Lethal effects of a hyperactive dGTP triphosphohydrolase in E. coli.

Escherichia coli possesses a unique class of dNTPases, known as deoxyguanosine triphosphohydrolase (dGTPase), which hydrolyses the DNA precursor dGTP into deoxyguanosine and tripolyphosphate. The enzyme has a fidelity role, as deletion of the encoding dgt gene results in a mutator effect. The Dgt enzyme is a protein hexamer subject to allosteric regulation by dGTP substrate itself and by ssDNA. In the present work, we describe a dGTPase mutant carrying a Cys273Ser amino acid substitution adjacent to the catalytically critical residue Tyr272. In vitro, the C273S enzyme was more active than wild type, its activity resembling the activated form. Cells harboring the mutant dgt-C273S gene as a single copy on the chromosome exhibited reduced viability. To further investigate this toxic effect, we expressed the mutant gene under controlled conditions from a plasmid in strain BL21-AI. Within 1 h after induction, the number of viable cells began declining rapidly, and after 2.5 h over 99.95% were dead. Microscopy showed that killing is associated with disruption of the bacterial nucleoid and eventual loss of chromosomal DNA. After maximal cell killing, the cultures were able to resume growth; however, this was due to the emergence of suppressor mutants that had lost the ability to express the mutant dGTPase. We infer that expression of the constitutively active dGTPase causes depletion of dGTP, which may lead to disrupted replication forks and eventual annihilation of the chromosome by nucleases. These studies highlight the critical importance of cellular dNTP control and support an additional mode of cell killing.