26695 Fur in G27 (the ‘‘Fur swap’’ strain) did not restore apo-Fur sodB regulation in G27 under either iron-limited growth or iron- depletion shock conditions (Fig. 4A and data not shown). However, apo-Fur regulation of pfr was as expected in all three strains (Fig. 4B and data not shown) [8]. Because the trends of the growth data for both the sodB and pfr RPA data were similar to the shock, the growth data has not been shown. While the AA difference in Fur was apparently not responsible for the difference in sodB regulation, we wondered if the levels of fur expression were similar between the different strains. To test this, RPAs were performed on RNA isolated from all three strains using a fur riboprobe. The basal level of fur expression in each strain was then compared to that of WT 26695 as shown in Fig. 4C. While the level of fur expression in the G27 strain was slightly higher than in 26695, no substantial differences in fur expression were found between the strains. As Fur has been shown to be autoregulatory, repressing its own expression in the presence of iron [25,26], we also compared Fur autoregulation between G27, 26695, and the ‘‘Fur swap’’ strain. fur RPAs were performed on RNA isolated from each strain, and an increase in fur expression was seen for G27, 26695, and the ‘‘Fur swap’’ strain under iron-depletion shock conditions while little to no increase was seen under iron-limited growth conditions (Fig. 4D and data not shown). This data shows that Fur autoregulation is consistent in each strain and further supports the notion that the AA difference in Fur is not responsible for the difference in sodB regulation between G27 and 26695. RPA determination of the role the 25 bp of the sodB promoter plays in sodB regulation Since the difference in sodB regulation between G27 and 26695 appeared not to be related to the difference in the Fur coding sequence, we next considered that there might be differences in the sodB promoter between the strains that could account for the discrepancy in regulation. Therefore, we sequenced the sodB promoter from G27 and compared it to the known sodB promoter sequence from 26695 [14]. As shown in Fig. 3B, a single base change was evident in the Fur Box. Previous DNA Footprint analysis showed that Fur protects a region that extends from sodB in H. pylori 25 bp to 247 bp within the sodB promoter [1]. At the 25 bp, G27 encodes a C while 26695 encodes an A. To determine if this nucleotide difference was important for sodB regulation, a ‘‘25 bp swap’’ strain was engineered such that the G27 promoter would encode an A at the 25 bp position. RPAs were then conducted on RNA isolated from the ‘‘25 bp swap’’ strain along with WT G27 and WT 26695, and results are shown in Fig. 5. While sodB expression remained unchanged in G27 under iron depletion shock conditions, a two-fold decrease in sodB expression was observed in the ‘‘25 bp swap’’ strain (Fig. 5A). The difference in fold decrease between G27 and the ‘‘25 bp swap’’ was statistically significant with a p-value of 0.006, as was the difference between G27 and 26695 with a p-value of 0.0001. While the fold decrease in sodB expression in the ‘‘25 bp swap’’ strain under iron-limited growth conditions did not reach 2-fold, it was consistently higher than its G27 counterpart (data not shown). apo-Fur regulation of pfr in each of these strains was similar and as expected [8] (Fig. 5B and data not shown). These data suggest that a single nucleotide difference within the sodB promoter is at least partially responsible for the difference in regulation of this gene between G27 and 26695. Comparison of sodB regulation in various strains of H. pylori Given the differences in sodB regulation in G27 and 26695, we wondered if other H. pylori strains exhibited apo-Fur regulation similar to G27 or 26695. Therefore, we also examined J99 and HPAG1. Analysis of the sodB promoter sequences of these two additional strains showed that at the 25 bp HPAG1 encodes a C similar to G27, and J99 encodes a G that is different from all other strains (Fig. 3B). Given that the A at the 25 bp seems to be crucial for apo-Fur regulation of sodB, we predicted that these strains would show Fur regulation of sodB similar to what was seen with G27. To test this, RPAs were performed on RNA isolated from J99 and HPAG1. As shown in Fig. 4, neither J99 nor HPAG1 displays the expected decrease in sodB expression [1]; both behave similarly to G27 (Fig. 4A). However, pfr expression (Fig. 4B), basal levels of fur expression (Fig. 4C), and fur autoregulation (Fig. 4D) are preserved in J99 and HPAG1. Taken together, these data Figure 3. Alignments of Fur and of the sodB promoters. Panel A contains the alignment of the predicted Fur amino acid sequences of G27 and 26695. As indicated by an arrow, amino acid 150 is different between the two strains. Panel B contains the sodB promoter alignment from G27, 26695, J99, and HPAG1 with essential promoter elements indicated. The predicted Fur Box ranges from bases 25 to 247 and is indicated by the dashed box [1]. The 25 bp difference between the strains is indicated with an arrow in Panel B. Alignments for both panels were constructed using MultAlin software [37]. doi:10.1371/journal.pone.0005369.g003 PLoS ONE | www.plosone.org 8 April 2009 | Volume 4 | Issue 4 | e5369