A gyrase mutant with low activity disrupts supercoiling at the replication terminus

Area of Science:

• Bacteriology
• Molecular Biology
• Genetics

Background:

• DNA gyrase, composed of GyrA and GyrB subunits, is essential for bacterial DNA replication.
• Temperature-sensitive mutations in essential genes typically imply protein inactivity at nonpermissive temperatures.

Purpose of the Study:

• To investigate the functional consequences of the gyrB652 mutation in DNA gyrase.
• To determine the in vivo and in vitro activity of the GyrB652 protein at permissive and nonpermissive temperatures.

Main Methods:

• Site-directed mutagenesis to create the gyrB652 allele (R436-S).
• Phenotypic analysis of bacterial strains carrying the gyrB652 allele at different temperatures.
• In vivo and in vitro enzyme activity assays for DNA gyrase.

Main Results:

• The gyrB652 mutation leads to temperature-sensitive growth inhibition of DNA replication at 42°C.
• At 37°C, gyrB652 strains exhibit SOS induction and DNA degradation.
• The GyrB652 protein is not inactive at 42°C but exhibits a reduced catalytic rate (kcat) and causes topological chaos.

Conclusions:

• The gyrB652 mutation is a hypomorphic allele, not a null mutation, affecting DNA gyrase function.
• The observed phenotypes are due to impaired supercoiling activity leading to replication fork collapse and DNA instability.