Shuttle plasmids for Escherichia coli and Clostridium perfringens

Abstract

Small plasmids which replicate in both Escherichia coli and Clostridium perfringens were made by recombining E. coli plasmid pBR322 with three different small (less than 4 kilobases) plasmids native to C. perfringens. Subsequently, two homologous, though distinct, tetracycline resistance determinants (tet) from other C. perfringens plasmids were cloned into them. Both tet systems made E. coli resistant to at least 5 ,g of tetracycline per ml when resident on the shuttle plasmids. The shuttle vectors have been used to transform Lphase variants and autoplasts of C. perfringens. In the latter case, the intact transforming plasmid could be isolated from walled cells after cell wall regeneration. Reciprocal transformation experiments in which plasmid DNAs derived from E. coli or C. perfringens were used suggest that restriction barriers exist between these two organisms. The plasmids contain restriction enzyme recognition sites in locations which are useful for cloning experiments.

Recent advances in the genetics of the clostridia

“…Vectors for use in C. perfringens Several C. perfringens-E, coli shuttle plasmids have been constructed in which both replicon and selectable marker are of homologous origin (Table 7). 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).…”

“…Autolysin activity might prevent regeneration of the bacterial cell wall and unless special precautions are taken to limit activity [13] transformation may be difficult to demonstrate. Nucleases, which might, perhaps, interfere more directly with protoplast transformation, have been documented [26][27][28][29] in strains of C. acetobutylicum, C. perfringens and C. thermohydrosulfuricum that have been successfully transformed (see Table 3). Restriction endonucleases have been detected in the last two species as well as several other commonly used laboratory strains (Table 2).…”

Transformation of Clostridium perfringens L forms with shuttle plasmid DNA

“…We have had success with transforming L-form cultures using some of the shuttle plasmids described by Squires et al (12) and Roberts et al (10). In this paper, we relate some of our experience with clostridial L-form transformation.…”

“…Although plasmid DNA from Clostridium perfringens has been introduced by transformation into Escherichia coli, where it may (1,2,10,12) or may not (3) be expressed, C. perfringens itself has been refractory to transformation with clostridial DNA. Since clostridial genes might best be studied by cloning into the homologous species, considerable interest centers around a transformation system for C. perfringens and plasmid vectors for cloning in this species.…”

“…Since clostridial genes might best be studied by cloning into the homologous species, considerable interest centers around a transformation system for C. perfringens and plasmid vectors for cloning in this species. The creation of shuttle vector plasmids that can replicate and express tetracycline resistance in both E. coli and C. perfringens has been described by Squires et al (12). These significant first reports of shuttle vectors and transformation (4) in C. perfringens confirm the unpublished results of others (D. E. Mahony, I. Roberts, and J. I. Rood, personal communication) showing that, unlike E. coli, the intact bacillus form of C. perfringens cannot be transformed easily.…”