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.     

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.     

Biodegradation of crude oil saturated fraction supported on clays

The role of clay minerals in crude oil saturated hydrocarbon removal during biodegradation was investigated in aqueous clay/saturated hydrocarbon microcosm experiments with a hydrocarbon degrading microorganism community. The clay minerals used for this study were montmorillonite, palygorskite, saponite and kaolinite. The clay mineral samples were treated with hydrochloric acid and didecyldimethylammonium bromide to produce acid activated- and organoclays respectively which were used in this study. The production of organoclay was restricted to only montmorillonite and saponite because of their relative high CEC. The study indicated that acid activated clays, organoclays and unmodified kaolinite, were inhibitory to biodegradation of the hydrocarbon saturates. Unmodified saponite was neutral to biodegradation of the hydrocarbon saturates. However, unmodified palygorskite and montmorillonite were stimulatory to biodegradation of the hydrocarbon saturated fraction and appears to do so as a result of the clays’ ability to provide high surface area for the accumulation of microbes and nutrients such that the nutrients were within the ‘vicinity’ of the microbes. Adsorption of the saturated hydrocarbons was not significant during biodegradation.     

Coagulation of bacteria by the action of montmorrillonite

Conditions were studied for the coagulation of aggregation-stable Bacillus subtilis, Flavobacterium rigens and Escherichia coli cell dispersions. Their aggregative stability decreased when montmorillonite, a clay mineral, was added to a weakly acid medium or in the presence of Al3+ ions. Bacterial heterocoagulation under the action of montmorillonite can be used as a universal strategy for biomass isolation from aqueous dispersion media.     

Clay minerals protect bacteriophage PBS1 of Bacillus subtilis against inactivation and loss of transducing ability by UV radiation

The effect of UV radiation on the survival of and transduction by phage PBS1 of Bacillus subtilis, free or adsorbed on the clay minerals montmorillonite (M) and kaolinite (K), was studied. After free or clay-associated phage (approximately 10(7) PFU.mL-1) was irradiated with UV light (254 nm) for 0, 1, 2, 5, 10, and 30 min and then allowed to infect B. subtilis FB300 (thiB4 metA29 argF4 Rfmr), the phage was titered, and Met+ transductants were enumerated on selective media. After 1 min of irradiation, the titer of free and clay-associated phage decreased significantly (approximately 1.6 times for free phage, and approximately 4.9 and 6.8 times for M and K, respectively), whereas the transduction frequency increased significantly (approximately 3 times for free phage and approximately 1.4 and 2.2 times for M and K, respectively). The titer and transduction frequency of clay-associated phage remain essentially constant between 1 and 10 min of irradiation, whereas the titer of free phage decreased by approximately 1 order of magnitude after 5 min of irradiation. When free phage was irradiated for 10 min, the titer and transduction frequency decreased by approximately 2 and 0.5 orders of magnitude, respectively, whereas 30 min of irradiation was necessary to obtain comparable decreases with clay-associated phage. These results indicated that phages are protected to some extent from UV radiation when adsorbed on clay minerals.     

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.     

Effect of bacteria on the inactivation and adsorption on clay minerals of reovirus

This investigation studied the antiviral activity of, and the utilization of viruses as substrates by, bacteria. Reovirus type 3 and bacterial species representative of those endemic to sewage, aquatic, and terrestrial habitats were used in the model systems. Culture supernatants from Bacillus subtilis maintained for 5 days in a minimal salts medium displayed antiviral activity, but supernatants from Escherichia coli or Serratia marcescens did not. Both live and toluene-killed cells reduced the inactivation of reovirus during 4 days of incubation at 23 +/- 2 degrees C. This protective effect was more pronounced with killed than with live cells of B. subtilis, confirming the presence of an antiviral component(s) in this species and indicating that the component(s) was metabolic in origin. When reovirus was presented to these bacteria as a sole source of carbon, some growth (determined spectrophotometrically) of B. subtilis and S. marcescens occurred with reovirus concentrations of 3.1 X 10(6) and 8.2 X 10(6) mean tissue culture infective dose-fifty X mL-1, respectively. Growth of S. marcescens did not occur with a reovirus concentration of 8.0 X 10(4) mean tissue culture infective dose-fifty X mL-1, nor did that of E. coli with any virus concentration used in this study. Adsorption of reovirus on kaolinite was enhanced by the culture supernatant from S. marcescens and on montmorillonite, albeit to a lesser extent, by that from E. coli. The effect of culture supernatants from B. subtilis on the adsorption of reovirus on clay minerals could not be determined, as a result of the antiviral component produced by these cells. The virus was not adsorbed on the bacteria.     

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.     

Effect of proteins on reovirus adsorption to clay minerals

Organic matter in sewage, soil, and aquatic systems may enhance or inhibit the infectivity of viruses associated with particulates (e.g., clay minerals, sediments). The purpose of this investigation was to identify the mechanisms whereby organic matter, in the form of defined proteins, affects the adsorption of reovirus to the clay minerals kaolinite and montmorillonite and its subsequent infectivity. Chymotrypsin and ovalbumin reduced the adsorption of reovirus to kaolinite and montmorillonite homoionic to sodium. Lysozyme did not reduce the adsorption of the virus to kaolinite, but it did reduce adsorption to montmorillonite. The proteins apparently competed with the reovirus for sites on the clay. As lysozyme does not adsorb to kaolinite by cation exchange, it did not inhibit the adsorption of reovirus to this clay. The amount of reovirus desorbed from lysozyme-coated montmorillonite was approximately 38% less (compared with the input population) than that from uncoated or chymotrypsin-coated montmorillonite after six washings with sterile distilled water. Chymotrypsin and lysozyme markedly decreased reovirus infectivity in distilled water, whereas infectivity of the virus was enhanced after recovery from an ovalbumin-distilled water-reovirus suspension (i.e., from the immiscible pelleted fraction plus supernatant). The results of these studies indicate that the persistence of reovirus in terrestrial and aquatic environments may vary with the type of organic matter and clay mineral with which the virus comes in contact.     

Effect of proteins on reovirus adsorption to clay minerals.

Organic matter in sewage, soil, and aquatic systems may enhance or inhibit the infectivity of viruses associated with particulates (e.g., clay minerals, sediments). The purpose of this investigation was to identify the mechanisms whereby organic matter, in the form of defined proteins, affects the adsorption of reovirus to the clay minerals kaolinite and montmorillonite and its subsequent infectivity. Chymotrypsin and ovalbumin reduced the adsorption of reovirus to kaolinite and montmorillonite homoionic to sodium. Lysozyme did not reduce the adsorption of the virus to kaolinite, but it did reduce adsorption to montmorillonite. The proteins apparently competed with the reovirus for sites on the clay. As lysozyme does not adsorb to kaolinite by cation exchange, it did not inhibit the adsorption of reovirus to this clay. The amount of reovirus desorbed from lysozyme-coated montmorillonite was approximately 38% less (compared with the input population) than that from uncoated or chymotrypsin-coated montmorillonite after six washings with sterile distilled water. Chymotrypsin and lysozyme markedly decreased reovirus infectivity in distilled water, whereas infectivity of the virus was enhanced after recovery from an ovalbumin-distilled water-reovirus suspension (i.e., from the immiscible pelleted fraction plus supernatant). The results of these studies indicate that the persistence of reovirus in terrestrial and aquatic environments may vary with the type of organic matter and clay mineral with which the virus comes in contact.     

Specificity of virus adsorption to clay minerals

Competitive adsorption studies indicated that reovirus type 3 and coliphage T1 did not share common adsorption sites on kaolinite and montmorillonite. Compounds in the minimal essential medium (e.g., fetal bovine serum, amino acids) in which the reovirus was maintained blocked adsorption of coliphage T1 to kaolinite and partially to montmorillonite in synthetic estuarine water, but they had no effect on coliphage adsorption to montmorillonite in distilled water or on the adsorption of the reovirus to either clay. The blockage of positively charged sites on kaolinite or montmorillonite by treatment of the clays with sodium metaphosphate or with the supernatants from montmorillonite or kaolinite, respectively, had no effect on adsorption of the reovirus. These data indicate that there was a specificity in adsorption sites for mixed populations of reovirus type 3 and coliphage T1 and emphasize the importance of using more than one type of virus, especially in combination, to predict virus behavior (e.g., adsorption, loss of infectivity) in soils and sediments containing clay minerals.     

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.     

Biodegradation and adsorption of C1- and C2-phenanthrenes and C1- and C2-dibenzothiophenes in the presence of clay minerals: effect on forensic diagnostic ratios

The impact of modified montmorillonites on adsorption and biodegradation of crude oil C1-phenanthrenes, C1-dibenzothiophenes, C2-phenanthrenes and C2-dibenzothiophenes was investigated in aqueous clay/oil microcosm experiments with a hydrocarbon degrading microorganism community. Consequently, the effect on C1-dibenzothiophenes/C1-phenanthrenes, C2-dibenzothiophenes/C2-phenanthrenes, 2+3-methyldibenzothiophene/4-methyldibenzothiophene and 1-methyldibenzothiophene/4-methyldibenzothiophene ratios commonly used as diagnostic ratios for oil forensic studies was evaluated. The clay mineral samples were treated to produce acid activated montmorillonite, organomontmorillonite and homoionic montmorillonite which were used in this study. The different clay minerals (modified and unmodified) showed varied degrees of biodegradation and adsorption of the C1-phenanthrenes, C1-dibenzothiophenes, C2-phenanthrenes and C2-dibenzothiophenes. The study indicated that as opposed to biodegradation, adsorption has no effect on the diagnostic ratios. Among the diagnostic ratios reviewed, only C2-dibenzothiophenes/C2-phenanthrenes ratio was neither affected by adsorption nor biodegradation making this ratio very useful in forensic studies of oil spills and oil–oil correlation.     

Effect of argillaceous minerals on the growth of phosphate-mobilizing bacteria Bacillus subtilis

It was shown that the argillaceous minerals montmorillonite and palygorskite at concentrations within 0.2-1.0% considerably accelerate the growth of phosphate-mobilizing bacteria Bacillus subtilis grown in media with hardly soluble Ca3(PO4)2 as the sole source of phosphorus. The most notable effect of these minerals was recorded at concentrations within 0.5-1.0%. The effect of argillaceous minerals in the colloidal form on bacterial growth was more pronounced than that of the powdered ones. An increase in montmorillonite or palygorskite concentrations to 2% is accompanied by the inhibition of the growth of the phosphate-mobilizing strain. At such concentrations the minerals adsorb ca. 22% of the glucose and 11.3% of the phosphate added to the nutrition medium.     

Studies on Plant Viruses-soil Colloids Interactions

Stability and infectivity of cucumber mosaic virus, strain D (CMV-D), associations with kaolinite and montmorillonite were determined, as affected by: i) nature of clay minerals; ii) nature of clay saturating cations; iii) exposure to dissociating salt solutions (2 M LiCl). Infectivity experiments carried out with sediments following centrifugation of the virus-clay mixtures (sd fractions), showed that, in absence of LiCl, the highest values were obtained with kaolinite, in the order Li⁺= K⁺ > NH₄⁺= Mg⁺⁺ > Na⁺ > Ca⁺⁺ clay saturating cations, ranging between 91 and 30 % of the untreated control, whereas comparable montmorillonite fractions gave infectivity values with all cations about 10–15 % of the control. In presence of 2 M LiCl, montmorillonite preserved infectivity of the same fraction (Lsd fraction), which, in the case of Li⁺- or Ca⁺⁺ -saturated samples, was higher when compared with the corresponding sd values, thus revealing for these cations an amplifying effect on infection. This did not occur with kaolinite which, however, gave a Lsd fraction more infectious than the other clay. The results confirmed that clay minerals preserve infectivity of virus preparations exposed to critical conditions, thus providing an explanation for the persistence in soil of infectivity of viruses which are normally not soil-borne. Under appropriate soil conditions these viruses may form complexes with clay minerals thus retaining an infectivity which may be enhanced by addition of cations as those contained in fertilizers.     

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 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.     

新疆其木干剖面中新世-上新世沉积物粘土矿物特征及其古气候指示意义

为重建新疆其木干地区中新世-上新世古气候,采用X射线衍射、扫描电子显微分析方法,对该区中新世-上新世沉积物中粘土矿物的相对含量、组合类型及显微形貌等进行了研究。结果显示:中新世早期-早中新世中期,沉积物中粘土矿物以伊利石和绿泥石为主,含少量的蒙脱石,表明以干旱气候为特征;晚中新世中期-早中新世晚期,伊利石的相对含量逐渐降低,且含有少量的蒙脱石和高岭石,指示相对温湿的气候条件;中新世晚期的粘土矿物组分与中新世早期相似,以伊利石和绿泥石为主,指示古气候以干旱为主导;晚中新世晚期至上新世伊利石相对含量降低,而蒙脱石和高岭石的相对含量升高,但由于粘土矿物中伊利石、绿泥石的含量仍然较高,指示古气候仍然以干旱为主导,但相对于中新世而言,这段时期为相对湿润期。以上结果表明,新疆其木干地区中新世-上新世古气候以干旱为主,并且气候经历了干旱-相对湿润-干旱-相对湿润的演化过程,但总体而言,本区中新世比上新世要更为干旱。     

Interaction of Rhizobium sp. with Toxin-Producing Fungus in Culture Medium and in a Tropical Soil

Experiments were conducted to determine the influence of a toxin-producing fungus on a rhizobial population in yeast-mannitol medium and in a tropical soil. The fungus, which was isolated from a highly weathered soil (Tropeptic Eutrustox), was identified as a Metarhizum sp. The density of rhizobial populations established in yeast-mannitol medium in the absence of the fungus was 10⁵ times higher than that established in its presence. However, the fungus did not exert similar antagonistic influence on the rhizobial population incubated with it in the sterilized test soil. Rhizobial growth activity in yeast-mannitol medium was also insensitive to the presence of the fungus when the medium was amended with 1% (wt/vol) kaolinite or montmorillonite. The results suggest that clay minerals may be responsible for protecting rhizobia against toxin-producing fungi in soil.