Genome engineering in Bacillus anthracis using tyrosine site-specific recombinases.

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Tyrosine site-specific recombinases (T-SSR) are enzymes crucial for DNA manipulation.
• Bacillus anthracis genetic engineering tools are needed for vaccine development and understanding virulence.

Purpose of the Study:

• To develop and evaluate three T-SSR systems for genetic modification in Bacillus anthracis.
• To create a B. anthracis strain lacking sporulation and proteases for recombinant protein production.
• To investigate the role of atxA gene copy number on virulence gene expression.

Main Methods:

• Utilized three T-SSR systems: Cre-loxP, Flp-FRT, and IntXO-PSL for B. anthracis genome editing.
• Constructed a B. anthracis strain with deletions in ten secretome proteases.
• Created strains with varying chromosomal copy numbers of the atxA gene.

Main Results:

• Successfully generated a sporulation-deficient, protease-deleted B. anthracis strain.
• This strain facilitated the production of recombinant proteins, including anthrax vaccine candidates.
• Demonstrated a correlation between atxA copy number and pagA gene expression under CO2 conditions.

Conclusions:

• The three T-SSR systems are effective tools for Bacillus anthracis genome editing.
• These engineered strains and tools advance B. anthracis research and vaccine development.
• The T-SSR systems show potential applicability in other prokaryotic and eukaryotic organisms.