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 identified 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 mutants of H. pylori. The fur homologue on a plasmid mediated full iron-dependent ferric uptake regulator activity in the fur-deficient mutant strains H1681 and H1780 of E. coli. Immunoblot analysis revealed that Fur from H. pylori cross-reacts with antibodies raised against Fur from E. coli. The fact that inactivation of the fur gene abolished the FURTA-positive phenotype in the E. coli indicator strain H1717, indicated that this phenotype is rather 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 protein. In summary, the results confirm the function of the H. pylori Fur homologue as iron-dependent transcriptional repressor by its ability to interact with the Fur-regulated promoters of the genes fiu and fhuF in E. coli.
Bereswill
S
S
University of Freiburg, Institute of Medical Microbiology and Hygiene, Department of Microbiology and Hygiene, Germany. bereswil@sun11.ukl.uni-freiburg.de
Lichte
F
F
Greiner
S
S
Waidner
B
B
Fassbinder
F
F
Kist
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D015139:Blotting, Southern; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D016480:Helicobacter pylori / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D007501:Iron / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D017422:Sequence Analysis, DNA; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D007501:Iron; D001616:beta-Galactosidase