Transformation of Clostridium acetobutylicum Protoplasts with Bacteriophage DNA

Techniques for the transformation of Clostridium acetobutylicum protoplasts with bacteriophage DNA are described. Transformation required regeneration of protoplasts and a 2-h eclipse period.     

Transformation of Streptococcus lactis Protoplasts by Plasmid DNA

Polyethylene glycol-treated protoplasts prepared from Streptococcus lactis LM3302, a lactose-negative (Lac⁻) derivative of S. lactis ML3, were transformed to lactose-fermenting ability by a transductionally shortened plasmid (pLM2103) coding for lactose utilization.     

Transformation of Bacillus Protoplasts by Plasmid pTP4 DNA

Polyethylene glycol (PEG)-induced protoplast transformation by plasmid pTP4 DNA encoded chloramphenicol resistance determinant was developed for Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium and B. pumilus. Protoplasts were formed by treatment of cells with lysozyme and the transformation frequencies (transformants per regenerants) were in the range of 1.3 × 10^?2 to 7.1 × 10^?1. Reisolated plasmid DNA prepared from transformants exhibited covalently closed and open circular forms similar to those of the donor DNA. These results indicate that PEG-induced protoplast transformation is an adequate method for plasmid transformation and pTP4 is a useful plasmid as a cloning vector in a wide range of varieties of the genus Bacillus.     

Transformation of protoplasts of nontransformableBacillus subtilis mutants by plasmid pUB 110 DNA

Protoplasts rather than intact cells of nontransformableB. subtilis mutants were transformed by plasmid pUB 110 DNA. Transformability of protoplasts of the NT mutants indicates that the mechanism of uptake of the donor DNA by protoplasts differs from that by competent intact cells.     

Stability of pUB110 and pUB110-derived plasmids in Bacillus sphaericus 2362

The segregational and structural stability of pUB110 and four derivatives of various insert size and location was examined in the mosquito pathogen Bacillus sphaericus 2362 grown under conditions relevant to use of the bacterium as a larval control agent. Plasmids pUB110 (4.5 kb), pLDT103 (7.6 kb), and pTST130 (6.5 kb) exhibited 95–100% segregational stability at growth temperatures of 28° C and 38° C and at generation times of 42 and 108 min during 40 generations in a chemostat. Plasmids pLDT117 (9.7 kb) and pTST112 (6.5 kb), which had deletions in the BA4 membrane-binding site of the plasmids, were almost as stable (92–99%) unter these conditions. However, under growth conditions that allowed sporulation in mosquito larval cadavers, in batch culture or in the chemostat, plasmids with BA4-region deletions exhibited only 77–88% (pLDT117) or 66–81% stability in the resulting spores. Whereas segregational instability was associated with interruption of the BA4 region of these plasmids, this instability was primarily associated with the sporulation phase of development rather than with vegetative growth. Structural instability was not detected. Correspondence to: A. Yousten     

Cryotransformation of Bacillus anthracis by the DNA of pUB110 plasmid

Possibility of cryotransformation of Bacillus anthracis cells by the DNA of pUB110 plasmid has been established. The parameters of cryotransformation process have been optimized permitting one to increase the efficiency of transformation up to 3.1 . 10(2) transformants per 1 mkg of transforming DNA. The factors affecting the efficiency of cryotransformation and its reproducibility have been studied including the treatment of recipient cells by glycine, the procedure of freeze-thawing, the composition of freezing medium. The recipient activity of Bacillus anthracis cells has been shown to depend on the set of their own plasmids.     

Transformation of Zymomonas mobilis with Plasmid DNA

A method for the transformation of Zymomonas mobilis with plasmid DNA was developed by using shuttle vectors, pZA31, pZA32 and pZA33, as a source of transforming DNA. Partial spheroplasts of Z. mobilis were prepared by growing cells in a hypertonic medium supplemented with a drug which inhibits the biosynthesis of bacterial cell walls, such as penicillin G and d-cycloserine. They were transformed with plasmid DNA in the presence of 15% polyethylene glycol 6,000, after treated with 100 mm CaCl₂. The frequency of transformation obtained was 10⁴ to 10⁵ transformants/μg of DNA for Z. mobilis IFO13756 (Z-6).     

Transformation and transduction in Bacillus subtilis strains with the BsuR restriction-modification system by modified and unmodified pUB110 plasmid DNA

Plasmid pUB110 DNA is restricted and modified during transformation of Bacillus subtilis cells possessing the BsuR system of restriction-modification. Restriction has comparatively little influence on the frequency of plasmid transformation only reducing it 20 times. The frequency of transduction of nonmodified plasmid DNA into modifying recipient cells, using phage Ar9 is also reduced a little. The frequency of transduction of chromosomal markers is much more lowered under the same experimental conditions.     

Transfer of Liposome-Sequestering Plasmid DNA into Daucus carota Protoplasts

Reverse-phase evaporation lipid vesicles (REV) liposomes, consisting of phosphatidyl choline and stearylamine in 1:3 molar ratio, encapsulated approximately 30% of exogenously supplied recombinant DNA vector, pBR322. The DNA sequestered in REV liposomes was highly tolerant to DNase.     

Improved Transformation Method for Acetobacter with Plasmid DNA

An improved method for transformation of derivative strains of A. aceti subsp. aceti No. 1023 with plasmid DNA has been developed. Addition of polyethylene glycol or dimethylsulfoxide increased the transformation efficiency by a factor of about ten. In the presence of PEG 4,000, various transformation conditions were examined. Cells were also made transformation competent by treatment with other divalent cations than Ca²⁺ . The pH of the buffer did not affect the efficiency significantly. The growth phase influenced the efficiency. Mutants showing high competence were derived by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. By the improved method using the highly transformable mutants, a transformation efficiency of approximately 105 transformants per γg plasmid DNA was achieved.     

Mutagenesis of Clostridium acetobutylicum

Mutagenesis of the obligate anaerobe Clostridium acetobutylicum was best accomplished using agents (e.g. ethyl methane sulphonate or N -methyl- N '-nitro- N -nitrosoguanidine) which are believed to act by a direct mutagenic mechanism. Other agents (e.g. u.v. radiation) whose effectiveness relies on misrepair of damaged DNA via an error-prone pathway, were poor mutagens of this organism. Procedures are described which readily yielded a variety of auxotrophic and other useful mutant strains of Cl. acetobutylicum and related saccharolytic clostridia.     

Mutagenesis of Clostridium acetobutylicum

Mutagenesis of the obligate anaerobe Clostridium acetobutylicum was best accomplished using agents (e.g. ethyl methane sulphonate or N-methyl-N'-nitro-N-nitrosoguanidine) which are believed to act by a direct mutagenic mechanism. Other agents (e.g. u.v. radiation) whose effectiveness relies on misrepair of damaged DNA via an error-prone pathway, were poor mutagens of this organism. Procedures are described which readily yielded a variety of auxotrophic and other useful mutant strains of Cl. acetobutylicum and related saccharolytic clostridia.     

Binding of ColE1-kan Plasmid DNA by Tobacco Protoplasts: Nonexpression of Plasmid Gene

Protoplasts prepared from cultured tobacco cells were treated with ColE1-kan plasmid DNA, a hybrid of ColE1 and pSC105 plasmids bearing a gene for kanamycin resistance. The conditions employed permitted the uptake or irreversible binding of 2.9% of the added DNA in acid-insoluble form. Upon commencement of division, the treated cells were plated in agar medium containing kanamycin and differentiating hormones. Plantlets or shoots obtained as presumptive transformants were further tested on kanamycin medium by subculturing small leaf pieces. No evidence was obtained for expression of the kanamycin resistance gene of ColE1-kan in tobacco tissue.     

Transformation of Acetobacter polyoxogenes with Plasmid DNA by Electroporation

Acetobacter polyoxogenes was transformed with plasmid DNA by electroporation. The following points were essential for transformation: (i) dilution of the culture broth with cold water and air bubbling of the culture broth for transformation at discharging from a jar fermentor, and (ii) selection of transformants by liquid cultivation. For shortening of the lag time in cultivation for selection of transformants, the following treatments were useful: (i) addition of sucrose to the cell suspension during transformation and to the broth for cultivation, and (ii) addition of 1 mm MgCl₂ to a mixture of cells and DNA during electroporation.     

Replication origins of single-stranded-DNA plasmid pUB110.

The two replication origins of plasmid pUB110 have been characterized. The site of initiation of DNA replication at the plus origin was mapped to within an 8-base-pair sequence. DNA synthesis initiated at the origin was made to terminate precociously in an inserted sequence of 18 base pairs that is homologous to a sequence in the origin. This suggests that pUB110 replicates as a rolling circle. The minus origin of plasmid pUB110 has been characterized, and the minimal sequence required for function has been determined. As with other minus origins, activity is orientation specific with respect to the direction of replication. Its activity is sensitive to rifampin in vivo, suggesting that RNA polymerase catalyzes single-strand to double-strand conversion. Unlike all other plasmids of gram-positive bacteria thus far described, the pUB110 minus origin is functional in more than one host.