Random and site-specific mutagenesis of the Helicobacter pylori ferric uptake regulator provides insight into Fur structure-function relationships

Jeremy J. Gilbreath, Oscar Q. Pich, Stéphane L. Benoit, Angelique N. Besold, JeongHeon Cha, Robert J. Maier, Sarah L.J. Michel, Ernest L. Maynard, D. Scott Merrell

Research output: Contribution to journalArticle

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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.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.

Original languageEnglish
Pages (from-to)304-323
Number of pages20
JournalMolecular Microbiology
Volume89
Issue number2
DOIs
Publication statusPublished - 2013 Jul 1

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Site-Directed Mutagenesis
Helicobacter pylori
Mutation
Iron
Regulator Genes
Metals
Binding Sites
Gene Expression
Atomic Spectrophotometry
Proteins
Fluorescence Polarization
Manganese
Circular Dichroism
Gastric Mucosa
Spectrum Analysis
Phenotype
Amino Acids
DNA

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

Cite this

Gilbreath, Jeremy J. ; Pich, Oscar Q. ; Benoit, Stéphane L. ; Besold, Angelique N. ; Cha, JeongHeon ; Maier, Robert J. ; Michel, Sarah L.J. ; Maynard, Ernest L. ; Merrell, D. Scott. / Random and site-specific mutagenesis of the Helicobacter pylori ferric uptake regulator provides insight into Fur structure-function relationships. In: Molecular Microbiology. 2013 ; Vol. 89, No. 2. pp. 304-323.
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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.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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Gilbreath, JJ, Pich, OQ, Benoit, SL, Besold, AN, Cha, J, Maier, RJ, Michel, SLJ, Maynard, EL & Merrell, DS 2013, 'Random and site-specific mutagenesis of the Helicobacter pylori ferric uptake regulator provides insight into Fur structure-function relationships', Molecular Microbiology, vol. 89, no. 2, pp. 304-323. https://doi.org/10.1111/mmi.12278

Random and site-specific mutagenesis of the Helicobacter pylori ferric uptake regulator provides insight into Fur structure-function relationships. / Gilbreath, Jeremy J.; Pich, Oscar Q.; Benoit, Stéphane L.; Besold, Angelique N.; Cha, JeongHeon; Maier, Robert J.; Michel, Sarah L.J.; Maynard, Ernest L.; Merrell, D. Scott.

In: Molecular Microbiology, Vol. 89, No. 2, 01.07.2013, p. 304-323.

Research output: Contribution to journalArticle

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, JeongHeon

AU - Maier, Robert J.

AU - Michel, Sarah L.J.

AU - Maynard, Ernest L.

AU - Merrell, D. Scott

PY - 2013/7/1

Y1 - 2013/7/1

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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