Engineering a reagentless biosensor for single-stranded DNA to measure real-time helicase activity in Bacillus

Area of Science:

• Bacterial molecular biology
• DNA metabolism and repair
• Biosensor development

Background:

• Single-stranded DNA-binding protein (SSB) is crucial for bacterial DNA metabolism, interacting with numerous proteins.
• SSB's partner protein interactions are often species-specific, necessitating tailored tools for different bacterial species.
• Existing methods for SSB biosensors, like those for E. coli, are not universally applicable to other bacteria.

Purpose of the Study:

• To generate a reagentless biosensor for Bacillus subtilis SSB.
• To demonstrate the utility of this biosensor as a helicase probe in gram-positive bacteria.
• To characterize SSB's DNA binding mode switching and stoichiometry in B. subtilis.

Main Methods:

• Production of a Bacillus subtilis SSB probe with a 9-fold fluorescence increase upon ssDNA binding.
• Application of the probe in DNA unwinding assays using a Bacillus helicase.
• Characterization of SSB binding stoichiometry and mode switching.

Main Results:

• A functional reagentless SSB biosensor was successfully created for B. subtilis.
• The probe was utilized to study DNA unwinding reactions and characterize SSB binding dynamics.
• The study revealed novel insights into B. subtilis SSB's DNA binding behavior and its role beyond DNA protection.

Conclusions:

• The developed B. subtilis SSB biosensor is effective for studying DNA metabolism in B. subtilis and related gram-positive bacteria.
• This reagentless probe offers a widely available alternative to previously published methods, especially for bacteria with distinct replication systems.
• The findings highlight the broader importance of SSB in coordinating DNA metabolism through interactions with partner proteins.