Soil colloids-bound plasmid DNA: Effect on transformation of E. coli and resistance to DNase I degradation

The adsorption and binding of plasmid p34S DNA on four different colloidal fractions from a Brown soil and clay minerals in the presence of various Ca²⁺ concentrations, the ability of bound DNA to transform competent cells of CaCl₂-treated Escherichia coli, and the resistance of bound DNA to degradation by DNase I were studied. DNA adsorption on soil colloids and clay minerals was promoted in the presence of Ca²⁺. Kaolinite exhibited the highest adsorption affinity for DNA among the examined soil colloids and clay minerals. In comparison with organo-mineral complexes (organic clays) and fine clays (<0.2 μm), DNA was tightly adsorbed by H₂O₂-treated clays (inorganic clays) and coarse clays (0.2-2 μm). The transformation efficiency of bound DNA increased with increasing concentrations of Ca²⁺ at which soil colloid or clay mineral-DNA complexes were formed. DNA bound by kaolinite showed the lowest transformation efficiency, and especially no transformants were observed with kaolinite-DNA complex prepared at 5-100 mM Ca²⁺. Compared to organic clays and fine clays, DNA bound on inorganic clays and coarse clays showed a lower capacity to transform E. coli at different Ca²⁺ concentrations. The presence of soil colloids and minerals provided protection to DNA against degradation by DNase I. Montmorillonite, organic clays and fine clays showed stronger protective effects for DNA than inorganic clays and coarse clays. The protection mechanisms as well as the differences in transforming efficiency of plasmid DNA molecules bound on various soil colloidal particles are discussed. The information obtained in this study is of fundamental significance for the understanding of the horizontal dissemination of recombinant DNA and the fate of extracellular DNA in soil environments.     

Reactions of nucleic acid bases with inorganic soil constituents

The adsorption at pH's 4, 6 and 8 of adenine, guanine, cytosine, thymine and uracil on clays (montmorillonite, illite and kaolinite), Fe- and Al-oxides (goethite, hematite and gibbsite), a soil, and on a laboratory-prepared fulvic acid-montmorillonite complex was investigated. Portions of the clays and soil were saturated with H⁺, Fe³⁺ and Ca²⁺.Quantitatively, the extent of adsorption of nucleic acid bases by the clays was proportional to their exchange capacities, but the nature of the dominant cation had only minor effects. By contrast, the adsorption was strongly affected by pH, tending to decrease with increase in pH. Adsorption on goethite and gibbsite was lower than that on clays, while adsorption of nucleic acid bases on soils was slightly lower than that on oxides. The fulvic acid-montmorillonite complex adsorbed substantial, although smaller amounts of purines and pyrimidines, than did montmorillonite alone. The main adsorption mechanism at pH 4 appeared to be cation exchange whereas at pH 8 complex formation between the nucleic acid bases and cations on inorganic surfaces seemed to occur.The results of this and earlier work show that both inorganic and organic soil constituents adsorb nucleic acid bases. Which adsorption reaction predominates will depend on the clay and organic matter content and on the pH.     

Adsorption and desorption of monomeric Bt (Bacillus thuringiensis) Cry1Aa toxin on montmorillonite and kaolinite

Genetically modified crops, which produce pesticidal proteins from Bacillus thuringiensis, release the toxins into soils through root exudates and upon decomposition of crop residues. Although the phenomena of gene transfer and emergence of resistance have been well documented, the fate of these toxins in soil has not yet been clearly elucidated. The aim of this study was to elucidate the adsorption and the desorbability of the Cry1Aa Bt insecticidal protein in contact with two sodium-saturated clays: montmorillonite and kaolinite. Because the toxin is released into soil in small quantities, it was assumed that it will be in a monomeric state in solution until it oligomerized on cell membranes. The originality of this study was to focus on the monomeric form of the protein. Specific sample conditions were required to avoid polymerisation. A pH above 6.5 and an ionic strength of at least 150 mM (NaCl) were necessary to keep the protein in solution and in a monomeric state. The adsorption isotherms obtained were of the L-type (low affinity) for both clays and fitted the Langmuir equation. The adsorption maximum of the toxin, calculated by the Langmuir nonlinear regression, decreased with increasing pH from 6.5, which was close to the isoelectric point, to 9. At pH 6.5, the calculated adsorption was 1.7 g g⁻¹ on montmorillonite and 0.04 g g⁻¹ on kaolinite. Desorbability measurements showed that a small fraction of toxin could be desorbed by water (up to 14%) and more by alkaline pH buffers (36 ± 7%), indicating that it was not tightly bound. Numerous surfactants were evaluated and the toxin was found to be easily desorbed from both clays when using zwitterionic and nonionic surfactants such as CHAPS, Triton-X-100, and Tween 20. This finding has important implications for the optimization of detection methods for Bt toxin in soil.     

Hydrolysis of cellobiose by β-glucosidase in the presence of soil minerals - Interactions at solid-liquid interfaces and effects on enzyme activity levels

Extracellular enzymatic activities in soils are essential for the cycling of organic matter. These activities take place in multiphase environments where solid phases profoundly affect biocatalytic activities. Aspergillus niger is ubiquitous in soils; its β-glucosidase plays an important role in the degradation of cellulose, and therefore in the global carbon cycle and in the turnover of soil organic matter. However, the information on the interactions of this protein with soil minerals is very limited, and even less is known about their consequences for the hydrolysis of the natural substrate cellobiose. We therefore characterised the sorptive interactions of this enzyme with the soil minerals montmorillonite, kaolinite and goethite and quantified the resulting changes in the hydrolysis rate of cellobiose. Fractions of adsorbed protein, and the resulting catalytic activity loss, were lower for montmorillonite than for kaolinite and goethite at given experimental conditions; adsorption was 9.7 ± 7.3% for montmorillonite, 70.3 ± 3.1% for kaolinite and 71.4 ± 1.8% for goethite, respectively. Adsorption of the protein to the minerals caused a total decrease in the catalytic activity of 18.8 ± 3.4% for kaolinite and 17.9 ± 4.7% for goethite whereas it was not significant for montmorillonite. The average catalytic activity lost by the pool of adsorbed molecules was 26.8% for kaolinite and 25.0% for goethite. Both the amount of adsorbed protein and the resulting loss of catalytic activity were found to be independent of the specific surface areas yet were influenced by the electrical properties of the mineral surfaces. Under the experimental conditions, montmorillonite and kaolinite are negatively charged whereas goethite is positively charged. However, because of the adsorption of phosphate anions from the buffer, a charge reversal took place at the surface of goethite. This was confirmed by zeta (ζ)-potential measurements in phosphate buffer, revealing negative values for all the tested minerals. Indeed goethite interacted with the enzyme as a negatively charged surface: the amount of adsorbed protein and the resulting catalytic activity loss were very similar to those of kaolinite. Our results show that, even if an important fraction of β-glucosidase is adsorbed to the minerals, the catalytic activity is largely retained. We suggest that this strong activity retention in presence of soil minerals results from a selective pressure on A. niger, which benefits from the activity of the adsorbed, and thus stabilized, enzyme pool.     

Sorption of L-methionine-sulphoximine on humic acids and clay minerals

 The sorption of L-methionine-sulphoximine (MSX) on soil-extracted humic fractions (MW>20,000 Da), and mined clays (kaolinite and montmorillonite) was studied by a reversed-phase high-performance liquid chromatography method. The molecule, acting as an inhibitor of glutamine synthetase activity, was recently used in soil incubation assays for the determination of gross rates of N mineralization. Equilibrium data of MSX adsorbed on humic fractions and clay minerals were described by both the Freundlich and Langmuir sorption isotherms. It was observed that humic fractions were more effective than mined clay minerals in removing MSX from water. Most isotherms were of Langmuir type, indicating a higher affinity of MSX for sorbing sites which become increasingly saturated at higher equilibrium concentrations. Analysis of Langmuir empirical constants revealed that different adsorbing mechanisms took place. Results showed that when MSX is applied in soil incubation assays, sorption of MSX on soil colloids considerably reduces the availability of the inhibitor to target microorganisms, and renders the method unsuitable for the determination of the gross rate of N mineralization, especially when high levels of both organic matter and clay minerals are present in the soil. Received: 19 April 1999     

Effects of Addition Sequence and pH of Oxalate on the Adsorption and Activity of Acid Phosphatase on Soil Colloids and Minerals

The adsorption and specific activities of acid phosphatase on soil colloids and minerals in oxalate systems with different pH values were studied using the batch method. Results indicated that the amount of phosphatase adsorption decreased in the following order: goethite ? yellow-brown soil (YBS) > kaolinite > latosol, and the specific activities of immobilized phosphatase were goethite > latosol > kaolinite > YBS. The profiles of enzymatic adsorption and specific activity on soil minerals were bell shaped, and their change tendencies were out of sync. The optimal pH for phosphatase adsorption was located between the isoelectric point of phosphatase and the zero point of charge of the studied soil minerals, and the optimal pH for the specific activity of the immobilized enzyme either did not change or shifted toward alkalinity. Enzymatic adsorption amounts and specific activities were greater when the enzyme was added before oxalate than when enzyme was added after oxalate.     

除草剂草甘膦在几种土壤和矿物上的吸附研究

通过批平衡实验考察了草甘膦在几种性质不同土壤和矿物上的吸附行为。研究发现土壤对草甘膦有较强的吸附能力，草甘膦在土壤上吸附量的大小与土壤理化性质密切相关。草甘膦在土壤和矿物上的吸附符合Freundlich吸附方程，其在土壤上的吸附常数K与土壤粘粒含量呈正相关，并随土壤氧化铁和氧化铝含量增加而增加，而与土壤的pH呈显著负相关。草甘膦在高岭石上的吸附量要比在蒙脱石上大，而草甘膦在金属离子饱和的蒙脱石和高岭石上的吸附研究结果表明，草甘膦在钠、钙、铁离子饱和的矿物上的吸附能力依次为Fe-蒙脱石〉Ca-蒙脱石〉Na-蒙脱石和Fe-高岭石〉Ca-高岭石〉Na-高岭石。     

Adsorption of pure and dirty bacterial DNA on clay minerals and their transformation frequency

Several studies have investigated the adsorption of pure DNA on soil particles and its transformation ability. However, the presence of not purified (dirty) rather than pure DNA is more common in the extracellular soil environment. For this reason, we have investigated the adsorption and binding of dirty DNA on montmorillonite (M) and kaolinite (K) and their transforming ability in comparison to pure DNA. After lysis of Bacillus subtilis cells, induced by KCl, dirty DNA was characterized by the presence of cellular wall debris (cwd) and other constitutional organic components (coc). The dirty DNA was then divided into two fractions, one with cellular wall debris (DNA +cwd) and the other without cellular wall debris (DNA −cwd). B. subtilis BD 1512 (Cm^r) and BD 170 (Cm^s) were selected as donor and recipient bacteria, respectively, for adsorption and transformation studies. Both cwd and coc seem to facilitate the adsorption of DNA to clay minerals, whereas only cwd promote the DNA binding on clays, protecting also the DNA fragments below 400 bp against nucleases. Both dirty DNA fractions adsorbed and bound on clay minerals were able to transform competent cells.     

有机酸对高岭石, 针铁矿和水铝英石吸附镉的影响

Effects of organic acids （oxalic, acetic, and citric） on adsorption characteristics of Cadmium （Cd） on soil clay minerals （kaolinite, goethite, and bayerite） were studied under different concentrations and different pH values. Although the types of organic acids and minerals were different, the effects of the organic acids on the adsorption of Cd on the minerals were similar, i.e., the amount of adsorbed Cd with an initial solution pH of 5.0 and initial Cd concentration of 35 mg L^-1 increased with increasing concentration of the organic acid in solution at lower concentrations, and decreased at higher concentrations. The percentage of Cd adsorbed on the minerals in the presence of the organic acids increased considerably with increasing pH of the solution. Meanwhile, different Cd adsorption in the presence of the organic acids, due to different properties on both organic acids and clay minerals, on kaolinite, goethite, or bayerite for different pHs or organic acid concentrations was found.     

Effects of physical and chemical properties of soils on adsorption of the insecticidal protein (Cry1Ab) from Bacillus thuringiensis at Cry1Ab protein concentrations relevant for experimental field sites

The adsorption of the insecticidal Cry1Ab protein of Bacillus thuringiensis (Bt) on Na-montmorillonite (M-Na) and soil clay fractions was studied. The aim of this study was not to find the adsorption capacity of the soils from the experimental field site, where Bt corn (MON810) was cultivated, but rather to characterize the adsorption behavior of the Cry1Ab protein at concentrations typically found at experimental field sites. In kinetic experiments, the Cry1Ab protein adsorbed rapidly (<60 min) on M-Na. As the concentration of M-Na was varied and the added Cry1Ab protein concentration was kept constant (20 and 45 ng ml⁻¹), the adsorption per unit weight of Cry1Ab protein decreased with increasing concentrations of M-Na. Adsorption of Cry1Ab protein on M-Na decreased as the pH value of the suspension increased. All adsorption isotherms could be described mathematically by a linear regression with the parameter k, the distribution coefficient, being the slope of the regression line. Although their mineralogical composition was nearly identical, the soil clay fractions showed different k values. The different k values were correlated with the physical and chemical properties of the soil clay fractions, such as the organic carbon content, the specific external surface area, and the electrokinetic charge of the external surfaces of the clays, as well as with the external surface charge density. An increase in the amount of soil organic matter, as well as an increase in the electrokinetic external surface charge of the soil clays, decreased the distribution coefficient k. An increase of the specific external surface areas of the soil clays resulted in a higher distribution coefficient k.Less than 10% of adsorbed Cry1Ab protein was reversibly adsorbed on the soil clays and, thus, desorbed. The desorption efficiency of distilled water was higher than that of a solution of CaCl₂ (2.25 mmol) and of dissolved organic carbon (50 mg C l⁻¹).