Isolation and initial characterization of a Bacillus subtilis mutant with novel protease secretion capability

Area of Science:

• Microbiology
• Biotechnology
• Enzymology

Background:

• Bacillus subtilis is a model organism for studying bacterial physiology and protein secretion.
• High-temperature enzyme production is crucial for various industrial processes.
• Understanding heat shock responses and protein secretion mechanisms is key to optimizing microbial production.

Purpose of the Study:

• To identify Bacillus subtilis mutants capable of protease production at elevated temperatures (48°C).
• To characterize the secretion profile of a novel high-temperature protease-producing mutant.
• To investigate the potential of this mutant as a hypersecretor of exported proteins at supra-optimal temperatures.

Main Methods:

• Generation of a mutant bank using pTV32 (Tn917 lacZ) transposition in Bacillus subtilis.
• Screening of mutants on milk agar for casein hydrolysis at 48°C.
• Comparative analysis of protease secretion at 48°C versus 37°C.
• Analysis of heat shock protein profiles in the identified mutant.

Main Results:

• Isolation of a single mutant, BUL786, exhibiting significant casein hydrolysis at 48°C.
• BUL786 demonstrated a 10-fold increase in protease secretion at 48°C compared to 37°C, with evidence of 48°C-specific activity.
• The mutant lacks the major 97K heat shock protein, and appears to secrete other proteins at 48°C.
• Other Bacillus subtilis strains, including hyper-protease secretors, failed to secrete proteases effectively at high temperatures.

Conclusions:

• The Bacillus subtilis BUL786 mutant exhibits enhanced and potentially specific protease secretion at 48°C.
• The absence of the 97K heat shock protein may be linked to the observed thermotolerant hypersecretion phenotype.
• BUL786 represents a promising candidate for a hypersecretor of exported proteins at temperatures above 45°C, with potential biotechnological applications.