Oxidation of phosphorothioate DNA modifications leads to lethal genomic instability

Genomic modification with sulfur as phosphorothioate (PT) is widespread among prokaryotes, including human pathogens. Apart from its physiological functions, the redox and nucleophilic properties of PT sulfur suggest effects on bacterial fitness in stressful environments. Here we show that PTs are dynamic and labile DNA modifications that cause genomic instability during oxidative stress. Using coupled isotopic labeling-mass spectrometry, we observed sulfur replacement in PTs at a rate of ~2%/h in unstressed Escherichia coli and Salmonella enterica. While PT levels were unaffected by exposure to hydrogen peroxide (H(2)O(2)) or hypochlorous acid (HOCl), PT turnover increased to 3.8–10%/h for HOCl and was unchanged for H(2)O(2), consistent with repair of HOCl-induced sulfur damage. PT-dependent HOCl sensitivity extended to cytotoxicity and DNA strand-breaks, which occurred at orders-of-magnitude lower doses of HOCl than H(2)O(2). The genotoxicity of HOCl in PT-containing bacteria suggests reduced fitness in competition with HOCl-producing organisms and during human infections.