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.     

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.     

Cloning and characterization of the fur gene from Moraxella bovis

A homologue of the ferric uptake regulator gene (fur) was isolated from Moraxella bovis by degenerate polymerase chain reaction and cloning. Fur protein of M. bovis exhibited 72.1% amino acid identity with Acinetobacter calcoaceticus Fur. Western blot analysis showed a decrease of Fur expression in response to sufficient-iron conditions compared with deficient-iron conditions. An electrophoretic mobility-shift assay indicated that Fur protein binds to DNA fragments containing a putative Fur-box derived from the upstream region of the M. bovis fur gene. Fur of M. bovis may regulate the expression of iron transport systems in response to iron limitation in the environment.     

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.     

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.     

The gonococcal fur regulon: identification of additional genes involved in major catabolic,recombination, and secretory pathways

In this study, we have characterized the in vitro binding of Neisseria gonorrhoeae Fur to several well-defined iron transport genes, as well as to additional genes involved in major catabolic, secretory, and recombination pathways of gonococci. The gonococcal Fur protein was recombinantly expressed in Escherichia coli HBMV119. Fur was isolated from inclusion bodies and partially purified by ion-exchange chromatography. Gonococcal Fur was found to bind to the promoter/operator region of a gene encoding the previously identified Fur-regulated periplasmic binding protein (FbpA) in a metal ion-dependent fashion, demonstrating that purified Fur is functional. In silico analysis of the partially completed gonococcal genome (FA1090) identified Fur boxes in the promoters of several genes, including tonB, fur, recN, secY, sodB, hemO, hmbR, fumC, a hypothetical gene (Fe-S homolog), and the opa family of genes. By using purified gonococcal Fur, we demonstrate binding to the operator regions of tonB, fur, recN, secY, sodB, hemO, hmbR, fumC, the Fe-S homolog gene, and the opa gene family as determined by an electrophoretic mobility shift assay. While gonococcal Fur was demonstrated to bind to the promoter regions of all 11 opa genes (opaA through -K), we did not detect binding of purified E. coli Fur with 8 of the 11 opa members, indicating that target DNA sequence specificities between these two closely related proteins exist. Furthermore, we observed differences in the relative strengths of binding of gonococcal Fur for these different genes, which most likely reflect a difference in affinity between gonococcal Fur and its DNA targets. This is the first report that definitively demonstrates the binding of gonococcal Fur to its own promoter/operator region, as well as to the opa family of genes that encode surface proteins. Our results demonstrate that the gonococcal Fur protein binds to the regulatory regions of a broad array of genes and indicates that the gonococcal Fur regulon is larger than originally proposed.     

Structural dynamics and functional domains of the fur protein

Proteolytic enzymes were used to detect metal-induced conformational changes in the ferric uptake regulation (Fur) protein of Escherichia coli K12. Metal binding results in enhanced cleavage of the N-terminal region of Fur by trypsin and chymotrypsin. Activation of both trypsinolysis sensitivity and DNA binding have similar metal ion specificity and concentration dependencies, suggesting that the conformational change detected is required for operator DNA binding. Isolation and characterization of biochemically generated fragments of Fur as well as other data indicate that the N-terminal region is necessary for the interaction of the repressor with DNA and that a C-terminal domain is sufficient for binding to metal ions.     

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.     

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.     

Cloning and Sequencing of the Vibrio parahaemolyticus fur Gene

A ferric uptake regulatory gene (fur) was cloned from Vibrio parahaemolyticus WP1 by a polymerase chain reaction-based technique followed by functional complementation of a fur mutation in Escherichia coli. A sequence analysis showed that, at the amino acid level, the V. parahaemolyticus Fur protein is 81% identical with the Fur protein from E. coli and over 90% identical with those of the Vibrio species.