Protoplast formation and regeneration of Streptococcus lactis cells.

Abstract

Conditions for protoplast formation and cell wall regeneration of Streptococcus lactis were described. Since S. lactis was relatively resistant to the action of lysozyme, cell wall digestion was insufficient whenthe cells were treated with lysozyme alone. Protoplasts were obtained by treatment with lysozyme and a bacterial a-amylase preparation in Tris-HCl buffer containing 3 mMMgCl2 and 20% sucrose. The number of remaining osmotically stable cells decreased to less than 9 x 10~7. Regeneration was accomplished with a hypertonic medium that contained CaCl2, MgCl2, gelatin and sucrose. The frequency of regeneration of protoplasts to normal cells was 3~10%after 5~7 days incubation in the hypertonic medium.Protoplast fusion is a useful technique for studying fundamental and applied genetics of bacteria for which the technique to exchange genetic material has not been well developed. A key factor in the use of this technique is the capability of protoplast formation and cell wall regeneration.In Bacillus subtilis,n)Bacillus megaterium12) and some other bacteria of industrial importance,15'17) methods of protoplast formation and cell wall regeneration are well established. With the exception of the work of Gasson,6) very little information regarding the protoplast formation and regeneration of lactic streptococci has been presented.In this communication, we describe the detailed conditions of protoplast formation and cell wall regeneration of Streptococcus lactis. MATERIALS AND METHODSBacterial strain and media. Streptococcus lactis MJ708 (Ade~Mal~)was used throughout this study. This mutant was derived from S. lactis 5271} by ethyl methanesulfonate treatment and was grown and maintained in basal medium. The basal mediumfor ordinary culturing had the following composition (per liter): tryptone (Difco), 5 g; yeast extract (Difco), 2.5 g; lactose, 5 g; sodium succinate, 10g; and adenine, lOmg, pH 6.8. The composition of the basal regeneration was tryptone (Difco), 1%; yeast extract (Difco), 0.5%; glucose, 1%; sucrose, 20%; and adenine, 10/jg/ml, pH 6.8. Osmotically stable cells were measured by plating with a regeneration medium with 20% sucrose omitted.Lytic activity of lysozyme. An overnight culture of S. lactis MJ708 was inoculated into lysis broth,2) and the culture was incubated at 30°C. At the mid-log phase, cells were harvested and washed once with 30mM Tris(hydroxymethyl)aminomethane-HCl buffer, pH 8.0(TH buffer), and then suspended in 0.5 volumes of the same buffer. Lysozyme (Sigma Chemical Corp.) and/or aamylase (Wako Pure Chemical Ind., Practical Grade, LotNo. SDL 6381 and LTF 5851) were added, and the suspension was incubated at 37°C for up to 3hr. Lysis of bacteria during incubation was monitored at intervals by measuring residual turbidity at 660 nm.Protoplast formation and regeneration. Washed cells of S. lactis MJ708were obtained as described above and resuspended in TH buffer containing 3mMMgCl2 and 20% sucrose (THMS buffer). Filter-sterilized lysozyme and a-amylase dissolved in THMSbuffer were added to...

Protoplast formation and cell wall regeneration in Clostridium perfringens

“…The role of CaCl2 and MgCI2 on the cell wall reversion of C. perfringens protoplasts was examined by omitting these compounds from PRM plates. This resulted in a fourfold decrease in regeneration frequency, in agreement with results obtained with other microorganisms (2,(15)(16)(17). The best cell wall regeneration frequencies were obtained when 25 mM CaC12 and MgCl2 were added to the regeneration medium, the protoplast production menstruum, and the dilution buffer.…”

Twofold Reduction of Phosphofructokinase Activity in Lactococcus lactis Results in Strong Decreases in Growth Rate and in Glycolytic Flux

“…L. lactis was transformed by electroporation as previously described (15) and plated on SR plates (31). E. coli was made competent using CaCl 2 (36) or made electrocompetent with 10% (vol/vol) glycerol.…”

Bacillus subtilis generates a major specific deletion in pAM beta 1

“…were prepared by the method of Okamoto et al (24) with some modifications (37). S. lactis IL1403 protoplasts were transformed as described by Kondo and McKay (14), except that protoplasts and DNA were incubated in 22.5% polyethylene glycol for 20 min at room temperature in the presence of liposomes, consisting of cardiolipin and phosphatidylcholine at a molar ratio of 1:6.…”