A single-stranded 6.6-kb DNA molecule complexed with protein was recovered from the supernatant of Clostridium acetobutylicum NCIB 6444. Electron microscopic examination of the DNA-protein complex revealed the presence of a ifiamentous viruslike particle, which was designated CAK1. The possible double-stranded plasmidlike replicative form and the single-stranded prophage were also recovered from the cell culture following alkaline lysis. CAK1 was released from the C. acetobutylicum cell culture in the absence of cell lysis. Polyethylene glycol-NaCl coprecipitation of the DNA-protein complex revealed the presence of single-stranded DNA complexed with protein in a manner rendering the DNA resistant to Bal 31 exonuclease. Proteinase treatment of CsCl density gradient-purified CAK1 resulted in recovery of DNase-sensitive single-stranded DNA. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CAK1 demonstrated the presence of a 5-kDa major coat protein. Hybridization data indicated that the single-stranded DNA from CAK1 has homology with the M13 phage of Escherichia coli. An examination of various physical properties of CAK1 suggests that it is similar to the filamentous phage recovered from gram-negative microorganisms. Although infectivity or inducibility of CAK1 could not be demonstrated, to our knowledge this represents the first report of a nonlytic filamentous viruslike particle containing single-stranded DNA being recovered from a gram-positive bacterium..

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid plB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 ,ug of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 106 and 104 transformants per ,ug of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.

A bacteriophage-plasmid hybrid (phagemid) designated pCAK1 was constructed by ligating 5-kbp Escherichia coli plasmid pAK102 (Apr Emr) and the 6.6-kbp HaeIII-linearized replicative form of the CAKi viruslike particle from Clostridium acetobutylicum NCIB 6444. Phagemid pCAK1 (11.6 kbp) replicated via the ColEl replication origin derived from pAK102 in E. coli. Single-stranded DNA (ssDNA) molecules complexed with protein 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 CAKi and associated gene expression are functional in E. coli DH11S. The single-stranded form of pCAK1 isolated from E. coli supernatant was transformed into E. coli DH5ct' or DH11S by electroporation. Isolation of ampicillin-resistant E. coli transformants following transformation suggests that the complementary-strand DNA synthesis replication origin of CAKi 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 chloroform), similar to the results obtained previously with CAK1. Following phagemid construction in E. coli, pCAK1 was transformed into C. acetobutylicum ATCC 824 and C.perfringens 13 by intact cell electroporation. Restriction enzyme analysis of pCAK1 isolated from erythromy.cin-resistant transformants of both C. acetobutylicum and C. pefringens suggested that it was identical to that present in E. coli transformants.