Oxidation of phosphorothioate DNA modifications leads to lethal genomic instability

Abstract

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 (H2O2) or hypochlorous acid (HOCl), PT turnover increased to 3.8–10%/h for HOCl and was unchanged for H2O2, 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 H2O2. The genotoxicity of HOCl in PT-containing bacteria suggests reduced fitness in competition with HOCl-producing organisms and during human infections.

NAIL‐MS in E. coli Determines the Source and Fate of Methylation in tRNA

“…The prerequisite for these studies was the complete labeling of the nucleic acid with heavy, non‐radioactive isotopes such as carbon‐13, nitrogen‐15, and sulfur‐34 and access to a mass spectrometer. The combination of the different labeling media allowed the creation of a pulse‐chase experiment, which was used to observe repair of phosphorothioates in bacterial DNA . In the same year, we adapted the approach to RNA modification analysis in yeast .…”

“…NAIL‐MS overcame this limitation. The prerequisite for a discriminatory NAIL‐MS experiment was the availability of a heavy isotope‐labeling medium, which could label either the damaged or the natural product (e.g., here, methylation or thiolation). Feeding organisms with CD 3 ‐methionine led to the formation of CD 3 ‐SAM and transfer of heavy methyl marks onto the RNA (Figure S2) by the respective enzymes.…”

Chemical Warfare at the Microorganismal Level: A Closer Look at the Superoxide Dismutase Enzymes of Pathogens

“…Additionally, in neutrophils, H 2 O 2 can be converted to the membrane permeable hypochlorous acid (HOCl) via myeloperoxidase 27 . HOCL can trigger oxidative unfolding of proteins 28 and is genotoxic to microbes 29 . Overall, through the production of these various reactive oxygen and nitrogen species, O 2 ·− lies at the heart of immune cell chemical warfare (Scheme 1).…”

Tight control of genomic phosphorothioate modification by the ATP‐modulated autoregulation and reusability of DndB

“…). This result is understandable because bleomycin causes an increase in reactive oxygen species (Mahmutoglu et al ., ; Chen et al ., ), which react with PT to cause desulfurization and DNA breakage (Xie et al ., ; Kellner et al ., ). Taken together, our results revealed that DndB serves as an ATP sensor to regulate the transcription of the dndBCDE operon and the resulting PT modifications in an unusual autoregulated and reusable manner.…”

“…found that PT bonds are selectively sensitive to hypochlorous acid (HOCl), suggesting reduced bacterial fitness under commonly encountered oxidative stresses, e.g. in competition with organisms producing hypohalous acid (Kellner et al ., ). Tong et al .…”