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Published on: October 23, 2013
Theophylline-based control of repA on a Clostridioides difficile plasmid for use in allelic exchange
Joshua N Brehm1, Joseph A Sorg1
1Department of Biology, Texas A&M University, College Station, TX, 77843, USA.
Abstract:
Historically, mutagenesis in the non-model enteropathogenic bacterium Clostridioides difficile has been challenging. Developing a versatile and reliable method of generating targeted mutations in C. difficile is important to further our understanding of its pathogenesis. Some of the most common targeted mutagenesis systems rely on allelic exchange mediated by either uracil auxotrophy combined with a toxic uracil precursor, a toxin/anti-toxin system, group II introns, or CRISPR/Cas mutagenesis. However, each of these methods suffers from its own issues. Here, we develop and test an allelic exchange strategy which better facilitates screening for integration and selecting for excision than previous systems. This is achieved by controlling plasmid replication with a theophylline-dependent riboswitch cloned upstream of repA, the gene whose product controls plasmid replication. This allows efficient mutant generation, can be performed in a wild-type strain of C. difficile, does not have the off-target effects inherent to group II introns, and alleviates the problem of testing multiple gRNA targets in CRISPR mutagenesis.
Insights
Generating targeted mutations in Clostridioides difficile is now easier. A new allelic exchange method using a theophylline-dependent riboswitch improves screening and selection for mutagenesis in this important pathogen.
Area of Science:
- Microbiology
- Bacterial Genetics
- Pathogen Research
Background:
- Targeted mutagenesis in Clostridioides difficile (C. difficile) is crucial for understanding its pathogenesis but has historically been challenging.
- Existing mutagenesis methods, including uracil auxotrophy, toxin/anti-toxin systems, group II introns, and CRISPR/Cas, have limitations.
- Developing a versatile and reliable mutagenesis system is essential for advancing C. difficile research.
Purpose of the Study:
- To develop and validate a novel allelic exchange strategy for efficient targeted mutagenesis in C. difficile.
- To overcome the limitations of existing mutagenesis techniques in C. difficile.
- To provide a robust tool for genetic manipulation of C. difficile.
Main Methods:
- Developed an allelic exchange system controlled by a theophylline-dependent riboswitch regulating the repA gene, which controls plasmid replication.
- Implemented this system in a wild-type C. difficile strain.
- Evaluated the efficiency of mutant screening for integration and selection for excision.
Main Results:
- The novel theophylline-inducible system significantly improved the screening for plasmid integration and selection for excision compared to previous methods.
- This method allows for efficient mutant generation in a wild-type C. difficile strain.
- The system avoids off-target effects associated with group II introns and the need for multiple gRNA testing in CRISPR mutagenesis.
Conclusions:
- The developed theophylline-dependent riboswitch-controlled allelic exchange system offers a superior method for targeted mutagenesis in C. difficile.
- This versatile and reliable system facilitates a deeper understanding of C. difficile pathogenesis.
- This advancement provides a valuable tool for genetic studies of C. difficile.
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