Iron-responsive regulation of the Helicobacter pylori iron-cofactored superoxide dismutase SodB is mediated by Fur

Abstract

Maintaining iron homeostasis is a necessity for all living organisms, as free iron augments the generation of reactive oxygen species like superoxide anions, at the risk of subsequent lethal cellular damage. The iron-responsive regulator Fur controls iron metabolism in many bacteria, including the important human pathogen Helicobacter pylori, and thus is directly or indirectly involved in regulation of oxidative stress defense. Here we demonstrate that Fur is a direct regulator of the H. pylori iron-cofactored superoxide dismutase SodB, which is essential for the defense against toxic superoxide radicals. Transcription of the sodB gene was iron induced in H. pylori wild-type strain 26695, resulting in expression of the SodB protein in iron-replete conditions but an absence of expression in iron-restricted conditions. Mutation of the fur gene resulted in constitutive, iron-independent expression of SodB. Recombinant H. pylori Fur protein bound with low affinity to the sodB promoter region, but addition of the iron substitute Mn2+ abolished binding. The operator sequence of the iron-free form of Fur, as identified by DNase I footprinting, was located directly upstream of the sodB gene at positions -5 to -47 from the transcription start site. The direct role of Fur in regulation of the H. pylori sodB gene contrasts with the small-RNA-mediated sodB regulation observed in Escherichia coli. In conclusion, H. pylori Fur is a versatile regulator involved in many pathways essential for gastric colonization, including superoxide stress defense.

FIG. 1.

Expression of the sodB gene in H. pylori strain 26695 is iron induced and Fur repressed. (A) Northern hybridization with a probe specific for the sodB gene and RNA purified from H. pylori wild-type strain 26695 (wt) and fur mutant (fur) cells grown in iron-restricted (−Fe) and iron-replete (+Fe) conditions. The positions of the specific mRNAs are indicated on the right, and the positions of the RNA size markers are indicated on the left. Staining of RNA by methylene blue was included for comparison of RNA amounts. (B) Identification of the H. pylori sodB transcription start site by primer extension analysis, using RNA purified from H. pylori wild-type strain 26695 (wt) and fur mutant (fur) cells grown in iron-restricted (−Fe) and iron-replete (+Fe) conditions. The sequence of the corresponding region is displayed on the left, with the + 1 nucleotide and the −10 promoter sequence indicated. (C) Iron- and Fur-responsive regulation of SodB at the protein level. Lysates of H. pylori wild-type strain 26695 (wt) and fur mutant (fur) cells, grown in iron-restricted (−Fe) and iron-replete (+Fe) conditions, were separated on 2-D protein gels. Only the relevant part of the protein gels (molecular masses [MW], 20 to 30 kDa; pI 6 to 7) is shown, and the position of the iron- and Fur-repressed SodB protein (as identified by MALDI-TOF mass spectrometry) is circled.

FIG. 2.

Regulation of sodB transcription is mediated by direct binding of the H. pylori Fur protein to the sodB promoter. (A) Electrophoretic mobility shift assay using recombinant H. pylori Fur protein and DIG-labeled sodB promoter DNA (P_sodB) isolated from H. pylori strain 26695. In the presence of the iron substitute manganese (+Mn²⁺) (bottom panel), Fur was unable to complex with the sodB promoter region, and a shift was not observed. Only in the absence of manganese (+EDTA) (top panel) was apo-Fur able to bind to the sodB promoter and cause a mobility shift (indicated as a Fur-P_sodB complex). The concentrations of Fur are indicated above the lanes, and the concentration of DNA was 22 pM. (B) Determination of the affinity of apo-Fur for the sodB promoter sequence. DIG-labeled P_sodB (22 pM) was mixed with increasing concentrations of Fur, with EDTA in the buffer. (C) Graphic representation of determination of the K_d of purified H. pylori Fur for the sodB promoter, using the data in panel B. The relative amounts of P_sodB and Fur-P_sodB were determined by densitometry.

FIG. 3.

Identification of the operator sequence for apo-Fur in the H. pylori 26695 sodB promoter. (A) DNase I footprinting assay using 440 pM DIG-labeled H. pylori 26695 sodB promoter DNA and increasing concentrations apo-Fur. The positions of the −10 and −35 promoter sequences located in the sodB promoter are indicated on the left, and the positions of the two protected regions (located at positions −5 to −23 and −25 to −47) and the DNase I hypersensitive site (at position −24) are indicated on the right. The concentrations of Fur protein used in lanes 1 to 6 were 0, 2.3, 4.6, 6.9, 9.2, and 16 μM, respectively. (B) Graphic representation of the sodB promoter with the location and sequence of the apo-Fur binding site indicated. The DNase I hypersensitivity residue is indicated between the two binding sites by a black background. The −10 and −35 promoter sequences are underlined in the binding sequence. RBS, ribosome binding sequence. (C) Alignment of the proposed binding sequence for apo-Fur in the H. pylori 26695 sodB promoter with the high-affinity binding sequences in the H. pylori pfr promoter (pfr boxes I and II) (12). Residues in the sodB sequence identical to residues in both pfr binding sequences are indicated by a black background, and residues identical to residues in only one of the pfr binding sites (12) are indicated by a grey background.