[Glycine-dependent cryotransformation of Bacillus anthracis by DNA of various plasmids]. / Glitsinzavisimaia kriotransformatsiia Bacillus anthracis DNK razlichnykh plazmid.

The possibility of the 1,8-60,0 Md plasmids DNA transfer into the cells of different Bacillus anthracis strains has been established. A number of parameters of the procedure has been optimized. The including of PEG6000 into the cryomixture in the final concentration 10% has been demonstrated to be vital for increase in transformants yields. Under the described conditions the maximal efficiency of transformation (1,2.10(3)) has been registered for the DNA of the plasmid pUB110. The efficiency of the process decreased concomittant with the size of the transformed plasmids representing 2-4.10(-1) for the plasmid perlicon pXO2 (60 Md). The plasmids obtained by Bacillus anthracis cells retained the functional and structural stability.

Glycine-induced cryotransformation of plasmids into Bacillus anthracis.

Different cloning vectors (pC194, pBC16, pUB110, pBD10, pBD8, pAM beta 1) and Bacillus anthracis plasmid pX02 were introduced into B. anthracis by a transformation method. To induce an artificial competence state for uptake of isolated plasmid DNA, the cultures were treated with glycine, to reduce cross-linking of peptidoglycan, followed by freezing and thawing. The procedure is extremely rapid and relatively efficient (maximum transformation efficiency about 10(3) c.f.u. per micrograms DNA) and allows different cloning vectors with molecular masses ranging from 1.8 to 17.7 MDa to be introduced into B. anthracis.

[Conjugation transfer of the pAMbeta1 plasmid to Bacillus anthracis]. / Kon''iugatsionnaia peredacha plazmidy pAMbeta1 Bacillus anthracis.

The possibility of conjugational transfer of the plasmid pAM beta 1 in the cells of different strains of Bacillus anthracis has been established. The efficiency of the plasmid replicon transfer in interspecies transfer B. thuringiensis X B. anthracis was n.10(-7), while in interspecies transfer it was n.10(-6). The capability of mobilization of extrachromosomal replicon pTG141 for conjugational transfer has been demonstrated. Bacillus anthracis transconjugants harbouring the pAM beta 1 plasmid have acquired the donor properties in conjugation.

[Cryotransformation of Bacillus anthracis by the DNA of pUB110 plasmid]. / Kriotransformatsiia sibireiazvennogo mikroba DNK plazmidy pUB110.

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.

[Transduction and conjugation transfer of the pXO2 plasmid in Bacillus anthracis]. / Transduktsionnaia i kon''iugatsionnaia peredacha plazmidy pXO2 Bacillus anthracis.

The plasmid pXO2 determining the capsule synthesis has been shown to be transfered into the cells of different strains of Bacillus anthracis (STI-1, Sterne, KM33, KM35) by the transducing bacteriophage CP54ant and by mobilization by pAM beta 1 replicon with the frequencies, consequently, n.10(-8) and n.10(-7). The optimal parameters for the selection of clones having acquired the pXO2 plasmid have been defined. Mobilization for conjugational transfer has been demonstrated for the plasmid pXO1 coding for the production of Bacillus anthracis toxin. The dramatic increase of virulence for white mice has been registered for Bacillus anthracis strains having acquired the pXO2 plasmid replicon.

[Plasmid transduction by Bacillus anthracis bacteriophage CP54]. / Transduktsiia plazmid bakteriofagom CP54 Bacisslus anthracis.

Possibility of plasmid transduction in Bacillus anthracis vaccine strains Sterne and STI-1 by bacteriophage CP54ant having an increased ability of adsorbtion and a shortened period of latent development in Bacillus anthracis cells has been isolated. The main parameters of plasmid transduction by the bacteriophage have been established for the plasmid pTG141 (TcR). They include the effect of multiplicity of infection, the level of UV-inactivation of bacteriophage, the presence of antiphage serum in the incubation medium. Plasmid transduction by the mutant phage CP54ant was found to be more efficient as compared with the one by the parent phage. The isolated transductants served as donors of the transduced plasmid for Bacillus anthracis and Bacillus thuringiensis strains.