available, since iron catalyzed oxidative damage could still be possible [1]. In keeping with this, some strains of H. pylori may have evolved to either inactivate apo-Fur regulation of sodB, or to weaken repression by decreasing the Fur/sodB binding affinity. Also of note, as shown in Fig. 2, in the absence of Fur, iron chelation results in slight increases in sodB (and pfr) perhaps suggesting the presence of additional regulatory proteins that ensure proper expression of this critical factor. sodB in H. pylori Furthermore, it is interesting to speculate that strains, which possess sequences similar to 26695, might actually show decreased in vivo fitness due to decreased expression of sodB in the iron limited environment of the stomach. Analysis of the sodB promoter sequence in the efficient gerbil colonizing strain B128 (isolate 7.13) [32] revealed that B128, similar to G27, encodes a C at the 25 bp (data not shown). Therefore, studies could potentially be designed with this strain that would allow for the determination of whether direct apo-Fur regulation of sodB provides a competitive advantage to H. pylori in vivo. Currently, little is understood about the sequences recognized by H. pylori Fur that dictate binding of the protein at target promoters. This is true of both iron-bound and apo forms of Fur. In E. coli, Fur binding has been shown to involve recognition of a well-conserved consensus sequence called a Fur Box. This Fur Box consists of two Figure 6. Fur binding to the sodB promoters. EMSAs were performed by incubating various concentrations of purified Fur with radiolabeled fragments of the WT G27, ‘‘25 bp swap,’’ and WT 26695 sodB promoters as well as the negative control promoter, rpoB, as detailed in the Materials and Methods. In the first four lanes, the Fur concentrations are indicated by the triangle from highest to lowest and range from 1.07 mg/mL to 0.026 mg/mL. A no protein control for each promoter is found in the fifth lanes. The last lane shows the 1006 cold (unlabeled) competition control for each promoter fragment, which were each performed with the highest concentration of Fur (1.07 mg/ mL). Fur exhibits specific interaction with each of the sodB promoters, and no interaction with the rpoB promoter except for very little non- specific binding at the highest Fur concentration. These data are representative of multiple independent EMSA experiments. doi:10.1371/journal.pone.0005369.g006 Figure 7. Competitive Binding Studies. To assess the relative affinity of Fur for each of the sodB promoter fragments (WT G27, ‘‘25 bp swap,’’ and WT 26695), Fur was incubated with each radiolabeled promoter and 56, 106, or 256 the amount of homologous or heterologous unlabeled sodB promoter fragments as described in the Materials and Methods. For each labeled promoter, lane one contains a no competition control. Lanes two to four, five to seven, and eight to ten contain the competition EMSAs with unlabeled WT G27, ‘‘25 bp swap,’’ and WT 26695 sodB fragments, respectively. The percent of labeled promoter that is outcompeted and remains unbound in each lane is given below each image. These data are representative of multiple independent experiments. doi:10.1371/journal.pone.0005369.g007 PLoS ONE | www.plosone.org 11 April 2009 | Volume 4 | Issue 4 | e5369