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

Abstract

AbstractClostridium botulinumcauses the disease botulism, and nontoxigenicClostridium sporogenesis closely related to group IC. botulinum.Despite its pathogenicity,C. botulinumremains poorly characterized. Genetic manipulation is critical for understanding bacterial physiology and disease. We compared the conjugal transformation efficiencies of seven strains, including group IC. botulinum(strains 62A, 7I03-H, Okra, Osaka05, 111) andC. sporogenes(strains JCM 1416T, 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 fromE. colidonor cells to recipient strains. In addition, we developed a novel conjugal suicide vector pXMTL that contains xylose-induciblemazFas a counter-selection marker, and can be transferred intoClostridiumspp. by conjugation. The allele-coupled exchange (ACE) system using pXMTL provides a rapid method for precise, markerless and scarless genome editing in group IC. botulinumandC. sporogenes.ImportanceGroup IC. botulinumandC. sporogenesexhibit 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-induciblemazFmarker, and can be transferred intoClostridiumspp. by conjugation. The combination of pXMTL and the optimized conjugation protocol provides a powerful tool for genetic manipulation of group IC. botulinumandC. sporogens.