Cloning the Bacillus subtilis 168 aroC gene encoding dehydroquinase

Area of Science:

• Molecular Biology
• Microbial Genetics
• Gene Expression

Background:

• Understanding gene regulation and function in Bacillus subtilis is crucial for microbial biotechnology.
• The aroC gene, involved in aromatic amino acid biosynthesis, and the ser-22 gene's function were previously less characterized in B. subtilis.
• Cloning and expression studies are essential for elucidating gene roles and developing genetic tools.

Purpose of the Study:

• To isolate and characterize the Bacillus subtilis aroC and ser-22 genes.
• To investigate the expression of the cloned aroC gene in both B. subtilis and Escherichia coli.
• To assess the functional activity of the cloned aroC gene product, dehydroquinase.

Main Methods:

• Isolation of a 14-kb Sau3A DNA fragment from Bacillus subtilis.
• Cloning of the DNA fragment into a suitable vector.
• Transformation and expression studies in both B. subtilis and E. coli.
• Enzyme activity assays to measure dehydroquinase-specific activity.

Main Results:

• A 14-kb DNA fragment containing Bacillus subtilis aroC and ser-22 genes was successfully isolated.
• The aroC gene demonstrated autonomous expression in B. subtilis and E. coli, indicating a functional promoter.
• Expression in E. coli was orientation-dependent, allowing determination of transcription direction.
• Dehydroquinase-specific activity increased 30- to 40-fold in both host organisms.
• Cloned genes were stable in both B. subtilis and E. coli, despite observed deletions during construction.

Conclusions:

• The cloned Bacillus subtilis aroC gene contains its own promoter and is functional in heterologous (E. coli) and homologous (B. subtilis) hosts.
• The study provides a method for enhancing dehydroquinase activity through gene cloning.
• The findings contribute to the genetic understanding of B. subtilis and offer potential for metabolic engineering applications.