Structural, functional and mutational analysis of the pfr gene encoding a ferritin from Helicobacter pylori.
The function of the pfr gene encoding the ferritin from Helicobacter pylori was investigated using the Fur titration assay (FURTA) in Escherichia coli, and by characterization of a pfr-deficient mutant strain of H. pylori. Nucleotide sequence analysis revealed that the pfr region is conserved among strains (> 95% nucleotide identity). Two transcriptional start sites, at least one of them preceded by a sigma 70-dependent promoter, were identified. Provision of the H. pylori pfr gene on a multicopy plasmid resulted in reversal of the Fur-mediated repression of the fhuF gene in E. coli, thus enabling the use of the FURTA for cloning of the ferritin gene. Inactivation of the pfr gene, either by insertion of a resistance cassette or by deletion of the up- and downstream segments, abolished this function. Immunoblot analysis with a Pfr-specific antiserum detected the Pfr protein in H. pylori and in E. coli carrying the pfr gene on a plasmid. Pfr-deficient mutants of H. pylori were generated by marker-exchange mutagenesis. These were more susceptible than the parental strain to killing by various metal ions including irons, copper and manganese, whereas conditions of oxidative stress or iron deprivation were not discriminative. Analysis by element-specific electron microscopy revealed that growth of H. pylori in the presence of iron induces the formation of two kinds of cytoplasmic aggregates: large vacuole-like bodies and smaller granules containing iron in association with oxygen or phosphorus. Neither of these structures was detected in the pfr-deficient mutant strain. Furthermore, the ferritin accumulated under iron overload and the pfr-deficient mutant strains lacked expression of a 12 kDa protein which was negatively regulated by iron in the parental strain. The results indicate that the nonhaem-iron ferritin is involved in the formation of iron-containing subcellular structures and contributes to metal resistance of H. pylori. Further evidence for an interaction of ferritin with iron-dependent regulation mechanisms is provided.
Bereswill
Stefan
S
University of Freiburg, Institute of Medical Microbiology and Hygiene, Department of Microbiology and Hygiene,Hermann-Herder-Str. 11, D-79104 Freiburg,Germany.
Waidner
Uta
U
University of Freiburg, Institute of Medical Microbiology and Hygiene, Department of Microbiology and Hygiene,Hermann-Herder-Str. 11, D-79104 Freiburg,Germany.
Odenbreit
Stefan
S
Max-von-Pettenkofer-Institute of Hygiene and Medical Microbiology, Department of Bacteriology,Pettenkoferstr. 9a, D-80336 Munich,Germany.
Lichte
Flavia
F
University of Freiburg, Institute of Medical Microbiology and Hygiene, Department of Microbiology and Hygiene,Hermann-Herder-Str. 11, D-79104 Freiburg,Germany.
Fassbinder
Frank
F
University of Freiburg, Institute of Medical Microbiology and Hygiene, Department of Microbiology and Hygiene,Hermann-Herder-Str. 11, D-79104 Freiburg,Germany.
Bode
G Nter
GN
University of Ulm, Department of Internal Medicine I,Robert Koch-Str. 8, D-89081 Ulm,Germany.
Kist
Manfred
M
University of Freiburg, Institute of Medical Microbiology and Hygiene, Department of Microbiology and Hygiene,Hermann-Herder-Str. 11, D-79104 Freiburg,Germany.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005293:Ferritins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D007501:Iron / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D012329:RNA, Bacterial / Q000235:genetics; D014158:Transcription, Genetic; D014617:Vacuoles / Q000378:metabolism / Q000648:ultrastructure
D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D005293:Ferritins; D007501:Iron