Figure 4. Strain specific differences in sodB regulation. Various H. pylori strains were grown to exponential phase as described in the sodB in H. pylori Materials and Methods, and RNA was isolated from iron replete and iron-depleted shock conditions. RPAs were performed using sodB, pfr, and fur riboprobes and results are displayed in Panels A, B, and D, respectively. Basal levels of fur expression relative to the level of expression in 26695 are depicted in Panel C. Fold decrease in expression for sodB and pfr, fold increase for fur, and relative levels of basal fur expression are plotted as single points for each strain with squares, diamonds, triangles, and circles. Each shape represents a biologically independent set of RNA. Median fold change is represented as a bar for each strain. The dotted-dashed line represents the 2-fold significance cut-off in Panels A, B, and D. In Panel A only, the triangles represent the average of two technical repeats on that independent set of RNA. doi:10.1371/journal.pone.0005369.g004 suggest that natural polymorphisms found at the 25 bp of the sodB promoter in different H. pylori strains affect the regulation of sodB by apo-Fur. In vitro binding of Fur to different sodB promoters Given that the 25 bp in the sodB promoter appears to play some role in the apo-Fur regulation of sodB, we next investigated the direct interaction of apo-Fur with the various sodB promoters. To assay the binding of apo-Fur, we performed Electrophoretic Mobility Shift Assays (EMSAs) and competition studies for each sodB promoter (WT G27, ‘‘25 bp swap,’’ and WT 26695) using purified Fur under apo reaction conditions [1]. As shown in Fig. 6, Fur binds to and retards the mobility of each of the three sodB promoters, but not the control rpoB promoter. Moreover, the addition of homologous unlabeled sodB promoter DNA was able to compete for Fur binding with each sodB promoter thus confirming specific interaction between Fur and the sodB promoters (Fig. 6). Because apo-Fur was able to bind to and shift each of the three sodB promoter fragments and because our expression data showed that the 25 bp was important for regulation, we reasoned that the various promoter fragments should show differences in their affinity for Fur. To test this, each labeled sodB promoter fragment was competed with varying concentrations of its own (homologous) unlabeled promoter fragment as well as with each of the other unlabeled promoter fragments. The success of the competition was then measured by quantitating the percent of unbound probe resulting from each competition reaction such that PDP32zD<PDzDP32, where P = Fur, DP32 = labeled DNA, and D = cold competitor. As shown in Fig. 7, the various promoter fragments showed differences in affinity such that 26695$25 bp.G27. In all cases, the 26695 and 25 bp promoter were better able to compete for Fur binding as the largest percentages of unbound labeled promoter fragment are observed with these two promoters in comparison to the WT G27 sodB promoter. Taken together with the expression data, these data indicate that the 25 bp is important for Fur interaction at the sodB promoter. Discussion Given how pleomorphic H. pylori is, it is not surprising that genes may be regulated differently in different strains. Indeed, there have been several instances of this reported in the literature in recent years involving acid-response and CrdRS [27], vacA regulation [28], virulence gene regulation in vivo [29], and cagA and vacA expression in response to salt [30]. In addition, a single nucleotide polymorphism upstream of the Fur-box was found to alter Fur regulation of IrgA in two different strains of E. coli [31] indicating that there may be more to Fur regulation in other organisms than just binding at the recognition sequence. This study adds to that body of knowledge and is the first to explore the differences in Fur regulation among different strains of H. pylori. PLoS ONE | www.plosone.org 9 April 2009 | Volume 4 | Issue 4 | e5369