title: Point mutations in Helicobacter pylori's fur regulatory gene that alter resistance to metronidazole, a prodrug activated by chemical reduction.
abstract: BACKGROUND
  Helicobacter pylori's Fur regulatory protein controls transcription of dozens of genes in response to iron availability, acidity and oxidative stress, and affects the vigor of infection and severity of disease. It is unusual among Fur family proteins in being active both when iron-loaded and iron-free. METHOLODOLGY/PRINCIPAL FINDINGS: We tested if H. pylori fur mutations could affect resistance to metronidazole (Mtz), an anti-H. pylori prodrug rendered bactericidal by chemical reduction. Point mutations were made by PCR in DNA containing fur and a downstream chloramphenicol resistance gene, and were placed in the H. pylori chromosome by transformation of a fur-deletion (Δfur) strain. Several substitutions affecting H. pylori Fur's ∼10 residue N terminal arm, which has no counterpart in prototype (E. coli-type) Fur proteins, increased Mtz resistance, as did mutations affecting the region between DNA binding and dimerization domains. Three types of mutations decreased resistance more than did Δfur: substitutions affecting the N-terminal arm; substitutions affecting the metal binding pocket; and nonsense mutations that resulted in a truncated Fur protein with no C-terminal dimerization domain. Most metal binding pocket mutations were obtained only in fur genes with additional inactivating mutations, and thus seemed deleterious or lethal because they.
  
  
  CONCLUSIONS
  These results establish that H. pylori Fur's distinctive N terminal arm is functional, and more generally illustrate that point mutations can confer informative phenotypes, distinct from those conferred by null mutations. We propose that fur mutations can affect Mtz susceptibility by altering the balance among Fur's several competing activities, and thereby the expression of genes that control cellular redox potential or elimination of bactericidal Mtz activation products. Further analyses of selected mutants should provide insights into Fur interactions with other cellular components, metabolic circuitry, and how H. pylori thrives in its special gastric niche.
authors.affiliation: Department of Molecular Microbiology, Washington University Medical School, St Louis, Missouri, United States of America.
authors.forename: Sung Sook
authors.initials: SS
authors.lastname: Choi
authors.forename: Peter T
authors.initials: PT
authors.lastname: Chivers
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Berg
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004252:DNA Mutational Analysis; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics*; D004843:Epistasis, Genetic / Q000187:drug effects; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial / Q000235:genetics*; D005809:Genes, Regulator; D005838:Genotype; D016480:Helicobacter pylori / Q000187:drug effects* / Q000235:genetics*; D007501:Iron / Q000378:metabolism; D008795:Metronidazole / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction / Q000187:drug effects; D010641:Phenotype; D017354:Point Mutation / Q000235:genetics*; D011355:Prodrugs / Q000494:pharmacology*; D012641:Selection, Genetic; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D001426:Bacterial Proteins; D011355:Prodrugs; D008795:Metronidazole; D007501:Iron