LAUSR

DNA phosphorothioate (PT) modification was recently identified to occur naturally in diverse bacteria and to be governed by DndABCDE proteins. The nuclease resistance as well as the redox and nucleophilic properties of PT sulfur make PT modification a versatile player in restriction‐modification (R‐M) defense, epigenetic regulation, environmental fitness and the maintenance of cellular redox homeostasis. In this study, we discovered that tight control of PT levels is mediated by the ATPase activity of DndB. The ATP‐binding activity of DndB stimulates the dissociation of the DndB‐DNA complex, allowing transcriptional initiation, whereas its ATP hydrolysis activity promotes the conversion of DndB‐ATP to free DndB that is capable of rebinding to promoter DNA for transcriptional inhibition. Since sulfur incorporation is an ATP‐consuming process, these activities provide an economical way to fine‐tune PT modification in an ATP‐sensing manner. To our knowledge, this ATP‐mediated regulation is a rare example among DNA epigenetic modification systems; the features of autoregulation and the repeated usage of DndB allow the dedicated regulation of PT levels in response to cellular ATP concentrations, providing insight into PT function and its role in physiology.