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Michael L. Vasil - One of the best experts on this subject based on the ideXlab platform.
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architecture of a protein central to iron homeostasis crystal structure and spectroscopic analysis of the Ferric Uptake Regulator
Molecular Microbiology, 2003Co-Authors: Ehmke Pohl, Jon C Haller, Ana Mijovilovich, Wolfram Meyerklaucke, Elspeth F Garman, Michael L. VasilAbstract:Iron is an essential element for almost all organisms, although an overload of this element results in toxicity because of the formation of hydroxyl radicals. Consequently, most living entities have developed sophisticated mechanisms to control their intracellular iron concentration. In many bacteria, including the opportunistic pathogen Pseudomonas aeruginosa, this task is performed by the Ferric Uptake Regulator (Fur). Fur controls a wide variety of basic physiological processes including iron Uptake systems and the expression of exotoxin A. Here, we present the first crystal structure of Fur from P. aeruginosa in complex with Zn2+ determined at a resolution of 1.8 A. Furthermore, X-ray absorption spectroscopic measurements and microPIXE analysis were performed in order to characterize the distinct zinc and iron binding sites in solution. The combination of these complementary techniques enables us to present a model for the activation and DNA binding of the Fur protein.
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Ferric Uptake Regulator mutants of pseudomonas aeruginosa with distinct alterations in the iron dependent repression of exotoxin a and siderophores in aerobic and microaerobic environments
Molecular Microbiology, 1996Co-Authors: Hazel A Barton, Adriana I Vasil, Zaiga Johnson, Charles D Cox, Michael L. VasilAbstract:Because the Ferric Uptake Regulator (fur ) appears to be an essential gene in Pseudomonas aeruginosa, resistance to manganese was used as an enrichment to isolate strains carrying point mutations in the fur gene in order to assess its role in the co-ordinate expression of siderophores and exotoxin A (ETA). This report describes a detailed molecular and phenotypic characterization of four mutants and one revertant, which carry point mutations in the fur gene. Two parental strains were used in this study. Three mutants were isolated from the widely used strain, PAO1. One of these, CS (cold sensitive), has a mutation in the 5′ non-coding region of the fur gene while the two other mutants derived from this parent have mutations resulting in the following deduced changes in Fur: mutant A2, H86 R; mutant A4, H86 Y. The other mutant (C6) and its revertant (C6Rv) were derived from PAO6261, a mutant of PAO1 with a deletion in the anr gene (anaerobic regulation of arginine deiminase and nitrate reduction) that controls anaerobic respiration in P. aeruginosa. Fur from the C6 mutant has an A10 G mutation while in the C6Rv spontaneous revertant the mutant Gly residue has been changed to Ser at this position. All mutants were examined for alterations in the iron-regulated expression of siderophores and ETA. The A2 and A4 mutants expressed higher levels of siderophores in iron-deficient media and in iron-replete media. The CS mutant constitutively expressed siderophores at 25°C. At 42°C siderophore biosynthesis was iron repressed as in the parental strain PAO1. The deletion of anr in PAO6261 had no apparent effect on the iron-mediated regulation of siderophore synthesis, but the C6 mutant derived from this strain produces siderophores constitutively. The iron-regulated production of siderophores by C6Rv was similar to the parental strain PAO6261 and PAO1. Because one of the parental strains used in this study is an Anr mutant, regulation of ETA production was assessed under aerobic and microaerobic conditions. Iron-dependent repression of ETA synthesis in both parental strains and A2 and A4 mutants was found to be 50–100-fold under aerobic and microaerobic conditions, as assayed by quantitative Western dot-blot assays. By contrast in the CS and C6 mutants, while iron-dependent repression of ETA synthesis was similar to both parental strains under aerobic conditions, ETA production in these mutants was constitutive in a microaerobic environment. RNase protection analysis of toxA and regAB transcription in PAO1, PAO6261 and the C6 mutant corroborated the results of quantitative dot-blot assays of ETA. The mutant Fur proteins were purified and examined for their ability to bind to the promoter of a gene (pvdS ) that positively regulates the expression of siderophores and ETA. Fur from the A2 and A4 mutants and from the C6Rv revertant was able to bind to the target DNA, but with reduced affinity by comparison to wild-type Fur. Fur from the C6 mutant in DNase I footprint experiments failed to protect the promoter region of the pvdS gene, but it retained some weak binding activity in gel mobility shift assays. The data presented in this study not only furnish some additional insights into the structure–function relationships of Fur, but also afford novel perspectives with regard to Fur and the iron-dependent regulation of virulence factors in P. aeruginosa under environmental conditions that have not previously been considered.
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Gene repression by the Ferric Uptake Regulator in Pseudomonas aeruginosa: cycle selection of iron-regulated genes.
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Urs A. Ochsner, Michael L. VasilAbstract: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.
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role of the Ferric Uptake Regulator of pseudomonas aeruginosa in the regulation of siderophores and exotoxin a expression purification and activity on iron regulated promoters
Journal of Bacteriology, 1995Co-Authors: Urs A. Ochsner, Adriana I Vasil, Michael L. VasilAbstract:The cloned Pseudomonas aeruginosa fur (Ferric Uptake Regulator) gene was overexpressed in P. aeruginosa by using a T7 expression system, and the Fur protein (PA-Fur) was purified by using a combination of ion-exchange chromatography and metal affinity chromatography. The DNA binding activity of the PA-Fur protein was confirmed by gel mobility shift assays and DNase I footprints of the synthetic DNA fragment GATAAT GATAATCATTATC, representing a perfect "Fur box". In addition, it was shown that PA-Fur is capable of binding to promoter and operator determinants of the tightly iron-regulated Escherichia coli fepA-fes enterobactin gene system. The activity of PA-Fur on the promoters of iron-regulated genes involved in the production of two siderophores, pyochelin and pyoverdin, and in the expression of exotoxin A was investigated. Data indicating that the promoters of the pchR gene, encoding a transcriptional activator for pyochelin synthesis, and of the pvdS gene, encoding a positive Regulator for pyoverdin production, are specifically recognized by Fur-Fe(II) are presented, suggesting that PA-Fur represses expression of pchR and pvdS during growth in an iron-replete environment. However, neither the promoter region of the gene encoding exotoxin A (toxA) nor the promoters of the regAB operon, required for toxA expression, interacted with high concentrations of purified PA-Fur. These data indicate that iron regulation of exotoxin A production involves additional factors which may ultimately be under the control of PA-Fur.
María F. Fillat - One of the best experts on this subject based on the ideXlab platform.
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fur like proteins beyond the Ferric Uptake Regulator fur paralog
Archives of Biochemistry and Biophysics, 2021Co-Authors: Emma Sevilla, Luisa M Peleato, Teresa M Bes, María F. FillatAbstract:Proteins belonging to the FUR (Ferric Uptake Regulator) family are the cornerstone of metalloregulation in most prokaryotes. Although numerous reviews have been devoted to these proteins, these reports are mainly focused on the Fur paralog that gives name to the family. In the last years, the increasing knowledge on the other, less ubiquitous members of this family has evidenced their importance in bacterial metabolism. As the Fur paralog, the major Regulator of iron homeostasis, Zur, Irr, BosR and PerR are tightly related to stress defenses and host-pathogen interaction being in many cases essential for virulence. Furthermore, the Nur and Mur paralogs largely contribute to control nickel and manganese homeostasis, which are cofactors of pivotal proteins for host colonization and bacterial redox homeostasis. The present review highlights the main features of FUR proteins that differ to the canonical Fur paralog either in the coRegulatory metal, such as Zur, Nur and Mur, or in the action mechanism to control target genes, such as PerR, Irr and BosR.
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the fur Ferric Uptake Regulator superfamily diversity and versatility of key transcriptional Regulators
Archives of Biochemistry and Biophysics, 2014Co-Authors: María F. FillatAbstract:Control of metal homeostasis is essential for life in all kingdoms. In most prokaryotic organisms the FUR (Ferric Uptake Regulator) family of transcriptional Regulators is involved in the regulation of iron and zinc metabolism through control by Fur and Zur proteins. A third member of this family, the peroxide-stress response PerR, is present in most Gram-positives, establishing a tight functional interaction with the global Regulator Fur. These proteins play a pivotal role for microbial survival under adverse conditions and in the expression of virulence in most pathogens. In this paper we present the current state of the art in the knowledge of the FUR family, including those members only present in more reduced numbers of bacteria, namely Mur, Nur and Irr. The huge amount of work done in the two last decades shows that FUR proteins present considerable diversity in their Regulatory mechanisms and interesting structural differences. However, much work needs to be done to obtain a more complete picture of this family, especially in connection with the roles of some members as gas and redox sensors as well as to fully characterize their participation in bacterial adaptative responses.
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identification of a Ferric Uptake Regulator from microcystis aeruginosa pcc7806
Fems Microbiology Letters, 2006Co-Authors: Beatriz Martinluna, Jose A. Hernandez, María F. Fillat, Teresa M Bes, Luisa M PeleatoAbstract:Ferric Uptake Regulator (Fur) proteins are widely recognized as repressors that in many prokaryotes regulate a large number of genes involved in iron homeostasis and oxidative stress response. In our study, we were able to identify the complete sequence of the fur gene from Microcystis aeruginosa using inverse-polymerase chain reaction. DNA sequence analysis confirmed the presence of a 183 amino-acid open reading frame that showed high identity with Fur proteins reported for cyanobacteria. The recombinant Fur protein has been purified and electrophoretical mobility shift assays shown to be active. Mn2+ and dithiothreitol enable Fur to bind to its promoter, with dithiothreitol being more potent. The expression of Fur in Microcystis was induced about twofold in iron-deficient conditions.
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the conformational stability and thermodynamics of fur a Ferric Uptake Regulator from anabaena sp pcc 7119
Biophysical Journal, 2005Co-Authors: Jose A. Hernandez, María F. Fillat, Teresa M Bes, Luisa M Peleato, Jorg Meier, Francisco N Barrera, Olga Ruiz De Los Panos, Estefania Hurtadogomez, Claudio N Cavasotto, José L. NeiraAbstract:Fur (Ferric Uptake Regulator) is a key bacterial protein that regulates iron acquisition and its storage, and modulates the expression of genes involved in the response to different environmental stresses. Although the protein is involved in several regulation mechanisms, and members of the Fur family have been identified in pathogen organisms, the stability and thermodynamic characterization of a Fur protein have not been described. In this work, the stability, thermodynamics and structure of the functional dimeric Fur A from Anabaena sp. PCC 7119 were studied by using computational methods and different biophysical techniques, namely, circular dichroism, fluorescence, Fourier-transform infrared, and nuclear magnetic resonance spectroscopies. The structure, as monitored by circular dichroism and Fourier-transform infrared, was composed of a 40% of α-helix. Chemical-denaturation experiments indicated that Fur A folded via a two-state mechanism, but its conformational stability was small with a value of ΔG = 5.3 ± 0.3 kcal mol−1 at 298 K. Conversely, Fur A was thermally a highly stable protein. The high melting temperature (Tm = 352 ± 5 K), despite its moderate conformational stability, can be ascribed to its low heat capacity change upon unfolding, ΔCp, which had a value of 0.8 ± 0.1 kcal mol−1 K−1. This small value is probably due to burial of polar residues in the Fur A structure. This feature can be used for the design of mutants of Fur A with impaired DNA-binding properties.
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Biochemical analysis of the recombinant Fur (Ferric Uptake Regulator) protein from Anabaena PCC 7119: factors affecting its oligomerization state.
Biochemical Journal, 2002Co-Authors: Jose A. Hernandez, M. Teresa Bes, María F. Fillat, José L. Neira, M. Luisa PeleatoAbstract:Fur (Ferric Uptake Regulator) protein is a DNA-binding protein which regulates iron-responsive genes. Recombinant Fur from the nitrogen-fixing cyanobacterium Anabaena PCC 7119 has been purified and characterized, and polyclonal antibodies obtained. The experimental data show that Fur from Anabaena dimerizes in solution with the involvement of disulphide bridges. Cross-linking experiments and MALDI-TOF (matrix-assisted laser desorption/ionization time of flight) MS also show several oligomerization states of Fur, and the equilibrium of these forms depends on protein concentration and ionic strength. In intact recombinant Fur, four cysteine residues out of five were inert towards DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)], and their modification required sodium borohydride. Metal analysis and electrospray ionization MS revealed that neither zinc nor other metals are present in this Fur protein. Purified recombinant Fur bound to its own promoter in gel-shift assays. Fur was shown to be a constitutive protein in Anabaena cells, with no significant difference in its expression in cells grown under iron-sufficient compared with iron-deficient conditions.
Yiquan Zhang - One of the best experts on this subject based on the ideXlab platform.
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transcriptional regulation of the mannitol phosphotransferase system operon by the Ferric Uptake Regulator fur in vibrio cholerae el tor serogroup o1
Research in Microbiology, 2021Co-Authors: He Gao, Han Wang, Qin Qin, Yue Gao, Yue Qiu, Jingyun Zhang, Jing Lou, Baowei Diao, Yiquan ZhangAbstract:Abstract The phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) allows bacteria to use various carbohydrates as energy resources including mannitol. The mannitol-specific PTS transporter in Vibrio cholerae is encoded by the mtlADR operon. Expression of the mtl operon has been shown to be strictly regulated by CRP, MtlS, and MtlR. In the present study, we investigated the regulation of mtlADR by the Ferric Uptake Regulator (Fur). The results showed that Fur binds to the promoter-proximal DNA region of mtlADR to repress its transcription independent of iron, in mannitol-containing growth medium. The capacity for mannitol fermentation was significantly increased in Δfur relative to that of WT for normal and iron-replete growth media. The level of organic acids produced by Δfur was significantly enhanced relative to that produced by the WT strain in the normal and iron-replete media but not in an iron-starved medium. The results provided for a deeper understanding of the regulation of mtlADR in V. cholerae.
Li Sun - One of the best experts on this subject based on the ideXlab platform.
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domain analysis of the edwardsiella tarda Ferric Uptake Regulator
Journal of General and Applied Microbiology, 2009Co-Authors: Kun Sun, Fang Wang, Shuang Cheng, Li SunAbstract:Recent studies have shown that the Ferric Uptake Regulator (Fur) of Edwardsiella tarda (Fur(Et)) shares high sequence identity with the Escherichia coli Fur (Fur(Ec)) at the N-terminal DNA-binding region. In the present study, the functional importance of the C-terminal region of Fur(Et) was investigated. It was found that Fur(Et) bearing deletion of the C-terminal 12 residues still possesses most of the repressor activity, whereas Fur(Et) bearing deletions of the C-terminal 16 and more than 16 residues are severely affected in activity. Domain swapping analyses indicated that the chimeric Fur proteins (Et75Ec73 and Et75Vh74) consisting of the N-terminal 1-75 region of Fur(Et) fused to the C-terminal 76-148 region of Fur(Ec) and the C-terminal 76-149 region of the Vibrio harveyi Fur (Fur(Vh)), respectively, are fully active. C92 of Fur(Ec) and C137 of Fur(Vh), which are functionally essential in Fur(Ec) and Fur(Vh), respectively, are also essential in Et75Ec73 and Et75074, respectively. Further study identified an artificial Fur protein, EtMF54, which is composed of the N-terminal 49 residues of Fur(Et) and five artificial residues. Compared to Fur(Et), EtMF54 possesses partial Fur activity that is iron-dependent. These results (I) indicate that there exist certain functional/structural compatibilities among Fur(Et), Fur(Ec), and Fur(Vh) at the C-terminal region; (ii) provide insights to the potential location of the Regulatory ion-binding site of Fur(Et).
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cys 92 cys 95 and the c terminal 12 residues of the vibrio harveyi Ferric Uptake Regulator fur are functionally inessential
Journal of Microbiology, 2008Co-Authors: Kun Sun, Fang Wang, Shuang Cheng, Min Zhang, Li SunAbstract:Ferric Uptake Regulator (Fur) is a global Regulator involved in multiple aspects of bacterial life. The gene encoding the Vibrio harveyi Fur (Fur(vh)) was cloned from a pathogenic V. harveyi strain isolated from diseased fish. Furvh shares 77% overall sequence identity with the Escherichia coli Fur (Fur(Ec)) and could complement a mutant of Fur(Ec). Like Fur(Ec), Fur(Vh), possesses two cysteine residues at positions 92 and 95, yet unlike Fur(Ec), in which these cysteine residues constitute part of the metal ion coordination site and hence are vital to the repressor activity, C92 and C95 of Fur(Vh) proved to be functionally inessential. Further study identified a Vibrio Fur signature sequence, which is preserved in all the ten Vibrio Fur proteins that have been discovered to date but in none of the non-vibrio Fur proteins. Site-directed and random mutation analyses of the signature residues, the cysteine residues, and seven highly charged amino acid residues indicated that D9, H32, C137, and K138 of Fur(vh) are functionally important but D9, C137, and K138 can be replaced by more than one functional substitutes. Systematic deletion analysis demonstrated that the C-terminal 12 residues of Fur(Vh) are functionally inessential. These results (i) indicated that the activation mechanism, or certain aspects of which, of Fur(Vh) is possibly different from that of Fur(Ec); and (ii) suggested that it is not very likely that the C-terminal 12 residues play any significant role in the activation or stability of Fur(Vh); and (iii) provided insights into the potential function of the local structure involving C137 and K138.
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molecular analysis of the fur Ferric Uptake Regulator gene of a pathogenic edwardsiella tarda strain
Journal of Microbiology, 2008Co-Authors: Fang Wang, Shuang Cheng, Kun Sun, Li SunAbstract:The gene encoding the Edwardsiella tarda Ferric Uptake Regulator (Fur(Et)) was cloned from a pathogenic E. tarda strain isolated from diseased fish. Fur(Et) shares 90% overall sequence identity with the Escherichia coli Fur (Fur(Ec)) and was able to complement the mutant phenotype of a fur(Ec)-defective E. coli strain. Mutational analysis indicated that C92S and C95S mutations inactivated Fur(Et) whereas E112K mutation resulted in a superactive Fur(Et) variant. Fur(Et) negatively regulated its own expression; interruption of this regulation impaired bacterial growth, altered the production of certain outer membrane proteins, and attenuated bacterial virulence.
Yukeung Mok - One of the best experts on this subject based on the ideXlab platform.
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two component phob phor Regulatory system and Ferric Uptake Regulator sense phosphate and iron to control virulence genes in type iii and vi secretion systems of edwardsiella tarda
Journal of Biological Chemistry, 2011Co-Authors: Smarajit Chakraborty, J Sivaraman, Ka Yin Leung, Yukeung MokAbstract:Inorganic phosphate (Pi) and iron are essential nutrients that are depleted by vertebrates as a protective mechanism against bacterial infection. This depletion, however, is sensed by some pathogens as a signal to turn on the expression of virulence genes. Here, we show that the PhoB-PhoR two-component system senses changes in Pi concentration, whereas the Ferric Uptake Regulator (Fur) senses changes in iron concentration in Edwardsiella tarda PPD130/91 to regulate the expression of type III and VI secretion systems (T3SS and T6SS) through an E. tarda secretion Regulator, EsrC. In sensing low Pi concentration, PhoB-PhoR autoregulates and activates the phosphate-specific transport operon, pstSCAB-phoU, by binding directly to the Pho box in the promoters of phoB and pstS. PhoB also binds with EsrC simultaneously on the promoter of an E. tarda virulence protein, evpA, to regulate directly the transcription of genes from T6SS. In addition, PhoB requires and interacts with PhoU to activate esrC and suppress fur indirectly through unidentified Regulators. Fur, on the other hand, senses high iron concentration and binds directly to the Fur box in the promoter of evpP to inhibit EsrC binding to the same region. In addition, Fur suppresses transcription of phoB, pstSCAB-phoU, and esrC indirectly via unidentified Regulators, suggesting negative cross-talk with the Pho regulon. Physical interactions exist between Fur and PhoU and between Fur and EsrC. Our findings suggest that T3SS and T6SS may carry out distinct roles in the pathogenicity of E. tarda by responding to different environmental factors.