The treatment of Xcc cultures with 50 mM H2O2 for 30 min resulted in approximately 10% survival (Fig. 2). The addition of CuSO4 (100 μM) to the H2O2 killing mixture was highly lethal to cells and reduced the per cent survival to 0.05% (Fig. 2). The synergistic
effect of CuSO4 and H2O2 was abolished when a Cu chelator (200 μM bathocuproine sulphonate) was added to the cell suspension before the combined treatment of CuSO4 and H2O2 (Fig. 2). This observation suggests the possibility that an elevated level of Cu ions could react with H2O2 to produce hydroxyl radicals, which lead to increased cell death. This speculation was supported by experiments in which the addition of hydroxyl scavengers DMSO (0.4 M) and glycerol (1.0 M) to bacterial cultures, before treatment with CuSO4 and H2O2, significantly protected bacterial cells from the killing effects (Fig. 2). We Selleck NVP-BEZ235 then determined whether lipid peroxidation contributes to CuSO4 and H2O2 toxicity. The ability of α-tocopherol (1 mM) to reduce the lethal effects of CuSO4 and H2O2 treatment was tested. As illustrated in Fig. 2, α-tocopherol was unable to alleviate CuSO4 and H2O2 this website killing. The evidence indicates that Cu ions potentiate H2O2 toxicity in a manner different
from tBOOH. While lipid peroxidation is a major factor responsible for the Cu ion-mediated enhancement of tBOOH toxicity, hydroxyl radicals likely account for Cu ion-dependent H2O2 toxicity. Alkyl hydroperoxide reductase, encoded by ahpC, is a member of the peroxiredoxin enzyme family. AhpC not only plays a role in the detoxification of organic hydroperoxides by converting them to their corresponding alcohols, but the enzyme is also necessary for the degradation of endogenously generated H2O2 due to its much lower kcat/Km almost compared with catalase (Seaver & Imlay, 2001). Thus, the ahpC mutant accumulates intracellular H2O2 and organic
hydroperoxides produced as byproducts of normal aerobic metabolism (Seaver & Imlay, 2001; Charoenlap et al., 2005; Wang et al., 2006). If Cu toxicity is partly due to the stimulation of oxidative stress production, we would expect that the Cu resistance level in the ahpC mutant might be altered. An Xcc ahpC mutant was constructed using the pKNOCK system (Alexeyev, 1999). The ahpC mutant was more sensitive to tBOOH killing treatment than the wild-type Xcc (data not shown). The Cu resistance of the ahpC mutant was measured using a killing assay (Sukchawalit et al., 2005), and the results showed that the mutant was more than 10-fold more sensitive to CuSO4 (1 mM) than the wild-type Xcc (Fig. 3). The ectopic expression of ahpC from the expression plasmid, pAhpC, complemented the CuSO4-sensitive phenotype of the ahpC mutant (Fig. 3, ahpC/pAhpC). The lack of a functional ahpC rendered Xcc vulnerable to elevated levels of CuSO4.