them. In consistent with the idea that most of the added H2O2 is consumed by catalase and other peroxidases, Imlay and colleagues have shown that, by using hydroperoxidase minus (Hpx ) strains of E. coli, simply shifting cells from anaerobic to aerobic growth conditions leads to sufﬁcient H2O2 production to impede growth, due mainly to DNA damage (42,43). It seems likely that, complementary to the known anti-oxidizing mechanisms, PT modiﬁcation, enhance the innate anti-oxidation capability of the hosting bacteria by protecting DNA against peroxide. It is also possible that the PT modiﬁcations may be quickly replaced when Figure 3. Consumption of H2O2 by d(GPSA). H2O2 incubated with increasing amounts of d(GPSA) or d(GA) (0.625, 1.25, 2.5 and 5 mM, respectively). The amount of H2O2 reduced was monitored using HPLC peak area. Nucleic Acids Research, 2012, Vol. 40, No. 18 9121 consumed by oxidation, providing for a high turnover rate that increases the overall reducing capacity for the cell. Figure 5 clearly shows that PT modiﬁed genomic DNA, both from the wild-type strain and the dndB mutant, is very quite resistant to DNA ds break damage caused by H2O2. This viewpoint was further tested by introducing dpt gene cluster into a DNA phosphorothioa- tion non proﬁcient strain E. coli DH10B. Figure 4S shows that introducing DNA PT modiﬁcation enhances the anti-oxidation capability of the bacteria. This explains why the dpt gene cluster resides in genetic mobile elements and is kept by diverse bacteria while the modiﬁ- cation is not essential. In vitro oxidation of the PT dinucleotide by PAA or H2O2 results in a mixture of DNA backbone cleavage and just removal of S from the DNA without backbone cleavage. Contrary to the expectation, PT DNA in vivo is more resistant to oxidative ds DNA breakage caused by H2O2 than unmodiﬁed DNA (Figure 5). The oxidative reaction is directed toward the exchange of sulfur with oxygen rather than to backbone cleavage. An explanation for this apparent paradox is that d(GHA), the intermediate of cleavage reaction pathway, is preferentially produced under high peroxide concentration (Figure 2B), which is less likely inside a bacterial cell. However even a single ds DNA breakage is lethal (or harmful), which should make the PT DNA hosting bacteria sensitive to oxidative envir- onments [especially considering the fact that there are approximately 6000 modiﬁcations per cell (6)]. An alter- natively explanation, is that the cell can choose the PT oxidation pathway. This possibility is currently under our intensive investigation. Figure 4. PT DNA hosting bacteria are resistant to H2O2. H2O2 treatment of S. enterica serovar 87 and its dpt mutant derivatives. The growth rates of the strains were monitored at OD600. The genotypes are indicated under the corresponding growth curves.