Secretion and processing of staphylococcal nuclease by Bacillus subtilis

Area of Science:

• Microbiology
• Molecular Biology
• Protein Secretion

Background:

• Staphylococcus aureus nuclease is a well-characterized extracellular enzyme.
• Understanding heterologous protein secretion in Bacillus subtilis is crucial for biotechnology.

Purpose of the Study:

• To elucidate the secretion and processing pathway of Staphylococcus aureus nuclease when expressed in Bacillus subtilis.
• To identify the specific processing steps and their relationship to secretion.

Main Methods:

• Pulse-chase radioactive labeling to track protein synthesis and secretion.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to analyze protein molecular weights.
• N-terminal microsequencing to determine protein identity and processing sites.

Main Results:

• An initial nuclease form (nuclease B) with a molecular weight of ~18,800 was detected extracellularly.
• Nuclease B was processed to mature nuclease A by a phenylmethylsulfonyl fluoride-sensitive protease.
• Processing occurred at a signal peptidase consensus site and was coupled to secretion.
• Mature nuclease A was two amino acids shorter than the S. aureus Foggi variant.
• Signal peptide length critically affected secretion, with removal of 72 amino acids abolishing secretion.

Conclusions:

• Bacillus subtilis can efficiently secrete and process Staphylococcus aureus nuclease.
• Extracellular processing by a specific protease is essential for nuclease secretion.
• Signal peptide processing and secretion are tightly regulated and dependent on signal peptide length.