Regulation of the Helicobacter pylori Fe-S cluster synthesis protein NifS by iron, oxidative stress conditions, and fur

Abstract

Transcription of both chromosomal and extrachromosomally introduced nifS was regulated (up-expressed) by oxygen or by supplemental iron conditions. This up-expression was not observed in a fur mutant strain background or when an iron chelator was added. Iron-bound Fur (but not apo-Fur) recognized the nifS promoter, and Fur bound significantly farther upstream (-155 bp to -190 bp and -210 to -240 bp) in the promoter than documented Helicobacter pylori Fur binding regions. This binding was stronger than Fur recognition of the flgE or napA promoter and includes a Fur recognition sequence common to the H. pylori pfr and sodB upstream areas. Studies of Fur-regulated genes in H. pylori have indicated that apo-Fur acts as a repressor, but our results demonstrate that iron-bound Fur activates (nifS) transcription.

FIG. 1.

Primer extension analysis of the nifS transcript. A primer extension system, the avian myeloblastosis virus reverse transcriptase kit (catalog no. E3030; Promega), was used to obtain the primer extension product of the nifS transcript. A 28-mer oligonucleotide (5′-TAAATTCGTTGTAACAAGGTTAATATTC-3′) complementary to the bases spanning the ATG start codon (TTG for nifS) was end labeled with [³²P]ATP, and RNA from H. pylori was used as the substrate for the primer extension reaction. The reaction was carried out according to the manufacturer's instructions, and the products were subjected to PAGE (6% polyacrylamide). Lane 1, primer extension product of the nifS transcript; lane 2, DNA ladder.

FIG. 2.

XylE activities as a measure of nifS induction under various stress conditions. Cells were grown at 2% partial pressure oxygen until logarithmic phase (an optical density at 600 nm of ∼0.4); oxygen was added to 12% partial pressure in a closed gas system, or the 2% O₂ atmosphere was maintained but the medium (see the text) was supplemented with 500 μM FeCl₃ or with a 75 μM concentration of the iron chelator 2,2-dipyridyl, to study the effects of oxygen stress, iron, and iron starvation on transcription of the gene. WT-xylE and fur-xylE, P_nifS-xylE fusion in the hp405 region of the genome; pHel3-xylE, P_nifS-xylE fusion on the shuttle vector. Simultaneous experiments were conducted in wild-type and fur mutant strain backgrounds. The means and standard deviations from five independent experiments are shown here, with three replicates for each experiment (a total of 15 samples for each mean). All wild-type results for both the added-O₂ and the iron stress conditions are significantly greater than for the 2% O₂ conditions (P < 0.01), and the results for the fur strain are significantly less (P < 0.01) than for the same stress condition (12% O₂ or supplemented iron) for the wild-type strain. The iron chelator conditions are not statistically different from the 2% O₂ conditions. One unit of xylE activity is equal to 1 μmol of oxidized catechol/min/10⁹ cells.

FIG. 3.

Purification of H. pylori Fur. The entire gene (hp1027) from H. pylori encoding Fur was introduced downstream of the T7 promoter in the pET21A vector (Novagen) and overexpressed in E. coli BL21 Rosetta by induction with 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside) (Sigma) at 30°C for 3 h. The cytoplasmic protein from crude extracts prepared from the IPTG-induced cells was obtained by ultracentrifugation (45,000 rpm for 2 h). The purification was performed by fast-protein liquid chromatography; the cytoplasmic protein was first passed through a HiTrap SP column for ion-exchange-based purification with a salt gradient of 50 mM to 1,000 mM NaCl (obtained by using buffer A [50 mM sodium phosphate-50 mM NaCl, pH 8.0] and buffer B [50 mM sodium phosphate-1,000 mM NaCl, pH 8.0]). Peak fractions containing Fur protein (from the ion-exchange procedure) were collected and further purified based on size exclusion by using a Sephacryl-200 column (buffer C [50 mM sodium phosphate-200 mM NaCl, pH 8.0]). UN, uninduced; IN, induced with 0.5 mM IPTG; Fur, purified Fur; M, molecular mass marker (masses, reading down from the top band, are 97.4, 66.2, 45.0, 31.5, 21.0, and 14.4 kDa).

FIG. 4.

Electrophoretic mobility shift assay. The Fur-P_nifS interaction was studied with the 300-bp radiolabeled P_nifS fragment and pure Fur protein. P_nifS (50 pM) was assayed with various MnCl₂ concentrations and constant Fur levels (A) or with increasing concentrations of Fur (0 to 1,000 nM) in the presence of 150 μM EDTA (B). Fur-P_nifS binding was determined with 100 μM MnCl₂ and various concentrations of Fur; the percent DNA bound was determined by phosphorimager scanning, and then the K_d value was calculated from all of the (bound and unbound) values (C). A 150-bp interior region of the gene encoding the 16S rRNA and pure thioredoxin reductase, representing nonspecific DNA and protein, respectively, were used as controls; these exhibited no shift (data not shown). The region of Fur recognition in P_nifS (in the presence of MnCl₂) was narrowed by use of a 138-bp non-Fur-binding fragment upstream of the start codon rather than the 300-bp fragment used in other experiments. The inability of this fragment to bind Fur is shown (D).

FIG.5.

(A) Competitive effects of P_flgE and P_napA on Fur-P_nifS (P_nifS at 50 pM) binding in the presence of 100 μM MnCl₂ determined by using increasing concentrations (0 to 400 pM) of competitor promoter DNA. (B) Effects of cold P_nifS (0 to 200 pM) on Fur binding to radiolabeled nifS promoter DNA.

FIG.5.

(A) Competitive effects of P_flgE and P_napA on Fur-P_nifS (P_nifS at 50 pM) binding in the presence of 100 μM MnCl₂ determined by using increasing concentrations (0 to 400 pM) of competitor promoter DNA. (B) Effects of cold P_nifS (0 to 200 pM) on Fur binding to radiolabeled nifS promoter DNA.