Facile autoplast generation and transformation in Bacillus thuringiensis subsp. kurstaki

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Bacillus thuringiensis subsp. kurstaki is an important insecticidal bacterium.
• Efficient genetic manipulation requires methods to introduce foreign DNA into bacterial cells.
• Current methods for generating protoplasts or spheroplasts often rely on exogenous enzymes.

Purpose of the Study:

• To develop an enzyme-free method for generating osmotically fragile forms of Bacillus thuringiensis subsp. kurstaki.
• To optimize conditions for maximizing the conversion rate and efficiency.
• To assess the viability and genetic transformation capacity of the generated forms.

Main Methods:

• Vegetative cells of Bacillus thuringiensis subsp. kurstaki were treated with polyethylene glycol (PEG) in a specific buffer.
• Optimal conditions for autoplast generation (buffer composition, pH, temperature, PEG concentration) were determined.
• Conversion rates were quantified by microscopy.
• Regeneration efficiency was assessed by plating on DM3-G medium.
• Plasmid DNA uptake was evaluated, and plasmid presence confirmed by Southern hybridization.

Main Results:

• An enzyme-free method was established for converting Bacillus thuringiensis subsp. kurstaki vegetative cells to autoplasts.
• Optimal conditions involved 50 mM sodium acetate buffer (pH 7.0) at 37°C with 10% PEG.
• Over 90% conversion to spherical autoplasts was achieved within 6 minutes.
• Autoplasts demonstrated efficient uptake of exogenous plasmid DNA.
• Regeneration frequencies ranged from 1.2 x 10^-1 to 5.3 x 10^-3.

Conclusions:

• An efficient, enzyme-free method for generating Bacillus thuringiensis subsp. kurstaki autoplasts was developed.
• These autoplasts are viable, capable of regeneration, and serve as efficient recipients for plasmid DNA transformation.
• This method offers a valuable tool for genetic engineering of Bacillus thuringiensis.