title: Random and site-specific mutagenesis of the Helicobacter pylori ferric uptake regulator provides insight into Fur structure-function relationships.
abstract: The ferric uptake regulator (Fur) of Helicobacter pylori is a global regulator that is important for colonization and survival within the gastric mucosa. H. pylori Fur is unique in its ability to activate and repress gene expression in both the iron-bound (Fe-Fur) and apo forms (apo-Fur). In the current study we combined random and site-specific mutagenesis to identify amino acid residues important for both Fe-Fur and apo-Fur function. We identified 25 mutations that affected Fe-Fur repression and 23 mutations that affected apo-Fur repression, as determined by transcriptional analyses of the Fe-Fur target gene amiE, and the apo-Fur target gene, pfr. In addition, eight of these mutations also significantly affected levels of Fur in the cell. Based on regulatory phenotypes, we selected several representative mutations to characterize further. Of those selected, we purified the wild-type (HpFurWT) and three mutant Fur proteins (HpFurE5A, HpFurA92T and HpFurH134Y), which represent mutations in the N-terminal extension, the regulatory metal binding site (S2) and the structural metal binding site (S3) respectively. Purified proteins were evaluated for secondary structure by circular dichroism spectroscopy, iron-binding by atomic absorption spectrophotometry, oligomerization in manganese-substituted and apo conditions by in vitro cross-linking assays, and DNA binding to Fe-Fur and apo-Fur target sequences by fluorescence anisotropy. The results showed that the N-terminal, S2 and S3 regions play distinct roles in terms of Fur structure-function relationships. Overall, these studies provide novel information regarding the role of these residues in Fur function, and provide mechanistic insight into how H. pylori Fur regulates gene expression in both the iron-bound and apo forms of the protein.
authors.affiliation: Department of Microbiology and Immunology, Uniformed Services University of Health Sciences, Bethesda, MD, USA.
authors.forename: Jeremy J
authors.initials: JJ
authors.lastname: Gilbreath
authors.forename: Oscar Q
authors.initials: OQ
authors.lastname: Pich
authors.forename: Stéphane L
authors.initials: SL
authors.lastname: Benoit
authors.forename: Angelique N
authors.initials: AN
authors.lastname: Besold
authors.forename: Jeong-Heon
authors.initials: JH
authors.lastname: Cha
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Maier
authors.forename: Sarah L J
authors.initials: SL
authors.lastname: Michel
authors.forename: Ernest L
authors.initials: EL
authors.lastname: Maynard
authors.forename: D Scott
authors.initials: DS
authors.lastname: Merrell
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D005753:Gastric Mucosa / Q000378:metabolism / Q000382:microbiology; D015964:Gene Expression Regulation, Bacterial; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial