2a). oxyR::CAT (chromosomal oxyR::CAT, mtoxyR+) showed a significant increase in CAT activity in response to both H2O2 and menadione (P= 0.005 and P= 0.009 respectively) while oxyR::CAT/rpoS− (chromosomal oxyR::CAT, moxyR+, rpoS) showed both a significantly lower basal amount (P= 0.022) and no induction of CAT expression Trametinib in response to pro-oxidants. Strain oxyR::CAT/rpoS−/RpoS, which

contains an isogenic replacement of rpoS, showed both a restored basal amount of CAT activity as well as induction of CAT activity in response to pro-oxidants. Collectively these results show that rpoS expression is required for the oxidative stress induction of OxyR. Our data therefore shows that expression of MAPK Inhibitor Library oxyR requires RpoS under both normal growth conditions and conditions of oxidative stress. Interestingly, catalase I, encoded by katG, has been shown to be repressed

by OxyR during normal growth and to be activated by OxyR during oxidative stress (6) as well as being regulated by RpoS (8). To further understand the interaction between OxyR, RpoS and katG, the B. pseudomallei strain katG::CAT (6) which has a chromosomal katG::CAT fusion, was used to generate three further strains containing katG::CAT and deletion of either oxyR (strain katG::CAT/oxyR−) or rpoS (strain katG::CAT/rpoS−) or deletion Avelestat (AZD9668) of both oxyR and rpoS (strain katG::CAT/oxyR−/rpoS−). The basal extent of expression of CAT during the mid-exponential growth phase was increased between 2- and 3-fold in the oxyR (katG::CAT/oxyR−), rpoS (katG::CAT/rpoS−) and oxyR-rpoS (katG::CAT/oxyR−/rpoS−) mutants as compared with the katG::CAT parental strain (Fig. 2b, black bars). A similar pattern was also observed in late log-phase cells of the mutants as compared to the wild-type strain (data not shown). These results suggest that both OxyR and RpoS repress katG transcription under normal growth

conditions and in the absence of oxidative stress. To understand if oxyR and rpoS are required for the induction of katG by pro-oxidants, katG expression was measured in the presence of oxidants in the parental strain, and in the single and double rpoS and oxyR mutants, as before. In the parental strain (katG::CAT) there were 5- and 3.5-fold inductions in CAT concentrations by 0.5 mM hydrogen peroxide and menadione, respectively (Fig. 2b). In contrast, the mutants without OxyR or RpoS or both failed to induce katG gene expression (Fig. 2b). From these results, it can be concluded that both OxyR and RpoS are required for the repression of katG during non-oxidative growth conditions, and the induction of katG expression during oxidative stress conditions. Similarly, the expression of dpsA in B. pseudomallei has been reported to be regulated by OxyR (10).