and biochemical evidence indicates that NADP(+) remains bound throughout the oxidative half-reaction, which is proposed to shelter the flavin intermediates from solvent and thereby prevent uncoupling of NADPH oxidation from hydroxylated product formation.
Project description:A novel outer membrane lipoprotein in Pseudomonas aeruginosa is encoded by the omlA gene, which was identified immediately upstream of the fur (ferric uptake regulator) gene. The omlA and fur genes were divergently transcribed and had overlapping promoter regions. The proximal fur P2 promoter and the omlA promoter shared a 5-bp DNA motif for their -10 promoter elements. The distal fur P1 promoter was located within the omlA coding sequence, and the omlA and fur T1 mRNAs overlapped by 154 nucleotides. Optimal expression of both fur and omlA required roughly 200 bp of DNA upstream of the promoter regions, suggesting the presence of cis-acting transcriptional activation elements located within the omlA and fur genes, respectively. The levels of Fur and OmlA proteins had no influence on omlA or fur expression, excluding any trans-acting cross-regulation between fur and omlA. Expression of omlA was constitutive regardless of growth phase, oxygen tension, iron concentration, pH, and temperature. OmlA contained a signal sequence typical of bacterial lipoproteins, with a cysteine as a putative cleavage and lipid attachment site. Inhibition of signal peptidase II by globomycin resulted in failure to process OmlA, thus giving strong evidence that OmlA is a lipoprotein. Cell fractionation followed by Western blot analysis indicated that all OmlA protein is localized in the outer membrane. Mature OmlA was an acidic (pI = 4. 5) protein of 17.3 kDa and had close to 40% amino acid sequence identity to SmpA (small protein A) of Escherichia coli, Vibrio cholerae, and Haemophilus influenzae, a protein of unknown function. All P. aeruginosa strains tested as well as Pseudomonas fluorescens were found to produce OmlA. A mutant strain with impaired production of OmlA but no change in the expression of the overlapping fur gene was constructed. The omlA mutant was hypersusceptible to anionic detergents such as sodium dodecyl sulfate and deoxycholate, and it showed increased susceptibility to various antibiotics, including nalidixic acid, rifampin, novobiocin, and chloramphenicol. A structural role of OmlA in maintaining the cell envelope integrity is proposed.
Project description:The 2-alkyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase HodC was previously described to cleave the Pseudomonas quinolone signal, PQS, which is exclusively used in the complex quorum sensing (QS) system of Pseudomonas aeruginosa, an opportunistic pathogen employing QS to regulate virulence and biofilm development. Degradation of PQS by exogenous addition of HodC to planktonic cells of P. aeruginosa attenuated production of virulence factors, and reduced virulence in planta. However, proteolytic cleavage reduced the efficacy of HodC. Here, we identified the secreted protease LasB of P. aeruginosa to be responsible for HodC degradation. In static biofilms of the P. aeruginosa PA14 lasB::Tn mutant, the catalytic activity of HodC led to an increase in viable biomass in newly formed but also in established biofilms, and reduced the expression of genes involved in iron metabolism and siderophore production, such as pvdS, pvdL, pvdA, and pvdQ. This is likely due to an increase in the levels of bioavailable iron by degradation of PQS, which is able to sequester iron from the surrounding environment. Thus, HodC, despite its ability to quench the production of virulence factors, is contraindicated for combating P. aeruginosa biofilms.
Project description:Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and chronic lung infections in cystic fibrosis patients. Iron is essential for bacterial growth, and P. aeruginosa expresses multiple iron uptake systems, whose role in lung infection deserves further investigation. P. aeruginosa Fe(3+) uptake systems include the pyoverdine and pyochelin siderophores and two systems for heme uptake, all of which are dependent on the TonB energy transducer. P. aeruginosa also has the FeoB transporter for Fe(2+) acquisition. To assess the roles of individual iron uptake systems in P. aeruginosa lung infection, single and double deletion mutants were generated in P. aeruginosa PAO1 and characterized in vitro, using iron-poor media and human serum, and in vivo, using a mouse model of lung infection. The iron uptake-null mutant (tonB1 feoB) and the Fe(3+) transport mutant (tonB1) did not grow aerobically under low-iron conditions and were avirulent in the mouse model. Conversely, the wild type and the feoB, hasR phuR (heme uptake), and pchD (pyochelin) mutants grew in vitro and caused 60 to 90% mortality in mice. The pyoverdine mutant (pvdA) and the siderophore-null mutant (pvdA pchD) grew aerobically in iron-poor media but not in human serum, and they caused low mortality in mice (10 to 20%). To differentiate the roles of pyoverdine in iron uptake and virulence regulation, a pvdA fpvR double mutant defective in pyoverdine production but expressing wild-type levels of pyoverdine-regulated virulence factors was generated. Deletion of fpvR in the pvdA background partially restored the lethal phenotype, indicating that pyoverdine contributes to the pathogenesis of P. aeruginosa lung infection by combining iron transport and virulence-inducing capabilities.
Project description:The expression of at least 24 distinct genes of Pseudomonas aeruginosa PAO1 is under direct control of the "ferric uptake regulator" (Fur). Novel targets of the Fur protein were isolated in a powerful SELEX (systematic evolution of ligands by exponential enrichment)-like cycle selection consisting of in vitro DNA-Fur interaction, binding to anti-Fur antibody, purification on protein G, and PCR amplification. DNA fragments obtained after at least three exponential enrichment cycles were cloned and subjected to DNA mobility-shift assays and DNase I footprint analyses to verify the specific interaction with the Fur protein in vitro. Iron-dependent expression of the corresponding genes in vivo was monitored by RNase protection analysis. In total, 20 different DNA fragments were identified which represent actual Pseudomonas iron-regulated genes (PIGs). While four PIGs are identical to already known genes (pfeR, pvdS, tonB, and fumC, respectively), 16 PIGs represent previously unknown genes. Homology studies of the putative proteins encoded by the PIGs allowed us to speculate about their possible function. Two PIG products were highly similar to siderophore receptors from various species, and three PIG products were significantly homologous to alternative sigma factors. Furthermore, homologs of the Escherichia coli ORF1-tolQ, nuoA, stringent starvation protein Ssp, and of a two-component regulatory system similar to the Pseudomonas syringae LemA sensor kinase were identified. The putative gene products of seven additional PIGs did not show significant homologies to any known proteins. The PIGs were mapped on the P.aeruginosa chromosome. Their possible role in iron metabolism and virulence of P. aeruginosa is discussed.