TY - JOUR
T1 - Random and site-specific mutagenesis of the Helicobacter pylori ferric uptake regulator provides insight into Fur structure-function relationships
AU - Gilbreath, Jeremy J.
AU - Pich, Oscar Q.
AU - Benoit, Stéphane L.
AU - Besold, Angelique N.
AU - Cha, Jeong Heon
AU - Maier, Robert J.
AU - Michel, Sarah L.J.
AU - Maynard, Ernest L.
AU - Merrell, D. Scott
PY - 2013/7
Y1 - 2013/7
N2 - The ferric uptake regulator (Fur) of Helicobacter pylori is a global regulator that is important for colonization and survival within the gastric mucosa. H.pyloriFur 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.pyloriFur regulates gene expression in both the iron-bound and apo forms of the protein.
AB - The ferric uptake regulator (Fur) of Helicobacter pylori is a global regulator that is important for colonization and survival within the gastric mucosa. H.pyloriFur 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.pyloriFur regulates gene expression in both the iron-bound and apo forms of the protein.
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U2 - 10.1111/mmi.12278
DO - 10.1111/mmi.12278
M3 - Article
C2 - 23710935
AN - SCOPUS:84880134730
SN - 0950-382X
VL - 89
SP - 304
EP - 323
JO - Molecular Microbiology
JF - Molecular Microbiology
IS - 2
ER -