Characterization and mutagenesis of fur gene from Burkholderia pseudomallei

Abstract

A homolog of the ferric uptake regulator gene ( fur) was isolated from Burkholderia pseudomallei ( Bp) by a reverse genetic technique. Sequencing of a 2.2 kb DNA fragment revealed an open reading frame with extensive homology to bacterial Fur proteins. The cloned gene encodes a 16 kDa protein that cross-reacts with a polyclonal anti- Escherichia coli Fur serum. The transcription 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 nucleotide change (G to A) converting a conserved amino acid arginine-69 to histidine. The fur missense mutant produced an elevated level of siderophore that could be complemented by a multicopy plasmid carrying the Bp fur. Interestingly, Fur was found to play roles as a positive regulator of FeSOD and peroxidase. The mutant showed a decreased activity of FeSOD and peroxidase, which could be important in its pathogenicity and survival in macrophages.