Vibrio cholerae fur mutations associated with loss of repressor activity: implications for the structural-functional relationships of fur.

We used the Vibrio cholerae Fur protein as a model of iron-sensitive repressor proteins in gram-negative bacteria. Utilizing manganese mutagenesis, we isolated twelve independent mutations in V. cholerae fur that resulted in partial or complete loss of Fur repressor function. The mutant fur genes were recovered by PCR and sequenced; 11 of the 12 contained point mutations (two of which were identical), and one contained a 7-bp insertion that resulted in premature truncation of Fur. All of the mutants, except that containing the prematurely truncated Fur, produced protein by Western blot (immunoblot) analysis, although several had substantially smaller amounts of Fur and two made an immunoreactive protein that migrated more rapidly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nine of the 11 point mutations altered amino acids that are identical in all of the fur genes sequenced so far, suggesting that these amino acids may play important structural or functional roles in Fur activity. Eight of the point mutations occurred in the amino-terminal half of Fur, which is thought to mediate DNA binding; most of these mutations occurred in conserved amino acids that have been previously suggested to play a role in the interaction between adjacent alpha-helices of the protein. Three of the point mutations occurred in the carboxy-terminal half of Fur, which is thought to bind iron. One mutation at histidine-90 was associated with complete loss of Fur function; this amino acid is within a motif previously suggested as being involved in iron binding by Fur. The fur allele mutant at histidine-90 interfered with iron regulation by wild-type fur in the same cell when the mutant allele was present at higher copy number; wild-type fur was dominant over all other fur mutant alleles studied. These results are analyzed with respect to previous models of the structure and function of Fur as an iron-sensitive repressor.     

Characterization of Vibrio parahaemolyticus manganese-resistant mutants in reference to the function of the ferric uptake regulatory protein

In many bacteria, the ferric uptake regulatory protein (Fur) has a central role in the negative regulation of genes affected by iron limitation. In this study, Vibrio parahaemolyticus strains carrying mutations in the fur gene encoding Fur were isolated by the manganese selection method to assess the function of Fur in connection with alternations in the coordinate expression of the siderophore vibrioferrin (VF) and iron-repressible outer membrane proteins (IROMPs). Ten out of 25 manganese-resistant mutants constitutively produced VF and expressed at least two IROMPs irrespective of the iron concentration in the medium. PCR-direct DNA sequencing of the fur genes in these mutants identified four different point mutations causing amino acid changes. Moreover, a fur overexpressing plasmid was constructed to prepare antiserum against V. parahaemolyticus Fur. Western blotting with this antiserum revealed that the intracellular abundance of the wild-type Fur was not significantly affected by the iron concentrations in the growth medium, and that the Fur proteins of the mutant strains occurred at substantially smaller amounts and/or migrated more rapidly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the wild-type Fur. These data afford an additional insight into the structure-function relationship of Fur and imply its involvement in the iron acquisition systems of V. parahaemolyticus, although it is yet unknown whether its action on the target genes is direct or indirect.     

Identification and analysis of a gene encoding a Fur-like protein of Staphylococcus epidermidis

Abstract A gene ( fur ) for a Fur-like protein was identified on a 1.1 kb chromosomal DNA fragment of Staphylococcus epidermidis BN 280; the fur gene is followed by an open reading frame coding for the N-terminus of a putative Superoxide dismutase. Within the − 35 promoter region of both genes, a sequence motif was detected with low similarity to Fur-binding regulatory DNA segments, the so-called Fur boxes. Fur titration in Escherichia coli strain H1717 demonstrated that the E. coli Fur protein binds to the Fur box of the promoter region of the S. epidermidis fur gene. The S. epidermidis Fur protein was expressed in E. coli as indicated by the formation of inactive dimers with the chimeric repressor CI(N)-Fur(C) (Stojiljkovic I. and Hantke. K. (1995) Mol. Gen. Genet. 247, 199–205), but was not able to complement the Fur mutation in E. coli H1681.     

The role of fur in the acid tolerance response of Salmonella typhimurium is physiologically and genetically separable from its role in iron acquisition.

The response of Salmonella typhimurium to low pH includes a low-pH protection system called the acid tolerance response (ATR). The iron-regulatory protein Fur has been implicated in the ATR since fur mutants are acid sensitive and cause altered expression of several acid shock proteins (J. W. Foster, J. Bacteriol. 173:6896-6902, 1991). We have determined that the acid-sensitive phenotype of fur mutations is indeed due to a defect in Fur that can be complemented by a fur(+)-containing plasmid. However, changes in cellular iron status alone did not trigger the ATR. Cells clearly required exposure to low pH in order to induce acid tolerance. The role of Fur in acid tolerance was found to extend beyond regulating iron acquisition. A mutation in fur converting histidine 90 to an arginine (H90R) eliminated Fur-mediated iron regulation of enterochelin production and deregulated an iroA-lacZ fusion but had no effect on acid tolerance. The H90R iron-blind Fur protein also mediated acid shock induction of several Fur-dependent acid shock proteins and acid control of the hyd locus. In addition, a Fur superrepressor that constitutively repressed iron-regulated genes mediated normal Fur-dependent acid tolerance and pH-controlled gene expression. The results indicate the acid-sensing and iron-sensing mechanisms of Fur are separable by mutation and reinforce the concept of Fur as a major global regulator in the cell.     

Effect of Salmonella typhimurium ferric uptake regulator (fur) mutations on iron- and pH-regulated protein synthesis.

Fur is an important regulatory protein known to function in the presence of iron as a repressor of iron-controlled genes. It was recently discovered that Fur is also essential to Salmonella typhimurium for mounting an adaptive acid tolerance response (J. W. Foster, J. Bacteriol 173:6896-6902, 1991). Because little is known about the effect of Fur on the physiology of this enteric pathogen, a systematic two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis was conducted to identify proteins whose synthesis is linked to iron levels. Mutations in the fur locus were identified and used to classify which proteins are controlled by Fur. Thirty-six proteins were overtly affected by iron availability, most of which were clearly under the control of Fur. Although most of the Fur-dependent proteins were under negative control, a significant portion (15 of 34) appeared to be under a form of positive control. Nine of the positively controlled proteins required Fur and iron for expression. However, Fur lacking iron was also required for the induction of six gene products. Surprisingly, not all iron-regulated proteins were controlled by Fur and not all Fur-dependent proteins were obviously regulated by iron status. Because fur mutants fail to mount an effective acid tolerance response, we made a comparative two-dimensional PAGE analysis of 100 total acid- and iron-regulated gene products. Production of most of these proteins was regulated by only one of the two stresses, yet a clear subset of seven genes were influenced by both acid and iron and were also controlled by fur. These proteins were also members of the acid tolerance response modulon. Consistent with the fur effect on pH-regulated protein synthesis, fur mutants lacked the inducible pH homeostasis system associated with the acid tolerance response. The results provide further evidence that Fur has an extensive impact on gene expression and cellular physiology and suggest an explanation for the acid-sensitive nature of fur mutants.     

Submicromolar hydrogen peroxide disrupts the ability of Fur protein to control free-iron levels in Escherichia coli

In aerobic environments, mutants of Escherichia coli that lack peroxidase and catalase activities (Hpx(-)) accumulate submicromolar concentrations of intracellular H(2)O(2). We observed that in defined medium these strains constitutively expressed members of the Fur regulon. Iron-import proteins, which Fur normally represses, were fully induced. H(2)O(2) may antagonize Fur function by oxidizing the Fur:Fe(2+) complex and inactivating its repressor function. This is a potential problem, as in iron-rich environments excessive iron uptake would endanger H(2)O(2)-stressed cells by accelerating hydroxyl-radical production through the Fenton reaction. However, the OxyR H(2)O(2)-response system restored Fur repression in iron-replete Luria-Bertani medium by upregulating the synthesis of Fur protein. Indeed, when the OxyR binding site upstream of fur was disrupted, Hpx(-) mutants failed to repress transporter synthesis, and they exhibited high levels of intracellular free iron. Mutagenesis and bacteriostasis resulted. These defects were eliminated by mutations or chelators that slowed iron import, confirming that dysregulation of iron uptake was the root problem. Thus, aerobic organisms must grapple with a conundrum: how to monitor iron levels in oxidizing environments that might perturb the valence of the analyte. The induction of Fur synthesis by the OxyR response comprises one evolutionary solution to that problem.     

Characterization of the Vibrio anguillarum fur gene: role in regulation of expression of the FatA outer membrane protein and catechols.

The chromosomally encoded Vibrio anguillarum fur gene was characterized. The amino acid sequence of the Fur protein showed a very high degree of homology with those of V. cholerae and V. vulnificus. The degree of homology was lower, although still high, with the Escherichia coli and Yersinia pestis Fur amino acid sequences, while the lowest degree of homology was found with the Pseudomonas aeruginosa Fur protein. The C-terminal portion of Fur is the least conserved region among these Fur proteins. Within this portion, two regions spanning amino acids 105 to 121 and 132 to the end are the least conserved. A certain degree of variation is also present in the N termini spanning amino acids 28 to 46. Regulation of expression of the V. anguillarum fur gene by iron was not detected by immunoblot analysis. Mutations in the cloned fur gene were generated either by site-directed mutagenesis (the Lys-77 was changed to a Gly to generate the derivative FurG77) or by insertion of a DNA fragment harboring the aph gene in the same position. FurG77 was impaired in its ability to regulate a reporter gene with the Fur box in its promoter, while the insertion mutant was completely inactive. V. anguillarum fur mutants were obtained by isolating manganese-resistant derivatives. In one of these mutants, which encoded a Fur protein with an apparent lower molecular weight, the regulation of the production of catechols and synthesis of the outer membrane protein FatA were partially lost. In the case of another mutant, no protein was detected by anti-Fur serum. This derivative showed a total lack of regulation of biosynthesis of catechols and FatA protein by iron.     

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.