iron-limited growth culture was allowed to grow overnight and was harvested the following morning for the stationary phase, iron- limited growth RNA samples. RNA was extracted as described previously [21]. RNase Protection Assays (RPAs) were performed as previously described [18] with 1.5 mg of RNA using sodB, pfr, amiE, and/or fur riboprobes that were generated using the primer pairs listed in Table 2. In brief, riboprobes were generated with 50mCi [32P]UTP (Perkin-Elmer) and a Maxiscript kit (Applied Biosystems). The RPA III kit (Applied Biosystems) was used for the RPA reactions that were resolved on 5% acrylamide-16 Tris- borate-EDTA-8M urea denaturing gels. The gels were exposed to phosphor screens, and the phosphor screens were scanned using a FLA-5100 multifunctional scanner (Fujifilm). Analyses and quantitation of the RPAs were performed using the Multi-Gauge software (version 3.0, Fujifilm). In all cases, three to four biological repeats of each experiment were performed. H. pylori Fur Expression and Purification H. pylori 26695 Fur coding sequence was amplified using primers HP_Fur_expression F2 (NdeI) and HP_Fur_expression R2 (XhoI), and the PCR product was cloned into the pGEM-T easy vector (Promega) to create plasmid pDSM429. pDSM430 was created by proper digestion of pET21A (Novagen) and pDSM429 with NdeI and XhoI and ligation of the gel purified fragments. The Fur coding region in pDSM430 was sequenced to verify the construct. To prevent cross contamination of H. pylori recombinant Fur with E. coli endogenous Fur, an E. coli BL21 Rosetta Dfur strain was constructed using the Wanner method [22]. Briefly, the Kan resistance cassette was amplified from pKD4 [22] with primers Red_EC_Fur_F and Red_EC_Fur_R. This PCR product was introduced into arabinose induced E. coli K-12 carrying the pKD46 plasmid [22] to create DSM355. DSM365 was created by transduction of DSM326 with P1L4 grown on DSM355. Endogenous E. coli Fur deletion was verified by PCR. pDSM430 was introduced into DSM365 to create DSM431, which was used for rFur induction. DSM431 was grown to mid log in Luria-Bertani (EMD Chemicals) medium and then induced with 0.5 mM IPTG (isopropyl-D-thiogalactopyranoside) (Sigma) at 30uC for 3 h. The cells were disrupted using French press (Amicon) and crude extracts were prepared from the IPTG- induced cells by centrifugation (5,000 rpm for 30 minutes). Protein purification was performed by fast-protein liquid chroma- tography; the cytoplasmic protein was first passed through a HiTrap SP column for ion-exchange-based purification with a salt gradient of 25 mM to 500 mM NaCl (obtained by using buffer A [50 mM sodium phosphate, 25 mM NaCl, pH 8.0] and buffer B [25 mM sodium phosphate, 500 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]). rFur was partially concen- trated using an Amicon Ultra Centrifugal Filter Device (Millipore) to remove a portion of buffer C. Then an equal volume of EMSA binding buffer (BB) was added to the partially concentrated rFur with an additional 50% glycerol. rFur was further concentrated before being quantitated and stored at 220uC. The final concentration of the rFur stock was 2 mg/mL. Electrophoretic Mobility Shift Assays (EMSAs) A 120 bp region of the sodB promoter (encompassing the Fur- box) [1] was PCR amplified using the following template and primer pairs: WT G27 and DSM491 (‘‘25 bp swap’’) with G27 sodB EMSA-F and sodB EMSA-R and WT 26695 with 26695 sodB EMSA-F and sodB EMSA-R. To serve as a negative control sodB in H. pylori in the EMSA studies, a 142 bp region of the rpoB promoter was amplified from WT G27 using the rpoB EMSA-F and rpoB EMSA-R primer pair. Each PCR product was acrylamide gel purified and resuspended in 16 Tris-EDTA (TE) buffer. 150 ng of each promoter region was end labeled with [32P] ATP (Perkin Elmer) using T4 polynucleotide kinase (New England Biolabs) as previously described [7]. The unincorporated nucleotide was removed using the MinElute Reaction Clean-up kit (Qiagen), and labeled promoter fragments were eluted twice with 10 mL EB, and 50 mL of apo-BB was added to the eluted product. EMSAs were performed under apo (iron-free) conditions as previously described for WT 26695 sodB [1]. Briefly, 1 ng of labeled sodB or rpoB promoter was mixed with 5 mL of the following dilutions of the Fur stock: 1:1,875, 1:3,125, 1:15,625, and 1:78,125 and combined with 10 mL of 26 apo-BB (24% glycerol, 40 mM Tris, pH 8.0, 150 mM KCl, 2 mM DTT, 600 mg/mL bovine serum albumin, 200 mM EDTA, and 0.1 mg/ mL sheared salmon sperm DNA). In addition, a no protein control reaction and a 100 ng cold (unlabeled) DNA competition reaction were performed. The cold competition reaction was performed with the highest concentration of Fur (1:1,875). All reactions were allowed to incubate at 37uC for 30 min. After the incubation, the reactions were separated on a 5% polyacrylamide gel (5% 19:1 acrylamide, 16 Tris Glycine EDTA (TGE) buffer, 2.5% glycerol) for 3 hours at 70 V in 16TGE buffer. The gels were then exposed to phosphor screens and scanned on a Storm 860 scanner (GE Healthcare). Analysis was performed using ImageQuant version 5.2 software (Molecular Dynamics). Competition EMSA Studies Competition studies were performed in a manner analogous to the EMSAs. Each labeled sodB promoter fragment was combined with the 1:1,875 dilution of rFur, apo-BB, and either 5 ng, 10 ng, or 25 ng of cold (unlabeled) sodB promoter from each of the three respective strains. A no competitor control was included for each labeled sodB promoter fragment. In this manner, each labeled sodB fragment (WT G27, ‘‘25 bp swap,’’ and WT 26695) competed for binding to Fur with its own unlabeled sodB fragment as well as to that of the other two strains. The incubations, electrophoresis, and analysis were performed as described for the EMSAs. Binding competition occurs as follows: PDP32zD<PDzDP32, where P = Fur, DP32 = labeled DNA, and D = cold competitor. Thus, if the competitor promoter fragment (D) can bind to Fur (P) with a higher affinity than the labeled promoter (DP32), then an increase in the amount of unbound, labeled promoter (DP32) would be seen. The percent of unbound, labeled sodB promoter was quantitated for each competition EMSA using densitometry as a means of comparing the relative affinity of each promoter fragment for Fur. Statistical Analysis Two-tailed Student’s t-tests were performed using Microsoft Office Excel 2003. Nucleotide sequence accession number The nucleotide sequence of the sodB promoter is available from GenBank under accession number EU888136. The G27 fur sequence was previously reported [18] and is available as GenBank accession number EF537051. Results apo-Fur Regulation in H. pylori In order to study apo-Fur dependent regulation in H. pylori, the sodB and pfr promoters from strain G27 were fused to the PLoS ONE | www.plosone.org 5 April 2009 | Volume 4 | Issue 4 | e5369