1887

Abstract

Iron is an essential nutrient for the survival and pathogenesis of bacteria, but relatively little is known regarding its transport and regulation in staphylococci. Based on the known sequences of ferric-uptake regulatory () genes from several Gram-positive and Gram-negative bacteria, a fragment containing the homologue was cloned from a genomic library of RN450. Nucleotide sequence analysis of this fragment revealed the presence of a 447 bp ORF that encodes a putative 149 aa polypeptide with an apparent molecular mass of 17 kDa. A putative ferrichrome-uptake () operon, containing the conserved Fur-binding sequences (Fur box) in the promoter region, was also cloned from the same library. To characterize the impact of Fur on the operon, was cloned, overexpressed as a His-tagged protein and purified by Ni-affinity column chromatography. The recombinant protein was digested with enterokinase to remove the His tag. Electrophoretic mobility-shift assays indicated that Fur binds to the promoter region of the operon in the presence of divalent cations. Fur also interacted with the promoter region of the recently reported operon that has been proposed to constitute a siderophore-transport system in . The DNase I-protection assay revealed that Fur specifically binds to the Fur box located in the promoter region of the operon. The primer-extension reaction indicated that the transcription-start site of the operon was located inside the Fur box. partially complemented a mutation in . The data suggest that Fur regulates iron-transport processes in .

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2000-03-01
2024-03-28
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