A dominant-negative fur mutation in Bradyrhizobium japonicum

In many bacteria, the ferric uptake regulator (Fur) protein plays a central role in the regulation of iron uptake genes. Because iron figures prominently in the agriculturally important symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, we wanted to assess the role of Fur in the interaction. We identified a fur mutant by selecting for manganese resistance. Manganese interacts with the Fur protein and represses iron uptake genes. In the presence of high levels of manganese, bacteria with a wild-type copy of the fur gene repress iron uptake systems and starve for iron, whereas fur mutants fail to repress iron uptake systems and survive. The B. japonicum fur mutant, as expected, fails to repress iron-regulated outer membrane proteins in the presence of iron. Unexpectedly, a wild-type copy of the fur gene cannot complement the fur mutant. Expression of the fur mutant allele in wild-type cells leads to a fur phenotype. Unlike a B. japonicum fur-null mutant, the strain carrying the dominant-negative fur mutation is unable to form functional, nitrogen-fixing nodules on soybean, mung bean, or cowpea, suggesting a role for a Fur-regulated protein or proteins in the symbiosis.     

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.     

Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene.

A 5.9-kb DNA fragment was cloned from Pseudomonas aeruginosa PA103 by its ability to functionally complement a fur mutation in Escherichia coli. A fur null mutant E. coli strain that contains multiple copies of the 5.9-kb DNA fragment produces a 15-kDa protein which cross-reacts with a polyclonal anti-E. coli Fur serum. Sequencing of a subclone of the 5.9-kb DNA fragment identified an open reading frame predicted to encode a protein 53% identical to E. coli Fur and 49% identical to Vibrio cholerae Fur and Yersinia pestis Fur. While there is extensive homology among these Fur proteins, Fur from P. aeruginosa differs markedly at its carboxy terminus from all of the other Fur proteins. It has been proposed that this region is a metal-binding domain in E. coli Fur. A positive selection procedure involving the isolation of manganese-resistant mutants was used to isolate mutants of strain PA103 that produce altered Fur proteins. These manganese-resistant Fur mutants constitutively produce siderophores and exotoxin A when grown in concentrations of iron that normally repress their production. A multicopy plasmid carrying the P. aeruginosa fur gene restores manganese susceptibility and wild-type regulation of exotoxin A and siderophore production in these Fur mutants.     

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.     

Agrobacterium tumefaciens fur has important physiological roles in iron and manganese homeostasis, the oxidative stress response, and full virulence

In Agrobacterium tumefaciens, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A fur mutant was constructed to address the physiological role of the regulator. Atypically, the mutant did not show alterations in the levels of siderophore biosynthesis and the expression of iron transport genes. However, the fur mutant was more sensitive than the wild type to an iron chelator, 2,2'-dipyridyl, and was also more resistant to an iron-activated antibiotic, streptonigrin, suggesting that Fur has a role in regulating iron concentrations. A. tumefaciens sitA, the periplasmic binding protein of a putative ABC-type iron and manganese transport system (sitABCD), was strongly repressed by Mn(2+) and, to a lesser extent, by Fe(2+), and this regulation was Fur dependent. Moreover, the fur mutant was more sensitive to manganese than the wild type. This was consistent with the fact that the fur mutant showed constitutive up-expression of the manganese uptake sit operon. Fur(At) showed a regulatory role under iron-limiting conditions. Furthermore, Fur has a role in determining oxidative resistance levels. The fur mutant was hypersensitive to hydrogen peroxide and had reduced catalase activity. The virulence assay showed that the fur mutant had a reduced ability to cause tumors on tobacco leaves compared to wild-type NTL4.     

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.     

Identification of a Functional fur Gene in Bradyrhizobium japonicum

The recent identification of the iron response regulator (Irr) in Bradyrhizobium japonicum raised the question of whether the global regulator Fur is present in that organism. A fur gene homolog was isolated by the functional complementation of an Escherichia coli fur mutant. The B. japonicum Fur bound to a Fur box DNA element in vitro, and a fur mutant grown in iron-replete medium was derepressed for iron uptake activity. Thus, B. japonicum expresses at least two regulators of iron metabolism.     

Derepression of the Bacillus subtilis PerR peroxide stress response leads to iron deficiency

The Bacillus subtilis PerR repressor regulates the adaptive response to peroxide stress. The PerR regulon includes the major vegetative catalase (katA), an iron storage protein (mrgA), an alkylhydroperoxide reductase (ahpCF), a zinc uptake system (zosA), heme biosynthesis enzymes (hemAXCDBL), the iron uptake repressor (fur), and perR itself. A perR null strain is resistant to hydrogen peroxide, accumulates a porphyrin-like compound, and grows very slowly. The poor growth of the perR mutant can be largely accounted for by the elevated expression of two proteins: the KatA catalase and Fur. Genetic studies support a model in which poor growth of the perR null mutant is due to elevated repression of iron uptake by Fur, exacerbated by heme sequestration by the abundant catalase protein. Analysis of the altered-function allele perR991 further supports a link between PerR and iron homeostasis. Strains containing perR991 are peroxide resistant but grow nearly as well as the wild type. Unlike a perR null allele, the perR991 allele (F51S) derepresses KatA, but not Fur, which likely accounts for its comparatively rapid growth.