Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens

Abstract

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.

The enterotoxin gene (cpe) of Clostridium perfringens can be chromosomal or plasmid‐borne

“…Gels were calibrated using concatemerized lambda DNA or yeast chromosomes as markers then processed for Southern blotting using Hybond C-extra membranes (Amersham) as outlined (Canard and Cole, 1989). Plasmid DNA was prepared from fresh overnight cultures of certain strains by the method of Kim and Blaschek (1989) involving alkaline lysis followed by caesium chloride gradient eentrifugation. The following probes were labelled with [^^P]-dCTP by nick translation or random priming (Sambrook et al.…”

Recent advances in the genetics of the clostridia

“…This transformation method is currently proving widely applicable to many different bacterial genera [42,43]. Reports of transformation of intact cells of C. acetobutylicum NCIB 8052 [36] and C. perfringens [40,41] with various plasmids have already appeared (Table 3) and doubtless other species of Clostridium will be added to the list in the very near future.…”

“…Among these are those reported by Squires et al [27], based on small indigenous C. perfringens plasmids into which a Tc R gene from pCW3 was incorporated; refined derivatives containing a multiple cloning site and a Cm R gene from plasmid pJIR62 instead of the Tc R gene originally present have also been constructed [39]. Kim and Blaschek [41] have recently described a vector, pAK201, based on the small caseinase plasmid, pHB101, and the Cm R gene of Tn4451 (piP401).…”

“…There have been few investigations of vector stability in clostridia and in no case so far has a systematic study been made, addressing problems of both segregational and structural instability. The limited data available to date [35,41] indicate that all vectors tested show some degree of segregational instability, as reflected by the accumulation of antibiotic-sensitive progeny during growth in the absence of selection. Much careful work remains to be done.…”

Genetic systems development in the clostridia

“…Recently, it was reported that only 44% of transformants containing the pMTL500 vector were resistant to erythromycin following 20 generations of subculture (N. Minton, Clostridium III). Clostridial vector constructs based on native DNA are expected to result in more stable genetic systems [21].…”