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Generating Targeted Mutants Using a Riboswitch to Control Plasmid Behavior During Allelic Exchange Mutagenesis.

Joshua N Brehm1, Joseph A Sorg2

  • 1Department of Biology, Texas A&M University, College Station, TX, USA.

Methods in Molecular Biology (Clifton, N.J.)
|May 7, 2026
PubMed
Summary

This study introduces a novel riboswitch method for efficient, markerless allele-coupled exchange (ACE) mutagenesis in Clostridioides difficile. This technique simplifies genetic manipulation by controlling plasmid replication, enabling reliable integration and excision events in wild-type strains.

Keywords:
Allelic exchange mutagenesisRiboswitchTargeted mutations

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Allele-coupled exchange (ACE) mutagenesis is a valuable tool for genetic manipulation in Clostridioides difficile.
  • Traditional ACE methods often rely on unstably replicating plasmids, toxic precursors, or toxin/antitoxin systems, posing limitations in ease of use and efficiency.
  • These limitations necessitate complementation of specific genes (e.g., pyrE) in newly generated mutants.

Purpose of the Study:

  • To develop an improved and more user-friendly method for markerless ACE mutagenesis in wild-type Clostridioides difficile.
  • To leverage a synthetic riboswitch to control mutagenesis plasmid replication, thereby enhancing the reliability of integration and excision events.
  • To streamline the mutagenesis process by eliminating the need for pyrE complementation and enabling growth on rich media.

Main Methods:

  • Utilized a synthetic riboswitch to indirectly regulate the replication of a mutagenesis plasmid in a wild-type C. difficile background.
  • Employed a pCD6-derived origin of replication for increased conjugal transfer efficiency and higher plasmid copy number.
  • Applied selective pressure to facilitate the sequential recombination events: plasmid integration into the chromosome and subsequent excision.

Main Results:

  • Successfully achieved efficient and markerless ACE mutagenesis in wild-type C. difficile.
  • Demonstrated reliable control over plasmid replication using the riboswitch system, leading to predictable integration and excision.
  • Showcased the ability to generate mutants on rich media, improving growth rates and overall experimental convenience.
  • Eliminated the requirement for pyrE complementation in newly created mutants.

Conclusions:

  • The developed riboswitch-controlled ACE system offers a significant advancement in C. difficile genetic engineering.
  • This method enhances ease of use, efficiency, and flexibility by enabling mutagenesis in wild-type strains on rich media.
  • The improved system facilitates more reliable and straightforward genetic manipulation of C. difficile for research purposes.