Transformation of Heat-Treated Clostridium acetobutylicum Protoplasts with pUB110 Plasmid DNA

Abstract

Heat treatment of Clostridium acetobutylicum SA-1 protoplasts at 55°C for 15 min before transformation resulted in expression in this microorganism of the kanamycin resistance determinant associated with plasmid pUB110. No heat treatment, or heat treatment at 65 or 44°C for various time intervals, resulted in no kanamycin resistance transformants being recovered on selective kanamycin-containing regeneration medium. DNase plate assay indicated that treatment at 55°C for 15 min completely inactivated the DNase activity associated with SA-1 protoplasts. Treatment of protoplasts at 65 or 55°C for various periods under simulated transformation conditions had an inhibitory effect, although prolonged treatment at 55 or 44°C appeared to stimulate DNase activity. Inactivation of protoplast-associated DNase activity by heat treatment at 550C for 15 min correlated with successful expression of kanamycin resistance and suggests that an extremely active, heatsensitive, protoplast-associated DNase may be a factor in the polyethylene glycol-induced transformation of C. acetobutylicum SA-1 protoplasts. Plasmid pUB110 DNA was isolated from C. acetobutylicum SA-1 kanamycinresistant (Kmr) transformant cultures by a modification of the procedure used for C. perfringens plasmids. Detection of pUB110 DNA was possible only when diethyl pyrocarbonate was incorporated into isolation protocols to inactivate DNase activity. Restriction studies further verified the presence of pUB110 DNA in C. acetobutylicum SA-1 Kmr transformants.

Recent advances in the genetics of the clostridia

“…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).…”

“…Plasmids containing the replication regions of pAMfll and pCB101 have been successfully transferred to C. acetobutylicum (see Table 7), but plasmids based on pUBll0 have not (cf. [26]).…”

Conjugative reporter system for the use in Bacillus licheniformis and closely related Bacilli

“…This vector was originally isolated from Staphylococcus aureus and can replicate in B. subtilis and B. licheniformis (Keggins et al 1978). Reports of a successful transfer of pUB110 to Clostridium acetobotylicum (Lin and Blaschek 1984) are promising for the use of our vector system in many species of the Firmicutes.…”

Genetic systems development in the clostridia

“…Gene transfer systems for the clostridia have been described [3,4,6,22]. 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].…”