Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Abstract An efficient protoplast transformation system and a procedure of plasmid transfer by means of protoplast fusion is described for Streptococcus lactis . Protoplasts of S. lactis IL1403 and S. lactis MG1363 were transformed by pGK12 [2.9 MDa erythromycin resistance (Em^r)] with an efficiency of 3 × 10⁵ transformants per μg plasmid DNA. This high efficiency was obtained by the inclusion in the transformation mixture of liposomes composed of cardiolipin and phosphatidyl choline in a molar ratio of 1 to 6 in the presence of 22.5% polyethylene glycol (PEG). This paper also reports an efficient plasmid transfer method between lactic and streptococci and Bacillus subtilis by means of protoplast fusion. When S. lactis and B. lactis protoplasts undergo fusion mediated by exposure to 37.5% polyethylene glycol, plasmid pGKV21 (3.2 MDa; Em^r) was transfered from one host to the other with a frequency of 10⁻³−10⁻⁵ transformants per regenerating recipient protoplast.     

Isolation and Identification of Ciliatine (2-Aminoethylphosphonic Acid) from Lipids of Edible Antarctic Krill,Euphausia superba

An improved method for regenerating Bacillus subtilis protoplasts at the frequency of 92～100% on a semi-synthetic medium was found. Protoplasts were preincubated in HCP-3 medium, an isotonic semi-synthetic medium supplemented with polyvinylpyrrolidone, and then plated on HCP-1.5 agar medium by overlaying. By this method, even on the regeneration medium supplemented with minimal nutritional requirements protoplasts regenerated at a frequency of as high as 20%. The modified method was applicable to the direct-selection of prototrophic recombinants after fusion (the highest recombination yield from the input protoplasts was 1.3%) and to protoplast transformation with plasmid DNA.     

Protoplast transformation of recalcitrant alkaliphilic Bacillus sp. with methylated plasmid DNA and a developed hard agar regeneration medium

Among the diverse alkaliphilic Bacillus strains, only a little have been reported to be genetically transformed. In this study, an efficient protoplast transformation procedure was developed for recalcitrant alkaliphilic Bacillus sp. N16-5. The procedure involved polyethylene glycol-induced DNA uptake by the protoplasts and subsequent protoplast regeneration with a developed hard agar regeneration medium. An in vivo methylation strategy was introduced to methylate the exogenous plasmid DNA for improving the transformation efficiency. The transformation efficiency reached to 1.1×10(5) transformants per μg plasmid DNA with methylated plasmid pHCMC04 and the developed hard agar regeneration medium. This procedure might also be applicable to the genetic transformation of other Bacillus strains.     

High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA.

Summary A highly efficient method for transformation of Bacillus subtilis by plasmid DNA is reported. The procedure, which involves polyethylene glycolinduced DNA uptake by protoplasts and subsequent regeneration of the bacterial cell wall, yields up to 80% transformants with an efficiency of 4x10⁷ transformants per g of supercoiled DNA. Plasmids constructed by in vitro ligation or endonuclease-generated fragments of linear plasmid DNA can also transform PEG-treated protoplasts, but at a lower frequency.     

Properties of protoplasts from the thermophile Bacillus coagulans and their significance for genetic studies

Regeneration of protoplasts of Bacillus coagulans was optimized by using low lysozyme concentrations and glycerol as the osmotic support. Protoplasts formed from cells grown at higher temperatures were thermostable and capable of regeneration at 55°C. Transfer of plasmids pAB224 and pUB110, using either whole cells or protoplast transformation was not achieved, despite using a variety of conditions. However, plasmid transfer was achieved by fusion with B. subtilis protoplasts containing plasmid pAB224.     

Protoplast transformation in coryneform bacteria and introduction of an alpha-amylase gene from Bacillus amyloliquefaciens into Brevibacterium lactofermentum.

The goal of this study was to investigate the likelihood of developing useful transformation systems for coryneform bacteria. Two species of coryneform bacteria, Brevibacterium lactofermentum and Corynebacterium lilium, were transformed with chimeras constructed from pUB110 and a cryptic coryneform plasmid (pGX1901). C. lilium protoplasts were also efficiently transfected with phage CS1 DNA. High transformation and transfection frequencies were obtained after only 2 min of lysozyme treatment of lysozyme-sensitive mutants. A series of experiments was also conducted to determine whether DNA from other species of important industrial microbes from the genus Bacillus could be expressed in coryneform bacteria. Evidence of restriction of Bacillus subtilis DNA by B. lactofermentum was observed but could be overcome. A Bacillus amyloliquefaciens alpha-amylase gene (amyEBamP) was subcloned onto a plasmid able to replicate in B. lactofermentum. B. lactofermentum transformants for this plasmid expressed amylase activity and produced material cross-reactive to amylase antibody.     

Protoplast transformation in coryneform bacteria and introduction of an alpha-amylase gene from Bacillus amyloliquefaciens into Brevibacterium lactofermentum

The goal of this study was to investigate the likelihood of developing useful transformation systems for coryneform bacteria. Two species of coryneform bacteria, Brevibacterium lactofermentum and Corynebacterium lilium, were transformed with chimeras constructed from pUB110 and a cryptic coryneform plasmid (pGX1901). C. lilium protoplasts were also efficiently transfected with phage CS1 DNA. High transformation and transfection frequencies were obtained after only 2 min of lysozyme treatment of lysozyme-sensitive mutants. A series of experiments was also conducted to determine whether DNA from other species of important industrial microbes from the genus Bacillus could be expressed in coryneform bacteria. Evidence of restriction of Bacillus subtilis DNA by B. lactofermentum was observed but could be overcome. A Bacillus amyloliquefaciens alpha-amylase gene (amyEBamP) was subcloned onto a plasmid able to replicate in B. lactofermentum. B. lactofermentum transformants for this plasmid expressed amylase activity and produced material cross-reactive to amylase antibody.     

Polyethyleneglycol-induced transformation of Bacillus subtilis protoplasts by bacterial chromosomal DNA

Summary Bacillus subtilis protoplasts, which in the presence of polyethyleneglycol (PEG) are transformed by plasmid DNA (Chang and Cohen 1979) can also be transformed under these conditions by chromosomal DNA. Transformation in this case occurs at a much lower frequency, not fully accounted for by the heterogeneity of this DNA. Another unexpected feature of the transformation studied, which may explain why it previously went unnoticed, is that DNA concentrations higher than 1–2 g/ml decrease the yield of transformants, without showing signs of general toxicity.PEG-induced protoplasts (PIP) transformation for chromosomal markers operates normally with protoplasts prepared from a non-transformable bacterial mutant. The evidence indicates that both native linear and plasmid DNAs must somehow be forced into the cells as a result of PEG action. Denatured chromosomal DNA however is almost inactive in PIP transformation. No competition between chromosomal and plasmid DNAs could be detected, when the DNA tested as inhibitor was in tenfold excess.     

Transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA

Abstract A method for efficient polyethylene glycol (PEG)-mediated transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA is described. The best conditions found for protoplast regeneration included using 0.45 M sucrose both during the cultivation of the cells and (as an osmotic stabilizer) during their treatment with lysozyme, whereas 0.25 M sodium-succinate was added to the regeneration plates. Under these conditions about 5–10% of input cells regenerated. The highest transformation frequency with plasmid DNA was obtained with a PEG 6000 concentration of 22.5% (w/v). Transforming B. amyloliquefaciens strains with the plasmid pUB110 isolated from B. amyloliquefaciens resulted in 2–4 · 10⁵ transformants/μg DNA, 100–1 000-times as high as with DNA from Bacillus subtilis , suggesting a restriction barrier between the two species. Transformation of B. amyloliquefaciens with plasmids pC194 or pE194 cop -6 gave poor yields and no restriction barrier could be demonstrated for these plasmids. However, by curing pC194 from one of the transformants, a mutant strain compatible to both the plasmids could be isolated, yielding 2–3·10⁴ transformants/μg DNA. Both laboratory and industrial B. amyloliquefaciens strains could be transformed with the procedure.     

Transformation of Bacillus polymyxa with plasmid DNA

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.     

Transformation of Bacillus polymyxa with plasmid DNA.

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.     

Transformation ofSchizophyllum commune: An analysis of specific properties

Several properties of transformation in the basidiomycete,Schizophyllum commune, were examined. The transformation efficiency of protoplasts made from germinating basidiospores is dependent upon the length of time that the spores are incubated under conditions that promote germination. Protoplasts prepared from ungerminated spores transform at least 10 times more efficiently than protoplasts prepared from germlings (25 μm in length) or from mycelium. Transformation frequencies of 1000 transformants/μg of control plasmid DNA and 10⁷ protoplasts are sufficient for obtaining transformants with 2 × 10⁷ protoplasts and 10 μg of bank DNA from a genomic plasmid library. The probability of cotransforming with two plasmids is dependent on the DNA concentrations of each; concentrations can be adjusted to yield nearly 100% cotrasformants. The presence of a nonselected plasmid in the reaction mix improves the transformation frequency of a selected marker carried on another plasmid; this is not true if linear fragments ofSchizophyllum genomic DNA are used as the nonselected DNA. Transformation of aSchizophyllum protoplast does not require its fusion to another protoplast.     

Spore-spheroplast transformation of Bacillus megaterium

A new method for transformation of Bacillus megaterium was developed by modification of Chang and Cohen's method. In our method, spore spheroplasts were used as recipient cells instead of the protoplasts of vegetative cells. Longer incubation (60 min) of spore spheroplasts and plasmid DNA before treatment with polyethylene glycol remarkably increased the efficiency of transformation. The frequency of transformation was about 10(4) per microgram of plasmid DNA. A shot-gun-type cloning of chromosome DNA of B. megaterium ATCC 12872 was available in B. megaterium ATCC 19213 strain by this transformation method.     

Protoplast fusion in Bacillus cereus

Polyethyleneglycol induced fusion of Bacillus cereus protoplasts and its genetic consequences have been investigated. The technique used allows the transfer of a small plasmid pBC16 between Bacillus cereus cells. Fusion has resulted in isolation of hybrid cells having acquired TcR phenotype (harbouring pBC16) with high frequencies (10(-2)-10(-3)). However, the genetic instability of hybrid cells and segregation effects have influenced dramatically the final results of plasmid transfer. Nevertheless, the fusion of protoplasts proves to be useful in construction of BAcillus cereus strains inheriting plasmid determinants.     

The role of DNA-gyrase from Bacillus subtilis during intermolecular irregular recombination]

The location of oxolinis acid-induced gyrase cleavage sites on pBR322 and pUB110 plasmid DNA in Bacillus subtilis cells has been studied and established. The treated Bacillus subtilis protoplasts were used in the study. Coordinates of the gyrase cleavage sites were compared to the location of the illegitimate recombination sites precisely mapped on the plasmid genomes. The obtained data indicate involvement of the DNA gyrase in formation of a fraction of recombinants in Bacillus subtilis.     

Plasmid transformation of Bacillus cereus protoplasts

The process of polyethyleneglycol-induced plasmid transformation of Bacillus cereus protoplasts was studied. Plasmid transfer into Bacillus cereus strains was demonstrated with the frequencies 1.3.10(1)-1.6.10(2) transformants per 1 mkg of plasmid DNA. The plasmids transferred are stably inherited by Bacillus cereus cells causing tetracycline resistance (pBC16) or kanamycin resistance (pUB110 and pBD64). The proposed method can be used for construction of Bacillus cereus strains having the plasmid determined characteristics.     

"Marker augmentation" phenomenon in the plasmid transformation and transduction of Bacillus subtilis

Transformation of Bacillus subtilis cells carrying pUB110 plasmid by DNA of homologous plasmid pBD12 results in the significant increase in the number of plasmid transformants. This phenomenon named "augmentation" was not observed when instead of intact cells, regenerating protoplasts were used, or if pBD12 DNA was introduced into the cells via transduction.     

Preparation and regeneration of protoplasts from Bacillus megaterium cells dried by sublimation]

Bacterial protoplasts are widely used in genetical research, for instance, in protoplasts fusion experiments and the transfer of heterologous DNA into bacterial cells. The usage of a new fresh grown culture of bacteria in every experiment restricts the reproducibility of the results preventing the technique becoming widespread. The use of antioxidants as components of stabilizing medium for sublimation drying of Bacillus megaterium cells supported cellular viability in bacterial culture. It also made possible preservation of such cellular fundamental properties as the ability to form protoplasts and regenerate the cell wall. Efficiencies of protoplasts formation and generation are similar for lyophilized and fresh grown cells. Cellular properties are conserved for 6 months of storage at least. Experiments with a lot of lyophilized biomass samples are highly reproducible. The potential of the technique was demonstrated in obtaining the hybrid Bacillus megaterium colonies by fusion of protoplasts derived from lyophilized genetically marked strains stored for up to 6 months.     

A Comparison of Methods for Plasmid Delivery into Plant Protoplasts

Three methods of plasmid delivery to mesophyll protoplasts ofNicotiana tabacum cv. Xanthi have been evaluated. Specifically;a) chemically stimulated uptake of isolated plasmid, b) deliveryof plasmid encapsulated in liposomes, and c) fusion of plasmid-containingspheroplasts, were combined with divalent cation (Ca²⁺ and Mg²⁺)or polyalcohol [polyethylene glycol (PEG) and polyvinyl alcohol(PVA)] treatments. The quantity and quality of plasmid associatedwith intact protoplasts, was assessed by DNA-DNA blot hybridisationanalysis, following stringent washing to separate intact protoplastsfrom non-viable protoplasts and debris. Treatments which increasedassociation of plasmid with protoplasts decreased protoplastviability. Optimum association of plasmid with protoplasts,in the context of acceptable loss of viability, was achievedwhen protoplasts were interacted with either naked plasmid orliposomeencapsulated DNA in the presence of 15% w/v PEG 6000,or with Escherichia coli spheroplasts containing chloramphenicol-amplifiedplasmid in the presence of 25% w/v PEG 6000. Divalent cationsdid not stimulate significant plasmid delivery without unacceptableloss of protoplast viability. Strategies to further increasethe efficiency of plasmid delivery are discussed. (Received June 21, 1984; Accepted August 20, 1984)     

Protoplast-mediated gene transfer into human leukemia (K562) cells

We have examined the usefulness of a protoplast fusion technique as a tool to transfer cloned genes into hematopoietic cells. Protoplasts carrying cloned plasmids, which would express specific markers when successfully transfected into human cells, were prepared and fused with human leukemic cell line K562 cells using polyethylene glycol as a fusogenic factor. As a result, K562 cells fused with protoplasts containing a plasmid pSV2-cat constructed to code for chloramphenicol acetyltransferase (CAT) expressed CAT activity efficiently. K562 cells were also readily transformed to geneticin-(G418) resistant cells following fusion with protoplasts carrying a plasmid pSV2-neo-SV-gpt, which confers the resistance of mammalian cells to G418 and mycophenolic acid. It was also demonstrated that the plasmid genome was stably integrated into the chromosomal DNA of G418-resistant K562 cells. Our results proved that protoplast fusion could be used to study the specific expression and the biologic activities of cloned genes in human hematopoietic cells.