Fur Homologue Research Articles

Identifying regulators of transcription in an obligate intracellular pathogen: a metal-dependent repressor in Chlamydia trachomatis.

A prominent feature exhibited by Chlamydia trachomatis growing in an iron-limiting environment is a differential pattern of protein expression. In many bacteria, iron-responsive proteins are regulated at the level of transcription by a family of repressors resembling the Escherichia coli ferric uptake regulator (Fur) protein. Although the chlamydial genome sequencing project did not unveil an obvious Fur homologue, a detailed examination indicated five unassigned open reading frames (ORFs) that would encode products with limited sequence homology to Fur. In this report, each chlamydial ORF was engineered in E. coli, and recombinant proteins were examined for functional characteristics resembling Fur. A Fur-specific polyclonal antiserum revealed that the protein encoded by ORF CT296 shares antigenic cross-recognition. Moreover, this protein forms dimers in solution in a fashion analogous to E. coli Fur. Further studies confirmed that the product of ORF CT296 is able to (i) complement Fur activity in a mutant strain of E. coli; and (ii) specifically bind to a 19 bp consensus sequence found in promoters of iron-regulated genes in E. coli. We propose a designation of dcrA (divalent cation-dependent regulator A) for ORF CT296, which encodes a protein distantly related to E. coli Fur. DcrA represents the first repressor described for this obligate intracellular bacterium.

Regulation of ferritin-mediated cytoplasmic iron storage by the ferric uptake regulator homolog (Fur) of Helicobacter pylori.

Homologs of the ferric uptake regulator Fur and the iron storage protein ferritin play a central role in maintaining iron homeostasis in bacteria. The gastric pathogen Helicobacter pylori contains an iron-induced prokaryotic ferritin (Pfr) which has been shown to be involved in protection against metal toxicity and a Fur homolog which has not been functionally characterized in H. pylori. Analysis of an isogenic fur-negative mutant revealed that H. pylori Fur is required for metal-dependent regulation of ferritin. Iron starvation, as well as medium supplementation with nickel, zinc, copper, and manganese at nontoxic concentrations, repressed synthesis of ferritin in the wild-type strain but not in the H. pylori fur mutant. Fur-mediated regulation of ferritin synthesis occurs at the mRNA level. With respect to the regulation of ferritin expression, Fur behaves like a global metal-dependent repressor which is activated under iron-restricted conditions but also responds to different metals. Downregulation of ferritin expression by Fur might secure the availability of free iron in the cytoplasm, especially if iron is scarce or titrated out by other metals.

Molecular characterization of the ferric-uptake regulator, fur, from Staphylococcus aureus.

Iron is an essential nutrient for the survival and pathogenesis of bacteria, but relatively little is known regarding its transport and regulation in staphylococci. Based on the known sequences of ferric-uptake regulatory (fur) genes from several Gram-positive and Gram-negative bacteria, a fragment containing the fur homologue was cloned from a genomic library of Staphylococcus aureus RN450. Nucleotide sequence analysis of this fragment revealed the presence of a 447 bp ORF that encodes a putative 149 aa polypeptide with an apparent molecular mass of 17 kDa. A putative ferrichrome-uptake (fhu) operon, containing the conserved Fur-binding sequences (Fur box) in the promoter region, was also cloned from the same S. aureus library. To characterize the impact of Fur on the fhu operon, fur was cloned, overexpressed as a His-tagged protein and purified by Ni2+-affinity column chromatography. The recombinant protein was digested with enterokinase to remove the His tag. Electrophoretic mobility-shift assays indicated that Fur binds to the promoter region of the fhu operon in the presence of divalent cations. Fur also interacted with the promoter region of the recently reported sir operon that has been proposed to constitute a siderophore-transport system in S. aureus. The DNase I-protection assay revealed that Fur specifically binds to the Fur box located in the promoter region of the fhu operon. The primer-extension reaction indicated that the transcription-start site of the fhu operon was located inside the Fur box. S. aureus fur partially complemented a fur- mutation in Bacillus subtilis. The data suggest that Fur regulates iron-transport processes in S. aureus.

Campylobacter jejuni contains two fur homologs: characterization of iron-responsive regulation of peroxide stress defense genes by the PerR repressor.

Expression of the peroxide stress genes alkyl hydroperoxide reductase (ahpC) and catalase (katA) of the microaerophile Campylobacter jejuni is repressed by iron. Whereas iron repression in gram-negative bacteria is usually carried out by the Fur protein, previous work showed that this is not the case in C. jejuni, as these genes are still iron repressed in a C. jejuni fur mutant. An open reading frame encoding a Fur homolog (designated PerR for "peroxide stress regulator") was identified in the genome sequence of C. jejuni. The perR gene was disrupted by a kanamycin resistance cassette in C. jejuni wild-type and fur mutant strains. Subsequent characterization of the C. jejuni perR mutants showed derepressed expression of both AhpC and KatA at a much higher level than that obtained by iron limitation, suggesting that expression of these genes is controlled by other regulatory factors in addition to the iron level. Other iron-regulated proteins were not affected by the perR mutation. The fur perR double mutant showed derepressed expression of known iron-repressed genes. Further phenotypic analysis of the perR mutant, fur mutant, and the fur perR double mutant showed that the perR mutation made C. jejuni hyperresistant to peroxide stress caused by hydrogen peroxide and cumene hydroperoxide, a finding consistent with the high levels of KatA and AhpC expression, and showed that these enzymes were functional. Quantitative analysis of KatA expression showed that both the perR mutation and the fur mutation had profound effects on catalase activity, suggesting additional non-iron-dependent regulation of KatA and, by inference, AhpC. The PerR protein is a functional but nonhomologous substitution for the OxyR protein, which regulates peroxide stress genes in other gram-negative bacteria. Regulation of peroxide stress genes by a Fur homolog has recently been described for the gram-positive bacterium Bacillus subtilis. C. jejuni is the first gram-negative bacterium where non-OxyR regulation of peroxide stress genes has been described and characterized.

The ferric uptake regulator (Fur) homologue of Helicobacter pylori: functional analysis of the coding gene and controlled production of the recombinant protein in Escherichia coli.

A homologue of the ferric uptake regulator protein Fur has recently been identified within the Helicobacter pylori genome. The promoterless gene on a plasmid did partially complement a fur-negative mutant of Escherichia coli, and was strongly positive in the Fur titration assay (FURTA). The genetic and functional characterization of the complete fur homologue performed in this study revealed that the gene is conserved among H. pylori strains ( > 95% identity), and does not carry nucleotide transitions in iron-resistant mutants of H. pylori. The fur homologue on a plasmid mediated full iron-dependent ferric uptake regulator activity in the fur-deficient mutant strains H1681 and H1780 of E. coli. Immunoblot analysis revealed that Fur from H. pylori cross-reacts with antibodies raised against Fur from E. coli. The fact that inactivation of the fur gene abolished the FURTA-positive phenotype in the E. coli indicator strain H1717, indicated that this phenotype is rather caused by the encoded protein than by real Fur titration. Subcloning of the fur gene into an expression vector allowed controlled production in E. coli, and purification of a recombinant version of the H. pylori Fur protein. In summary, the results confirm the function of the H. pylori Fur homologue as iron-dependent transcriptional repressor by its ability to interact with the Fur-regulated promoters of the genes fiu and fhuF in E. coli.

Interaction of Bacillus subtilis Fur (ferric uptake repressor) with the dhb operator in vitro and in vivo.

Bacillus subtilis contains three metalloregulatory proteins belonging to the ferric uptake repressor (Fur) family: Fur, Zur, and PerR. We have overproduced and purified Fur protein and analyzed its interaction with the operator region controlling the expression of the dihydroxybenzoate siderophore biosynthesis (dhb) operon. The purified protein binds with high affinity and selectivity to the dhb regulatory region. DNA binding does not require added iron, nor is binding reduced by dialysis of Fur against EDTA or treatment with Chelex. Fur selectively inhibits transcription from the dhb promoter by sigmaA RNA polymerase, even if Fur is added after RNA polymerase holoenzyme. Since neither DNA binding nor inhibition of transcription requires the addition of ferrous ion in vitro, the mechanism by which iron regulates Fur function in vivo is not obvious. Mutagenesis of the fur gene reveals that in vivo repression of the dhb operon by iron requires His97, a residue thought to be involved in iron sensing in other Fur homologs. Moreover, we identify His96 as a second likely iron ligand, since a His96Ala mutant mediates repression at 50 microM but not at 5 microM iron. Our data lead us to suggest that Fur is able to bind DNA independently of bound iron and that the in vivo role of iron is to counteract the effect of an inhibitory factor, perhaps another metal ion, that antagonizes this DNA-binding activity.

Fur and iron transport proteins in the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius.

The Brazilian purpuric fever (BPF) clone of Haemophilus influenzae biogroup aegyptius causes a fatal septicaemic disease, resembling fulminant meningococcal sepsis, in children. When isolate F3031 was grown under iron-limiting conditions, the presence of several iron-regulated proteins of 38-110 kDa was revealed by electrophoretic analysis and a Fur homologue was shown by immunoblotting. Dot-blot assays and immunoblotting indicated that BPF cells bound human transferrin and contained transferrin-binding proteins in the outer membrane. However, the binding activity and the biosynthesis of these proteins were detected even under iron-rich conditions. Immunoblot analysis demonstrated the presence of a periplasmic protein related to the ferric iron-binding protein A (FbpA), the major iron-binding protein described in Neisseria spp. However, the FbpA homologue in strain F3031 was constitutively expressed and was smaller than the periplasmic protein detected in H. influenzae type b strain Eagan. The periplasm of strain F3031 also contained a protein related to the Streptococcus parasanguis FimA protein which recently has been shown to be involved in iron acquisition in Yersinia pestis. Although the Eagan and F3031 FimA homologues had a similar mol. wt, of 31 kDa, the expression of the BPF fimA-like gene was not regulated by the iron concentration of the culture medium.

The ferric uptake regulation (Fur) repressor is a zinc metalloprotein.

The Fur protein regulates the expression of a wide variety of iron-responsive genes; however, the interaction of this repressor with its cognate metal ion remains controversial. The iron-bound form of Fur has proved difficult to obtain, and conflicting results have been published using Mn(II) as a probe for in vitro DNA-binding studies. We report here that the purified protein contains tightly bound zinc and propose that Zn(II) is bound to the protein in vivo. Upon purification, Fur retains ca. 2.1 mol of Zn(II)/mol of Fur monomer (Zn2Fur). One zinc is easily removed by treatment of Zn2Fur with zinc chelating agents, resulting in Zn1Fur with ca. 0.9 mol of Zn(II)/mol of protein. The remaining zinc in Zn1Fur can only be removed under denaturing conditions to yield apo-Fur with ca. 0.1 mol of Zn(II)/mol of protein. Our results suggest that many literature descriptions of purified Fur protein do not correspond to the apo-protein, but to Zn1Fur or Zn2Fur. Dissociation constants (Kd) of protein-DNA complexes are ca. 20 nM for both Zn2Fur and Zn1Fur as determined by electrophoretic mobility shift assays and DNase I footprinting assays. The two metalated forms, however, show qualitative differences in the footprinting assays while apo-Fur does not bind specifically to the operator. The existence of these Zn(II) binding sites in Fur may resolve some discrepancies in the literature and have implications concerning Zur, a Fur homologue in E. coli that regulates zinc-responsive genes.

Characterization of the Acinetobacter baumannii Fur regulator: cloning and sequencing of the fur homolog gene

Growth kinetics, siderophore activity and iron-regulated bacterial proteins of Acinetobacter baumannii BM2580 were studied in iron-restricted and iron-supplemented chemically defined media. Iron-regulated outer membrane proteins of 75 kDa and 80 kDa were expressed under iron-restricted conditions. Cloning and sequencing of the complete iron-uptake regulatory ( fur) gene from A. baumannii BM2580 is reported for the first time. This gene is preceded by a single autoregulated promoter whose −10 region overlaps the Fur binding site. The open reading frame identified encodes a polypeptide consisting of 145 amino acids. The fur gene is followed by a divergent open reading frame coding for the C-terminus of a putative PilU protein. Sequence analysis indicates that the Fur protein of A. baumannii was 63% identical to the Escherichia coli Fur protein.

Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors.

Fur (ferric uptake regulator) proteins control iron uptake in many Gram-negative bacteria. Although Fur homologues have been identified in Gram-positive bacteria, their roles in gene regulation are unknown. Genome sequencing has revealed three fur homologues in Bacillus subtilis: yqkL, yqfV and ygaG. We demonstrate that yqkL encodes an iron uptake repressor: both siderophore biosynthesis and transcription of ferri-siderophore uptake genes is constitutive in the yqkL mutant. Thus, yqkL encodes a repressor that is functionally as well as structurally related to Fur. B. subtilis peroxide stress genes are induced by either H2O2 or by metal ion limitation. Previous genetic studies defined a regulatory locus, perR, postulated to encode the peroxide regulon repressor. We demonstrate that a ygaG mutant has the perR phenotype: It is highly resistant to peroxides and overexpresses catalase, alkyl hydroperoxide reductase and the DNA binding protein MrgA. Nine spontaneous perR mutations, isolated by virtue of their ability to derepress mrgA transcription in the presence of managanous ion, all contain sequence changes in the ygaG locus and can be complemented by the cloned ygaG gene. Thus, ygaG encodes the peroxide regulon repressor and is allelic with perR.

Cloning and characterization of the fur gene from Helicobacter pylori

The fur homologue of Helicobacter pylori was isolated by screening a plasmid-based, genomic DNA library using the Fur titration assay (FURTA). The analysis of the DNA sequence revealed significant homology with Fur proteins from various other bacterial species. The highest degree of homology was observed for the Fur protein from Campylobacter jejuni. The H. pylori fur gene on a plasmid could partially complement the fur mutation in Escherichia coli strain H1681. The repressor activity depended on addition of iron to the medium indicating that iron acts as a co-repressor for the H. pylori protein similar to Fur from other bacteria. Comparison of Fur from H. pylori strain NCTC11638 with the recently published genomic DNA sequence of another strain (26695) confirmed the identity of the fur homologue and revealed that the fur locus is highly conserved in both strains.

Cloning and characterization of the fur gene from Helicobacter pylori.

The fur homologue of Helicobacter pylori was isolated by screening a plasmid-based, genomic DNA library using the Fur titration assay (FURTA). The analysis of the DNA sequence revealed significant homology with Fur proteins from various other bacterial species. The highest degree of homology was observed for the Fur protein from Campylobacter jejuni. The H. pylori fur gene on a plasmid could partially complement the fur mutation in Escherichia coli strain H1681. The repressor activity depended on addition of iron to the medium indicating that iron acts as a co-repressor for the H. pylori protein similar to Fur from other bacteria. Comparison of Fur from H. pylori strain NCTC11638 with the recently published genomic DNA sequence of another strain (26695) confirmed the identity of the fur homologue and revealed that the fur locus is highly conserved in both strains.

Cloning and sequencing of a Haemophilus ducreyi fur homolog

Iron regulation in a growing number of bacterial species is being attributed to the presence of a fur (ferric uptake regulation) regulatory system. In the presence of iron, Fur acts as a classical negative regulator, binding conserved sequences within the promoter of iron-repressible genes and blocking transcription. Western blot analysis utilizing Escherichia coli Fur antisera detected a band of approximately 17 kDa in soluble extracts of Haemophilus ducreyi. Additionally, Southern blot hybridization of the H. ducreyi chromosome with a meningococcal fur probe indicated that H. ducreyi might contain a fur homolog. This putative fur homolog was cloned into the E. coli vector pACYC184. This clone was capable of repressing expression of a normally Fur-regulated lacZ fusion in the fur-background of E. coli strain H1780. The deduced amino acid sequence shows H. ducreyi fur to be 54% identical and 73% similar to E. coli fur, containing putative DNA-binding and metal-binding domains. These data demonstrate that H. ducreyi has a functional fur system.

Characterization of Campylobacter upsaliensis fur and its localization in a highly conserved region of the Campylobacter genome

Despite increasing recognition of the importance of Campylobacter upsaliensis in human disease little is known about either the virulence properties or genetics of this enteric pathogen. The complete coding sequence of a C. upsaliensis gene has yet to be published. We have cloned and sequenced the complete iron-uptake regulatory ( fur) gene from the type strain of this species. The C. upsaliensis fur homolog was isolated from a genomic library of C. upsaliensis ATCC 43954 constructed in phage lambdaGEM-11. The open reading frame identified encodes a polypeptide consisting of 156 amino acids. The 5′-flanking region of the C. upsaliensis fur gene contains 3 putative Fur-binding sequences and two catabolite activator-binding sequences indicating the potential for autogenous and cAMP-mediated regulation, respectively. Primer extension analysis identified a single transcription start site 262 nt upstream from the AUG initiation codon. Sequence analysis indicates that the Fur protein of C. upsaliensis is highly homologous (87% amino acid identity) to Campylobacter jejuni Fur. Furthermore, the arrangement of the lysS and glyA genes downstream of fur is precisely conserved in both C. upsaliensis ATCC 43954 and C. jejuni TGH9011. Using the polymerase chain reaction close linkage of fur with lysS and glyA was also observed among multiple isolates of C. upsaliensis, C. jejuni and C. coli suggesting a possible functional relevance for this conserved genetic arrangement in campylobacteria.

Iron-responsive genetic regulation in Campylobacter jejuni: cloning and characterization of a fur homolog.

The Fur protein of Escherichia coli represses transcription from Fur-responsive genes in an iron-dependent manner. We have demonstrated a Fur-like iron-responsive genetic regulatory activity operating in Campylobacter jejuni by using a chloramphenicol acetyl transferase reporter gene separated from its promoter by a synthetic Fur-responsive operator. A fur-like gene has been cloned from C. jejuni by partial functional complementation of an E. coli fur mutation. Sequence analysis has shown that, at the amino acid level, the C. jejuni Fur protein is 35% identical with its E. coli counterpart.

Identification and cloning of a fur homologue from Neisseria meningitidis

The iron response in a number of bacterial systems is mediated by fur (ferric uptake regulation)-like regulatory systems. We have cloned and characterized a gene from Neisseria meningitidis that was homologous to Escherichia coli fur. This clone was capable of modulating expression from both E. coli and neisserial iron-regulated promoters in response to iron, and it produced a protein that reacted with anti-E. coli fur serum. Although the DNA and predicted amino acid sequences were very similar to those of four other published fur homologues, meningococcal fur was the most divergent of the group. Inability to construct a meningococcal fur mutant suggested that fur may be essential in this species.

Identification and cloning of a fur homolog from Neisseria gonorrhoeae.

The promoter region of the major iron-regulated protein of Neisseria gonorrhoeae, Fbp, has two regions that exhibit homology with the Escherichia coli consensus Fur-binding sequences. Gel retardation assays suggested that purified E. coli Fur bound to two sites within the Fbp promoter. The presence of a gonococcal Fur homolog was suggested by Southern hybridization under conditions of low stringency, which revealed a DNA locus that exhibited homology to the E. coli fur gene. Oligonucleotides derived from the conserved regions of fur genes of extremely diverse bacteria were used to amplify a 140-bp fragment of a putative gonococcal fur gene. This fragment was used to identify clones containing the entire gonococcal fur gene. After sequencing the gonococcal fur gene and its promoter region, we found that gonococcal Fur exhibited 50% identity with E. coli Fur at the amino acid level; however, it complemented two E. coli Fur- mutants. The presence of a Fur homolog in N. gonorrhoeae suggests that Fur-regulated genes are widely distributed among extremely diverse bacteria.

Regulation of toxA and regA by the Escherichia coli fur gene and identification of a Fur homologue in Pseudomonas aeruginosa PA103 and PA01

A multicopy plasmid containing the Escherichia coli fur gene was introduced into Pseudomonas aeruginosa strain PA103C. This strain contains a toxA-lacZ fusion integrated into its chromosome at the toxA locus. Beta-galactosidase synthesis in this strain is regulated by iron, as is seen for exotoxin A production. Beta-galactosidase synthesis and exotoxin A production in PA103C containing multiple copies of E. coli fur was still repressed in low iron conditions. The transcription of regA, a positive regulator of toxA, was also found to be inhibited by multiple copies of the E. coli fur gene. In addition, the ability of PA103C containing multiple copies of E. coli fur to produce protease was greatly reduced relative to PA103C containing a vector control. A polyclonal rabbit serum containing antibodies that recognize E. coli Fur was used to screen whole-cell extracts from Vibrio cholerae, Shigella flexneri, Salmonella typhimurium and Pseudomonas aeruginosa. All strains tested expressed a protein that was specifically recognized by the anti-Fur serum. These results and those described above suggest that Fur structure and function are conserved in a variety of distinct bacterial genera and that at least some of these different genera use this regulatory protein to control genes encoding virulence factors.