Construction and characterization of a phage-plasmid hybrid (phagemid), pCAK1, containing the replicative form of viruslike particle CAK1 isolated from Clostridium acetobutylicum NCIB 6444

Abstract

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.

Microbial Cellulose Utilization: Fundamentals and Biotechnology

“…Suicide vectors designed for either single-or double (gene replacement)-crossover events can be used for gene knockout studies as discussed above (see "Metabolic engineering"). A filamentous virus-like particle from C. acetobutylicum NCIB 6444 has been isolated (329) and used to construct a replicative phagemid (330). Conjugative transposons with selectable antibiotic resistance markers may be used for gene isolation and characterization by insertional mutagenesis, as reviewed for clostridia by Young et al (771).…”

Genetic systems development in the clostridia

“…1) was constructed by ligating the 5-kb pAK102 E. coli plasmid (Amp r and Erm r) and the 6.6-kb HaelII linearized replicative form (RF) of the CAK1 virus-like particle from C. acetobutylicum NCIB 6444 [9]. Phagemid pCAK1 (11.6 kb) replicated via the ColE1 replication origin derived from pAK102 in E. coli.…”

“…As an alternative to pMTL500E, a plasmid-based shuttle vector based on the only indigenous plasmid recovered from C. acetobutylicum NCIB 6443 (P. Rogers, personal communication), namely pDMll, is under development in the laboratory of H.P.B. Preliminary work has involved the excision of the chloramphenicol resistance gene from the pAK201 C. perfringens-E, coli vector [21] and its ligation into the E. coli replicon derived from pAK102 [9] to generate pJF102, an erythromycin-and chloramphenicol-encoding plasmid which was subsequently ligated with pDMll to form pJF201 (Fig. 2).…”

“…However, the strain specific nature of transformation and the instability of various shuttle vectors necessitated the development of a more versatile genetic system for Clostridium [3,4,23]. Progress has been made in the development of simple electroporation protocols for the clostridia using a 10% PEG solution [9,24,25]. We suggested earlier [21] that the cell wall structure represents an additional barrier to DNA uptake in C. perfringens.…”

“…Shaded areas on pCAK1 indicate the separate deleted regions• tionality in Clostridium acetobutylicum [3,7]. The identification of the CAK1 filamentous virus-like particle from C. acetobutylicum NCIB 6444 [8] together with the more recent construction of the 11.6-kb pCAK1 phagemid Eseherichia coli-C, acetobutylicum expression vector [9] provides the foundation for the development of a versatile genetic system for Clostridium spp. resembling that of E. coli filamentous bacteriophage M13 [10][11][12].…”