Characterization of the Vibrio alginolyticusfur Gene and Localization of Essential Amino Acid Sites in Fur by Site-Directed Mutagenesis

The expression of iron-regulated genes in bacteria is typically controlled by the ferric uptake regulator (Fur) protein, a global transcriptional repressor that regulates functions as diverse as iron acquisition, oxidative stress, virulence and acid tolerance. We have identified a fur homologue in Vibrio alginolyticus and shown that it complements an Escherichia coli fur mutant. Reverse transcriptase PCR (RT-PCR) analysis proved that unlike many other fur homologues, V. alginolyticusfur is not under the iron-response Fur autoregulation. Homology modeling of the V. alginolyticus Fur protein with the recently solved crystal structure of Fur from Pseudomonas aeruginosa indicated extensive structural conservation. Based on the highly conserved DNA-binding sites and metal-binding sites in Fur protein, a series of site-directed mutations were respectively introduced into the cloned V. alginolyticus fur gene and resulted in partial or complete loss of Fur repressor function. Mutations in H33 and H90 were associated with complete loss of Fur function, mutations in Y56, R57, H87, C93 and H125 were related to partial loss of Fur function, and mutations in C96 and C133 did not show obvious change of Fur function. Our studies allowed the localization of some essential amino acid sites which may play important structural or functional roles in V. alginolyticus Fur activity.