Protoplast formation and cell wall regeneration in Clostridium perfringens

Abstract

A protocol was developed for the efficient production and regeneration of Clostridium perfringens protoplasts. Cell wall regeneration frequencies of up to 5% were obtained.

Recent advances in the genetics of the clostridia

“…Trans-Reference formation C acetobutylicum P262 + [19] C. acetobutylicum N1-4 a - [20] C acetobutylicum N1-4081 b + [13] C. pasteurianum ATCC 6013 - [21] C tertium ATCC 19405 - [22] C perfringens ATCC 3626B - [23] C thermohydrosulfuricum DSM 568 - [16] C thermopapyrolyticum c - [24] a This strain was formerly called C. saccharoperbutylacetonicurn [13];…”

Induction and Regeneration of Autoplasts from Clostridium thermohydrosulfuricum JW102 and Thermoanaerobacter ethanolicus JW200

“…However, for cells incubated at 55°C (data not shown), the plating efficiency was only 0.01%. A small percentage of autoplasts from both strains, JW102 and JW200, exhibited unusual osmostability (12,13,15). When wall-less cells of JW102 or JW200 were diluted in distilled water, approximately 3% of the L-phase CFU (based on a control using isosmotic medium) survived and when subsequently plated out they formed typical autoplast colonies, with the "fried egg" structure ( Fig.…”

“…Autoplasts were plated on minimal medium (solidified with 0.8% [wt/vol] agar) suitable for the growth of both organisms (20), except that 1.9 mM MgCl2-0.45 mM CaCl2-0.6% bovine serum albumin was added (after autoclaving) for stabilization of the autoplasts (Table 2) (10,15) and glycerol (0.3 M) or sucrose (0.29 M) was added as an osmostabilizer. However, addition of the cell wall precursor 0.1 M N-acetylglucosamine (15) or 0.5% (wt/vol) gelatin as an osmostabilizer (6,7) to the basal medium repressed growth of colonies. The L-phase colonies (i.e., colonies consisting only of autoplast-shaped cells) were compact and usually had a diameter of less than 1 mm after 5 days of incubation at 60°C, whereas the colonies of walled rodshaped cells were about 4 mm in diameter (Fig.…”

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

“…Attempts at regenerating the cell wall of L-phase variants of C. perfringens have not been successful (9,12,16,19). However, the cell wall of lysozyme-induced protoplasts of C. perfringens can be regenerated at low frequency (20).…”