Functional specialization within the Fur family of metalloregulators

The ferric uptake regulator (Fur) protein, as originally described in Escherichia coli, is an iron-sensing repressor that controls the expression of genes for siderophore biosynthesis and iron transport. Although Fur is commonly thought of as a metal-dependent repressor, Fur also activates the expression of many genes by either indirect or direct mechanisms. In the best studied model systems, Fur functions as a global regulator of iron homeostasis controlling both the induction of iron uptake functions (under iron limitation) and the expression of iron storage proteins and iron-utilizing enzymes (under iron sufficiency). We now appreciate that there is a tremendous diversity in metal selectivity and biological function within the Fur family which includes sensors of iron (Fur), zinc (Zur), manganese (Mur), and nickel (Nur). Despite numerous studies, the mechanism of metal ion sensing by Fur family proteins is still controversial. Other family members use metal catalyzed oxidation reactions to sense peroxide-stress (PerR) or the availability of heme (Irr).     

Predicting regulons and their cis-regulatory motifs by comparative genomics

We have combined and compared three techniques for predicting functional interactions based on comparative genomics (methods based on conserved operons, protein fusions and correlated evolution) and optimized these methods to predict coregulated sets of genes in 24 complete genomes, including Saccharomyces cerevisiae, Caernorhabditis elegans and 22 prokaryotes. The method based on conserved operons was the most useful for this purpose. Upstream regions of the genes comprising these predicted regulons were then used to search for regulatory motifs in 22 prokaryotic genomes using the motif-discovery program AlignACE. Many significant upstream motifs, including five known Escherichia coli regulatory motifs, were identified in this manner. The presence of a significant regulatory motif was used to refine the members of the predicted regulons to generate a final set of predicted regulons that share significant regulatory elements.     

Prediction and experimental testing of ferric uptake regulator regulons in vibrios

Iron homeostasis is in many bacteria regulated by the ferric uptake regulator (Fur). Despite the available information on Fur regulons, it is likely that there are Fur-regulated genes and operons that are unique to vibrios, and knowledge into these can potentially provide new insights into vibrio virulence and pathogenesis. We constructed a vibrio-specific alignment matrix based on Fur-binding sites from the literature and used existing software (Patser) to search five published vibrio genomes and the Vibrio salmonicida draft genome for Fur-regulated genes. The consensus Fur-binding site from our matrix is 5'-AATGANAATNATTNTCATT-3'. Fur-binding motifs were found associated with 50-61 single genes and 16-20 operons in each genome. Predictions were tested by monitoring the expression of a subset of genes and operons in V. salmonicida. Six previously undescribed Fur-regulated genes showed increased expression under iron-restrictive conditions. Our work provides a comprehensive list of predicted Fur regulons in six vibrio genomes, which may be used to generate new hypotheses for future experiments.     

Crystal structure of the Vibrio cholerae ferric uptake regulator (Fur) reveals insights into metal co-ordination

The ferric uptake regulator (Fur) is a metal-dependent DNA-binding protein that acts as both a repressor and an activator of numerous genes involved in maintaining iron homeostasis in bacteria. It has also been demonstrated in Vibrio cholerae that Fur plays an additional role in pathogenesis, opening up the potential of Fur as a drug target for cholera. Here we present the crystal structure of V. cholerae Fur that reveals a very different orientation of the DNA-binding domains compared with that observed in Pseudomonas aeruginosa Fur . Each monomer of the dimeric Fur protein contains two metal binding sites occupied by zinc in the crystal structure. In the P. aeruginosa study these were designated as the regulatory site (Zn1) and structural site (Zn2). This V. cholerae Fur study, together with studies on Fur homologues and paralogues, suggests that in fact the Zn2 site is the regulatory iron binding site and the Zn1 site plays an auxiliary role. There is no evidence of metal binding to the cysteines that are conserved in many Fur homologues, including Escherichia coli Fur. An analysis of the metal binding properties shows that V. cholerae Fur can be activated by a range of divalent metals.     

Isolation and analysis of a fur mutant of Neisseria gonorrhoeae.

The pathogenic Neisseria spp. produce a number of iron-regulated gene products that are thought to be important in virulence. Iron-responsive regulation of these gene products has been attributed to the presence in Neisseria spp. of the Fur (ferric uptake regulation) protein. Evidence for the role of Fur in neisserial iron regulation has been indirect because of the inability to make fur null mutations. To circumvent this problem, we used manganese selection to isolate missense mutations of Neisseria gonorrhoeae fur. We show that a mutation in gonococcal fur resulted in reduced modulation of expression of four well-studied iron-repressed genes and affected the iron regulation of a broad range of other genes as judged by two-dimensional polyacrylamide gel electrophoresis (PAGE). All 15 of the iron-repressed spots observed by two-dimensional PAGE were at least partially derepressed in the fur mutant, and 17 of the 45 iron-induced spots were affected by the fur mutation. Thus, Fur plays a central role in regulation of iron-repressed gonococcal genes and appears to be involved in regulation of many iron-induced genes. The size and complexity of the iron regulons in N. gonorrhoeae are much greater than previously recognized.     

Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus

In most bacteria, the ferric uptake regulator (Fur) is a global regulator that controls iron homeostasis and other cellular processes, such as oxidative stress defense. In this work, we apply a combination of bioinformatics, in vitro and in vivo assays to identify the Caulobacter crescentus Fur regulon. A C. crescentus fur deletion mutant showed a slow growth phenotype, and was hypersensitive to H₂O₂ and organic peroxide. Using a position weight matrix approach, several predicted Fur-binding sites were detected in the genome of C. crescentus, located in regulatory regions of genes not only involved in iron uptake and usage but also in other functions. Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis. Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency. Furthermore, several genes that are regulated via small RNAs in other bacteria were found to be directly regulated by Fur in C. crescentus. In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.     

Fur from Microcystis aeruginosa binds in vitro promoter regions of the microcystin biosynthesis gene cluster

Promoter regions of the mcy operon from Microcystis aeruginosa PCC7806, which is responsible for microcystin synthesis in this organism, exhibit sequences that are similar to the sequences recognized by Fur (ferric uptake regulator). This DNA-binding protein is a sensor of iron availability and oxidative stress. In the presence of Fe(2+), a dimer of Fur binds the iron-boxes in their target genes, repressing their expression. When iron is absent the expression of those gene products is allowed. Here, we show that Fur from M. aeruginosa binds in vitro promoter regions of several mcy genes, which suggests that Fur might regulate, among other factors, microcystin synthesis. The binding affinity is increased by the presence of metal and DTT, suggesting a response to iron availability and redox status of the cell.