sodB in H. pylori Figure 2. Direct Comparison of sodB Regulation in H. pylori Strains G27 and 26695. WT and Dfur strains of G27 and 26695 were grown to exponential (A) and stationary (B) phase in iron replete and iron-limited (growth) media (60 mM dpp). After growth overnight, one-half of the exponential phase, iron replete culture was removed for RNA isolation. 200 mM dpp (final concentration) was added to create an iron-depletion shock condition to the remaining half of the iron replete cultures, and those cultures were grown for an additional hour prior to RNA isolation. The same procedure was applied the following day to the iron replete, stationary phase culture. After overnight growth, one-half of the iron-limited growth culture was removed for RNA isolation in exponential phase while the remaining half was allowed to grow into stationary phase, and RNA was isolated the following day. RNase Protection Assays (RPAs) were performed on RNA isolated from these strains using sodB, pfr, and amiE riboprobes. Data for Exponential phase cultures are shown in Panel A, and data for Stationary phase cultures are shown in Panel B. Fold-changes are indicated below each pair and were calculated by comparing either the relative amount of protected riboprobe in the iron-depletion shock environment (S) or the relative amount of protected riboprobe in the iron limited growth environment (G) to the iron replete lane (N). These data are representative of multiple independent experiments. doi:10.1371/journal.pone.0005369.g002 conditions (Fig 2A and 2B). Furthermore, iron-bound Fur regulation of amiE was as expected [24] for both G27 and 26695; amiE expression was increased under both iron limited conditions (Fig. 2A and 2B). Taken in total, these data indicate that apo-Fur regulation of sodB is altered in G27 as compared to 26695. Analysis of the role an amino acid (AA) difference in Fur plays in sodB regulation Given the difference in sodB regulation between the two strains, we reasoned that either a difference in Fur or a difference in sodB between the two strains was likely to be responsible for the change. We therefore aligned the predicted Fur amino acid sequence from G27 and 26695 to determine if there were any obvious differences between the two strains that might account for the differences in sodB regulation. As shown in Fig. 3A, the last AA was found to differ between the strains. In G27 AA 150 is a Tyr while in 26695 it is a Cys. To determine if this AA difference had any role in Fur- dependent regulation of sodB, a ‘‘Fur swap’’ strain was created, which completely replaced the G27 fur coding sequence with the coding sequence from 26695. RPAs were then conducted on RNA harvested from WT G27, WT 26695, and the ‘‘Fur swap’’ strain. Results are shown in Fig. 4. In order to show the reproducibility of the data, RPA data is represented in a graphical format. In this manner the fold change for each strain and biological repeat is displayed as a point on the graph. Additionally, the median fold change is depicted as a bar to allow for easy comparison between the strains. Because the decrease in sodB expression in 26695 is most pronounced in exponential phase, only results of RPAs performed using exponential phase RNA are shown. Expressing PLoS ONE | www.plosone.org 7 April 2009 | Volume 4 | Issue 4 | e5369