Transformation of Bacillus subtilis by DNA bound on montmorillonite and effect of DNase on the transforming ability of bound DNA.

The equilibrium adsorption and binding of DNA from Bacillus subtilis on the clay mineral montmorillonite, the ability of bound DNA to transform competent cells, and the resistance of bound DNA to degradation by DNase I are reported. Maximum adsorption of DNA on the clay occurred after 90 min of contact and was followed by a plateau. Adsorption was pH dependent and was greatest at pH 1.0 (19.9 micrograms of DNA mg of clay-1) and least at pH 9.0 (10.7 micrograms of DNA mg of clay-1). The transformation frequency increased as the pH at which the clay-DNA complexes were prepared increased, and there was no transformation by clay-DNA complexes prepared at pH 1. After extensive washing with deionized distilled water (pH 5.5) or DNA buffer (pH 7.5), 21 and 28%, respectively, of the DNA remained bound. Bound DNA was capable of transforming competent cells (as was the desorbed DNA), indicating that adsorption, desorption, and binding did not alter the transforming ability of the DNA. Maximum transformation by bound DNA occurred at 37 degrees C (the other temperatures evaluated were 0, 25, and 45 degrees C). DNA bound on montmorillonite was protected against degradation by DNase, supporting the concept that "cryptic genes" may persist in the environment when bound on particulates. The concentration of DNase required to inhibit transformation by bound DNA was higher than that required to inhibit transformation by comparable amounts of free DNA, and considerably more bound than free DNase was required to inhibit transformation by the same amount of free DNA. Similarly, when DNA and DNase were bound on the same or separate samples of montmorillonite, the bound DNA was protected from the activity of DNase.     

Transformation of Bacillus subtilis by DNA bound on clay in non-sterile soil

Abstract Chromosomal DNA from Bacillus subtilis and different forms of plasmid pHV14 (covalently closed circular (CCC), linear monomer (M), and linear multimer (LM)) were adsorbed and bound on the clay mineral montmorillonite. After extensive washing of the clay-DNA complexes with DNA buffer (pH 7.5), approx. 25% of the chromosomal DNA, and approx. 30, 90, and 5%, respectively, of the CCC, M and LM form remained bound. Chromosomal and plasmid DNA bound on clay were capable of transforming competent cells, with different specific activities. The clay-DNA complexes persisted in non-sterile soil and retained transforming ability up to 15 days after their addition to the soil. DNA bound on montmortillonite was protected from the activity of Eco RI, supporting the evidence that DNA adsorbed on soil components was resistant to degradation by nucleases.     

Transformation in Bacillus subtilis. Fate of newly introduced transforming DNA

Summary Donor deoxyribonucleic acid (molecular weight 5-8×10⁷) introduced into competent cells of Bacillus subtilis is converted to molecules with a weight average molecular weight of 9×10⁶. These molecules, having little transforming activity, constitute in all probability eclipse phase DNA. At least part of the DNA is transiently complexed with a cellular component, changing its buoyant behaviour in CsCl gradients. When shortly after uptake of donor DNA the total DNA extracted from recipient cells is sheared to a molecular weight of 8×10⁶ or less, no eclipse phase is discernable. Donor marker frequencies in sheared, reisolated DNA mixtures decrease by a factor of 4 as a function of time of incubation of the transforming cells. This indicates that only 25% of the irreversibly absorbed DNA is finally integrated into the recipient genome.     

Transformation of Bacillus subtilis by single-stranded plasmid DNA.

The single-stranded form of a pE194-based plasmid transformed Bacillus subtilis protoplasts at least as efficiently as did the double-stranded plasmid, but the single-stranded form did not detectably transform B. subtilis competent cells.     

Transformation of Bacillus subtilis: transforming ability of deoxyribonucleic acid in lysates of L-forms or protoplasts.

The transformation of Bacillus subtilis by homologous deoxyribonucleic acid (DNA) made available by gently lysing a stable L-form or protoplast suspension was 3 to 10-fold more efficient than DNA isolated by conventional procedures. This increased transformation was not influenced by digestion with pronase, trypsin, or ribonuclease. Preincubation of isolated DNA with L-form lysates did not increase the transformation efficiency above that achieved with untreated, isolated DNA. In addition to displaying a higher efficiency of transformation, the DNA found in these gently prepared lysates was also able to co-transform heretofore unlinked markers at frequencies in excess of those found by congression. Comparison of the frequency of multiple marker transformations to single marker events as a function of DNA dilution conclusively proves that these markers originated from the same continuous strand of DNA.     

The initial attachment of transforming DNA to competent Bacillus subtilis.

Summary The initial attachment of transforming DNA to competent Bacillus subtilis is temperature independent between 25° and 45°. However, below 15° there is a significant reduction in the amount of DNA attached to competent cells. The DNA that is attached at 4° can lead to transformation or interfere effectively with the subsequent attachment of a distinctive DNA when the cells are shifted to a permissive temperature (37°). These data suggest that the attachment of DNA at 4° is to sites normally involved in the transformation process. The amount of DNA that is initially attached to the bacteria at 4° or 37° after perturbation of the cells by ionic strength changes, repetitive washings, or periodate oxidation varies with the temperature at which the treatment occurs. These results are consistent with a reorientation of the DNA attachment sites upon lowering the temperature to 4°, such that their affinity for DNA and susceptibility inhibitory treatments are reduced.National Institutes of Health Research Career Program Awardee, CA-K3-6487 during a portion of this investigation.     

Integration of Deoxyribonuclease-Treated DNA in Bacillus subtilis Transformation

Normal preparations of B. subtilis DNA have weight average native molecular weights of 10 to 30 x 10⁶. For any given preparation the upper and lower 95% size limits may differ by a factor of ten or more. Single-stranded molecular weights indicate an average of 1 to 4 breaks per single strand of the native DNA. The reduction in transforming activity and viscosity following DNAase I digestion can be accounted for by a direct relationship between the transforming activity of a DNA and its single-stranded molecular weight. Uptake studies with DNAase I treated heavy (²H¹⁵N ³H) DNA show that single strand breaks inhibit integration less than transformation. A provisional estimate of the size of the integrated region based on correlating the single strand size of the donor-recipient complex with the donor-recipient density differences following alkali denaturation came to 1530 nucleotides. Using a competent, nonleaky thymine-requiring strain of B. subtilis grown in 5-BU medium before and after transformation, it was shown that (a) No detectable amount of DNA synthesis is necessary for the initial stages of integration, (b) Cells which have recently been replicating DNA are not competent. (c) Cells containing donor DNA show a lag in DNA replication following transformation, (d) When donor DNA is replicated it initially appears in a density region between light and hybrid. This indicates that it includes the transition point formed at the time of reinitiation of DNA synthesis in the presence of 5-BU following transformation. A model is proposed in which donor DNA is integrated at the stationary growing point of the competent cell, which is in a state of suspended DNA synthesis.     

Transformation of Bacillus subtilis by electroporation

Summary Optimal conditions for the transformation of Bacillus subtilis by electroporation were achieved. Frequencies of greater than 10⁵ transformants/μg of plasmid DNA were obtained for a number of strains and plasmids. Increased transformation efficiency of mini-prep DNA from B. subtilis and Escherichia coli was obtained after microdialysis.     

Transformation of Bacillus subtilis by crude lysates containing staphylococcal plasmid DNA

Crude lysates from staphylococcal strains, containing DNA, were capable of transforming Bacillus subtilis at a rate of 1.68 X 10(-10) - 20.6 X 10(-10) depending on the marker according to which the transformers were selected. In a new host, plasmids showed the same behavior pattern as in the staphylococcus but their spontaneous loss was in all the cases recorded significantly more often.     

Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex

Following uptake by competent Bacillus subtilis, transforming DNA is converted to two distinct slowly sedimenting molecular forms which possess little transforming activity (eclipse). A few minutes after uptake is initiated, a physical complex of donor and recipient DNA begins to form. The recovery of donor transforming activity following eclipse, and the appearance of recombinant activity, previously reported by Venema, Pritchard &; Venema-Schröder (1965), is shown to be due to changes occurring in the donor—recipient complex. This complex exists transiently in a form with low recombinant-type transforming activity. This transient form may be one in which the donor and recipient components are joined non-covalently. The donor-recipient complex is shown to be a heteroduplex structure in which the donor moiety has an approximate molecular weight of 750,000.