Nucleic Acids Research, 2012, Vol. 40, No. 18 9119 cellular levels of H2O2 reach 2 mM (33). If PT DNA func- tions as a reducing thiol in vivo, one concern is whether the PT DNA react with physiological scale peroxide concen- tration. The lower limit of PAA reaction that can be detected by ﬂuorescence is 0.14 mM, while as low as a 10 mM PAA reaction can still be detected to react with the dinucleotide using mass monitoring (Supplementary Figure S3). Another concern is whether the in vivo consumption of H2O2 by PT DNA is comparable to the consumption by catalases, peroxidases, peroxiredoxins, plus the thiols on proteins and small molecules such as glutathione and Bacillithiol (34–36), and is therefore physiologically sig- niﬁcant? There are around 6000 PT sulfur molecules per S. enterica cell (6), correspond to a 6–10 mM of concentra- tion, which is substantially lower than the concentration of glutathione, estimated to be 3–7 mM in bacteria (36). However, the concentration of PT’s is comparable to that of the protein antioxidant thioredoxin, which is estimated to be 10 mM (37). Glutathione is present in most of the gram-negative bacteria, but absent in most of the gram-positive bacteria. Glutathione is involved in multiple physiological functions, including protecting the bacteria against antibiotics, oxidative stress and Figure 2. PT dinucleotide is oxidized by H2O2 and PAA. (A) Conversion of d(GPSA) to d(GA) by H2O2. d(GPSA) was incubated with (110 mM) H2O2, generating reaction products 2–7 (a). The main product is 3. d(GA) (b) and d(GPSA) (c) were run as control. Note the remaining H2O2 peak can be observed. (B) HPLC traces showing the conversion of PT to H–phosphonate linkage by increasing concentrations of per-acetic acid (PAA). Note the amount of reaction product 2, H–phosphonate dinucleotide [d(GHA)], increased corresponding to increasing concentration of PAA. At 30 mM concentration of PAA, d(GPSA) is converted mainly to d(GHA) ( 67.5% of reaction products under the reaction condition). (C) High resolution mass spectrum analysis of H–phosphonate dinucleotides. Mass fragmentations of d(GPSA) (1), d(GHA) (2) and d(GA) (3). Characteristic mass peaks m/z = 348, 316 and 332, indicating the linkages of PT, H–phosphonate and phosphodiester were marked. Fragmentation peaks corres- ponding to G (m/z = 152) and A (m/z = 136) were also indicated. (D) HPLC traces showing the hydrolyzed products of synthesized d(GHA) standard and the lyophilized d(GHA) generated by oxidizing d(GPSA) using PAA. Note, after release from the synthesizing column by ammonium hydroxide, all d(GHA) was hydrolyzed, generating products 4–7 mainly. Peak 3 was barely detectable. d(GPSA) oxidized by PAA generated d(GA) (peak 3) as a main product. For mass fragmentation of peak 4–7 (see Supplementary Figure 2S2). (continued)