Genetic transformation of Bacillus brevis 47, a protein-secreting bacterium, by plasmid DNA

Methods for the production and regeneration of Lactobacillus casei protoplasts are described. Protoplasts of L. casei strains were obtained by treatment with mutanolysin or with mutanolysin and lysozyme together in a protoplast formation buffer containing 0.02 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.0), 1 mM MgCI2, 0.5% gelatin, and 0.3 M raffinose. Cells were regenerated on a complex medium supplemented with bovine serum albumin, MgC92, CaC12, gelatin, and raffinose. Lengthy digestion with lytic enzymes inhibited the capacity of protoplasts to regenerate. The optimum conditions of protoplast formation varied from strain to strain. Using predetermined optimal conditions it was possible to prepare protoplasts of several L. casei strains and regenerate them with 10 to 40% efficiency. The methods were applicable to other species of lactobacilli as well.

mentioning

confidence: 99%

Production and Regeneration of Lactobacillus casei Protoplasts

Lee-Wickner

Chassy

1984

“…This study constitutes the first report, to our knowledge, of a nonprotoplast transformation procedure for the lactic acid bacteria. PEG-dependent, nonprotoplast transformation procedures have been described for such diverse microbes as Bacillus brevis (27), E. coli (11), Clostridium thermohydrosulfuricum (26), Saccharomyces cerevisiae (11), Rhodopseudomonas sphaeroides (6), and Kluyveromyces lactis (8). The specifics of these protocols vary, but in several cases, preconditioning of the cells in certain buffers was essential for transformation (6,26,27).…”

Section: Discussionmentioning

confidence: 99%

“…PEG-dependent, nonprotoplast transformation procedures have been described for such diverse microbes as Bacillus brevis (27), E. coli (11), Clostridium thermohydrosulfuricum (26), Saccharomyces cerevisiae (11), Rhodopseudomonas sphaeroides (6), and Kluyveromyces lactis (8). The specifics of these protocols vary, but in several cases, preconditioning of the cells in certain buffers was essential for transformation (6,26,27). Also, each procedure exhibited an absolute requirement for PEG treatment, distinguishing this group of procedures from wholecell procedures which require treatment with alkaline cations, but not with PEG, and from protoplast procedures which require cell wall digestion.…”

Section: Discussionmentioning

confidence: 99%

A method for genetic transformation of nonprotoplasted Streptococcus lactis

Sanders

Nicholson

1987

Plasmid transformation of whole cells of Streptococcus lactis LM0230 was demonstrated. The procedure required polyethylene glycol and incubation in hypertonic media, but did not require enzymatic cell wall digestion. Conditions were optimized, yielding 5 x 105 transformants per ,ug of pSA3 DNA. Variables tested for effect on transformation efficiency included molecular weight, concentration, and pH of polyethylene glycol; cell density; plating media; DNA concentration; heat shock; and incubation of cells in hypertonic buffer. DNAs transformed included pSA3, pVA856, pTV1, and c2(. Transformation from DNA-DNA ligation mixes, with DNA not purified through density gradients, and with previously frozen cells was also achieved. The method described here for transformation of nonprotoplasted cells of LM0230 is unique, and to date has not been applied successfully to other lactic acid bacteria. * Corresponding author. were obtained for intact, cesium chloride gradient-purified pSA3 plasmid DNA into S. lactis LM0230. This paper describes the first report of a whole-cell plasmid transformation system for lactic acid bacteria.

“…have been reported. These include transduction (20,25,34), spontaneous transfer by a conjugationlike process (15,21,32), competent-cell plasmid transformation procedures for B. subtilis (10, 11), protoplast transformation procedures (5,7,18,19,27,28), and a procedure developed for B. brevis involving exposing the cells to alkaline Tris hydrochloride (39). However, no useful procedure for plasmid transfer has been reported for B. polymyxa.…”

mentioning

confidence: 99%

Transformation of Bacillus polymyxa with plasmid DNA

Mallonee

Speckman

1989

A plasmid transformation system was developed for Bacillus polymyxa ATCC 12321 and derivatives of this strain. The method utilizes a penicillin-treated-cell technique to facilitate uptake of the plasmid DNA. Low-frequency transformation (10-6 per recipient cell) of plasmids pC194, pBD64, and pBC16 was accomplished with this method. Selection for the transformants was accomplished on both hypertonic and nonhypertonic selective media, with the highest rates of recovery occurring on a peptone-glucose-yeast extract medium containing 0.25 M sucrose. Several additional plasmids were shown to be capable of transferring their antibiotic resistance phenotypes to B. polymyxa through the use of a protoplast transformation procedure which allowed for a more efficient transfer of the plasmid DNA. However, cell walls could not be regenerated on the transformed protoplasts, and the transformants could not be subcultured from the original selective media.