Characterization of the DNA- and Metal-Binding Properties of Vibrio anguillarum Fur Reveals Conservation of a Structural Zn ²⁺ Ion

Abstract

The ferric uptake regulator, Fur, represses iron uptake and siderophore biosynthetic genes under ironreplete conditions. Here we report in vitro solution studies on Vibrio anguillarum Fur binding to the consensus 19-bp Escherichia coli iron box in the presence of several divalent metals. We found that V. anguillarum Fur binds the iron box in the presence of Mn 2؉ , Co 2؉ , Cd 2؉ , and to a lesser extent Ni 2؉ but, unlike E. coli Fur, not in the presence of Zn 2؉ . We also found that V. anguillarum Fur contains a structural zinc ion that is necessary yet alone is insufficient for DNA binding.Iron is essential for survival and virulence of bacterial pathogens; however, its concentration in host tissues is limited. To acquire iron, bacterial pathogens rely on iron uptake systems that consist of an iron siderophore (a small-molecule chelator) and membrane transport proteins, which import Fe 3ϩ -siderophore complexes into the cell (7, 18). These systems are regulated by the DNA-binding protein Fur (ferric uptake regulator) in response to iron availability (3,11,20). When the intracellular concentration of iron increases above a certain level, Fur represses the transcription of genes encoding components of the membrane transport system as well as enzymes involved in siderophore biosynthesis.To date, the most extensive biochemical studies have been limited to the Escherichia coli Fur protein, which in the presence of various divalent metals that act as corepressors binds a conserved 19-bp operator sequence, the iron box, which is located in the promoter region of iron uptake genes (4, 5, 9). Moreover, these studies have indicated that E. coli Fur possesses two metal ion-binding sites. One, the corepressor binding site, uses histidines and carboxylate ligands to coordinate binding of Fe 2ϩ (and two of its functional mimics, Mn 2ϩ and Co 2ϩ ) (1). This event promotes a conformational change that leads to DNA binding. The other cation-binding site is involved in protein structure and stability and binds Zn 2ϩ with high affinity, using the thiols of Cys92 and Cys95 as two of the four coordinating ligands (2,3,14). These cysteines are essential for E. coli Fur activity both in vivo and in vitro (6).Fur homologues have been identified in multiple bacteria, where they also regulate iron acquisition. In the fish pathogen Vibrio anguillarum strain 775, Fur represses production of a siderophore, anguibactin, and of Fe 3ϩ -anguibactin transport proteins in response to abundant iron (21). V. anguillarum Fur is closely related to its counterparts from other Vibrio species, including Vibrio cholerae (93% sequence identity), Vibrio vulnificus (91%), and Vibrio parahaemolyticus (91%) (Fig. 1). Yet V. anguillarum Fur is less homologous to E. coli Fur (76% sequence identity), and the two proteins have different numbers of cysteines and histidines (Fig. 1) The V. anguillarum fur gene subcloned into the isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible pT7-5 vector (21) was kindly provided by A. M. Wertheimer. E. coli BL21(DE3) cells t...

cis- and trans-acting elements involved in regulation of norB (norZ), the gene encoding nitric oxide reductase in Neisseria gonorrhoeae

“…Beyond this function, for which its role in regulation was initially defined, Fur has been implicated in the regulation of genes important in oxidative stress and acid resistance, as well as in virulence (Hantke, 2001). Fur acts to repress transcription by binding to a 19 bp consensus sequence (the Fur Box) and blocking RNA polymerase entrance to the promoter or promoter clearance (Ochsner & Vasil, 1996;Escolar et al, 1999;Zheleznova et al, 2000). Though generally thought of as a repressor, there are a few examples showing that Fur acts as an activator of transcription (Escolar et al, 1999;Hantke, 2001).…”

Iron-Dependent Transcription of the frpB Gene of Helicobacter pylori Is Controlled by the Fur Repressor Protein

“…Boxes 2 and 3 also contain sequences resembling the Fur box consensus, although to lesser extents, showing 11 and 9 out of 19 matches, respectively. Therefore, as reported for other bacteria (23,32,33), Fur from H. pylori binds to sequences sharing similarity to the E. coli Fur box.…”

Vibrio Iron Transport: Evolutionary Adaptation to Life in Multiple Environments

“…Fur has been shown to play similar roles in V. vulnificus (214)(215)(216), V. anguillarum (217,218), and V. salmonicida (219). In each of these vibrios, Fur negatively regulates the expression of genes for iron acquisition systems, thereby reducing the chance of toxicity due to iron overload.…”