Interactions Of Soil Active Particles With DNA And Effects On DNA Stability, Transformation Of Cells And PCR Amplification

Typical zonal soil such as Brown soil was sampled from Tianwai village, Taishan,Shandong province in China. Two soil colloidal components i.e. fine clay (＜0.2μm) andcoarse clay (0.2-2μm) were separated by centrifugation. The two treatments applied tofine and coarse clays were organic matter left on the samples (organic clays) and organicmatter removed from the samples by H₂O₂ (inorganic clays). Brown soil was divided intofour types of clays: coarse organic clay (0.2-2μm, organo-mineral complexes), coarseinorganic clay (0.2-2μm, H₂O₂-treated clays), fine organic clay (＜0.2μm,organo-mineral complexes) and fine inorganic clay (＜0.2μm, H₂O₂-treated clays). Theadsorption, desorption and binding mechanism of DNA on Brown soil colloid or mineral(montmorillonite, hydroxyaluminum-montmorillonite, kaolinite or goethite), the abilityof bound DNA to transform competent cells and the resistance to degradation by DNase I,the amplification of bound plasmid DNA by PCR and the effect of soil active particles onmicrobial metabolic activity were investigated in this study. The main results were listedas following:1. Adsorption-desorption of DNA, FTIR, circular dichroism (CD), fluorescencespectroscopy, microcalorimetry were carded out to clarify the adsorption mechanism ofDNA on permanent-charge soil active particles. The maximum amount of DNA adsorbedfollowed the order: montmorillonite(?)fine inorganic clay＞fine organic clay＞kaolinite＞coarse inorganic clay＞coarse organic clay. A marked decrease in the adsorption ofDNA on organic clays and montmorillonite was observed with the increase of pH from2.0 to 5.0. Negligible DNA was adsorbed by organic clays above pH 5.0. As for inorganicclays and kaolinite, a slow decrease in DNA adsorption was found with increasing pHfrom 2.0 to 9.0. Magnesium ion was more efficient than sodium ion in promoting DNAadsorption on soil colloids and minerals. DNA molecules on soil colloids and mineralswere desorbed by sequential washing with 10 mM Tris, 100 mM NaCl and 100 mMsodium phosphate buffer at pH 7.0. A percentage of 53.7-64.4% of adsorbed DNA onorganic clays and montmorillonite was released, while only 10.7-15.2% of DNA oninorganic clays and kaolinite was desorbed by Tris and NaCl. The percent desorption ofDNA from inorganic clays, organic clays, montmorillonite and kaolinite by sodiumphosphate buffer was 39.7-42.2, 23.6-28.8, 29.7 and 11.4%, respectively. DNAadsorption on organic clays was endothermic (1.1＜△H_ads＜3.5 kJ/g), whereas that oninorganic clays was exothermic (-0.3＜△H_ads＜-0.1 k J/g). Dehydration effects andelectrostatic interactions dominated DNA adsorption on organic clays andmontmorillonite, and DNA was adsorbed predominantly by ligand exchange and possibly hydrogen bonding on inorganic clays and kaolinite. FTIR spectra showed that the bindingof DNA on kaolinite and inorganic clays changed its conformation from the B-form to theZ-form, whereas montmorillonite and organic clays retained the original B-form of DNA.A structural change from B-to C-form in DNA molecules desorbed from kaolinite wasobserved by CD spectroscopy and confirmed by fluorescence spectroscopy and DNAmolecules desorbed from soil colloid or montmorillonite were still B-form.2. The effects of various organic and inorganic ligands on DNA adsorption on soilactive particles were studied. A marked decrease in DNA adsorption was observed onmontmorillonite and kaolinite with increasing anion concentrations from 0 to 5 mM.However, the amount of DNA adsorbed by montmorillonite and kaolinite was enhancedwhen ligand concentration was higher than 5 mM. In the system of soil colloids, with theincrease of anion concentrations, a steady decrease was found and the ability of ligands indepressing DNA adsorption followed the sequence of phosphate＞citrate＞tartrate.Compared to inorganic clays, a sharp decrease in DNA adsorption was observed onorganic clays with increasing ligand concentrations. The results suggest that the influenceof anions on DNA adsorption varies with the type and concentration of anion as well asthe surface properties of soil components. Introduction of DNA into the system before theaddition of ligands had the greatest amount of DNA adsorption on soil colloids. Organicand inorganic ligands promoted DNA adsorption on montmorillonite and kaolinite whenligands were introduced into the system before the addition of DNA.3. Adsorption and desorption of DNA on Bacillus thuringiensis and Pseudomonasputida and their composites with soil colloids or minerals were investigated. B.thuringiensis and P. putida did not show significant difference in the amount of DNAadsorption although the two bacterial cells have different surface properties. DNA wasadsorbed on bacteria mainly through van der Waals and electrostatic forces. Comparedwith B. thuringiensis, DNA adsorbed by P. putida was more easily desorbed. There wasno significant difference in the amount of DNA adsorption on kaolinite between theabsence and the presence of P. putida. Excepte for the effect of P. putida on DNAadsorption on kaolinite, the presence of B. thuringiensis and P. putida significantlypromoted DNA adsorption on soil colloids and minerals, and the stimulative effect of B.thuringiensis was stronger than that ofP. putida in the system of soil colloids.4. Adsorption of DNA on different hydroxyaluminum-montmorillonite complexes(Al(OH)_x-M) containing 2.5, 10.0 and 20.0 mmoi coated Al/g clay (AM_2.5, AM₁₀ andAM₂₀) was studied in Tris-HCl and sodium phosphate buffers. The coatings ofmontmorillonite by hydroxyaluminum species decreased the amount of DNA adsorption,but increased the affinity of DNA adsorption. At the same pH, the amount of DNA adsorption on montmorillonite or Al(OH)_x-M complexes in sodium phosphate wasgreater than that in Tris-HCl, which suggested that the nature of a buffer solution stronglyaffected DNA adsorption on clays. As for Al(OH)_x-M complexes, the higher the level ofAI(OH)_x coatings, the lesser the amount of DNA was adsorbed in sodium phosphatebuffer. The reduction of DNA adsorption in phosphate buffer with the increase of thelevel of AI(OH)_x coatings may be ascribed to the strong competition of phosphate anionswith DNA molecules on surface sites of Al(OH)_x-M complexes. An increase in theconcentration of Ca²⁺ and/or a decrease in the values of pH favored DNA adsorption onmontmorillonite and Al(OH)_x-M complexes. The percent desorption of DNA frommontmorillonite, AM_2.5, AM₁₀ and AM₂₀ was 65.01, 30.00, 8.04 and 5.18% by Tris-HClbuffer. It suggested that the larger the OH-Al loading on M surface, the greater thebinding energy of DNA. DNA adsorption on montmoriUonite was endothermic (â–³H_ads=1.15 J/g), whereas that on Al(OH)_x-M complexes was exothermic (-9.50＜△H_ads＜-6.64 J/g). The bases and phosphate groups of DNA are involved in DNA adsorption onclays and DNA changes its conformation from the B-form to the Z-form as the result ofits binding on AM₁₀ and AM₂₀. Electrostatic forces, hydrogen bonding and ligandexchange dominated DNA adsorption on Al(OH)_x-M complexes. SEM showed that a filmwas formed on the surface of AM₁₀ after the binding of DNA, while that was notobserved on montmorillonite surface.5. Electrophoresis and thermometric TAMâ…¢were used to investigate thedegradation of chromosomal DNA in the system of soil active particles or bound on soilcolloid or mineral by DNase I. When nuclease concentration was 2μg ml^-1, DNA wastotally degraded. In systems of kaolinite and coarse and fine inorganic clays, DNA wasdegraded to 2-4 kb segments at 20μg ml^-1 of nuclease. For DNA in systems ofmontmorillonite and coarse and fine organic clays, no evident change was observed in thepatterns with 40μg ml^-1 nuclease in comparison with no nuclease. The heat released fromthe hydrolysis of DNA, free or bound on coarse inorganic clay, coarse organic clay,kaolinite and montmorillonite by nuclease was-4.7561,-4.0553,-2.3792,-2.3647,-0.2243 mJ, respectively. These results indicate that soil colloids and minerals can exertan effective protection for DNA to resist degradation by the nuclease. Among the soilcolloids and minerals studied, montmorillonite and organic clays provide more protectionfor DNA against degradation by DNase I than kaolinite and inorganic clays. Theprotection of DNA was not a result of the adsorption affinity of DNA for soil colloid ormineral and the changes in DNA structure. The presence of organic matter and anefficient adsorption of nucleases on soil colloids and minerals appear to be responsiblefor the lower degradation of DNA in soil ecosystems. 6. The ability of bound plasmid p34S DNA on soil colloids and minerals totransform competent cells of CaCl₂-treated Escherichia coli, and the resistance of boundplasmid DNA to degradation by DNase I were studied. The transformation efficiency ofbound plasmid DNA increased with increasing concentrations of Ca²⁺ at which soilcolloid or clay mineral-plasmid DNA complexes were formed. Plasmid DNA bound bykaolinite showed the lowest transformation efficiency, and especially no transformantswere observed with kaolinite-plasmid DNA complex prepared at 5-100 mM Ca²⁺.Compared to organic clays and fine clays, plasmid DNA bound on inorganic clays andcoarse clays showed a lower capacity to transform E. coli at different Ca²⁺ concentrations.Transformation by 10μg of free plasmid DNA was inhibited 99.8% by 10 ng of DNase I.As for the same amount ofplasmid DNA bound by soil colloids and montmorillonite, 100ng of DNase I resulted in 92.3-93.8% inhibition of transformation by plasmid DNAbotmd on inorganic clays, whereas 2000 ng of DNase I caused only 64.0-98.0%inhibition of transformation by that on organic clays and montmorillonite. The percentageof reduction of transformants by plasmid DNA bound on coarse clays was higher thanthat on fine clays. Montmorillonite, organic clays and fine clays showed strongerprotective effects for plasmid DNA than inorganic clays and coarse clays. The adsorptionaffinity of plasmid DNA for soil colloid or mineral and a conformational change in theplasmid DNA molecule when bound on clays may determine the efficiencies oftransformation.7. We have used the polymerase chain reaction (PCR) to amplify a 600-base pair (bp)sequence of plasmid pGEX-2T DNA bound on soil colloidal particles and three differentminerals (goethite, kaolinite, montmorillonite). DNA bound on soil colloids, kaolinite,and montmorillonite was not amplified when the complexes were used directly butamplification occurred when the soil colloid or kaolinite-DNA complex was diluted 10-and 20-fold. The montmorillonite-DNA complex required at least 100-fold dilutionbefore amplification was detectable. DNA bound on goethite was amplified whether thecomplex was used directly, or diluted 10-and 20-fold. The amplification ofmineral-bound plasmid DNA by PCR is, therefore, markedly influenced by the type andconcentration of minerals used.8. The thermodynamic data of the metabolic activity of E. coli as influenced by soilcolloids and minerals were provided. The growth rate constant (k) of E. coli in LB was0.07394 min^-1, and the k values of E. coli in the system of coarse inorganic clay, kaolinite,coarse organic clay, montmorillonite and goethite were 0.07273, 0.05754, 0.05418,0.04541 and 0.02032 min^-1, respectively. It suggested that the selected soil colloids andminerals inhibited significantly the exponential growth of E. coli. The inhibitory ability of the three minerals on metabolic activity of E. coli followed the sequence of goethite＞montmorillonite＞kaolinite. Compared with inorganic clay, organic clay showed thehigher inhibitory effect.