pylori that is not directly linked to iron metabolism, but is involved with the oxidative stress response, is superoxide dismutase (sodB) [1]. SodB was first identified in H. pylori in 1993 and was shown to be iron co-factored like the Escherichia coli FeSod with 53.5% identity between the two proteins [9]. However, unlike E. coli FeSod, which is localized within the cytosol of the bacterium, H. pylori SodB is associated with the cell surface [9]. SodB is the only identified Sod in H. pylori and has been shown to be critical for survival in vivo [10]. Also, sodB deficient mutants are more sensitive to O2 as well as exhibit a higher rate of spontaneous mutation [10,11]. Interestingly, H. pylori sodB mutants have been shown to harbor more free iron within their cells than WT bacteria [11]. Globally, Sods are responsible for combating oxidative stress (both internal and external) by converting superoxide radicals into hydrogen peroxide and oxygen. Superoxide radicals are formed as a by-product of metabolism and, if left unchecked, can react with ferric iron (Fe3+) to form hydrogen peroxide, which in turn feeds the Fenton Reaction [12] and is detrimental to the cell. Sods prevent the interaction of iron and superoxide radicals as well as block the formation of hydroxyl radicals from hydrogen peroxide [12]. In this way, the role of Fur as the primary regulator of iron uptake and the role of SodB as the primary defense against superoxide radicals in H. pylori are linked. In keeping with this, sodB has been shown to be regulated by apo-Fur such that it is repressed under circumstances where iron is severely limited [1]. This regulation appears to be direct since Electrophoretic Mobility Shift Assays showed that Fur specifically binds to the sodB promoter in the absence of iron [1]. Herein we describe a series of experiments that define a single polymorphic nucleotide within the H. pylori sodB promoter that is important for apo-Fur dependent regulation. Moreover, we show that alterations in this single base result in strain specific responses to iron limitation. Materials and Methods Bacterial strains and growth Strains and plasmids used in this study are listed in Table 1, and primer sequences are listed in Table 2. Strains of H. pylori were maintained as frozen stocks at 280uC in brain heart infusion broth (BD) supplemented with 10% fetal bovine serum (Gibco) and 20% glycerol (EMD Chemicals, Inc.). Bacterial strains were grown on horse blood agar (HBA) plates which contained 4% Columbia agar base (Neogen Corporation), 5% defibrinated horse blood (HemoStat Laboratories, Dixon, CA), 0.2% b-cyclodextrin (Sigma), 10 mg/ml vancomycin (Amresco), 5 mg/ml cefsulodin (Sigma), 2.5 U/ml polymyxin B (Sigma), 5 mg/ml trimethoprim (Sigma), and 8 mg/ml amphotericin B (Amresco). Liquid cultures of H. pylori were grown in brucella broth (Neogen Corporation) supplemented with 10% fetal bovine serum and 10 mg/ml vancomycin at 37uC with shaking at 100 rpm. As noted in Table 1, where appropriate, cultures and plates were supplement- ed with 8 mg/ml chloramphenicol (Cm) (EMD Chemicals, Inc.) and/or 25 mg/ml kanamycin (Kan) (Gibco). In addition, where detailed in the Materials and Methods, some HBA plates contained 5% sucrose (Suc) (Sigma). Both liquid and plate cultures were grown under microaerophilic conditions (5% O2, 10% CO2, and 85% N2) generated with an Anoxomat gas evacuation and replacement system (Spiral Biotech) in gas evacuation jars. H. pylori strains used in this study are all derivatives of G27 [13] and 26695 [14,15], with the exception of WT H. pylori J99 [16] and HPAG1 [17]. A fur (HP1027) mutant of G27, DSM300, was utilized in this work and contains a deletion insertion of the fur coding sequence with the cat gene from Campylobacter coli conferring Cm resistance as previously described [18]. This DHP1027::cat H. pylori strains pDSM236 pDSM368 pKSF-II pDSM386 pDSM387 pDSM469 pDSM475 pDSM481 pDSM429 pDSM430 pKD4 pKD46 G27 DSM300 26695 DSM357 DSM238 DSM308 DSM369 DSM370 DSM391 DSM403 DSM480 DSM491 J99 HPAG1 DSM328 DSM355 DSM326 DSM365 DSM431 E. coli strains doi:10.1371/journal.pone.0005369.t001 construct was also naturally transformed into 26695 to create an analogous fur mutation in this strain background and is called DSM357. Exponential phase cultures were grown for 20 hrs, and stationary phase cultures were grown for 44 hrs. Creation of the sodB promoter fusion plasmid A transcriptional fusion of the sodB (HP0389) promoter to the promoterless gfpmut3 on the transcriptional reporter plasmid, pTM117, was constructed as previously described [18]. Briefly, the sodB promoter of WT G27 was PCR amplified using sodB-F1 and sodB-R1 primers, which incorporate SacII and BamHI restriction sites, respectively. The resulting PCR fragment was subcloned into pGEM-T Easy (Promega) and digested with SacII (New England PLoS ONE | www.plosone.org 2 April 2009 | Volume 4 | Issue 4 | e5369 sodB in H. pylori Table 1. Plasmids and strains used in this study. Plasmid or strain Description Plasmids pTM117 Modified pHP666 to include E. coli origin and rop gene, aphA-3 cassette (Kanr), multiple cloning site, and a promoterless gfpmut3 gene pTM117 sodB promoter::gfpmut3fusion This study pTM117 pfr promoter::gfpmut3fusion pEK::kan-sacB pGEM-T Easy::Dfur pGEM-T Easy::Dfur::kan-sacB pGEM-T Easy::DsodB pGEM-T Easy::DsodB::kan-sacB pGEM-T Easy::sodB C-5A pGEM-T Easy::26695 fur pET21A::26695 fur kan template plasmid Red recombinase expression plasmid WT H. pylori G27 Dfur::cat, Cmr WT H. pylori 26695 Dfur::cat, Cmr G27 (pDSM236), Kanr DSM300 (pDSM236), Kanr Cmr G27 (pDSM368), Kanr DSM300 (pDSM368), Kanr Cmr G27 Dfur::kan-sacB, Kanr Sucs G27, fur 26695, Sucr Kans G27 DsodB::kan-sacB, Kanr Sucs G27 sodB C-5A, Sucr Kans WT H. pylori WT H. pylori K12 (pKD46), Ampr, Temps K12 Dfur, Kanr BL21 DE3 Rosetta/pLysS, Cmr BL21 DE3 Rosetta/pLysS Dfur, Kanr, Cmr BL21Dfur (pDSM430) Ampr, Cmr, Kanr Reference [18] [18] [19,20] This study This study This study This study This study This study This study [22] [22] [13] [18] [14,15] This study This study This study [18] [18] This study This study This study This study [16] [17] [22] This study This study This study This study