[Start codon in the serine proteinase gene from Bacillus intermedius]

Area of Science:

• Microbiology
• Molecular Biology
• Enzymology

Background:

• Bacillus intermedius secretes an extracellular serine subtilisin-like proteinase (aprBi).
• Understanding the aprBi gene's translation initiation is crucial for optimizing protein production.
• The aprBi gene has three potential start codons: TTG, GTG, and ATG.

Purpose of the Study:

• To determine the precise translation initiation site of the Bacillus intermedius aprBi gene.
• To investigate the functional activity of putative start codons within the aprBi gene.

Main Methods:

• Bioinformatic analysis using SignalP to predict start codon function.
• Site-directed mutagenesis to alter putative start codons (TTG, GTG, ATG).
• Gene expression analysis in a protease-deficient Bacillus subtilis strain (AJ73).

Main Results:

• SignalP analysis indicated functional probabilities for the three putative start sites.
• Mutagenesis experiments confirmed the functional start codon.
• Expression studies demonstrated that translation initiates at the GTG codon.

Conclusions:

• The translation of the Bacillus intermedius aprBi gene initiates from the GTG codon.
• This finding clarifies the genetic regulation of aprBi expression in Bacillus species.