Development and Optimization of Genetic Manipulation Systems in Group I Clostridium botulinum

Clostridium botulinum causes the disease botulism, and nontoxigenic Clostridium sporogenes is closely related to group I C. botulinum. Despite its pathogenicity, C. botulinum remains poorly characterized. Genetic manipulation is critical for understanding bacterial physiology and disease. We compared the conjugal transformation efficiencies of seven strains, including group I C. botulinum (strains 62A, 7I03-H, Okra, Osaka05, 111) and C. sporogenes (strains JCM 1416 T , ATCC 15579), and our results showed that few or no transformants were obtained in certain strains. In the present study, we demonstrate that our optimized protocol increases the efficiency of DNA transfer from E. coli donor cells to recipient strains. In addition, we developed a novel conjugal suicide vector pXMTL that contains xylose-inducible mazF as a counter-selection marker, and can be transferred into Clostridium spp. by conjugation. The allele-coupled exchange (ACE) system using pXMTL provides a rapid method for precise, markerless and scarless genome editing in group I C. botulinum and C. sporogenes . Importance Group I C. botulinum and C. sporogenes exhibit low transformation efficiencies, and few or no transformants are yielded by some strains. In this study, we optimized the conjugation protocol to improve transformation efficiency. In addition, we developed a novel suicide vector pXMTL harboring a xylose-inducible mazF marker, and can be transferred into Clostridium spp. by conjugation. The combination of pXMTL and the optimized conjugation protocol provides a powerful tool for genetic manipulation of group I C. botulinum and C. sporogens .