Langmuir 2000, 16, 1233-1238
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Differences between Linear Chromosomal and Supercoiled Plasmid DNA in Their Mechanisms and Extent of Adsorption on Clay Minerals Franck Poly,† Claire Chenu,‡ Pascal Simonet,† James Rouiller,§ and Lucile Jocteur Monrozier*,† Laboratoire d’Ecologie Microbienne du Sol-UMR-CNRS 5557- UCB Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France, Unite´ de Science du Sol-INRA, Route de St Cyr, 78026 Versailles Cedex, France, and Centre de Pe´ dologie Biologique-CNRS, 17 rue Notre Dame des Pauvres, 54500 Vandoeuvre Les Nancy, France Received April 26, 1999 Plasmid (mainly as the supercoiled form) and linear chromosomal DNA were compared in terms of their mechanisms and degree of adsorption on three clay minerals, kaolinite, montmorillonite, and illite. Based on adsorption isotherms on Ca-clays, adsorption was complete for both plasmid and linear DNA at low concentrations of DNA. Amounts of DNA adsorbed on illite in water were at least 2-fold greater than the amounts adsorbed on kaolinite and montmorillonite, regardless of whether excess divalent Ca (5 mM) was present in the solution. Increasing the concentration of DNA (>25 µg mL-1) increased the adsorption of linear DNA, whereas the adsorption of plasmid DNA molecules decreased, probably as the result of selfaggregation in solution. Titration of acidic groups of DNA showed a narrow range of strong acidity for the plasmid form, whereas the pH of linear chromosomal acidic groups ranged from very low to neutral or slightly alkaline pKa values. The amount of acidic groups per gram of DNA was higher in linear DNA (13.4 cmol g-1) than in supercoiled plasmid DNA (1.8 cmol g-1). Direct observations of plasmid DNA adsorbed on clay minerals by low temperature scanning electron microscopy (LTSEM) indicated that these molecules could act as bridges between clay domains by the ends of the supercoiled molecule. The location and strength of the acidic groups of DNA determine the interaction between clay and DNA. Supercoiled plasmid DNA interacts by a low number of strongly acidic groups, presumably located at the maximum of bending of the double strand where a high charge density exists. Linear chromosomal molecules appear to attach on the clay surface and edges, as demonstrated by previous observations (Paget, E.; et al. FEMS Microbiol Letters 1992, 97, 31), through acidic groups distributed along the DNA molecules. Such differences in interactions between clay and DNA should influence the accessibility to nucleases and persistence of DNA in soil environments.
Introduction The aim of this study was to investigate the mechanisms that enable extracellular DNA to survive in soils. One of the mechanisms that could be involved in the unwanted spread of genes from transgenic plants, or any genetically modified microorganisms, introduced to soils to the indigenous microflora is natural transformation, i.e., the uptake of naked DNA by competent bacteria.2,3 The initial steps of gene transfer include the release of DNA in soil and its adsorption on soil minerals and colloids, thereby protecting the DNA against degradation by nucleases.1,4-9 DNA of bacterial origin in soil includes chromosomal DNA * To whom correspondence should be addressed. Tel.: 33 (0) 4 72 43 13 80. Fax: 33 (0)4 72 43 12 23. E-mail: joke@ biomserv.univ-lyon1.fr. † Laboratoire d’Ecologie Microbienne du Sol. ‡ Unite ´ de Science du Sol. § Centre de Pe ´ dologie Biologique. (1) Paget, E.; Jocteur Monrozier, L.; Simonet, P. FEMS Microbiol. Lett. 1992, 97, 31-40. (2) Bertolla, F.; van Gijsegem, F.; Nesme, X.; Simonet, P. Appl. Environ. Microbiol. 1997, 63, 4965-4968. (3) Stewart, G. J. In Gene Transfer in the Environment; Levy, S. B., Miller, R. V. Eds.; McGraw-Hill: New York, 1989; p 139-164. (4) Khanna, M.; Stotzky, G. Appl. Environ. Microbiol. 1992, 58, 19301939. (5) Lorenz, M. G.; Wackernagel, W. In Gauthier, M. J., Ed.; SpringerVerlag: Berlin 1992; pp 103-114. (6) Recorbet, G.; Picard, C.; Normand, P.; Simonet, P. Appl. Environ. Microbiol. 1993, 53, 4289-4293. (7) Romanowski, G.; Lorenz, M. G.; Wackernagel, W. Appl. Environ. Microbiol. 1991, 57 1057-1061.
in linear form, as well as supercoiled plasmids, i.e., the covalently closed circular (CCC) form of plasmids. These two types of DNA molecules differ in their ability to transform bacteria10 with efficiencies depending on whether the species of recipient bacteria can integrate the genetic information via a homologous recombination process2 or directly replicate the plasmid DNA. Not much is known about the specific reactivity of the linear chromosomal form of DNA toward soil components compared with plasmid supercoiled molecules.11 In this study the extent to which these two types of DNA molecules adsorb on clay particles and the adsorption mechanisms were compared. The adsorption of each of these DNA molecules on three pure clay minerals homoionic to Ca frequently encountered in soils at midlatitudes12 was determined. Ca is known to act as cationic bridge between anionic groups of organic molecules and negative charges on the clay13 and favors DNA adsorption. The adsorption isotherm technique was used to provide (8) Widmer, F.; Seidler, R. J.; Watrud, L. S. Mol. Ecol. 1996, 5, 603613. (9) Widmer, F.; Seidler, R. J.; Donegan, K. K.; Reed, G. L. Mol. Ecol. 1997. 6, 1-7. (10) Lorenz, M. G.; Wackernagel, W. Microbiol. Rev. 1994, 58, 563602. (11) Gallori, E.; Bazzicalupo, M.; Dal Canto, L.; Fani, R.; Nannipieri, R.; Vettori, C.; Stotzky, G. FEMS Microbiol. Ecol. 1994, 15, 119-126. (12) Brady, N. C. The Nature and Properties of Soils, 9th ed.; Macmillan: NewYork, 1984. (13) Theng, B. K. G. In Formation and Properties of Clay-Polymer Complexes; Elsevier Science: Amsterdam, 1979; p 227-236.
10.1021/la990506z CCC: $19.00 © 2000 American Chemical Society Published on Web 11/27/1999
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Poly et al. Table 1. General Properties of Clays Used
clay mineral
origin
montmorillonite
Wyoming (U. S.A.) Le Puy (France) Saint Austell (U.K.)
illite kaolinite
cation exchange capacity (CEC) (cmol kg-1)
estimated positive charges (Brady et al., 12) (cmol kg-1)
exchangeable calcium (ECa) (cmol kg-1)
exchangeable sodium (cmol kg-1)
exchangeable aluminum (cmol kg-1)
specific surface area (SSA) (m2 g-1)
90
0.7
53
0.08
0
800
24.5
3.9
25
0.03
0.1
122
4
2.3
0
0.1
12
qualitative information on the adsorption process and quantitative data about the extent of adsorption. Direct observation of the complexes using low-temperature scanning electron microscopy (LTSEM) was used to visualize the spatial organization of clay-DNA complexes.14 Materials and Methods Plasmid and Chromosomal DNA. The pKS Ω Cla8 (9 Kb) plasmid15 used in this study was extracted and purified with the Qiagen kit (Qiagen Inc., Chatsworth, CA) according to the recommendations of the manufacturer. As estimated on agarose gels, more than 85% of the molecules consisted of supercoiled and open-circular forms. Considering a length of 1 µm for ≈3 Kb,16 the total length of the linear form of this 9 Kb plasmid would be near 3 µm; consequently the circular form could not have a diameter longer than 1.5 µm, and the length of the CCC form should be even shorter. A commercial solution of calf thymus DNA (>23 kb) (Boehringer, Mannheim, Germany) was used as a source of chromosomal linear DNA. To prevent the chelating properties of EDTA from biasing the adsorption of DNA on mineral surfaces, the usual storage buffers were replaced by ultrapure, sterile distilled water. The DNA concentration was determined by a colorimetric method17 adapted to microtitration plates for routine analysis. To each well of the plate containing 62 µL of perchloric acid (3 N), 62 µL of the DNA solution was added, followed by 75 µL of a diphenylamine reagent (acetic acid 100 mL, paraldehyde 10 µL, diphenylamine 4 g). The reaction was allowed to proceed in the dark for 20 h at room temperature before absorbance at 590 nm was determined with a microplate reader (Thermomax, Molecular Devices, BraunScienceTec). Clay Minerals. General properties of the clay minerals used are presented in Table 1. After the clay was saturated with Na+, homoionic clays were prepared by sedimentation of the Na+saturated clay in water. The fraction of the supernatant containing particles smaller than 2 µm was collected, saturated with Ca2+ by shaking in 1 M CaCl2 solution for 16 h, centrifuged, and resuspended in 1 M CaCl2.. The saturation step was repeated twice. The resulting homoionic clays were then washed several times with 10-3 M CaCl2 and autoclaved twice for each 1 h. The clays were then washed three times with either sterile, purified water or 10-3 M CaCl2 solution to obtain clay suspensions (containing 16 mg clay mL-1) in H2O and in CaCl2. These clay suspensions were stored at 4 °C until used. Clay DNA Adsorption Isotherms. A 200 µL amount of plasmid or calf-thymus DNA solution at each of eight concentrations (approximately 1.5, 3, 6, 12, 25, 50, 100, and 200 µg mL-1) was mixed in a 2-ml polyallomer centrifugation tube with 200 µL of the homoionic clay suspension to provide a final clay concentration of 8 mg mL-1 and a final DNA concentration ranging from 0.75 to 100 µg mL-1. Controls were prepared by replacing either the DNA solutions or the clay suspensions with ultrapure sterilized H2O or CaCl2 solutions. Adsorption was (14) Khanna, M.; Yoder, M.; Calamai, L.; Stotzky, G. Sci. Soils 1998, 3, 1-8 (http:// link.springer.de/link/service/journals/10112/fpapers/ 8003001/). (15) Arlat, M.; van Gijsegem, F.; Huet, J. C.; Pernollet, J. C.; Boucher, C. A. EMBO J. 13, 1994, 543-553. (16) Burkardt, H. J.; Pu¨hler, A. 1984. In Methods in Microbiology 17; Academic Press: London, 1984; pp 133-155. (17) Richards, G. M. Anal. Biochem. 1974, 57, 369-376.
5.6
achieved by rotating the tubes containing the DNA and clay mixtures on a wheel at 15 rpm for 2 h at room temperature4,5 before centrifugation at 4 °C for 20 min at 15000g to sediment clay particles. The supernatant was recovered, and the concentration of nonadsorbed DNA determined according to the colorimetric method described earlier. For each DNA concentration, the amount of adsorbed DNA was calculated by subtracting the amounts present in the supernatant from the amounts initially added to the clays. Adsorption isotherms were established by plotting the amount of DNA adsorbed on clay versus the amount remaining in solution at equilibrium.18 All experiments were run in triplicate for linear chromosomal DNA and in duplicate for CCC plasmid DNA. Surface Acidity of DNA Molecules. As an ionic macromolecule, DNA exhibits titrable anionic groups. To determine the amount and the pKa of acidic DNA groups, the DNAs were neutralized with NaOH and back-titrated with HCl: plasmid DNA or linear DNA (430 µg) was dissolved in 20 mL of 0.025 M NaOH to which 5 mL of 1 M NaClO4 had been added, as an ionic strength buffer, and deionized water was added to a final volume of 50 mL. The resulting solution was titrated by adding 0.110 M HCl under N2 atmosphere to establish ∆(∆pH)/∆V curves according to Gran’s method.19 Results are given as pH values of ∆(∆pH)/∆V peaks (mean pKa) and in cmol of the acidic functional groups forming the respective peak. Usually three zones of pKa can be distinguished: strong acidity values (pKa ≈ pH 4), medium values (pKa ≈ pH 6), and weak values (pKa ≈ pH 7). Measurements were done in duplicate, and results are expressed as the mean of these duplicates. Titration of Ca2+ in Solution. The concentration of Ca2+ in supernatants desorbed from suspensions of illite and kaolinite in water after DNA adsorption was measured by ICPMS (Ionized Cold Plasma/Mass Spectrometry). Low-Temperature Scanning Electron Microscopy (LTSEM). Low-Temperature Scanning Electron Microscopy, which keeps the structure of the sample in its hydrated state, involves several steps: cryofixation, fracturing, partial freeze-drying and coating, examination, and analysis at low temperature in the SEM.20 Pellets of undried (H2O max ) 90%) clay-DNA complexes recovered after centrifugation were mounted onto a nitrocellulose membrane (0.05 µm). DNA solution was then dropped on a small piece of aluminum paper and covered with another small sheet of aluminum. Each sample was cryofixed by immersion in melting nitrogen at - 210 °C,20 fractured and transferred to the preparation chamber of the microscope at -180 °C where the water was partially sublimated under vacuum at -90 °C.20 After gold coating, the sample was introduced into the refrigerated column of the microscope, a Philips SM 525, to be observed at a temperature below -160 °C. Statistics. Using Statview-SE software, one way ANOVA and Fisher PLSD were used for comparison of the means of the amount of adsorbed DNA.
Results Adsorption of Linear and Plasmid DNA. The adsorption isotherm curves, first delineated for each combination of Ca-clay (three types) and DNA (two types) (18) Giles, C. H.; MacEwan, T. H.; Nakhwa, S. N.; Smith, D. J. Chem. Soc. Part III 1960, 3973-3993. (19) Gran, G. The Analyst, Part II 1952, 17, 661-671. (20) Chenu, C.; Tessier, D. Scan. Microsc. 1995, 9, 989-1010.
Mechanisms and Clay Absorption of Linear and Supercoiled DNA
Figure 1. Adsorption isotherm of chromosomal DNA on Caillite, Ca-kaolinite, and Ca-montmorillonite in H2O (A) and in 5 mM CaCl2 (B). Standard deviation calculated on triplicates is indicated by bars.
tested in pure water (Figures 1A and 2A), indicated that the adsorption of DNA depended on its initial concentration in solution. At the lowest concentrations of introduced DNA (e10 µg mL-1), adsorption was always characterized by a high affinity for the clays (i.e., no DNA remaining in solution), regardless of the type of DNA (plasmid or linear DNA). This part of the curve where the points lined up on the Y axis is called the high affinity stage.18 When the DNA concentration increased, some DNA characteristically remained in solution, although the amounts of adsorbed DNA still increased. However, from this stage onward, CCC plasmid and linear DNA exhibited contrasting behavior. Adsorption of linear DNA on illite and kaolinite either continued to increase gradually (Figure 1A), whereas increasing the concentration of CCC plasmid DNA led to a decrease in adsorption (Figure 2A). This discrepancy was particularly marked in the case of illite, whereas it was weaker in kaolinite and montmorillonite where the amounts of plasmid DNA molecules adsorbed decreased slightly as the DNA concentration increased (>25 µg mL-1). Quantifying the charges of adsorbed DNA (Table 2) at the maximum of the high affinity stage and quantifying the charges of the clay (Table 1) clearly showed that the number of plasmid DNA charges adsorbed on clay was always below the number of clay charges, regardless of environmental conditions (water or CaCl2) (Table 1), and this was also true regardless of the quantity of DNA adsorbed (data not shown). By contrast, the charges of adsorbed linear DNA exceeded the number of charges
Langmuir, Vol. 16, No. 3, 2000 1235
Figure 2. Adsorption isotherm of plasmid DNA on Ca-illite, Ca-kaolinite, and Ca-montmorillonite in H2O (A) and in 5 mM CaCl2 (B). Standard deviation calculated on duplicates is indicated by bars.
provided by clays at maximal adsorption in water (data not shown) and at the maximum of the high affinity phase in CaCl2 (Table 2), except for montmorillonite. Titration of the supernatants showed that desorption of Ca2+ and adsorption of linear DNA occurred concomitantly (Figure 3). The proportion of Ca2+ desorbed varied depending, specifically, on the calcium exchange capacity of each clay. A concentration of adsorbed linear DNA of g1300 µg g-1 of illite and g400 µg g-1 of kaolinite enhanced the solubilization of Ca2+, causing the concentration of Ca2+ in solution to increase to 27 mg L-1 and 4.5 mg L-1, respectively. A greater amount of exchangeable Ca2+ was released to the solution from illite (67%) than from kaolinite (50%). Once the amount of adsorbed DNA reached 2000 µg g-1 of kaolinite, no further desorption of Ca was observed, whereas desorption of Ca from illite was still active even for the highest values of adsorbed linear DNA. Plasmid DNA did not significantly modify the equilibrium between adsorbed and soluble calcium. Calcium Effect on DNA Sorption. The presence of soluble 5 mM CaCl2 enhanced the amount of DNA adsorbed compared with pure water (Figures 1 and 2), and this increase varied depending on the type of DNA and clay (Table 2). With illite, in the high affinity phase, the maximum effect of calcium on DNA sorption was stronger for linear DNA (×4) than for plasmid DNA (×2.2), whereas with kaolinite, the increase was higher for plasmid DNA (×4) than for linear DNA (×2). The adsorption isotherm curves for linear DNA diverged strongly for kaolinite and illite in the presence of CaCl2. The specific effect of Ca on DNA sorption to illite was mainly the result of the strong increase in adsorption (×4)
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Poly et al.
Table 2. Comparison of Amount (µg) and Charges (cmol) of Plasmid and Chromosomal DNA Adsorbed on Ca-Clay Minerals, at the Maximum of the High Affinity Phase, in Water and in 5 mM CaCl2 H2O mg kg-1 ( SDa cmol kg-1 ( SD
CaCl2 mg kg-1 ( SD cmol kg-1 ( SD
ratio CaCl2/H2O
plasmid DNA adsorbed on
Ca-montmorillonite Ca-illite Ca-kaolinite
600 ( 105 1200 ( 45 300 ( 25
1.08 ( 0.19 2.16 ( 0.08 0.54 ( 0.05
1200 ( 40 2600 ( 40 1200 ( 20
2.16 ( 0.07 4.68 ( 0.07 2.16 ( 0.04
2 2.2 4
chromosomal DNA adsorbed on
Ca-montmorillonite Ca-illite Ca-kaolinite
400 ( 20 1000 ( 90 400 ( 35
5.36 ( 0.04 13.4 ( 0.16 5.36 ( 0.06
1000 ( 90 4000 ( 20 800 ( 45
13.4 ( 0.16 53.6 ( 0.04 10.7 ( 0.08
2.5 4 2
a
SD ) Standard deviation.
Figure 3. Ca concentration in solution during adsorption of CCC plasmid DNA on kaolinite and illite and during the adsorption of chromosomal DNA on kaolinite and illite. Values are expressed as the difference between the Ca concentration in the clay-DNA mixture and in the clay (control).
in the high affinity phase, whereas adsorption on kaolinite was only weakly affected by CaCl2 (×2). The effect of Ca on adsorption on montmorillonite remained low and was similar for plasmid (×2) and chromosomal linear DNA (×2.5) in the high affinity phase. Acidity of DNAs. Total acidity was higher for linear DNA (13.4 × 103 cmol kg-1) than for plasmid DNA (1.8 × 103 cmol kg-1) (Table 3). Moreover, the distribution of acidic functional groups differed between the two DNA types: for linear DNA, about 80% of the functional groups had a medium or low acidity, and 20% were strongly acidic; whereas for plasmid DNA, there were no functional groups with weak acidity, only a few with moderate acidity (13%), and the majority (87%) were strongly acidic. LTSEM Observation of Plasmid DNA. LTSEM was used to observe free plasmid DNA deposited on aluminum foil (Figure 4A) or DNA adsorbed on kaolinite (Figure 4B). Observation of pure DNA showed the presence of toroidal filaments (F), about 50 nm thick and up to 5 µm long, each of which consisted of several CCC plasmid molecules associated together to form such structures. Plasmid DNA also aggregated to form a weblike structure (N), and only few individual plasmid molecules were observed. With this method, kaolinite was visualized as a disordered staking of hexagonally shaped domains. The size (1-1.5 µm) of the DNA structures observed extending between the clay platelets (Figure 4B) indicated that the CCC plasmids reached a maximal stretch, the extremities of which were adsorbed either to the edges or to the surfaces of the kaolinite. Discussion Regardless of whether extracellular DNA is naturally released from plant, animal, or bacterial cells or intentionally inoculated into microcosms, it could still be detected after several months in soils.1,8,9 Although
numerous experiments have demonstrated the importance of soil colloids in protecting nucleic acids against nuclease degradation,1,4-9 few quantitative data on DNA persistence and levels protection have been presented.4,21 Romanowski et al.21 showed that most molecules were rapidly degraded in soil microcosms and those remaining detectable rapidly lost their transformation capacities. Field experiments22 showing that DNA from transgenic tobacco could be detected in soils by the polymerase chain reaction (PCR) 1 year, but not 3 years, after harvesting the plants confirm that the duration of protection is limited. However, determining whether chromosomal linear and CCC plasmid DNA are adsorbed on clay minerals in the same way is of particular interest, as the way of adsorption can contribute to understanding of the mechanisms protecting the DNA against nuclease degradation. Such knowledge, in turn, should aid an understanding of the factors that affect transformation potentials under natural conditions. As the result of high populations of bacteria in soils23 as well as of their generally high content of extrachromosomal, high-copy-number replicons24 and active mechanisms releasing plasmids in supercoiled form,25-27 bacterial DNA must account for a significant proportion of the DNA released into the soils. Moreover, the various clay minerals, that possess strong adsorption capacities for DNA1,4,13,28 among soil colloids differ in terms of their physical and chemical properties,12 suggesting that their adsorption potentials differ as well. Clay Properties. Although the results clearly confirmed this differing potential of adsorption, illite which was intermediate between kaolinite and montmorillonite in terms of cation exchange capacity (CEC), specific surface area (SSA), and exchangeable calcium (ECa) (Table 1), exhibited the highest adsorption. Thus, several additional factors have to be considered to explain the low adsorption of DNA by montmorillonite. The difference of 40% between the CEC and ECa (Table 1) of montmorillonite indicated that the exchangeable sites of this clay were not completely expressed. The number of layers of Ca-smectites (montmorillonite) varies depending on the ionic strength (Ca in solution), and the (21) Romanowski, G.; Lorenz, M. G.; Sayler, G.; Wackernagel, W. Appl. Environ. Microbiol. 1992, 58, 3012-3019. (22) Paget, E.; Lebrun, M. ; Freyssinet, G.; Simonet, P. Eur. J. Soil Biol. 1998, (in press). (23) Harris, D. In Beyond the Biomass; Ritz, K., Dighton, J., Giller, K. E. Eds.; BSSS. Wiley-Sayce Publication: New York, 1994; pp 111112. (24) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, MA, 1989. (25) Lorenz, M. G.; Gerjets, D.; Wackernagel, W. Arch. Microbiol. 1991, 156, 319-326. (26) Paul, J. H.; David, A. W. Appl. Environ. Microbiol. 1989, 55, 1865-1869. (27) Paul, J. H.; Jeffrey, W. H.; DeFlaun, M. F. Appl. Environ. Microbiol. 1987, 53, 170-179. (28) Creechio, C.; Stotzky, G. Soil Biol. Biochem. 1998, 8/9, 10611067.
Mechanisms and Clay Absorption of Linear and Supercoiled DNA
Langmuir, Vol. 16, No. 3, 2000 1237
Table 3. Available Acidic Functional Groups of Chromosomal and Plasmid DNA acidity (cmol kg-1 DNA)
a
strong
medium
low
total
chromosomal DNA
2.69 × 103
6.27 × 103
4.42 × 103
13.4 × 103
plasmid DNA
(20)a 1.58 × 103 (87)
(47) 0.25 × 103 (13)
(33) 0 (0)
(100) 1.8 × 103 (100)
() ) Percent of total acidity of each DNA.
Figure 4. Cryoscanning electron micrographs showing Blue script plasmid DNA. (A) Plasmid DNA in 5 mM CaCl2, DNA molecules aggregated to form a net (N) or filaments consisting of several plasmid molecules (F) (bars ) 10 µm). (B) Plasmid DNA adsorbed on homoionic kaolinite and acting as a bridge (arrows) between clay platelets (bars ) 1 µm).
stresses to which the clay has been subjected.29 The Camontmorillonite used in this study30 was composed of domains of about 10 sheets, thus providing an external surface area of about 80 m2 g-1 instead of theoretical SSA of 800 m2 g-1 (Table 1) which explains differences between the CEC (measured on fully dispersed Na-montmorillonite) and ECa (measured on incompletely dispersed Camontmorillonite). As Ca2+ acted as a cationic bridge between anionic groups on DNA and negative charges on the clay,5,13 it can be concluded that this low level of exchangeable Ca2+ limited adsorption of DNA to montmorillonite. The clay minerals also differed in terms of surface area available for adsorption of DNA. This difference is related (29) Tessier, D. 1990. In Soil Colloids and their Associations in Aggregates; De Boot, M. F., Hayes, M. H. B., Herbillon, A. Eds.; pp 387-416. (30) Chenu, C. Jaunet, A. M. 1990. C. R. Acad. Sci. 310 (II): 975980.
to the physical structure of the clay minerals, in which the aluminosilicate layers cluster together to form particles rather than remaining separate. The number of layers of illite is generally low and constant and it can be assumed that the external surface area available for adsorption is roughly equal to the total surface area. As DNA is unlikely to penetrate interlayer sites,14 which expand only 0.40.5 nm, the external surface of montmorillonite accessible to DNA is much lower than its total theoretical surface area. As the number of charges involved in adsorption of DNA depends on the accessible surface, adsorption of DNA on montmorillonite remained lower than the expected theoretical value. The ratio of positive to negative charges (crystal edges/ crystal surfaces) was greater for illite (0.16) and kaolinite (0.57) than for montmorillonite (0.0008).12,31 These positive charges emanate from the broken edges of the crystalline lattice that expose free Al3+ groups. Therefore, these positive charges are involved in the adsorption of anions,32 mainly the phosphate anions of DNA responsible for adsorption. Ben-Hur et al.33 found that anionic polymers absorbed more strongly to Ca-illite than to Ca-montmorillonite owing to the presence of positively charged edges on illite. The fact that linear DNA adsorbed on illite and kaolinite to a greater extent than on montmorillonite, despite their lower CEC, SSA, and ECa values, emphasizes the influence that positive charges of the lattice edges have, according to Alvarez et al.,34 and the microorganization of clay particles have on the mechanism of DNA adsorption. Briefly, DNA adsorbs on clays through positive charges, either Ca2+ on CEC or positive charges on lattice edges. Calcium Effect. The amount of adsorption of DNA to clay also depends on additional soil parameters, including valency and cation concentrations. According to Lindsay,35 Ca2+ is the predominant cation in soil, with concentrations ranging from 2.5 to 12.4 mM in acidic and calcareous soils, respectively. To simulate actual soil conditions as closely as possible, the adsorption of DNA to the various clays in the presence of 5 mM CaCl2 which represented the environmental value in neutral soils, was investigated. The way with which divalent ions, such as Ca2+, bind to DNA can be ascribed to the presence of the phosphate groups.36 The electrostatic attraction of Ca2+ 37 neutralizes
(31) Stotzky, G. In Interactions of Soil Minerals with Natural Organics and Microbes; Huang, P. M., Schnitzer, M., Eds.; Soil Science Society of America: Madison, WI, 1986; pp 305-428. (32) Soltner, D. In Les bases de la Production Ve´ ge´ tale, 20th ed. Sciences et techniques agricoles, tome I;. Sainte-Gemmes sur Loire: Loire, France, 1994; pp 17-84. (33) Ben-Hur, M.; Malik, M.; Letey, J.; Mingelgrin. U. Soil Sci. 1992, 153, 349-356. (34) Alvarez, A. J.; Khanna, M.; Toranzos, G. A.; Stotzky, G. Mol. Ecol. 1998, 7, 775-778. (35) Lindsay, W. L. In Chemical Equilibria in Soils. John Wiley and Sons: New York, 1980. (36) Langlais, M.; Tajmir-Riahi, H. A.; Savoie, R. Biopolymers 1990, 30, 743-752. (37) Braunlin, W. H.; Drakenberg, T.; Nordenskio¨ld, L. J. Biomol. Struct. Dyn. 1992, 10, 333-343.
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the negative charges on DNA,38-40 reducing inter and intrarepulsion forces, thereby leading to a more compact molecular conformation.13 This compact conformation was confirmed with the LTSEM observations. Isolated plasmids corresponding to a single molecule were not observed as they self-aggregated in the presence of CaCl2 as described by Khanna et al.,14 to form structures resembling filaments or even nets. In the presence of kaolinite, the same structures were observed, indicating that DNA released into soils can be aggregated in the same way. According to Aylmore and Quirk41 the presence of diluted CaCl2 solution tend to suppress the formation of diffuse electrical double layers around clay particles, leading to increased contact between DNA and mineral surface. A comparison of the adsorption isotherms in the presence of water and 5 mM CaCl2 indicated that CaCl2, because of its neutralizing capacity and ionic strength, positively affected adsorption regardless of the type of DNA and clay mineral. Plasmid Versus Linear Chromosomal DNA. The adsorption isotherms showed that the two types of DNA exhibited different patterns of adsorption process. When approaching a concentration of plasmid DNA that provided approximately 100 × 10-6 cmol mL-1 ()55 µg mL-1) of acidic functional groups, adsorption decreased, which could have resulted from self-aggregation of molecules of CCC plasmid DNA. Solute/solute (plasmid DNA/plasmid DNA) attraction increased more rapidly than solute/ substrate (plasmid DNA/clay) attraction18 when the concentration of acidic groups on plasmid DNA in solution was too high. Of the key factors that affect the adsorption of DNA to minerals,42 the ones that differ the most between linear and CCC plasmid DNA molecules are the density and availability of the free phosphate groups. Furthermore, acidity resulting from these functional groups varies in intensity (distribution of pKa on a pH scale) and in concentration (number of functional groups per gram). Plasmid DNA had stronger acidity values than linear DNA. It can be hypothesized that in CCC plasmid DNA, only acidic groups concentrated at the maximum bending of supercoiled molecules would be available and involved in adsorption, whereas most of the phosphate groups localized within the supercoiled molecule cannot influence (38) Ma, C.; Bloomfield, V. A. Biophys. J. 1994, 67, 1678-1681. (39) Manning, G. S. Q. Rev. Biophys. 1978, 11, 179-246. (40) Tajmir-Riahi, H. A.; Naoui, M.; Ahmad, R. Biopolymers 1993, 33, 1819-1827. (41) Aylmore, L. A. G.; Quirk, J. P. Nature 1960, 187, 1046-1048. (42) Bezanilla, M.; Manne, S.; Laney, D. E.; Lyubchenko, Y. L.; Hansma, H. G. Langmuir 1995, 11, 665-669. (43) Paget, E.; Simonet, P. FEMS Microbiol. Ecol. 1994, 15, 109118.
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adsorption. In contrast to plasmid DNA, linear DNA exposes a much higher number of available acidic functional groups, with many groups of weak and medium acidity (80%) (Table 3). These anionic charges should have been located all along the linear double-stranded molecules and established a multi-loci contact with the clay. This different distribution and accessibility of charges on DNA may explain the difference between adsorption of CCC and linear DNA. Comparison between the number of charges offered by the clay surfaces (Table 1) and the number of acidic groups brought by adsorbed linear DNA (Table 2) showed that part of the charges on adsorbed DNA remains free. This is clearly shown at the top of the high affinity phase in CaCl2 solution (Table 2) and at the maximum of adsorption in water. The number of still free charges when linear DNA was adsorbed could explain the transfer of exchangeable Ca2+ to the solution (Figure 3): some of the acidic groups of the linear DNA molecule not involved in the adsorption process were not neutralized and remained available for desorption of Ca2+. When plasmid DNA molecules were adsorbed, all accessible charges of the organic molecules were involved in adsorption, and there was no Ca2+ desorption. LTSEM showed aggregated CCC plasmid molecules extending between the edges of the clay particles, indicating that adsorption was limited to certain areas of the plasmid. In contrast to CCC plasmid molecules acting as bridges between kaolinite crystals, linear DNA was observed lying on several montmorillonite domains where it was absorbed partly or completely to the clay edges and surface,1,43 indicating that CCC plasmids and linear DNA molecules do not adsorb on soil particles in the same way. The adsorption process results from the electrostatic interaction between accessible anionic charges of DNA and cations of either CEC or edges of clays, which depend on DNA configuration and on the physicochemical properties of clay. The adsorption process could influence the actual level of protection of each DNA type against nuclease degradation and their availability for transforming competent strains through two different ways: (1) adsorbed DNA accessibility to the enzyme should be highest for CCC forms, since the molecule is naked except for a few strongly acidic areas; (2) the capacity to remain in solution would favor more degradation of the supercoiled form than the linear chromosomal form by nuclease activity. Acknowledgment. The authors thank Anne Marie Jaunet (INRA, Versailles, France) for technical assistance with the LTSEM. LA990506Z
