previously [18]. Briefly, DSM238 was grown overnight in liquid culture with and without the iron chelator, 2,29-dipyridyl (dpp) (Sigma) at a final concentration of 60 mM, and DSM308 was grown overnight in the absence of chelator. As a comparison, the previously characterized strains, DSM369 and DSM370, which bear pfr (nonheme iron-containing ferritin) transcriptional fusion plasmids in WT and Dfur G27, respectively, were grown in the same manner [18]. Following overnight growth, 1.5 ml of each culture were pelleted and resuspended in 1 ml of sterile 16 phosphate-buffered saline. Bacterial clumps and culture debris were subsequently removed by passing the resuspended culture through a 1.2-mm Acrodisc PSF syringe filter (Pall). Flow cytometry analysis was performed using a Beckman Coulter Epics XL-MCL flow cytometer with a laser setting of 750 V for the pfr fusion construct and 900 V for the sodB fusion construct. 100,000 events were collected for each assay. WinList 3D, version 6.0 (Verity Software House) was used to analyze the flow cytometry data. Creation of the ‘‘Fur swap’’ Strain To exchange the fur coding sequence, we first created a G27 strain containing the counter-selectable kan-sacB cassette previously described by Copass, et al [19]. This cassette contains the sacB gene from Bacillus subtilis, which confers Suc sensitivity and is expressed under the control of the flaA promoter of H. pylori, and the aphA3 gene from Campylobacter coli, which confers Kan resistance. A 340 bp region upstream of the G27 fur coding sequence was PCR amplified using primers FurCF1 [18] and HpUKanSacR, and a 339 bp region downstream of the fur coding sequence was PCR amplified using primers HpDKanSacF and HpDKanSacR. HpUKanSacR and HpDKanSacF were designed to incorporate XhoI and SmaI restriction endonuclease sites. Each of these products were purified and mixed in a Splicing by Overlap Extension (SOE) PCR reaction using the FurCF1 and HpDkanSacR primers. The resulting 679 bp product was subcloned into pGEM-T Easy creating pDSM386. The kan-sacB cassette was liberated from pKSF-II [19,20] by sequential double digestion with XhoI (New England Biolabs) and SmaI (New England Biolabs), and this fragment was ligated to the appropri- ately digested pDSM386 to create pDSM387. This plasmid was naturally transformed into WT G27, and transformants were selected on HBA plates containing Kan. Double crossover homologous recombination of pDSM387 with the WT chromo- some results in the complete deletion of the fur (HP1027) coding sequence and replacement with the upstream fur-kan-sacB- downstream fur product. The resulting transformants were patched on 5% Suc HBA plates to ensure Suc sensitivity, and proper integration into the chromosome was confirmed by PCR with sacBSCN-F2 and HpKanSacSCN-R primers, which lie within the sacB gene and downstream of fur, respectively. One such transformant was named DSM391. To create the ‘‘Fur swap’’ strain, a 923 bp product of the H. pylori 26695 genome was amplified using the FurCF and FurCR primers. This product, which includes the fur coding sequence and a portion of the upstream and downstream regions, was purified and naturally transformed into DSM391. Transformants were selected on 5% Suc HBA plates and patched onto Kan HBA plates to ensure Kan sensitivity. Double crossover homologous recombination resulted in the replacement of the kan-sacB cassette with the fur coding sequence of 26695, and this strain was named DSM403. Proper integration was confirmed by PCR with the FurCF and FurCR primers and by sequencing with the FurCR primer. DSM403 expresses 26695 fur from the native fur locus in a G27 strain background. sodB in H. pylori Creation of a ‘‘25 bp swap’’ mutation in the sodB promoter The sodB promoter from G27 was sequenced using primers USod-F and DSod-R and compared to the known sequence of the sodB promoter from 26695 [14]. This comparison revealed a single base pair (bp) difference within the predicted Fur Box [1] at the 25 position relative to the start of transcription. The ‘‘25 bp swap’’ mutation within the sodB promoter of G27 was created using SOE PCR and the kan-sacB cassette from pKSF-II. A 297 bp region upstream and a 329 bp region downstream of sodB were PCR amplified from G27 using primer pairs USod-F and USod-R and DSod-F and DSod-R, respectively. USod-R and DSod-F contain XhoI and SmaI restriction endonuclease sites to allow for the directional cloning of the kan-sacB fragment. The upstream and downstream products were purified and mixed in a SOE PCR reaction with the USod-F and DSod-R primers. The resulting 626 bp SOE PCR product was subcloned into pGEM-T Easy to create pDSM469. pDSM469 and pKSF-II were each sequentially double digested with XhoI and SmaI, and the resulting fragments were ligated to create pDSM475. pDSM475 was naturally transformed into WT G27, and transformants were selected on Kan and then patched to verify sucrose sensitivity. Double crossover homologous recombination of pDSM475 into the G27 chromo- some results in the deletion of the sodB gene and replacement with the kan-sacB cassette. The resulting Kan resistant, sucrose sensitive strain, DSM480, was confirmed by PCR with sacBSCN-F2 and HpsodBSCN-R primers, the latter of which lies downstream of sodB. The 25 bp in the G27 sodB promoter was mutated from a C to an A using SOE PCR. First, primers USod-F and SodBMt-R were used to PCR amplify upstream of the sodB promoter through to the 25 bp and incorporate the C-5A mutation. Second, primers DSod-R and SodBMt-F were used to PCR amplify from the 25 bp through to downstream of the sodB gene and to incorporate the C-5A mutation. These products were purified and combined in SOE PCR reaction using the USod-F and DSod-R primers. The resulting SOE PCR product was sublconed into pGEM-T Easy. The subcloned sodB 25 bp promoter mutation construct was designated pDSM481 and was confirmed by sequencing with the USod-F and DSod-R primers. pDSM481 was naturally transformed into DSM480 to integrate the sodB 25 bp promoter mutation into the chromosome in place of the kan-sacB cassette. Transformants were selected as detailed above for the creation of DSM403. The resulting Suc resistant, Kan sensitive strain was named DSM491. Proper recombination was confirmed by PCR with the USod-F and DSod-R primers (yielding a 1,262 bp fragment) and by sequencing with both of those primers. DSM491 expresses sodB with the C-5A mutation from its native locus within the G27 chromosome. RNase protection assays (RPAs) RPAs were utilized to characterize apo-Fur regulation of sodB in various strains of H. pylori. Two normal (iron replete) media cultures were started for each strain, one for exponential and one for stationary growth phase. Following overnight growth, one half of each exponential phase culture was removed for RNA isolation. To the remaining half of the iron-replete exponential phase cultures, 200 mM dpp (final concentration) was added to create an iron-depleted shock condition. Those cultures were grown for an additional hour prior to RNA isolation. The iron-replete stationary phase cultures were grown for an additional night, and on the following morning one half of the culture was removed for RNA isolation while the other was exposed to 200 mM dpp for an additional hour before RNA isolation. In addition, one culture for each strain was grown in iron limited media (60 mM dpp). After overnight growth, one-half of each culture was removed for RNA isolation in exponential phase. The remaining half of the PLoS ONE | www.plosone.org 4 April 2009 | Volume 4 | Issue 4 | e5369