The ferric uptake regulator (Fur) homologue of Helicobacter pylori: functional analysis of the coding gene and controlled production of the recombinant protein in Escherichia coli

A homologue of the ferric uptake regulator protein Fur has recently been id entified within the Helicobacter pylori genome. The promoterless gene on a plasmid did partially complement a fur-negative mutant of Escherichia coli, and was strongly positive in the Fur titration assay (FURTA). The genetic and functional characterization of the complete fur homologue performed in this study revealed that the gene is conserved among H. pylori strains (>95 % identity), and does not carry nucleotide transitions in iron-resistant mu tants of H. pylori. The fur homologue on a plasmid mediated full iron-depen dent ferric uptake regulator activity in the fur-deficient mutant strains H 1681 and H1780 of E. coli. Immunoblot analysis revealed that Fur from H. py lori cross-reacts with antibodies raised against Fur from E. coli. The fact that inactivation of the fur gene abolished the FURTA-positive phenotype i n the E. coli indicator strain H1717, indicated that this phenotype is rath er caused by the encoded protein than by real Fur titration. Subcloning of the fur gene into an expression vector allowed controlled production in E. coli, and purification of a recombinant version of the H. pylori Fur protei n. In summary, the results confirm the function of the H. pylori Fur homolo gue as iron-dependent transcriptional repressor by its ability to interact with the Fur-regulated promoters of the genes fiu and fhuF in E. coli.