[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.

[The genetic transfer, heredity and phenotypic expression of plasmid RP4::Mu cts genes in Bacillus cereus]. / Geneticheskii perenos, nasledovanie i fenotipicheskoe vyrazhenie genov plazmidy RP4::Mu cts v Bacillus cereus.

The transcipients were obtained in intrageneric matings of E.coli donor harbouring the plasmid PR4::Mu cts 62 with Bac. cereus GP7 recipient cells with the frequency 10(-9). The transcipient clone Bac. cereus 682 was selected having stably inherited and expressed the hybrid plasmid PR4::Mu cts 62 genes for antibiotic resistance and temperature sensitivity. Production of the bacteriophage Mu cts 62 particles was not registered in the bacillary transcipient cells. The plasmid RP4::Mu cts 62 genes were localized in the chromosome of Bac. cereus 682 transcipient by the blot-hybridization technique with 32P-labelled DNA of the bacteriophage Mu cts 62 and the plasmid PR4. The transcipient of Bac. cereus 682 has the donor properties and transfers the RP4::Mu cts 62 genes to recipient cells of Bac. cereus DSM 318 with the frequency of 10(-6)-10(-7). The expression and transfer of the gram-negative plasmid genes in gram-positive bacterial cells are discussed.

[Glycine-dependent cryotransformation of Bacillus cereus by plasmid DNA]. / Glitsinzavisimaia kriotransformatsiia Bacillus cereus plazmidnoi DNK.

A new technique for transformation of naturally noncompetent strains of Bac. cereus is proposed. Penetration of the DNA into recipient cells is based on two-step effect. At the first step of the process bacilli are affected by glycine in the early logarithmic phase of growth of the common periodic culture. At the second step the mixed DNA and recipient cells are frozen-thawed. The process permits the transforming DNA penetration via the outer membrane layer of the recipient cells having the affected permeability under the conditions of keeping bacillar recipient cells intact. The efficiency of transformation of Bac. cereus by the plasmids pUB110 and pBC16 DNA by the proposed technique is 1.10(4) and 3.10(3) of transformants per 1 mkg of the plasmid DNA.

[Effect of the molecular weight of DNA on the effectiveness of plasmid transformation of E. coli K12]. / Vliianie molekuliarnoi massy DNK na éffektivnost' plazmidnoi transformatsii E. coli K12.

The level of plasmid transformation and transfection by the high molecular mass DNA was studied for Escherichia coli mutants having increased efficiency of plasmid transformation by low molecular mass DNA. Decreased level of plasmid transformation and transfection registered in some mutants as compared to the one in wild type strain suggests the specificity of Escherichia coli cells penetration for DNA of different molecular mass.

[In vivo restriction of Escherichia coli-transforming DNA by endonuclease R.M.EcoRI]. / Restriktsiia transformiruiushchei DNK Escherichia coli éndonukleazoi R.M.EcoRI in vivo.

Transformation of Escherichia coli K-12 for various chromosomal markers was accomplished by using AB1157 recBC+ strain as a recipient. The yield of transformants was reduced 10-fold, as compared with that obtained in JC7623 recBC sbcB recipient. Elimination of transformation has been obtained for arg, pro, his markers in AB1157 (pSA14) harbouring the R.M.EcoRI coding plasmid. Production of restriction endonuclease in this strain did not affect the efficiency of transformation for thr, leu markers. The presence of pSA25 which is isogenic to pSA14 but devoid of R.M.EcoRI genes has been irrelevant to transformation for leu, arg, pro, his, thr markers. Correlation between the restriction of transformed markers in vivo and in vitro is discussed.

[Reaction between isolated lambda phage DNA and membrane structures of Escherichia coli cells]. / Vzaimodeistvie izolirovannoi DNK faga lambda s membrannymi strukturami kletok Escherichia coli.

The saccharose density gradient (30--55%) centrifugation technique applied to E. coli membrane preparations was used to show that treatment of the bacteria with Ca2+ in the cold results in the redistribution of the absorbed phage DNA from the cell wall to the cytoplasmic membrane while freezing-thawing of the bacteria leads to equal distribution of the infectious DNA among all membrane fractions. Quantitative estimation of such a redistribution is reported.

[Adsorption of lambda phage DNA onto Escherichia coli cells treated with Ca2+ ions and onto frozen--thawed bacteria]. / Adsorbtsiia DNK faga lambda na kletkakh Escherichia coli, obrabotannykh ionami Ca2+, i na zamorozhennykh-ottaiannykh bakteriiakh.

The study of the biologically active tritium-labeled phage lambda DNA adosrption on Ca2+-treated and frozen--thawed E. coli cells showed the absence of a correlation between the adsorption level and transfection efficiency. Thus the infectious phage lambda DNA adsorption level does not change, while frozing--thawing of E. coli cells but it increases ten-fold when treating the cells with Ca2+ in ice, the transfection efficiency level with this DNA being equal for both types of recipients.

[Frozen and thawed bacteria as recipients of isolated phages and plasmid DNA]. / Zamorozhennye i ottaiannye bakterii kak retsipienty izolirovannykh fagovykh i plazmidnykh DNK.

Bacteria subjected to freezing and thawing are effective recipients of phage 1 phi 7 DNA, lambda DNA, and plasmid pMB9 DNA. The effectiveness of transfection and plasmid transformation of frozen and thawed bacteria is determined by the joint action of 3 factors: 1) the conditions of freezing and thawing of a recipient and DNA mixture with freezing carried out at a rate of 400 degrees C/min to--76 degrees C or--196 degrees C and with subsequent thawing at 42 degrees C; 2) a transitory character of recipient competence preservation in respect of phage and plasmid DNA; 3) the degree of recipient cryolability depending, in particular, on the genotype of the recipient. The maximum indices of transfection effectiveness and plasmid transformation have been obtained with bacterial concentration equal to 1--5 X 10(9) cells/ml, phage and plasmid DNA concentration equal to 0.05--0.5 mcg/ml in the reaction mixture containing 0.5--1% of Spofa bactopeptone, PH 7.4--7.6.

[Plasmid transformation of frozen and thawed bacteria]. / Plazmidnaia transformatsiia zamorozhennykh i ottaiannykh bakterii.

Plasmid DNA transformation efficiency depends on three essential factors: 1) the optimal regime of the recipients freezing-thawing; 2) the period of the recipients competence preservation; 3) individual sensitivity of microorganisms to freezing-thawing. It is demonstrated that plasmid DNA pMB9 activity indices are of maximal value during freezing at -70 degrees C or -196 degrees C and thawing at 42 degrees C. The short period of the competence, about 15 seconds, determines the rate of its infection. In this case it was achieved by mutual freezing-thawing of bacteria and DNA pMB9. The optimal yield of transformants is obtained in the following conditions: the concentration of bacteria - 1 - 5.10(9) cells/ml, the concentration of DNA pMB9 - 0.05--0.5 mcg/ml in the reaction mixture containing 0.5--1% of bactopeptone ("Spofa") and at pH 7.4--7.6.

[Interaction of the isolated DNA of lambda phage with spheroplasts of E. coli treated with sturine]. / O vzaimodeistvii izolirovannoi DNK faga lambda so sferoplastami E. coli, obrabotannymi sturinom.

The method of centrifugation in sucrose density gradient (30-55%) of the spheroplast membrane preparations treated and untreated with sturine and infected with phage lambda DNA demonstrated that sturine, treatment increased the phage lambda DNA absorption three-fold. About 50% of the lambda DNA molecules adsorbed by spheroplasts are bound with the cytoplasmic membrane of spheroplasts treated with sturine; 50% of the lambda DNA molecules are bound with the cell wall membrane on the sturine-untreated spheroplasts. The data obtained allow to conclude that the stimulating effect of sturine in E. coli spheroplasts transfection by lambda DNA is connected with redistribution of phage DNA absorbed on spheroplasts from the cell wall to the cytoplasmic membrane facilitating the penetration of DNA and its fastening on the membrane.

[Use of transformation for studying the nature of formation of stable bacterial L forms]. / Primenenie transformatsii dlia izucheniia prirody obrazovaniia stabil'nykh L-form bakterii

The authors obtained a stable variant of the L-forms of Bacillus subtilis capable of exponential growth of the minimal and synthetic medium. An electron-microscopic study of different stages of the L-form formation was carried out by the method of ultra-thin sections. A possibility was shown of the transfer of the L-form formation sign by the method of transformation. DNA isolation from the L-forms by soft lysis considerably facilitated and simplified the genetic analysis of the L-form formation by the transformation method.