Fur Protein Research Articles

Iron Binding Site in a Global Regulator in Bacteria–Ferric Uptake Regulator (Fur) Protein: Structure, Mössbauer Properties, and Functional Implication

Fur protein plays key roles in regulating numerous genes in bacteria and is essential for intracellular iron concentration regulation. However, atomic level pictures of the iron binding site and its functional mechanism remain to be established. Here we present results of the first quantum chemical investigation of various first- and second-shell models and experimental Mössbauer data of E. Coli Fur, including 1) the first robust evidence that site 2 is the Fe binding site with a 3His/2Glu ligand set, being the first case in non-heme proteins, with computed Mössbauer data in excellent accord with experiment; 2) the first discovery of a conservative hydrogen bonding interaction in the iron binding site based on X-ray and homology structures; 3) the first atomic level hypothesis of active site reorganization upon iron concentration increase, triggering the conformational change needed for its function. These results shall facilitate structural and functional studies of Fur family proteins.

Analysis of a ferric uptake regulator (Fur) knockout mutant in Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis; a serious infectious disease in aquaculture raised salmonids. Iron acquisition has been shown to be critical for the survival of pathogenic bacteria during the course of infection. Previous work has demonstrated that A. salmonicida expresses iron-repressible IROMP proteins, suggesting the presence of iron acquisition systems that are under the control of a ferric uptake regulator (Fur). In this study, the A. salmonicida fur has been sequenced and a fur deletion strain generated. The A. salmonicida fur gene has an open reading frame of 428bp, coding for a protein of 143 amino acids, and with high homology to previously described Fur proteins. The Fur protein product had a 94% sequence identity and 96% sequence similarity to the Aeromonas hydrophila Fur protein product. Transcription of the A. salmonicida fur gene was not regulated by the iron status of the bacterium and is not autoregulated, as in Escherichia coli. Proteomic analysis of the A. salmonicida fur mutant, fails to repress iron-regulated outer membrane proteins in the presence of iron. The A. salmonicida fur::KO mutant shows significantly reduced pathogenicity compared to the wild-type parental strain. In addition, the A. salmonicida fur mutant provides an important tool for further investigation of the iron acquisition mechanisms utilized by A. salmonicida.

Fur−mutation increases the survival time ofEscherichia coliunder photooxidative stress in aquatic environments

We investigated the survival of the wild type Escherichia coli (QC771) and fur- mutant strain (QC1732) under photooxidative stress in different water sources. The survival of fur- mutant and wild type E. coli was seen as a significant decrease in the visible light samples in the presence of methylene blue (MB). The fur-E. coli strain lived longer than the wild type E. coli strain on exposure to MB and visible light, which generates singlet oxygen, in both lake water (48-h) and pure water (16-h). It is interesting to note that the survival of both wild type and the fur- mutant strain was more protected at 24 °C than at other temperatures. The Fur protein does not have any relation to the entry of E. coli into the viable but nonculturable state (VBNC) under photooxidative stress. This is the first study which shows that fur- mutation increases the resistance of E. coli to photooxidative stress in aquatic environments, and the Fur protein does not have any relation to the entry of E. coli into the VBNC state.

Fur-Mediated Global Regulatory Circuits in Pathogenic Neisseria Species

The ferric uptake regulator (Fur) protein has been shown to function as a repressor of transcription in a number of diverse microorganisms. However, recent studies have established that Fur can function at a global level as both an activator and a repressor of transcription through both direct and indirect mechanisms. Fur-mediated indirect activation occurs via the repression of additional repressor proteins, or small regulatory RNAs, thereby activating transcription of a previously silent gene. Fur mediates direct activation through binding of Fur to the promoter regions of genes. Whereas the repressive mechanism of Fur has been thoroughly investigated, emerging studies on direct and indirect Fur-mediated activation mechanisms have revealed novel global regulatory circuits.

Fur-Regulated Iron Uptake System of Edwardsiella ictaluri and Its Influence on Pathogenesis and Immunogenicity in the Catfish Host

The ability of bacterial pathogens to take up iron from the host during infection is necessary for their multiplication within the host. However, host high-affinity iron binding proteins limit levels of free iron in fluids and tissues. To overcome this deficiency of iron during infection, bacterial pathogens have developed iron uptake systems that are upregulated in the absence of iron, typically tightly controlled by the ferric uptake regulator (Fur) protein. The iron uptake system of Edwardsiella ictaluri, a host-restricted pathogen of channel catfish (Ictalurus punctatus) and the main pathogen of this fish in aquaculture, is unknown. Here we describe the E. ictaluri Fur protein, the iron uptake machinery controlled by Fur, and the effects of fur gene deletion on virulence and immunogenicity in the fish host. Analysis of the E. ictaluri Fur protein shows that it lacks the N-terminal region found in the majority of pathogen-encoded Fur proteins. However, it is fully functional in regulated genes encoding iron uptake proteins. E. ictaluri grown under iron-limited conditions upregulates an outer membrane protein (HemR) that shows heme-hemoglobin transport activity and is tightly regulated by Fur. In vivo studies showed that an E. ictaluri Δfur mutant is attenuated and immune protective in zebrafish (Danio rerio) and catfish (Ictalurus punctatus), triggering systemic immunity. We conclude that an E. ictaluri Δfur mutant could be an effective component of an immersion-oral vaccine for the catfish industry.

Effect of iron limitation and fur gene inactivation on the transcriptional profile of the strict anaerobe Clostridium acetobutylicum

Iron is a nutrient of critical importance for the strict anaerobe Clostridium acetobutylicum, as it is involved in numerous basic cellular functions and metabolic pathways. A gene encoding a putative ferric uptake regulator (Fur) has been identified in the genome of C. acetobutylicum. In this work, we inactivated the fur gene by using insertional mutagenesis. The resultant mutant showed a slow-growing phenotype and enhanced sensitivity to oxidative stress, but essentially no dramatic change in its fermentation pattern. A unique feature of its physiology was the overflowing production of riboflavin. To gain further insights into the role of the Fur protein and the mechanisms for establishment of iron balance in C. acetobutylicum, we characterized and compared the gene-expression profile of the fur mutant and the iron-limitation stimulon of the parental strain. Not surprisingly, a repertoire of iron-transport systems was upregulated in both microarray datasets, suggesting that they are regulated by Fur according to the availability of iron. In addition, iron limitation and inactivation of fur affected the expression of several genes involved in energy metabolism. Among them, two genes, encoding a lactate dehydrogenase and a flavodoxin, were highly induced. In order to support the function of the latter, the ribDBAH operon responsible for riboflavin biosynthesis was also upregulated significantly. Furthermore, the iron-starvation response of C. acetobutylicum involved transcriptional modifications that were not detected in the fur mutant, suggesting that there exist additional mechanisms for adaptation to low-iron environments. Collectively, these results demonstrate that the strict anaerobe C. acetobutylicum senses and responds to availability of iron on multiple levels using a sophisticated system, and that Fur plays an important role in this process.

The FsrA sRNA and FbpB Protein Mediate the Iron-Dependent Induction of the Bacillus subtilis LutABC Iron-Sulfur-Containing Oxidases

The Bacillus subtilis ferric uptake regulator (Fur) protein regulates iron homeostasis and directly represses more than 20 operons. Fur indirectly regulates many more genes, including those controlled by the small, noncoding RNA FsrA. FsrA translationally represses numerous target genes and, for at least some targets, appears to function in conjunction with one or more of three small, basic proteins, known as FbpA, FbpB, and FbpC. The lactate-inducible lutABC operon encodes iron sulfur-containing enzymes required for growth on lactate. We here demonstrate that a fur mutant strain grows poorly on lactate due to FsrA-dependent repression of LutABC synthesis. Growth is restored in an fsrA mutant and also partially restored by mutation of the fbpAB operon. Genetic studies indicate that the 48-amino-acid FbpB protein but not FbpA contributes to regulation of lutABC. FbpB may function, at least in part, by increasing the efficiency of FsrA targeting to the lutABC mRNA, since the role of FbpB can be bypassed by modest upregulation of FsrA. These results provide support for a model in which FbpB, and perhaps other Fbp proteins, contributes along with FsrA to the translational regulation of gene expression.

Non-Heme Iron Sensors of Reactive Oxygen and Nitrogen Species

In bacteria, transcriptional responses to reactive oxygen and nitrogen species (ROS and RNS, respectively) are typically coordinated by regulatory proteins that employ metal centers or reactive thiols to detect the presence of those species. This review is focused on the structure, function and mechanism of three regulatory proteins (Fur, PerR, and NorR) that contain non-heme iron and regulate the transcription of target genes in response to ROS and/or RNS. The targets for regulation include genes encoding detoxification activities, and genes encoding proteins involved in the repair of the damage caused by ROS and RNS. Three-dimensional structures of several Fur proteins and of PerR are revealing important details of the metal binding sites of these proteins, showing a surprising degree of structural diversity in the Fur family. Discussion of the interaction of Fur with ROS and RNS will illustrate the difficulty that sometimes exists in distinguishing between true physiological responses and adventitious reactions of a regulatory protein with a reactive ligand. Consideration of these three sensor proteins illuminates some of the key questions that remain unanswered, for example, the nature of the biochemical determinants that dictate the sensitivity and specificity of the interaction of the sensor proteins with their cognate signals.

A Global Investigation of the Bacillus subtilis Iron-Sparing Response Identifies Major Changes in Metabolism

The Bacillus subtilis ferric uptake regulator (Fur) protein is the major sensor of cellular iron status. When iron is limiting for growth, derepression of the Fur regulon increases the cellular capacity for iron uptake and mobilizes an iron-sparing response mediated in large part by a small noncoding RNA named FsrA. FsrA functions, in collaboration with three small basic proteins (FbpABC), to repress many "low-priority" iron-containing enzymes. We have used transcriptome analyses to gain insights into the scope of the iron-sparing response and to define subsets of genes dependent for their repression on FsrA, FbpAB, and/or FbpC. Enzymes of the tricarboxylic acid (TCA) cycle, including aconitase and succinate dehydrogenase (SDH), are major targets of FsrA-mediated repression, and as a consequence, flux through this pathway is significantly decreased in a fur mutant. FsrA also represses the DctP dicarboxylate permease and the iron-sulfur-containing enzyme glutamate synthase (GltAB), which serves as a central link between carbon and nitrogen metabolism. Allele-specific suppression analysis was used to document a direct RNA-RNA interaction between the FsrA small RNA (sRNA) and the gltAB leader region. We further demonstrated that distinct regions of FsrA are required for the translational repression of the GltAB and SDH enzyme complexes.

Suppression of pleiotropic phenotypes of a Burkholderia multivorans fur mutant by oxyR mutation

Fur (ferric uptake regulator) is an iron-responsive transcriptional regulator in many bacterial species, and the fur mutant of Burkholderia multivorans ATCC 17616 exhibits pleiotropic phenotypes, such as an inability to efficiently use several carbon sources, as well as high sensitivity to hydrogen peroxide (H(2)O(2)), paraquat (a superoxide-producing compound) and nitric oxide (NO). To gain more insight into the pleiotropic role of the Fur protein of ATCC 17616, spontaneous suppressor mutants of the ATCC 17616 fur mutant that restored tolerance to NO were isolated and characterized in this study. The microarray-based comparative genomic analysis and subsequent sequencing analysis indicated that such suppressor mutants had a 2 bp deletion in the oxyR gene, whose orthologues encode H(2)O(2)-responsive transcriptional regulators in other bacterial species. The suppressor mutants and the reconstructed fur-oxyR double-deletion mutant showed indistinguishable phenotypes in that they were all (i) more resistant than the fur mutant to H(2)O(2), superoxide, NO and streptonigrin (an iron-activated antibiotic) and (ii) able to use carbon sources that cannot efficiently support the growth of the fur mutant. These results clearly indicate that the oxyR mutation suppressed the pleiotropic effect of the B. multivorans fur mutant. The fur-oxyR double mutants were found to overexpress the KatG (catalase/peroxidase) and AhpC1 and AhpD (alkyl hydroperoxide reductase subunits C and D) proteins, and their enzymic activities to remove reactive oxygen and nitrogen species were suggested to be responsible for the suppression of phenotypes caused by the fur mutation.

Fur in Magnetospirillum gryphiswaldense influences magnetosomes formation and directly regulates the genes involved in iron and oxygen metabolism.

Magnetospirillum gryphiswaldense strain MSR-1 has the unique capability of taking up large amounts of iron and synthesizing magnetosomes (intracellular magnetic particles composed of Fe3O4). The unusual high iron content of MSR-1 makes it a useful model for studying biological mechanisms of iron uptake and homeostasis. The ferric uptake regulator (Fur) protein plays a key role in maintaining iron homeostasis in many bacteria. We identified and characterized a fur-homologous gene (MGR_1314) in MSR-1. MGR_1314 was able to complement a fur mutant of E. coli in iron-responsive manner in vivo. We constructed a fur mutant strain of MSR-1. In comparison to wild-type MSR-1, the mutant strain had lower magnetosome formation, and was more sensitive to hydrogen peroxide and streptonigrin, indicating higher intracellular free iron content. Quantitative real-time RT-PCR and chromatin immunoprecipitation analyses indicated that Fur protein directly regulates expression of several key genes involved in iron transport and oxygen metabolism, in addition it also functions in magnetosome formation in M. gryphiswaldense.

Arginine 66 Residue of Fur is Required for the Regulatory Function of this Protein in the Acid Adaptation Mechanism of Helicobacter pylori

Helicobacter pylori colonizes the gastric mucosa and must survive the acid pH of that environment. Like other enteric bacterial pathogens, including Salmonella enterica, H. pylori develops an acid tolerance response that is dependent on the function of the transcriptional regulator protein Fur. To explore by site-directed mutagenesis whether two particular amino acid residues in the amino acid sequence of the H. pylori Fur protein, arginine 66 and histidine 99, are involved in the acid response mechanism in this bacterium. Complementation assays in Escherichia coli H1780 (fur null mutant) both with plasmids carrying the H. pylori fur gene bearing substitution mutations R66A or H99A or R66A/H99A and with the H. pylori Fur-R66A mutant were conducted. Wild-type and mutated Fur proteins from H. pylori were assayed by using the fiu::lacZ reporter gene in the E. coli H1780 heterologous system at various pH and iron concentrations. Both bacterial growth and repression of the reporter gene were impaired under acid conditions in E. coli H1780 complemented with pUC19-fur-R66A. Also, in the H. pylori Fur-R66 strain bacterial growth and speA gene expression were impaired under acid conditions. Arginine 66 but not histidine 99 in H. pylori Fur is required for the regulatory function of the Fur protein in the acid adaptation mechanism of the bacterium.

Transcriptional factor Fur from Thermoplasma volcanium binds its own promoter DNA in a divalent cation-dependent manner

Because archaea possess many respiratory enzymes or radical scavengers with catalytic domains that contain iron, the expression of the genes encoding these enzymes might be regulated by iron acquisition. The genome of an archaeon, Thermoplasma volcanium contains a gene that encodes Fur (TVN0292). The fur gene of T. volcanium was amplified by PCR, and cloned into plasmid pET28a. TvFur (T. volcanium Fur protein) was expressed in E. coli cells and then purified. EMSA revealed that TvFur binds to its own promoter DNA. The binding to its own promoter was in an Mn(2+)-, Zn(2+)-, and Ni(2+)-dependent manner. DNase I footprinting analysis revealed that the binding sequence of tvfur promoter was 5'-G TTATTAT G TTTATAT A TTAATTA G-3'. An analysis utilizing oligonucleotides in TvFur-binding sequences revealed that TvFur binds to the TATA-box or regions in the vicinity of the TATA-box in the promoter. These results indicated that TvFur regulates transcription depending on the availability of environmental divalent cations.

Inhibition and dispersal of Agrobacterium tumefaciens biofilms by a small diffusible Pseudomonas aeruginosa exoproduct(s)

Environmental biofilms often contain mixed populations of different species. In these dense communities, competition between biofilm residents for limited nutrients such as iron can be fierce, leading to the evolution of competitive factors that affect the ability of competitors to grow or form biofilms. We have discovered a compound(s) present in the conditioned culture fluids of Pseudomonas aeruginosa that disperses and inhibits the formation of biofilms produced by the facultative plant pathogen Agrobacterium tumefaciens. The inhibitory activity is strongly induced when P. aeruginosa is cultivated in iron-limited conditions, but it does not function through iron sequestration. In addition, the production of the biofilm inhibitory activity is not regulated by the global iron regulatory protein Fur, the iron-responsive extracytoplasmic function σ factor PvdS, or three of the recognized P. aeruginosa quorum-sensing systems. In addition, the compound(s) responsible for the inhibition and dispersal of A. tumefaciens biofilm formation is likely distinct from the recently identified P. aeruginosa dispersal factor, cis-2-decenoic acid (CDA), as dialysis of the culture fluids showed that the inhibitory compound was larger than CDA and culture fluids that dispersed and inhibited biofilm formation by A. tumefaciens had no effect on biofilm formation by P. aeruginosa.

The Neisseria meningitidis ZnuD Zinc Receptor Contributes to Interactions with Epithelial Cells and Supports Heme Utilization when Expressed in Escherichia coli

Neisseria meningitidis employs redundant heme acquisition mechanisms, including TonB receptor-dependent and receptor-independent uptakes. The TonB-dependent zinc receptor ZnuD shares significant sequence similarity to HumA, a heme receptor of Moraxella catarrhalis, and contains conserved motifs found in many heme utilization proteins. We present data showing that, when expressed in Escherichia coli, ZnuD allowed heme capture on the cell surface and supported the heme-dependent growth of an E. coli hemA strain. Heme agarose captured ZnuD in enriched outer membrane fractions, and this binding was inhibited by excess free heme, supporting ZnuD's specific interaction with heme. However, no heme utilization defect was detected in the meningococcal znuD mutant, likely due to unknown redundant TonB-independent heme uptake mechanisms. Meningococcal replication within epithelial cells requires a functional TonB, and we found that both the znuD and tonB mutants were defective not only in survival within epithelial cells but also in adherence to and invasion of epithelial cells. Ectopic complementation rescued these phenotypes. Interestingly, while znuD expression was repressed by Zur with zinc as a cofactor, it also was induced by iron in a Zur-independent manner. A specific interaction of meningococcal Fur protein with the znuD promoter was demonstrated by electrophoretic mobility shift assay (EMSA). Thus, the meningococcal ZnuD receptor likely participates in both zinc and heme acquisition, is regulated by both Zur and Fur, and is important for meningococcal interaction with epithelial cells.

Expression of fur and its antisense α-fur from Microcystis aeruginosa PCC7806 as response to light and oxidative stress

Ferric uptake regulation (Fur) proteins are prokaryotic transcriptional regulators that integrate signaling of iron metabolism and oxidative stress responses with several environmental stresses. In photosynthetic organisms, Fur proteins regulate many genes involved in photosynthesis, nitrogen metabolism and other key processes. Also, Fur triggers the expression of virulence factors in many bacterial pathogens, and Fur from Microcystis aeruginosa has been shown to bind promoter regions of the microcystin synthesis gene cluster. In this work, we studied transcriptional responses of fur genes under different light intensities and oxidative stress. An antisense of fur, the α-fur RNA, plays an important role in regulating fur expression under oxidative stress, affecting levels of Fur protein in cells. Importantly, an active photosynthetic electron chain is required for the expression of the fur gene.

Identification of three novel antisense RNAs in the fur locus from unicellular cyanobacteria

The interplay between Fur (ferric uptake regulator) proteins and small, non-coding RNAs has been described as a key regulatory loop in several bacteria. In the filamentous cyanobacterium Anabaena sp. PCC 7120, a large dicistronic transcript encoding the putative membrane protein Alr1690 and an α-furA RNA is involved in the modulation of the global regulator FurA. In this work we report the existence of three novel antisense RNAs in cyanobacteria and show that a cis α-furA RNA is conserved in very different genomic contexts, namely in the unicellular cyanobacteria Microcystis aeruginosa PCC 7806 and Synechocystis sp. PCC 6803. Syα-fur RNA covers only part of the coding sequence of the fur orthologue sll0567, whose flanking genes encode two hypothetical proteins. Transcriptional analysis of fur and its adjacent genes in Microcystis unravels a highly compact organization of this locus involving overlapping transcripts. Maα-fur RNA spans the whole Mafur CDS and part of the flanking dnaJ and sufE sequences. In addition, Mafur seems to be part of a dicistronic operon encoding this regulator and an α-sufE RNA. These results allow new insights into the transcriptomes of two unicellular cyanobacteria and suggest that in M. aeruginosa PCC 7806, the α-fur and α-sufE RNAs might participate in a regulatory connection between the genes of the dnaJ-fur-sufE locus.

Bacillus subtilis Fur represses one of two paralogous haem-degrading monooxygenases

Identification of genes regulated by the ferric uptake regulator (Fur) protein has provided insights into the diverse mechanisms of adaptation to iron limitation. In the soil bacterium Bacillus subtilis, Fur senses iron sufficiency and represses genes that enable iron uptake, including biosynthetic and transport genes for the siderophore bacillibactin and uptake systems for siderophores produced by other organisms. We here demonstrate that Fur regulates hmoA (formerly yetG), which encodes a haem monooxygenase. HmoA is the first characterized member of a divergent group of putative monooxygenases that cluster separately from the well-characterized IsdG family. B. subtilis also encodes an IsdG family protein designated HmoB (formerly YhgC). Unlike hmoA, hmoB is constitutively expressed and not under Fur control. HmoA and HmoB both bind haemin in vitro with approximately 1 : 1 stoichiometry and degrade haemin in the presence of an electron donor. Mutational and spectroscopic analyses indicate that HmoA and HmoB have distinct active site architectures and interact differently with haem. We further show that B. subtilis can use haem as an iron source, but that this ability is independent of HmoA and HmoB.

Characterization of the Fur Regulon in Pseudomonas syringae pv. tomato DC3000

The plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) is found in a wide variety of environments and must monitor and respond to various environmental signals such as the availability of iron, an essential element for bacterial growth. An important regulator of iron homeostasis is Fur (ferric uptake regulator), and here we present the first study of the Fur regulon in DC3000. Using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq), 312 chromosomal regions were highly enriched by coimmunoprecipitation with a C-terminally tagged Fur protein. Integration of these data with previous microarray and global transcriptome analyses allowed us to expand the putative DC3000 Fur regulon to include genes both repressed and activated in the presence of bioavailable iron. Using nonradioactive DNase I footprinting, we confirmed Fur binding in 41 regions, including upstream of 11 iron-repressed genes and the iron-activated genes encoding two bacterioferritins (PSPTO_0653 and PSPTO_4160), a ParA protein (PSPTO_0855), and a two-component system (TCS) (PSPTO_3382 to PSPTO_3380).

Helicobacter pylori Exhibits a Fur-Dependent Acid Tolerance Response

Helicobacter pylori colonizes the acid environment of the gastric mucosa. Like other enteric bacterial pathogens, including Salmonella enterica, which must survive a brief exposure to that environment, H. pylori displays a rapid response to subtle changes in pH, which confers an increased ability to survive at more extreme acidic pH. This two-step acid tolerance response (ATR) requires de novo protein synthesis and is dependent on the function of the global regulatory protein Fur. We have explored the physiological bases of the ATR in H. pylori. Proteomic analysis of phenotypes of H. pylori and fur mutant strains show that subtle pH changes elicit significant changes in the pattern of proteins synthesized. A loss-of-function mutation in the fur gene, obtained by insertion of an antibiotic resistance cassette, indicated that Fur regulates the expression of a fraction of H. pylori proteins. A subset of proteins is involved in the ATR and confer a negative ATR phenotype.