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.     

Amplification of bacterial DNA bound on clay minerals by the random amplified polymorphic DNA (RAPD) technique

Abstract: Chromosomal DNA from Bacillus subtilis , bound on the clay minerals, montmorillonite (Wyoming (W) and Apache County (Ap)) and kaolinite (K), was subjected to the random amplified polymorphic DNA (RAPD) technique. DNA bound on the clays was not amplified with 0.625, 1.875, 6.25, and 12.5 U of Taq DNA polymerase, but amplification occurred when the clay-DNA complexes were diluted 10- and 20-fold or when 21 U of Taq DNA polymerase was added. DNA desorbed from the Ap-DNA and K-DNA equilibrium complexes was amplified with 0.625 U of Taq DNA polymerase, whereas amplification of DNA desorbed from the W-DNA complex occurred only after a 10-fold dilution or when 1.875 U of Taq DNA polymerase was used. These observations indicate that clay minerals differentially affect the amplification process, probably by inhibiting the activity of Taq DNA polymerase.     

Protection of Sediment-Adsorbed Transforming DNA Against Enzymatic Inactivation

The action of DNase I on transforming DNA, both adsorbed to marine sediment and in solution, was investigated. DNase I reduced the transformation frequencies of free DNA more than of adsorbed DNA. Changes in salt concentration or pH did not have a significant influence on the DNA-sediment complex. Soil components other than organic materials and clay minerals can bind DNA and retard its enzymatic degradation.     

Amplification of DNA bound on clay minerals

DNA adsorbs and binds on clay minerals, which provides protection to the DNA against degradation by nucleases but does not eliminate the ability of bound DNA to transform cells. These observations support the concept that 'cryptic genes' can persist in the environment when bound on particles and that the genes could subsequently be expressed if an appropriate host was transformed. The polymerase chain reaction (PCR) was used to amplify free and bound DNA from Bacillus subtilis and calf thymus. DNA bound on montmorillonite, but not on kaolinite, was amplified. However, amplification occurred when kaolinite was pretreated with sodium metaphosphate. DNA was not released from the clays during the amplification procedure. The type of clay (e.g. its structure and charges) affected amplification. Because DNA bound on clay is protected against biodegradation, the ability to amplify DNA bound on clay by the PCR has palaeontological, archaeological, and anthropological implications for the detection of 'ancient' DNA, as well as for monitoring the persistence of recombinant DNA introduced to the environment in genetically modified organisms.     

Adsorption of DNA to sand and variable degradation rates of adsorbed DNA

Adsorption and desorption of DNA and degradation of adsorbed DNA by DNase I were studied by using a flowthrough system of sand-filled glass columns. Maximum adsorption at 23 degrees C occurred within 2 h. The amounts of DNA which adsorbed to sand increased with the salt concentration (0.1 to 4 M NaCl and 1 mM to 0.2 M MgCl2), salt valency (Na+ less than Mg2+ and Ca2+), and pH (5 to 9). Maximum desorption of DNA from sand (43 to 59%) was achieved when columns were eluted with NaPO4 and NaCl for 6 h or with EDTA for 1 h. DNA did not desorb in the presence of detergents. It is concluded that adsorption proceeded by physical and chemical (Mg2+ bridging) interaction between the DNA and sand surfaces. Degradability by DNase I decreased upon adsorption of transforming DNA. When DNA adsorbed in the presence of 50 mM MgCl2, the degradation rate was higher than when it adsorbed in the presence of 20 mM MgCl2. The sensitivity to degradation of DNA adsorbed to sand at 50 mM MgCl2 decreased when the columns were eluted with 0.1 mM MgCl2 or 100 mM EDTA before application of DNase I. This indicates that at least two types of DNA-sand complexes with different accessibilities of adsorbed DNA to DNase I existed. The degradability of DNA adsorbed to minor mineral fractions (feldspar and heavy minerals) of the sand differed from that of quartz-adsorbed DNA.     

Adsorption of DNA to sand and variable degradation rates of adsorbed DNA.

Adsorption and desorption of DNA and degradation of adsorbed DNA by DNase I were studied by using a flowthrough system of sand-filled glass columns. Maximum adsorption at 23 degrees C occurred within 2 h. The amounts of DNA which adsorbed to sand increased with the salt concentration (0.1 to 4 M NaCl and 1 mM to 0.2 M MgCl2), salt valency (Na+ less than Mg2+ and Ca2+), and pH (5 to 9). Maximum desorption of DNA from sand (43 to 59%) was achieved when columns were eluted with NaPO4 and NaCl for 6 h or with EDTA for 1 h. DNA did not desorb in the presence of detergents. It is concluded that adsorption proceeded by physical and chemical (Mg2+ bridging) interaction between the DNA and sand surfaces. Degradability by DNase I decreased upon adsorption of transforming DNA. When DNA adsorbed in the presence of 50 mM MgCl2, the degradation rate was higher than when it adsorbed in the presence of 20 mM MgCl2. The sensitivity to degradation of DNA adsorbed to sand at 50 mM MgCl2 decreased when the columns were eluted with 0.1 mM MgCl2 or 100 mM EDTA before application of DNase I. This indicates that at least two types of DNA-sand complexes with different accessibilities of adsorbed DNA to DNase I existed. The degradability of DNA adsorbed to minor mineral fractions (feldspar and heavy minerals) of the sand differed from that of quartz-adsorbed DNA.     

Adsorption of DNA on clay minerals: protection against DNaseI and influence on gene transfer

Abstract Numerous authors have investigated DNA relationships with sandy soil. A model composed of various DNAs adsorbed on montmorillonite clay was developed to assay enzyme (DNaseI) activity on clay-adsorbed nucleic acids. The extent of DNA adsorption was affected by the concentration and valency of the cations used (Mg²⁺, Ca²⁺, Na⁺), indicating a charge-dependent process. Calf thymus DNA was found to be highly adsorbed by smectite (up to 30 mg g⁻¹ of dry clay). Adsorbed DNA was shown to be more resistant to degradation by DNaseI than free DNA. Experimental data with plasmid and short linear amplified (through polymerase chain reaction) DNA showed that protection against nucleases was only partial. Nevertheless, clay-adsorbed DNA was found to be still able, even after a strong DNaseI treatment, to artificially transform competent Escherichia coli cells. The results show that persistance of DNA and gene transfer by genetic transformation may occur in soil.     

Adsorption of plasmid DNA to mineral surfaces and protection against DNase I.

The adsorption of [3H]thymidine-labeled plasmid DNA (pHC314; 2.4 kb) of different conformations to chemically pure sand was studied in a flowthrough microenvironment. The extent of adsorption was affected by the concentration and valency of cations, indicating a charge-dependent process. Bivalent cations (Mg2+, Ca2+) were 100-fold more effective than monovalent cations (Na+, K+, NH4+). Quantitative adsorption of up to 1 microgram of negatively supercoiled or linearized plasmid DNA to 0.7 g of sand was observed in the presence of 5 mM MgCl2 at pH 7. Under these conditions, more than 85% of DNA adsorbed within 60 s. Maximum adsorption was 4 micrograms of DNA to 0.7 g of sand. Supercoil molecules adsorbed slightly less than linearized or open circular plasmids. An increase of the pH from 5 to 9 decreased adsorption at 0.5 mM MgCl2 about eightfold. It is concluded that adsorption of plasmid DNA to sand depends on the neutralization of negative charges on the DNA molecules and the mineral surfaces by cations. The results are discussed on the grounds of the polyelectrolyte adsorption model. Sand-adsorbed DNA was 100 times more resistant against DNase I than was DNA free in solution. The data support the idea that plasmid DNA can enter the extracellular bacterial gene pool which is located at mineral surfaces in natural bacterial habitats.     

Adsorption of plasmid DNA to mineral surfaces and protection against DNase I.

The adsorption of [3H]thymidine-labeled plasmid DNA (pHC314; 2.4 kb) of different conformations to chemically pure sand was studied in a flowthrough microenvironment. The extent of adsorption was affected by the concentration and valency of cations, indicating a charge-dependent process. Bivalent cations (Mg2+, Ca2+) were 100-fold more effective than monovalent cations (Na+, K+, NH4+). Quantitative adsorption of up to 1 microgram of negatively supercoiled or linearized plasmid DNA to 0.7 g of sand was observed in the presence of 5 mM MgCl2 at pH 7. Under these conditions, more than 85% of DNA adsorbed within 60 s. Maximum adsorption was 4 micrograms of DNA to 0.7 g of sand. Supercoil molecules adsorbed slightly less than linearized or open circular plasmids. An increase of the pH from 5 to 9 decreased adsorption at 0.5 mM MgCl2 about eightfold. It is concluded that adsorption of plasmid DNA to sand depends on the neutralization of negative charges on the DNA molecules and the mineral surfaces by cations. The results are discussed on the grounds of the polyelectrolyte adsorption model. Sand-adsorbed DNA was 100 times more resistant against DNase I than was DNA free in solution. The data support the idea that plasmid DNA can enter the extracellular bacterial gene pool which is located at mineral surfaces in natural bacterial habitats.     

Genetic transformation of Bacillus subtilis cells in the presence of clay minerals montmorillonite and kaolinite

The effect of the clay minerals montmorillonite and kaolinite on the transformation of competent Bacillus subtilis cells with chromosomal DNA was studied. Clay particles were found to substantially increase the transformation frequency of competent cells, as well as the rate of their spontaneous chromosomal and plasmid transformation. The effect was ascribed to the adsorption of bacterial cells on the surface of mineral particles.     

Adsorption, desorption, and activity of glucose oxidase on selected clay species.

The adsorption of the enzyme glucose oxidase (EC 1.1.3.4) to clays followed the pattern described for other proteins as being pH dependent. Maximum adsorption occurred at or below the isoelectric point of the enzyme. The amount of enzyme adsorbed to clay was influenced by the type of clay used, and also the saturating cations. Initially adsorbed enzyme showed low specific activities, and as amounts of enzyme adsorbed approached maximum stauration of clay, specific activities increased approaching that determined for free enzyme. The adsorption of glucose oxidase involved a temperature-independent cation-exchange mechanism, and enzyme adsorbed to surfaces of clay could be desorbed in active form by elevation of pH of suspending solution. This was followed by a slower temperature-dependent fixation, probably by hydrogen bonding, which resulted in protein being irreversibly adsorbed to clay surfaces. It is proposed that on adsorption of glucose oxidase to clay surfaces unravelling of the protein structure occurred, which allowed penetration of protein into the interlamellar spaces of montmorillonite. This proposal was based on the observed expansion of montmorillonite to 23 A, and the decreases in amount of a second-protein lysozyme adsorbed with extended incubation times of glucose oxidase - clay complexes at pH 4.5.     

Effect of the clay minerals montmorillonite and kaolinite on the genetic transformation of competentBacillus subtilis cells

The effect of the clay minerals montmorillonite and kaolinite on the transformation of competentBacillus subtilis cells with chromosomal DNA was studied. Clay particles were found to substantially increase the transformation frequency of competent cells, as well as the rate of their spontaneous chromosomal and plasmid transformation. The effect was ascribed to the adsorption of bacterial cells on the surface of mineral particles.     

Plasmid DNA in a groundwater aquifer microcosm -adsorption, DNAase resistance and natural genetic transformation of Bacillus subtilis

Prokaryotes can exchange chromosomal and plasmid genes via extracellular DNA in a process termed genetic transformation. This process has been observed in the test tube for several bacterial species living in the environment but it is not clear whether transformation occurs in natural bacterial habitats. A major constituent of terrestrial environments are solid particles such as quartz, silt and clay, which have considerable surface areas and which make up the solid-liquid interfaces of the habitat. In previous experiments the adsorption of DNA to chemically purified quartz and clay minerals was shown and the partial protection of adsorbed DNA against DNAase I. In a microcosm consisting of natural groundwater aquifer material (GWA) sampled directly from the environment and groundwater (GW) both linear duplex and supercoiled plasmid DNA molecules bound rapidly and quantitatively to the minerals. The divalent cations required to form the association were those present in the GWA/GW microcosm. The association was stable to extended elution over one week at 23°C. Upon adsorption, the DNA became highly resistant against enzymatic degradation. About 1000 times higher DNAase I concentrations were needed to degrade bound DNA to the same extent as DNA dissolved in GW. Furthermore, chromosomal and plasmid DNA bound on GWA transformed competent cells of Bacillus subtilis. However, in contrast to DNA in solution, on GWA the chromosomal DNA was more active in transformation than the plasmid DNA. The studies also revealed that in the transformation of B. subtilis Mg²⁺ can be replaced by Na⁺, K⁺ or NH₄ The observations suggest that in soil and sediment environments, mineral material with inorganic precipitates and organic matter can harbour extracellular DNA leaving it available for genetic transformation.     

Evaluation of biological and physical protection against nuclease degradation of clay-bound plasmid DNA

In order to determine the mechanisms involved in the persistence of extracellular DNA in soils and to monitor whether bacterial transformation could occur in such an environment, we developed artificial models composed of plasmid DNA adsorbed on clay particles. We determined that clay-bound DNA submitted to an increasing range of nuclease concentrations was physically protected. The protection mechanism was mainly related to the adsorption of the nuclease on the clay mineral. The biological potential of the resulting DNA was monitored by transforming the naturally competent proteobacterium Acinetobacter sp. strain BD413, allowing us to demonstrate that adsorbed DNA was only partially available for transformation. This part of the clay-bound DNA which was available for bacteria, was also accessible to nucleases, while the remaining fraction escaped both transformation and degradation. Finally, transformation efficiency was related to the perpetuation mechanism, with homologous recombination being less sensitive to nucleases than autonomous replication, which requires intact molecules.     

Binding of the protoxin and toxin proteins ofBacillus thuringiensis subsp.kurstaki on clay minerals

The equilibrium adsorption and binding of the delta-endotoxin proteins, i.e., the protoxins (M_r=132 kDa) and toxins (M_r=66 kDa), fromBacillus thuringiensis subsp.kurstaki were greater on montmorillonite than on kaolinite (five-fold more protoxin and three-fold more toxin were adsorbed on montmorillonite). Approximately two- to three-fold more toxin than protoxin was adsorbed on these clay minerals. Maximum adsorption occurred within 30 min (the shortest interval measured), and adsorption was not significantly affected by temperatures between 7° and 50°C. The proteins were more easily desorbed from kaolinite than from montmorillonite; they could not be desorbed from montmorillonite with water or 0.2% Na₂CO₃, but they could be removed with Tris-SDS (sodium dodecyl sulfate) buffer. Adsorption was higher at low pH and decreased as the pH increased. Adsorption on kaolinite was also dependent on the ionic nature of the buffers. The molecular mass of the proteins was unaltered after adsorption on montmorillonite, as shown by SDS-PAGE (polyacrylamide gel electrophoresis) of the desorbed proteins; no significant modifications occurred in their structure as the result of binding on the clay, as indicated by infrared analysis; and there was no significant expansion of the clay by the proteins, as shown by x-ray diffraction analysis. The bound proteins appeared to retain their insecticidal activity against the third instar larvae ofTrichoplusia ni.     

Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.

A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 microliters from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 micrograms/microliters, depending on the type of soil extracted. The coextracted humic acid fraction of a clay silt was similar to a commercially available standard humic acid mixture, as determined by electrophoretic mobility in agarose gels, UV fluorescence, and inhibition assays with DNA-transforming enzymes. Restriction endonucleases were inhibited at humic acid concentrations of 0.5 to 17.2 micrograms/ml for the commercial product and 0.8 to 51.7 micrograms/ml for the coextracted humic acids. DNase I was less susceptible (MIC of standard humic acids, 912 micrograms/ml), and RNase could not be inhibited at all (MIC, > 7.6 mg/ml). High inhibitory susceptibilities for humic acids were observed with Taq polymerase. For three Taq polymerases from different commercial sources, MICs were 0.08 to 0.64 micrograms of the standard humic acids per ml and 0.24 to 0.48 micrograms of the coextracted humic acids per ml. The addition of T4 gene 32 protein increased the MIC for one Taq polymerase to 5.12 micrograms/ml. Humic acids decreased nonradioactive detection in DNA-DNA slot blot hybridizations at amounts of 0.1 micrograms and inhibited transformation of competent Escherichia coli HB101 with a broad-host-range plasmid, pUN1, at concentrations of 100 micrograms/ml. Purification of crude DNA with ion-exchange chromatography resulted in removal of 97% of the initially coextracted humic acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of an arginine-specific protein kinase tightly bound to rat liver DNA

A new protein kinase has been characterized among the proteins tightly bound to rat liver DNA and released by DNase I and RNase A treatment. This enzyme was separated by gel filtration from this released material. Its apparent molecular mass was found to be 34 kDa and it is made of a single unit. The main characteristic of this protein kinase is that it is arginine-specific. Isolation of phosphoarginine required the use of proteolytic enzymes at alkaline pH since the phosphate bond is highly acid-labile. This protein kinase is able to autophosphorylate and to phosphorylate a single chromosomal protein of 11 kDa also tightly bound to DNA. It uses ATP and dATP as phosphate donors and is cAMP-independent. Its optimal activity requires Mn2+ ions. Vanadate, spermine and heparin have no effect on its activity.     

Insecticidal Activity of the Toxins from Bacillus thuringiensis subspecies kurstaki and tenebrionis Adsorbed and Bound on Pure and Soil Clays

The release of transgenic plants and microorganisms expressing truncated genes from various subspecies of Bacillus thuringiensis that encode active insecticidal toxins rather than inactive protoxins could result in the accumulation of these active proteins in soil, especially when bound on clays and other soil particles. Toxins from B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. tenebrionis, either free or adsorbed at equilibrium or bound on pure clay minerals (montmorillonite or kaolinite) or on the clay size fraction of soil, were toxic to larvae of the tobacco hornworm (Manduca sexta) and the Colorado potato beetle (Leptinotarsa decemlineata), respectively. The 50% lethal concentrations (LC(inf50)) of free toxins from B. thuringiensis subsp. kurstaki were higher than those of both bound and adsorbed complexes of these toxins with clays, indicating that adsorption and binding of these toxins on clays increase their toxicity in diet bioassays. The LC(inf50) of the toxin from B. thuringiensis subsp. tenebrionis that was either free or adsorbed on montmorillonite were comparable, whereas the toxin bound on this clay had higher LC(inf50) and the toxin bound on kaolinite had lower LC(inf50) than when adsorbed on this clay. Results obtained with the clay size fraction separated from unamended soil or soil amended with montmorillonite or kaolinite were similar to those obtained with the respective pure clay minerals. Therefore, insecticidal activity of these toxins is retained and sometimes enhanced by adsorption and binding on clays.     

Formation of Single-Stranded DNA during DNA Transformation of Neisseria gonorrhoeae

Neisseria gonorrhoeae is naturally competent for DNA transformation. In contrast to other natural prokaryotic DNA transformation systems, single-stranded donor DNA (ssDNA) has not previously been detected during transformation of N. gonorrhoeae. We have reassessed the physical nature of gonococcal transforming DNA by using a sensitive nondenaturing native blotting technique that detects ssDNA. Consistent with previous analyses, we found that the majority of donor DNA remained in the double-stranded form, and only plasmid DNAs that carried the genus-specific DNA uptake sequence were sequestered in a DNase I-resistant state. However, when the DNA was examined under native conditions, S1 nuclease-sensitive ssDNA was identified in all strains tested except for those bacteria that carried the dud-1 mutation. Surprisingly, ssDNA was also found during transformation of N. gonorrhoeae comA mutants, which suggested that ssDNA was initially formed within the periplasm.