CONSTRUCTION AND CHARACTERIZATION OF A PHAGE-PLASMID HYBRID (PHAGEMID), PCAK1, CONTAINING THE REPLICATIVE FORM OF VIRUS-LIKE PARTICLE CAK1 ISOLATED FROM CLOSTRIDIUM-ACETOBUTYLICUM NCIB-6444

A bacteriophage-plasmid hybrid (phagemid) designated pCAK1 was constru cted by ligating 5-kbp Escherichia coli plasmid pAK102 (Ap(r) EM(r)) a nd the 6.6-kbp HaeIII-linearized replicative form of the CAK1 viruslik e particle from Clostridium acetobutylicum NCIB 6444. Phagemid pCAK1 ( 11.6 kbp) replicated via the ColE1 replication origin derived from pAK 102 in E. coli. Single-stranded DNA (ssDNA) molecules complexed with p rotein in a manner which protected ssDNA from nucleases were recovered from the supernatant of E. coli DH11S transformants containing pCAK1 in the absence of cell lysis. This suggests that the viral-strand DNA synthesis replication origin of CAK1 and associated gene expression ar e functional in E. coli DH11S. The single-stranded form of pCAK1 isola ted from E. coli supernatant was transformed into E. coli DH5alpha' or DH11S by electroporation. Isolation of ampicillin-resistant E. coli t ransformants following transformation suggests that the complementary- strand DNA synthesis replication origin of CAK1 is also functional in E. coli. The coat proteins associated with ssDNA of pCAK1 demonstrated sensitivity to proteinase K and various solvents (i.e., phenol and ch loroform), similar to the results obtained previously with CAK1. Follo wing phagemid construction in E. coli, pCAK1 was transformed into C. a cetobutylicum ATCC 824 and C. peperfringens 13 by intact cell electrop oration. Restriction enzyme analysis of pCAK1 isolated from erythromyc in-resistant transformants of both C. acetobutylicum and C. perfringen s suggested that it was identical to that present in E. coli transform ants.