Efficient and markerless allele-coupled exchange (ACE) mutagenesis is possible in a wild-type C. difficile background by using a synthetic riboswitch to indirectly control replication of the mutagenesis plasmid. Controlling plasmid replication gives the user the ability to introduce selective pressure for recombination of the mutagenesis plasmid into the chromosome (integration) followed by recombination of plasmid DNA out of the chromosome (excision) reliably. ACE has been successfully used in C. difficile research for over a decade, mostly with unstably replicating plasmids using toxic pyrimidine precursors or toxin/antitoxin systems to select for recombination events. The advantages of using a riboswitch to control plasmid replication include the ability to begin with a wild-type parent strain, use of a higher copy number origin of replication, and generation of mutants on rich medium. A wild-type progenitor means there is no need to complement pyrE into newly made mutants, increasing ease of use. The inclusion of the more stable, pCD6-derived origin of replication in the mutagenesis vector should increase efficiency of conjugal transfer, and its higher plasmid copy number should increase the frequency of crossover events. The efficacy of the riboswitch in rich media also avoids the use of minimal or defined media, increasing growth rates and ease of use.