sodB in H. pylori Figure 5. Role of the 25 bp in sodB regulation. WT G27, WT 26695, and the ‘‘25 bp swap’’ strain were grown as described in the Materials and Methods, and RNA was isolated under iron replete and iron-depletion shock conditions. RPAs were performed on RNA isolated from 4 biologically independent experiments using sodB and pfr riboprobes. Data from sodB RPAs are presented in Panel A, and data from pfr RPAs are presented in Panel B. Each square, diamond, triangle, and circle represent the average fold decrease calculated from three technical repeats with each independent set of RNA for each strain and growth condition combination. Median fold decrease is represented as a bar for each combination, and the dotted-dashed line represents the 2-fold significance cut-off. *p-value of 0.0001. #p-value of 0.006. doi:10.1371/journal.pone.0005369.g005 apo-Fur regulation remains a unique form of Fur regulation found only in H. pylori. Additionally, our understanding of this type of regulation is currently limited as only two apo-Fur repressed genes, sodB [1] and pfr [8], have been characterized. Here we present evidence that H. pylori shows strain specific differences in sodB apo-regulation that are partially controlled by a natural polymorphism found at the 25 bp of the sodB promoter. Alteration of this single nucleotide in the G27 promoter to resemble the residue found in 26695 resulted in alteration of G27 sodB regulation that mimicked regulation seen in 26695. Based on this observation, we accurately predicted that two other commonly used strains of H. pylori, J99 and HPAG1, would show altered sodB regulation since they each encode a different nucleotide at the 25 position within the sodB promoter. The importance of the 25 bp within the sodB promoter is further supported by our EMSA competition data. At low concentrations of competitor DNA, the ‘‘25 bp swap’’ promoter is able to bind to apo-Fur with an affinity similar to WT 26695 while WT G27 exhibits weaker binding. At higher concentrations of competitor, the affinity of the ‘‘25 bp swap’’ promoter for apo- Fur is still greater than WT G27 but slightly less than WT 26695. Thus, it appears that strain specific regulation of sodB is due to differences in the affinity of Fur for the various promoters and that natural polymorphisms at the 25 bp are largely responsible for this differential regulation. The significance of the sodB polymorphism in H. pylori fitness, especially in vivo, is currently unclear. However, the affinity of apo- Fur for the sodB promoter in 26695 was reported to be relatively weak (Kd = 260 nM) [1], and based upon our competition data it is likely even weaker in G27. As Ernst, et al. suggested, a weak affinity between apo-Fur and the sodB promoter makes physiolog- ical sense, as SodB is the only defense H. pylori has against superoxide radical damage [1,10]. Therefore, it would be ill- advised to repress sodB under conditions where any iron is still PLoS ONE | www.plosone.org 10 April 2009 | Volume 4 | Issue 4 | e5369