Identification of a Bacillus subtilis secretion mutant using a beta-galactosidase screening procedure

Area of Science:

• Microbiology
• Molecular Biology
• Protein Secretion

Background:

• High-level synthesis of exportable beta-galactosidase (LacZ) fusion proteins in Bacillus subtilis is lethal.
• This lethal phenotype has been proposed as a method for selecting secretion mutants.
• Previous attempts using inducible lacZ fusion systems were hindered by cis-acting mutations reducing gene expression.

Purpose of the Study:

• To develop and implement a screening strategy for identifying Bacillus subtilis secretion mutants.
• To characterize a novel conditional secretion mutant of Bacillus subtilis.

Main Methods:

• Development of a modified indicator plasmid for screening increased basal LacZ activity.
• Nitrosoguanidine mutagenesis to generate mutants.
• Growth assays and protein secretion analysis at permissive (30°C) and non-permissive (42°C) temperatures.
• Analysis of processing kinetics and secretion of fusion proteins.

Main Results:

• A conditional Bacillus subtilis secretion mutant was identified.
• The mutant exhibited extreme growth retardation at 42°C when producing LacZ fusion protein.
• The mutant was defective in the secretion of subtilisin Carlsberg at 42°C.
• Processing and secretion of a subtilisin Carlsberg-alkaline phosphatase fusion were impaired at the non-permissive temperature.
• The identified secretion defect was not associated with the secA/div locus.

Conclusions:

• A viable screening method for B. subtilis secretion mutants was established by assessing basal LacZ activity.
• A novel conditional secretion mutant was characterized, demonstrating temperature-sensitive defects in protein processing and secretion.
• The findings provide insights into the complex machinery of protein secretion in Bacillus subtilis.