Development of genetic systems for Clostridia spp. by utilization of extrachromosomal DNA elements

An Escherichia coli-Clostridium perfringens shuttle vector (pAK201) was constructed by ligating a 3.6 Kb fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 Kb). Plasmid pKA201 is 8 kb and codes for resistance to chloramphenicol in E. coli and C. perfringens. Plasmid pAK201 was transformed into E. coli and C. perfringens using electroporation. The transformation frequencies were 10$\sp6$ and 10$\sp4$ transformants/ug DNA in E. coli and C. perfringens.Transposon Tn916 was transformed into C. perfringens using electroporation of plasmid pAM120. The transformation frequency was 10$\sp2$ transformants/ug DNA. Southern hybridization indicated that transposon Tn916 was inserted into C. perfringens chromosome.The 7.6 Kb pVA677 plasmid was introduced into Clostridium acetobutylicum using electroporation. The transformation frequency was 2.8 $\times$ 10$\sp3$ transformants/ug DNA. C. acetobutylicum transformants did not produce butanol. The loss of butanol-productivity may be due to a physiological response to electroporation-induced stress rather than a genetic alteration.The plasmid pDM6 was isolated from C. acetobutylicum NCIB 6444. Plasmid pDM6 was recovered as ds- and ssDNA. A 6.6 kb ssDNA complexed with protein was recovered from the supernatant. The DNA-protein complex revealed the presence of a filamentous viruslike particle (CAK1). Tricine-SDS-PAGE of CAK1 demonstrated the presence of a 5 KDa major coat protein. Hybridization data indicated that CAK1 has homology with M13 phage. Physical properties of CAK1 are similar to the filamentous phages.A phage-plasmid hybrid (phasmid) pCAK1 was constructed by ligating the 5 kb pAK102 plasmid and the 6.6 kb RF of CAK1. The phasmid pCAK1 (11.6 kb) replicates using an ColE1 origin. ssDNA complexed with protein were recovered from the supernatant of E. coli. The ds- and ssDNA or pCAK1 were transformed into E. coli using electroporation. The transformation frequencies were 3.0 $\times$ 10$\sp4$ transformants/ug of RF DNA and 4.0 $\times$ 10$\sp3$ transformants/ug of ssDNA. These suggest that the replication origins of VS and CS of CAK1 are functional in E. coli. Tricine-SDS-PAGE of viruslike structure of pCAK1 demonstrated the presence of three major coat proteins including a 5 KDa major protein of CAK1.U of I OnlyETDs are only available to UIUC Users without author permissio