Figure 5. Quantification of PT turnover by isotope-labeling and mass spectrometry.

(a) Principle of PT turnover assay. Bacteria were grown in minimal medium containing [¹⁵N]/[³⁴S]-labeled nutrients, with resulting bacterial DNA containing [¹⁵N]-labeled nucleobases with [³⁴S]-labeled PTs (Original PT). To initiate an experiment, cells are transferred to minimal medium with [¹⁴N]/[³²S]-labeled constituents and subjected to stresses. Newly synthesized PT-linked dinucleotides (New PT) are labeled with [¹⁴N] and [³²S]. The [³⁴S] in the original PTs was found to undergo replacement with [³²S] (Turnover PT) in both untreated and oxidant-treated cells. We also observed low levels of PT dinucleotides labeled with [³⁴S] and [¹⁴N] (Cryptic PT) resulting from reuse of [³⁴S] from nutrient pools or other PT modification sites. In all cases, isolated DNA was hydrolyzed to canonical mononucleotides and PT-containing dinucleotides, and quantities of various isotope-labeled dinucleotides were determined by LC-MS/MS analysis. (b) Validation of PT turnover method. Cultures of wild-type E. coli B7A were grown with [¹⁵N]/[³⁴S]-containing medium, and then cells were transferred to [¹⁴N]/[³²S]-containing medium. At various times, genomic DNA was isolated and subjected to LC-MS/MS analysis of PT-containing dinucleotides. The plot shows cell growth (OD₆₀₀; blue line), original PT (red line) and new PT (black line). (c) The level of [¹⁵N]/[³³S]-labeled, PT-containing dinucleotides (“turnover” PT) were determined in wild-type, untreated E. coli B7A as a function of time. The level of this turnover PT as a function of total PT was used to calculate the %PT turnover. (d) Estimates of the rate of PT turnover per hour were calculated by performing linear regression analysis of plots of %PT turnover as a function of time in unstressed E. coli B7A and S. enterica Cerro 87 (black) and for bacteria exposed to HOCl (red) and H₂O₂ (blue), with regression analysis illustrated in Supplementary Figure 7 for wild-type, untreated Cerro 87. Data represent mean ± SD for 3 biological replicates and values for HOCl-treated samples are significantly different from untreated and H2)2-treated by Student’s t-test at P < 0.05.