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

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• E. coli has a unique deoxyguanosine triphosphohydrolase (dGTPase) enzyme that hydrolyzes dGTP, a DNA precursor, playing a crucial role in DNA fidelity.
• The dGTPase enzyme (Dgt) is a hexameric protein regulated by dGTP and single-stranded DNA (ssDNA).
• Loss of the dgt gene results in a mutator effect, indicating the enzyme's importance in maintaining genomic stability.

Purpose of the Study:

• To characterize a novel dGTPase mutant (Cys273Ser) with altered activity.
• To investigate the in vivo effects of expressing the constitutively active dGTPase mutant.
• To elucidate the mechanism of cell death induced by the mutant dGTPase.

Main Methods:

• Site-directed mutagenesis to create the Cys273Ser dGTPase mutant.
• In vitro enzyme activity assays comparing mutant and wild-type enzymes.
• Controlled gene expression in E. coli BL21-AI using a plasmid system.
• Microscopy to observe cellular changes during mutant expression.
• Analysis of suppressor mutant emergence.

Main Results:

• The Cys273Ser mutant exhibited higher in vitro activity than wild-type dGTPase, similar to an allosterically activated form.
• Expression of the mutant dgt-C273S gene in E. coli led to rapid cell death (>99.95% loss of viability within 2.5 hours).
• Cell death was associated with nucleoid disruption and chromosomal DNA loss, and subsequent growth resumption was due to suppressor mutants that silenced mutant dGTPase expression.

Conclusions:

• Constitutively active dGTPase expression causes dGTP depletion, potentially leading to replication fork disruption and DNA degradation.
• The study demonstrates a novel mechanism of cell killing mediated by uncontrolled dNTP levels.
• Maintaining precise cellular dNTP pools is critical for bacterial survival and genomic integrity.