promoterless gpfmut3 gene in pTM117. Currently, these promoters represent the only known targets of apo-Fur [1,8]. Given this apo- regulation and since promoter activity can be measured by changes in fluorescence with our system, we expected to see a decrease in GFP fluorescence under iron limited conditions for both promoter fusions. However, as shown in Fig. 1A, the addition of iron chelator resulted in no change in the level of sodB expression. This is in contrast to pfr, where iron depletion resulted in strong repression of pfr expression (Fig. 1B). Both sodB and pfr were upregulated in a fur mutant (Fig. 1A and 1B) suggesting that both genes are repressed by Fur. However, the lack of responsiveness to iron chelation suggested that sodB apo-regulation is not as expected in G27. Since apo-Fur has been shown to have a lower affinity for the sodB promoter than the pfr promoter, and since the gfpmut3 allele encodes a long-lived GFP variant [23], we reasoned that we might not be able to detect small changes in GFP expression under the control of the sodB promoter under iron limited conditions. Therefore, we performed RPAs to further investigate the discrepancy between our results and results previously reported for sodB regulation in strain 26695 [1]. Additionally, we considered the fact that strain specific differences might be responsible for the discrepancy. Therefore, RPAs using a sodB riboprobe were performed on RNA isolated from WT and Dfur derivatives from both G27 and 26695. pfr and amiE (aliphatic amidase) riboprobes were also used as control apo-Fur and iron-bound Fur regulated target genes, respectively. Fig. 2A shows results for all three riboprobes using RNA isolated from exponential phase cultures. Again, we observed that for G27 the level of sodB expression did sodB in H. pylori not change under iron-limited growth conditions (G) or under a harsher iron-depletion shock condition (S) that was added to ensure robust chelation as compared to normal (N) iron replete conditions. Examination of sodB expression in 26695 revealed a smaller protected fragment than originally expected. However, sequence analysis revealed that the smaller fragment is due to a small region of mismatch between the sodB mRNA sequence in 26695 and the G27 template DNA used to generate the riboprobe. This mismatch causes a bubble of single stranded RNA to form and thus is subjected to RNase cleavage in the region of mismatch (data not shown). For WT 26695, a 2-fold decrease in sodB expression was achieved under both iron-limited growth and iron- depletion shock conditions, which agrees with the previous report [1]. This change is Fur-dependent as there is no change in sodB expression under either iron depletion condition in the absence of fur. Since it has been shown that growth phase strongly affects gene expression in H. pylori [21], we performed similar experiments on RNA harvested from stationary phase cultures. As shown in Fig. 2B, we obtained identical results with the exception that the fold decrease seen in sodB expression was less pronounced in 26695 in this growth phase. Again, there was no decrease in sodB expression in G27, indicating that growth phase is not responsible for the differences in our results. Moreover, the difference in sodB regulation between the two strains is not the result of a generalized difference in apo-Fur regulation between G27 and 26695 since the appropriate decrease in pfr expression [8] was observed in both strains under iron-limited growth and iron-depletion shock Figure 1. Flow Cytometry analysis of sodB and pfr GFP reporters. Strains bearing sodB::gfpmut3 or pfr::gfpmut3 promoter fusions were grown overnight in either iron replete or iron depleted media. Changes in fluorescence were analyzed as described in the Materials and Methods. Results for the sodB promoter fusions are displayed in Panel 1A, and results for the pfr promoter fusions are displayed in Panel 1B. For both A and B, solid lines indicate the plasmid in WT H. pylori G27 grown in iron replete conditions, dotted lines indicate the plasmid in WT bacteria grown in iron deplete conditions, and dashed lines indicate the plasmid in Dfur bacteria grown in iron replete conditions. Fluorescence is measured in relative units, and the data are representative of multiple independent flow analyses. doi:10.1371/journal.pone.0005369.g001 PLoS ONE | www.plosone.org 6 April 2009 | Volume 4 | Issue 4 | e5369