Characterization and mutagenesis of fur gene from Burkholderia pseudomallei

A homolog of the ferric uptake regulator gene (fur) was isolated from Burkh olderia pseudomallei (Bp) by a reverse genetic technique. Sequencing of a 2 .2 kb DNA fragment revealed an open reading frame with extensive homology t o bacterial Fur proteins. The cloned gene encodes a 16 kDa protein that cro ss-reacts with a polyclonal anti-Escherichia coli Fur serum. The transcript ion start site was determined by the primer extension technique. Expression analysis of fur showed no increased fur mRNA levels in response to various stresses and iron conditions. A positive selection procedure involving the isolation of manganese-resistant mutants was used to isolate mutants that produce altered Fur proteins. Sequencing of a fur mutant revealed a nucleot ide change (G to A) converting a conserved amino acid arginine-69 to histid ine. The fur missense mutant produced an elevated level of siderophore that could be complemented by a multicopy plasmid carrying the Bp fur. Interest ingly, Fur was found to play roles as a positive regulator of FeSOD and per oxidase. The mutant showed a decreased activity of FeSOD and peroxidase, wh ich could be important in its pathogenicity and survival in macrophages. (C ) 2000 Elsevier Science B.V. All rights reserved.