A bacteria-specific 2[4Fe-4S] ferredoxin is essential in Pseudomonas aeruginosa (original) (raw)
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Primary structure of protein B from Pseudomonas putida, member of a new class of 2Fe-2S ferredoxins
FEBS Letters, 1988
The primary structure of the 2Fe-2S ferredoxin (protein B) from the benzene dioxygenase system of Pseudomonas purida strain NCIB 12190 was determined by gas-phase sequencing of the protein and its fragments. Fast atom bombardment mass spectrometry indicated a molecular mass of 11860 Da. The sequence contained five cysteine residues, four of which would be required to coordinate the iron-sulphur cluster. The amino acid sequence determined in the present study is compared to that of a protein deduced from the DNA sequence from another strain of Pseudomonasputidu. Little sequencc homology was observed when protein B was compared to 2Fe-2S ferredoxins from plant and cyanobacterial sources. The novel sequence determined here suggests a new class of ferredoxin, which is consistent with the observed mid-point redox potential being significantly less negative (-155 mV) than those of the 2Fe-2S ferredoxins involved in photosynthesis (-310 to -455 mV) Ferredoxin; Protein sequence; Benzene dioxygenase; Iron-sulfur protein
The ferrichrome receptor A as a new target for Pseudomonas aeruginosa virulence attenuation
FEMS Microbiology Letters, 2016
Pseudomonas aeruginosa is an opportunistic pathogen, known to develop robust biofilms. Its biofilm development increases when antibiotics are presented at subminimal inhibitory concentrations (MICs) for reasons that remain unclear. In order to identify genes that affect biofilm development under such a sublethal antibiotic stress condition, we screened a transposon (Tn) mutant library of PAO1, a prototype P. aeruginosa strain. Among ∼5000 mutants, a fiuA gene mutant was verified to form very defective biofilms in the presence of sub-MIC carbenicillin. The fiuA gene encodes ferrichrome receptor A, involved in the iron acquisition process. Of note, biofilm formation was not decreased in the ΔpchΔpvd mutant defective in the production of pyochelin and pyoverdine, two well-characterized P. aeruginosa siderophore molecules. Moreover, ΔfiuA, a non-polar fiuA deletion mutant, produced a significantly decreased level of elastase, a major virulence determinant. Mouse airway infection experiments revealed that the mutant expressed significantly less pathogenicity. Our results suggest that the fiuA gene has pleiotropic functions that affect P. aeruginosa biofilm development and virulence. The targeting of FiuA could enable the attenuation of P. aeruginosa virulence and may be suitable for the development of a drug that specifically controls the virulence of this important pathogen.
Ferredoxin I specifically controls soxs-like activation of FPR in Azotobacter vinelandii
Journal of Inorganic Biochemistry, 1997
Previous studies have established that disruption of the fdxA gene that encodes Azotobacter vinelandii ferredoxin I (AvFdI) leads to overexpression of NADPH: ferredoxin reductase (FPR). Investigation of the mechanism of this regulatory system has shown that, in response to fdxA disruption, FPR levels are increased because of transcriptional activation in a manner dependent upon a specific DNA sequence. That sequence is similar to the defined SoxS binding site of Escherichia coli. Using A. vinelandii fpr-luciferase fusion reporter constructs here we show that, like E. coli SoxRS-regulated genes, the A. vinelandii fpr gene is activated by the superoxide propagator paraquat and that the response occurs via the same DNA sequence that is responsive to fdxA disruption. Using a DsoxR strain of E. coli and the same reporter constructs we show that, when introduced into E. coli, the A. vinelandii fpr promoter is controlled by SoxRS. Although the two systems are functionally similar and must have structural similarities at the level of DNA-binding specificity for the SoxS-like recognition sequence, data are presented to show that they are likely to have differences at the protein/gene level. The results suggest that in A. vinelandii the specific function of FdI is to serve as the redox sensor that inactivates the paraquat-responsive system and that other organisms (e.g. Pseudomonas, Caulobacter) that have homologous ferredoxins might have similar regulatory systems.
Journal of Bacteriology, 2006
Pseudomonas aeruginosa secretes two siderophores, pyoverdine and pyochelin, under iron-limiting conditions. These siderophores are recognized at the cell surface by specific outer membrane receptors, also known as TonB-dependent receptors. In addition, this bacterium is also able to incorporate many heterologous siderophores of bacterial or fungal origin, which is reflected by the presence of 32 additional genes encoding putative TonB-dependent receptors. In this work, we have used a proteomic approach to identify the inducing conditions for P. aeruginosa TonB-dependent receptors. In total, 11 of these receptors could be discerned under various conditions. Two of them are only produced in the presence of the hydroxamate siderophores ferrioxamine B and ferrichrome. Regulation of their synthesis is affected by both iron and the presence of a cognate siderophore. Analysis of the P. aeruginosa genome showed that both receptor genes are located next to a regulatory locus encoding an extracytoplasmic function sigma factor and a transmembrane sensor. The involvement of this putative regulatory locus in the specific induction of the ferrioxamine B and ferrichrome receptors has been demonstrated. These results show that P. aeruginosa has evolved multiple specific regulatory systems to allow the regulation of TonB-dependent receptors.
Journal of Biological Inorganic Chemistry, 1998
Previous studies have established that disruption of the fdxA gene that encodes Azotobacter vinelandii ferredoxin I (AvFdI) leads to overexpression of NADPH: ferredoxin reductase (FPR). Investigation of the mechanism of this regulatory system has shown that, in response to fdxA disruption, FPR levels are increased because of transcriptional activation in a manner dependent upon a specific DNA sequence. That sequence is similar to the defined SoxS binding site of Escherichia coli. Using A. vinelandii fpr-luciferase fusion reporter constructs here we show that, like E. coli SoxRS-regulated genes, the A. vinelandii fpr gene is activated by the superoxide propagator paraquat and that the response occurs via the same DNA sequence that is responsive to fdxA disruption. Using a DsoxR strain of E. coli and the same reporter constructs we show that, when introduced into E. coli, the A. vinelandii fpr promoter is controlled by SoxRS. Although the two systems are functionally similar and must have structural similarities at the level of DNA-binding specificity for the SoxS-like recognition sequence, data are presented to show that they are likely to have differences at the protein/gene level. The results suggest that in A. vinelandii the specific function of FdI is to serve as the redox sensor that inactivates the paraquat-responsive system and that other organisms (e.g. Pseudomonas, Caulobacter) that have homologous ferredoxins might have similar regulatory systems.
Biochemistry, 1999
Terpredoxin (Tdx) is a 105-residue bacterial ferredoxin consisting of a single polypeptide chain and a single Fe 2 S 2 prosthetic group. Tdx was first identified in a strain of Pseudomonas sp. capable of using R-terpineol as sole carbon source. The Tdx gene, previously cloned from the plasmid-encoded terp operon, that carries genes encoding for proteins involved in terpineol catabolism, has been subcloned and expressed as the holoprotein in E. coli. Physical characterization of the expressed Tdx has been performed, and a model for the solution structure of oxidized Tdx (Tdx o ) has been determined. Highresolution homo-and heteronuclear NMR data have been used for structure determination in diamagnetic regions of the protein. The structure of the metal binding site (which cannot be determined directly by NMR methods due to paramagnetic broadening of resonances) was modeled using restraints obtained from a crystal structure of the homologous ferredoxin adrenodoxin (Adx) and loose restraints determined from paramagnetic broadening patterns in NMR spectra. Essentially complete 1 H and 15 N NMR resonance assignments have been made for the diamagnetic region of Tdx o (ca. 80% of the protein). A large fivestranded -sheet and a smaller two-stranded -sheet were identified, along with three R-helices. A high degree of structural homology was observed between Tdx and two other ferredoxins with sequence and functional homology to Tdx for which structures have been determined, Adx and putidaredoxin (Pdx), a homologous Pseudomonas protein. 1 H/ 2 H exchange rates for Tdx backbone NH groups were measured for both oxidation states and are rationalized in the context of the Tdx structure. In particular, an argument is made for the importance of the residue following the third ligand of the metal cluster (Arg49 in Tdx, His49 in Pdx, His56 in Adx) in modulating protein dynamics as a function of oxidation state. Some differences between Tdx and Pdx are detected by UV-visible spectroscopy, and structural differences at the C-terminal region were also observed. Tdx exhibits only 2% of the activity of Pdx in turnover assays performed using the reconstituted camphor hydroxylase system of which Pdx is the natural component. † This work was supported in part by a grant from the National Institutes of Health (GM44191, T.C.P.). S.S.P. acknowledges support from Grant P41-RR00995 (MIT FBML). ‡ A set of 15 structures have been deposited with the Brookhaven PDB database with the access code 1b9r. § Brandeis University. | Massachusetts Institute of Technology.
FEMS Microbiology Letters, 2005
Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low affinity siderophore, pyochelin. Uptake of the iron-siderophore complexes is an active process that requires specific outer membrane located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins, ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, apparently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA (PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterobactin. Ferric-enterobactin also failed to stimulate growth of the double pfeA-pirA mutant although, like its synthetic analogues, it stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferricenterobactin receptor. The example of these two enterobactin receptors probably illustrates a more general phenomenon of siderophore receptor redundancy in P. aeruginosa.
Acta crystallographica, 2007
The iron-sulfur (Fe-S) cluster-biosynthesis (ISC) system of the-proteobacterium Pseudomonas putida JCM 20004 contains a constitutively expressed vertebrate-type [2Fe-2S] ferredoxin, FdxB, which lacks the conserved free cysteine residue near the Fe-S cluster site that has been proposed to function in the catalysis of biological Fe-S cluster assembly in other bacterial homologues. Recombinant FdxB was heterologously overproduced in Escherichia coli, purified and crystallized in its oxidized form by the hanging-drop vapourdiffusion and streak-seeding methods using 1.6 M trisodium citrate dihydrate pH 6.5. The thin needle-shaped crystals diffract to 1.90 Å resolution and belong to the hexagonal space group P6 1 22, with unit-cell parameters a = 87.58, c = 73.14 Å. The asymmetric unit contains one protein molecule.
Open Forum Infectious Diseases
Background Pseudomonas aeruginosa is an aerobic Gram-negative bacterium that causes life-threatening acute and chronic infections in humans. Genotypic mutations and phenotypic variations are key features of its antimicrobial resistance and adaptation to the host environment. Pyoverdine associated genes and divergent receptors play a key role in acute Pseudomonas infections. This study seeks to address the heterogeneity of ferrichrome-iron receptor (fpvA) expression, its effect on pathogenicity and its propensity to cause acute infections clinically. Genetic and phenotypic variation of a clinical isolates of P. aeruginosa (PA097 and PA115) were identified by complete genome sequencing method. Methods An IRB-approved prospective study collected 38 P. aeruginosa clinical isolates and stored at Carilion Medical Center. Two genetically unrelated clinical strains were selected from tracheal aspirates: PA097 and PA115. These isolates were characterized by pyoverdine (pvd) quantification in...