Binding of DNA from Bacillus subtilis on

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Binding of DNA from Bacillus subtilis on Montmorillonite–Humic Acids–Aluminum ...... Blum, S.A.E., M.G. Lorenz, and W. Wackernagel. 1997. ... 25:319–329.

Reproduced from Soil Science Society of America Journal. Published by Soil Science Society of America. All copyrights reserved.

Binding of DNA from Bacillus subtilis on Montmorillonite–Humic Acids–Aluminum or Iron Hydroxypolymers: Effects on Transformation and Protection against DNase Carmine Crecchio,* Pacifico Ruggiero, Maddalena Curci, Claudio Colombo, Giuseppe Palumbo, and Guenther Stotzky ABSTRACT

Despite the relatively large number of papers dealing with the adsorption of DNA on clays and HA, essentially no information is available about the adsorption of DNA on organomineral particles, probably the dominant form of clays and HA in soil. The influence of Al and Fe in the intercalation of HA in the swelling clay and in the sorptive properties is still not fully understood, despite many studies of sorption/desorption of organic compound by synthetic HA-clay model sorbents (Mortland, 1970; Murphy et al., 1990; Goldberg et al., 1999). The release of DNA from plants, animals, and microorganisms can occur by lysis after their death, after infection of bacteria by phages (Redfield, 1988), and by active release of plasmid and chromosomal DNA by living bacteria (Lorenz et al., 1991). Such extracellular DNA can attain concentrations that could result in horizontal gene transfer (HGT) by transformation. Numerous bacterial species have been reported to be capable of natural transformation and of active or passive release of extracellular DNA (e.g., Lorenz and Wackernagel, 1994; Paget and Simonet, 1994). When DNA enters the soil environment, it may be rapidly hydrolyzed as the result of the ubiquitous presence of cell-bound or free DNases produced by microorganisms (Blum et al., 1997). However, some DNA may be tightly bound on soil particles and not be easily degraded by DNases (Khanna and Stotzky, 1992). A fundamental aspect of natural transformation in soils is the biodegradation of extracellular DNA: if degradation is fast, DNA is unlikely to be taken up by bacteria, as rapid degradation will result in its inactivation before transformation could occur; on the other hand, the persistence of DNA will enhance the probability of transformation. Therefore, one of the primary factors controlling the fate of extracellular DNA and transformation in soil is the adsorption of DNA on soil particles (Trevors, 1996; Pietramellara et al., 1997; Yin and Stotzky, 1997). In addition to the vertical transfer of genes, bacteria can also acquire genes by horizontal transfer. Studies of HGT in bacteria have been reviewed (e.g., Prozorov, 1999; Arber, 2000; Eisen, 2000), including concerns about HGT in soil systems (e.g., Hill and Top, 1998; Dro¨ge et al., 1999). Of the three mechanisms of HGT in bacteria (conjugation, transduction, and transformation), transformation results in the acquisition of new genetic material by the uptake of extracellular naked DNA, followed by its interaction and stable incorporation into a competent cell (Smith et al., 1981; Davison, 1999). In soil, the

The equilibrium adsorption and binding of DNA from Bacillus subtilis on complexes of montmorillonite–humic acids Al or Fe hydroxypolymers (Al–M–HA or Fe–M–HA) at different M/HA ratios, the desorption of DNA, the capacity of bound DNA to transform competent cells of B. subtilis in vitro, and the protection of bound DNA from degradation by free and organomineral-bound DNase I are reported. Adsorption was rapid (maximal after 2 h), occurred from pH 3 to 10, and was higher on Al–M–HA than on Fe–M–HA. Saturation of the sites on the surface or between the layers of Al– or Fe–M–HA occurred with only some complexes, depending on how the complexes were prepared. Essentially no desorption under stringent conditions was observed. Bound DNA transformed auxotrophic competent cells of B. subtilis, although at a lower frequency than free DNA. Bound DNA was protected more than free DNA against degradation by DNase I, and differences in resistance to degradation between free and bound DNA were more evident when DNase was also bound on the organomineral complexes.

C

lay minerals (Khanna and Stotzky, 1992; Paget et al., 1992; Khanna et al., 1998), sand (Lorenz et al., 1988), HAs (Crecchio and Stotzky, 1998), and nonsterile soil (Blum et al., 1997; Lee and Stotzky, 1999) display a high capacity to adsorb DNA and to protect it against nucleolytic degradation. Under most environmental conditions, DNA molecules are net negativelycharged, and they can adsorb to net positively-charged surfaces, such as the edges of clay minerals (Khanna et al., 1998), as well as to net negatively-charged surfaces, such as the surfaces of clays, by electrostatic bridges with the water of hydration of charge-compensating cations (Stotzky, 1986; Lorenz and Wackernagel, 1994; Paget and Simonet, 1994). Or, they can be introduced between Al hydroxide layers and clay minerals by simple ion-exchange reactions (Choy et al., 1999). Under acidic conditions (generally below pH 5), DNA becomes positively charged by protonation of adenine and cytosine, followed by guanine, and by protonation of the negative charges of phosphate groups (Theng, 1979). This protonation produces cationic groups in the DNA molecule that can bind to negatively-charged sites on clays.

C. Crecchio, P. Ruggiero, and M. Curci, Dipartimento di Biologia e Chimica Agroforestale ed Ambietale, Univ. di Bari, Via Amendola 165/A, 70126 Bari, Italy; C. Colombo and G. Palumbo, Dipartimento di Scienze Animali, Vegetali, e dell’Ambiente, Univ. del Molise, Via De Sancits s/n, 86100 Campobasso, Italy; G. Stotzky, Lab. of Microbial Ecology, Dep. of Biology, New York Univ., 100 Washington Sq. East, New York, NY 10003, USA. Received 12 May 2004. Soil Biology & Biochemistry. *Corresponding author ([email protected]).

Abbreviations: Al–M–HA, montmorillonite–humic acids–Al hydroxypolymers; CEC, cation-exchange capacity; Fe–M–HA, montmorillonite–humic acids–Fe hydroxypolymers; HA, humic acid; HGT, horizontal gene transfer; M, montmorillonite; SSA, specific surface area; TBAB, tryptose blood agar base; XRD, x-ray powder diffractogram.

Published in Soil Sci. Soc. Am. J. 69:834–841 (2005). doi:10.2136/sssaj2004.0166 © Soil Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA

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CRECCHIO ET AL.: BINDING OF DNA FROM BACILLUS SUBTILIS

presence of microniches wherein competence factors can accumulate, as well as the stressful conditions that usually predominate in soil (Stotzky, 1989), enhance the development of competence in situ. The purposes of these studies were to investigate (i) the adsorption and binding of chromosomal DNA on complexes of Al– or Fe–M–HA as examples of surfaceactive particles more representative of colloids in situ than of the pure clays, HA, and their complexes previously investigated; (ii) the ability of bound DNA to transform bacterial cells; and (iii) the resistance of bound DNA to DNase. Bacillus subtilis was used as a model with which to study HGT by transformation in these systems, as many species of Bacillus are indigenous to soil, most do not conjugate, and the strains used lacked plasmids and transducing phages. MATERIALS AND METHODS Growth and Maintenance of Bacteria Strains of B. subtilis [donor: BD1512, resistant to chloramphenicol (Cmr); recipient: BD54, susceptible to chloramphenicol (Cms)] were grown and maintained on slants of tryptose blood agar base (TBAB) supplemented with 5 mL of 1 M MgSO4·7H2O L⫺1, 0.2 mL of 0.1 M MnCl2·4H2O L⫺1, and Cm (10 ␮g mL⫺1), when resistant to the antibiotic, at room temperature (24 ⫾ 2⬚C), at which the recipients maintained their competence better than at 4⬚C (Khanna and Stotzky, 1992).

Preparation of DNA DNA was prepared according to Dubnau and DavidoffAbelson (1971). Cells were grown overnight (18 h) in VY broth [veal infusion broth containing 0.5% yeast extract], and 5 mL was transferred to 95 mL of fresh VY broth and grown to ≈1010 cells mL⫺1. The cells were harvested by centrifugation at 9000 g, washed with TES buffer [30 mM Tris, 5 mM EDTA, and 50 mM NaCl (pH 7.5)], and resuspended in 10 mL of TES containing 1 mg of lysozyme (Sigma) and 50 ␮g of pancreatic RNase (Sigma). After shaking for 30 min at 37⬚C, 10 mL of TES containing 160 mg of Sarkosyl (Sigma) and 0.5 mg of Pronase (Sigma) was added, and the mixture was incubated with continuous shaking (100 rev min⫺1) at 37⬚C for 2 h and then brought to 24⬚C. An equal volume (20 mL) of phenol (saturated with TES) was added, the mixture was centrifuged at 12 000 g for 10 min, the phenol phase was discarded, and the aqueous phase was extracted a second time with phenol. Two volumes (40 mL) of ice-cold ethanol (100%) were added to the aqueous phase to precipitate the DNA, which was

spooled on a glass rod and dissolved in DNA buffer [10 mM Tris, 0.1 mM EDTA, and 4 mM NaCl (pH 7.5)]. The DNA concentration was determined spectrophotometrically at 260 nm (A260), and the purity was assessed by the 260/280-nm ratio (which was higher than 1.8) and by electrophoresis on a 0.7% agarose gel (a single band was detected by staining with ethidium bromide) (Stotzky et al., 1996).

Preparation and Characterization of the Organomineral Complexes The ⬍0.2-␮m fraction of a M (Swy-1, Crook County, Wyoming) was separated by centrifugation (Jackson, 1979). The clay was saturated with Na⫹ (1 M NaCl), and excess Cl⫺ was removed by repeated washings with deionized distilled water (ddH2O), followed by dialysis, until a test for Cl⫺ (AgNO3) was negative. Humic acids were extracted from an Andisol (a forest soil from Mount Vulture, Italy; organic C 9.8%; total N 0.7%; pH 5.25), sampled in the A1 horizon, with a mixture of 0.5 M NaOH and 0.1 M Na4P2O7 under a N2 atmosphere for 24 h at 25⬚C with vigorous shaking (10 L of solution kg⫺1 of soil). The suspension was centrifuged at 6000 g for 20 min, the sediment was reextracted overnight, and the pooled supernatants were precipitated by bringing the alkaline extract to pH 2.0 with 12 M HCl (Schnitzer, 1978). The HA were centrifuged at 8000 g for 20 min, the pellet was reextracted, reprecipitated, and treated with a mixture of 0.1 M HCl and 0.1 M HF (Piccolo, 1988), dialyzed against ddH2O, and lyophilized. The main characteristics of HAs are reported by Crecchio and Stotzky (1998). The A1 to A5 and F1 to F5 samples were obtained by adding 0.5 M NaOH to pH 5.0 to mixtures of Na-M ⫹ Al(NO3)3 (Al) or Fe(NO3)3 (Fe) at concentration of 0.3 mmol g⫺1 of clay; after 1 h, HA (25, 50, or 100 mg g⫺1 clay) were added separately (M/HA) or together (M ⫹ HA), and each suspension was brought to pH 7.0 by adding 0.25 M NaOH (method Al or Fe/OH/M/OH; Table 1). The suspensions were diluted to 1 L with ddH2O, aged for 24 h at 25⬚C, and centrifuged at 3000 g for 15 min; the organomineral samples were then washed twice with ddH2O, ultrasonically dispersed, and lyophilized (Violante et al., 1999). Cation-exchange capacity (CEC) was determined by washing 100 mg of the complexes in 50 mL of 0.4 M BaCl2 in the presence of triethanolamine buffer (pH 8). Barium was exchanged with magnesium (0.05 M MgSO4·7H2O), and the amount of magnesium was determined by titration with 0.005 M EDTA (Jackson, 1979). Organic C was determined with an EA 1108 CHN Analyzer (Fisons Instrument, Lucino di Rodano, Italy). Specific surface area was determined by adsorption of ddH2O at 19% relative humidity obtained with ammonium acetate, assuming that the weight of water re-

Table 1. Components of organomineral complexes and modalities of their preparation. Sample

Cation

Montmorillonite (M)

Humic acids (HA)

M/HA

A1 A2 A3 A4 A5 F1 F2 F3 F4 F5

mmol Al (0.3) Al (0.3) Al (0.3) Al (0.3) Al (0.3) Fe (0.3) Fe (0.3) Fe (0.3) Fe (0.3) Fe (0.3)

g 1 1 1 1 1 1 1 1 1 1

mg – 25 25 50 100 – 25 50 50 100

– 40 40 20 10 – 40 20 20 10

Modality of preparation† Al/OH/M/OH Al/OH/(M ⫹ HA)/OH Al/OH/HA/M/OH Al/OH/(M ⫹ HA)/OH Al/OH/(M ⫹ HA)/OH Fe/OH/M/OH Fe/OH/(M ⫹ HA)/OH Fe/OH/(M ⫹ HA)/OH Fe/OH/HA/M/OH Fe/OH/(M ⫹ HA)/OH

† Al and Fe ⫽ Al(NO3)3 and Fe(NO3)3, respectively; OH ⫽ addition of 0.25 M NaOH to titrate the Al or Fe hydroxypolymers to pH 5.0 and the organomineral complexes to pH 7.0; HA/M ⫽ separate addition of HA to Al or Fe hydroxypolymers followed by M; (M ⫹ HA) ⫽ simultaneous addition of HA and M to Al or Fe hydroxypolymers.

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quired to form a monolayer on 1 m2 of surface is 2.78 ⫻ 10⫺4 g (Quirk, 1955). X-ray powder diffractograms (XRDs) of oriented specimens were recorded using an x-ray diffractometer equipped with iron-filtered Co-K␣ radiation generated at 40 kV and 30 mA, at a scanning speed of 5 2␪ min⫺1. The oriented specimens were stored for 24 h at 20⬚C in a desiccator containing CaCl2 before XRD analysis. About 10 mg of the complexes were treated with 10 mL of ammonium-oxalate at pH 3.0 (Schwertmann, 1964). The solubilized Fe and Al were subsequently determined by atomic absorption spectroscopy with a spectrophotometer.

Preparation of Organomineral-DNA Complexes All interactions between DNA and Al– or Fe–M–HA were done in 1 mL of DNA buffer in Eppendorf centrifuge tubes by rotating the tubes on a motorized wheel (70 rev min⫺1) at 24⬚C, centrifuging at 13 000 g for 15 min, and the DNA content of the supernatants measured at A260. The contact time for maximal equilibrium adsorption of DNA (25 ␮g) on organomineral complexes (10 mg) was determined for 1 to 16 h. A constant amount of DNA (25 ␮g) and variable amounts of the organomineral complexes (0.5 to 25 mg) were used to determine the saturation of organomineral complexes by DNA. A constant amount of organomineral complexes (15 mg for Al–M–HA and 1 mg for Fe–M–HA) and variable concentrations of DNA (10 to 400 ␮g for Al–M–HA and 10 to 150 ␮g for Fe–M–HA) were used to construct equilibrium adsorption isotherms. In the case of Fe–M–HA, it was not possible to add more DNA, as suspensions with DNA ⬎ 150 ␮g were not homogeneous and not well mixed by rotating the tubes. The adsorption data were used to fit Langmuir, Freundlich, and linear equations. The effect of pH on adsorption was evaluated by mixing DNA (25 ␮g) with Al– or Fe–M–HA (15 mg and 1 mg, respectively) in water adjusted from pH 3 to 10 with diluted HCl or NaOH. Desorption studies were done by resuspending the pellets of organomineral-DNA complexes in 1 mL of a washing solution (ddH2O, 0.1 M NaCl, or 0.1 M Na4P2O7), rotating the tubes on a motorized wheel (70 rev min⫺1) at 24⬚C, centrifuging at 13 000 g for 15 min, and analyzing the DNA content of the supernatants at A260.

Preparation of Competent Cells Cultures of recipient cells were grown overnight at 37⬚C in a competence medium (Spizizen II), which consisted of Spizizen salts [1.4% K2HPO4, 0.6% KH2PO4, 1% sodium citrate, 0.2% (NH4)2SO4], 0.2% Bacto Casamino Acids, 0.1% yeast extract (Difco), 0.5% glucose, and 2.5 ␮L of 1 M MgCl2 mL⫺1 (each autoclaved separately). Recipient cells were made competent by the method of Dubnau and Davidoff-Abelson (1971): a 1:100 dilution of recipient cells grown overnight in the competence medium was inoculated into fresh competence medium and shaken (200 rev min⫺1) at 37⬚C for 6.5 h, when the culture reached the late log-early stationary phase. The cells were harvested by centrifugation at 9000 g for 10 min and resuspended in 5 mL of the competence medium (Stotzky et al., 1996).

Transformation Procedure Freshly prepared competent cells [108 colony-forming units (CFU)] were incubated with 10 ␮g of free or bound DNA in 1 mL of competence medium on a motorized wheel (40 rev min⫺1) at 37⬚C for 30 min. Serial decade dilutions of the suspensions diluted with 0.85% NaCl were plated on TBAB supplemented with 10 ␮g mL⫺1 of chloramphenicol. Competent cells that received no DNA or only organomineral particles

were plated on the selective medium and served as negative controls to distinguish revertant mutants from transformants. No revertants were detected. The total number of viable cells was determined by plating the diluted cultures on TBAB. All plates were incubated at 37⬚C for 24 to 36 h. Details on methods for the study of transformation by free and bound DNA have been published (Stotzky et al., 1996).

Effect of DNase on Transformation by Free and Bound DNA Free or bound DNA (10 ␮g) was incubated with 10 ng of DNase I (Sigma) in 1 mL of DNase buffer [50 mM Tris, 10 mM MgCl2, and 50 ␮g of bovine serum albumin (Sigma) (pH 7.5)] at 24 ⫾ 2⬚C for 15 min, when 1 mL of competent cells (108 CFU) was added and the mixture incubated at 40 rev min⫺1 and 37⬚C for 30 min. The number of transformants and total viable cells were enumerated on the selective and nonselective media. The same experiments were done with bound DNase: 100 ng of DNase I was bound on Al–M–HA, which was washed with ddH2O (three times) until no more DNase was desorbed (Khanna and Stotzky, 1992).

Statistics All experiments were performed in triplicate. The data, normalized to 1 mg of organomineral particles or 1 ␮g of DNA, are expressed as the means ⫾ the standard error of the means (x ⫾ SEM).

RESULTS AND DISCUSSION To study the adsorption of DNA on surface-active particles presumably more representative of soil colloids in situ than the more simple complexes previously investigated (e.g., Greaves and Wilson, 1969; Lorenz et al., 1988; Paget et al., 1992; Crecchio and Stotzky, 1998; Stotzky, 2000) organomineral complexes consisting of HA, M, and Al or Fe hydroxypolymers were prepared at different M/HA ratios. Two different modalities of preparation were used: the simultaneous addition of M and HA to Al or Fe hydroxypolymers or the separate addition of HA followed by M. Table 1 summarizes the components comprising the organomineral complexes and the modalities of their preparation. Table 2 reports the main properties of the complexes determined as described in Materials and Methods. The CECs (14 to 46 cmol (⫹) kg⫺1) of the Al–M–HA complexes were lower than those of the Fe–M–HA complexes (48 to 69 cmol(⫹) kg⫺1) (Table 2), and the CECs of all complexes were lower than that of the M SWy-1 (80 ⫾ 4 cmol(⫹) kg⫺1) (Violante et al., 1999). The decreasing of CEC has to be related to the higher degree of interlayering by OH-Al (Colombo and Violante, 1997; Violante et al., 1999). Sample A1 (Al–M) had a CEC (14 cmol kg⫺1) lower than that (62 cmol(⫹) kg⫺1) determined for similar complexes formed in the presence of 5 mmol of Al species after 7 d of aging, probably because the Al species were aged for only 1 d and were instratificated into the interlayers of the M. With aging, larger polymers of OH-Al were formed that did not intercalate the interlamellar space, resulting in a higher CEC (Colombo and Violante, 1997). The higher CECs of the F1 to F5 complexes indicated the presence of small

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Table 2. Some properties of the organomineral complexes.

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Sample A1 A2 A3 A4 A5 F1 F2 F3 F4 F5

Organic C % – 0.92 0.98 2.71 3.16 – 0.76 1.91 3.33 4.11

Specific surface area 2

g⫺1

m 209 139 180 112 121 143 134 129 121 132

Charge (⫹) m⫺2

cmol 0.07 0.15 0.14 0.34 0.38 0.33 0.39 0.51 0.40 0.52

Cationexchange capacity cmol(ⴙ) 14 21 25 38 46 48 53 65 49 69

kg⫺1

amounts of irregular intercalated material, as suggested by the broadening of the peaks of the basal spacing (Table 2). The Fe–M–HA complexes had d001 spacings similar to the complexes formed in the presence of Al (A1–A5) at 20⬚C, but they collapsed very rapidly to 1.14–1.01 nm and 0.97 nm after exposure to 300 and 550⬚C, respectively, indicating the formation of an asymmetrical layer of hydroxy-Fe (Table 2). The specific surface area (SSA) of HA–metal–clay complexes depends on the methods of coprecipitation of Fe and Al and the HAs (Violante et al., 1999). The SSA of the Al–M–HA complexes ranged from 112 to 209 m2 g⫺1, whereas those of the Fe–M–HA complexes ranged from 121 to 143 m2 g⫺1. The SSA of the Al–M–HA complexes decreased as the organic C content increased (Table 2), perhaps as the result of the formation of aggregate particle related with HA-clay structures (Murphy et al., 1990). In addition, the freeze-drying and air-drying treatments during the preparation of the solid phases may have produced different pore and surface structures of the particle clusters (Laird et al., 2001). The aggregation of suspended Al phases may have been the most important mechanism of aggregation of the larger-size Al–M–HA aggregates (Goldberg et al., 2001). The concentration of the organomineral particles at which saturation by a constant amount of DNA occurred and the optimal contact time with DNA were determined (data not shown). The equilibrium adsorption isotherms of a constant amount of DNA added to different amounts of the organomineral particles (0.5–25.0 mg) showed an increase in the adsorption of DNA with increasing amounts of the particles until a plateau was reached at 1 and 15 mg of Fe–M–HA and Al–M–HA, respectively (isotherms not shown). Equilibrium adsorption of DNA on the complexes was maximal after 2 h and constant up to 16 h, the longest contact time investigated, with no significant differences among different DNA complexes. The adsorption isotherms of DNA on a constant amount of Al–M–HA (Fig. 1a) differed in slopes depending on the concentration of DNA in solution. The slopes were steeper when the concentration of DNA was ⱕ10 to 30 ␮g mL⫺1, and significantly less steep at a concentration of DNA ⬎ 50 to 100 ␮g mL⫺1; in two cases, A2 and A4, adsorption increased linearly with concentration. This trend is typical of molecules whose affinity for adsorbents changes as the concentration of

Basal spacing Fe mg 0.23 0.31 0.30 0.32 0.26 30.45 28.73 28.88 28.65 28.83

Al

100ⴗC

36.31 36.90 37.15 38.10 37.11 1.71 0.67 1.25 0.64 1.45

1.45 1.52 1.56 1.51 1.56 1.53 1.44–1.22 1.59 1.47 1.58

g⫺1

200ⴗC nm 1.42 1.39 1.50 1.42 1.43 1.01 1.16–1.01 1.01 1.26 1.22

300ⴗC

500ⴗC

1.41 1.39 1.43 1.42 1.43 1.01 1.16–1.01 1.01 1.01 1.25–1.01

1.23 1.32 1.27 1.33 1.42 0.97 0.97 0.97 0.97 1.01

the adsorbate in solution increases, indicating different mechanisms of adsorption. The data were also tested for their fit to the main adsorption equations (Langmuir, Freundlich, linear), and the relevant values for the constants and regression coefficients are reported in Table 3. The isotherms fitted the Langmuir equation only for complex A1 (which did not contain HA), with a plateau at about 50 ␮g DNA mL⫺1, and for complex A3, although in this case saturation was less evident at the equilibrium concentrations used; all other complexes

Fig. 1. (a) Equilibrium adsorption of DNA on Al–M–HA. (b) Equilibrium adsorption of DNA on Fe–M–Ha. Data expressed as the means ⫾ the SEs of the means (SEMs). Where not reported, SEMs are within the dimensions of the symbols.

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Table 3. Best fitting adsorption isotherm parameters, regression coefficients, and DNA adsorbed at two equilibrium concentrations. Langmuir equation

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Sample

K1

K2

Freundlich equation R

2

n

kf

R2

Linear equation R2

DNA adsorbed†

DNA adsorbed‡

␮g mg⫺1 complex A1 A2 A3 A4 A5 F1 F2 F3 F4 F5

0.950 – 0.665 – – 3.906 5.780 – – –

0.187 – 0.051 – – 0.145 0.092 – – –

0.9903 – 0.9869 – – 0.9727 0.9963 – – –

– – – – 0.552 – – – – 0.577

– – – – 0.365 – – – – 6.226

– – – – 0.8238 – – – – 0.9367

– 0.9254 – 0.9309 – – – 0.9373 0.9361 –

2.85 2.00 5.39 1.20 1.32 15.83 30.24 20.35 15.07 26.70

4.88 7.60 12.88 8.98 4.49 25.63 58.58 46.46 43.30 75.39

† Equilibrium concentration: 10 ␮g DNA mL⫺1 ‡ Equilibrium concentration: 100 ␮g DNA mL⫺1.

showed a continuous increase in adsorption as the DNA concentration increased and no saturation occurred. In particular, the adsorption of DNA on complex A5 decreased progressively as the equilibrium concentration of DNA increased and fitted the Freundlich equation, whereas the adsorption of DNA on complexes A4 and A5 showed a linear trend (Fig. 1a and Table 3). The adsorption isotherms of DNA on a constant amount of Fe–M–HA are shown in Fig. 1b, and the corresponding equations and parameters are presented in Table 3. Saturation was reached at low concentrations of DNA on sample F1, as was also observed with complex A1, indicating that the absence of HA in the Fe and Al complexes limits the number of sites available for adsorption even at low equilibrium concentrations of DNA. The isotherm of complex F2 fitted the Langmuir equation, although a plateau was suggested only at the highest concentrations of DNA used, indicating that more DNA was needed to saturate the adsorption capacity of complex F2. All other samples showed a continuous increase of adsorption (e.g., complexes F3 and F4), although there appeared to be a decrease in the number of sites available for adsorption on complex F5 (Fig. 1b and Table 3). The adsorption of DNA on the Fe–M–HA complexes was up to 20 times higher than on the Al–M–HA complexes, when complexes with the same amounts of components and modality of preparation were compared. This is clearly demonstrated in Table 3, which reports the micrograms of DNA adsorbed per milligram of complex calculated by interpolating the adsorption equations at two equilibrium concentrations, 10 and 100 ␮g DNA mL⫺1, where the slopes of the isotherms were significantly different. The higher capacity of the Fe complexes to adsorb DNA was probably a result of the larger size of the Fe–OH precipitation products than of the Al–OH polymers, which affected the internal layering of swelling phyllosilicates (Huang and Violante, 1986; Colombo and Violante, 1997; Violante et al., 1999). Thus, as more Fe3⫹ remains outside coating the M-HA complexes than enters the interlayers, Fe3⫹ has more opportunity to interact with DNA. This was evident when the DNA adsorbed per milligram of complex, calculated at equilibrium concentrations of 10 and 100 ␮g

DNA mL⫺1, were related to the CEC (Fig. 2a). Iron complexes, whose CECs ranged from 48 to 69 cmol(⫹) kg⫺1, adsorbed significantly more DNA mg⫺1 of complex than did Al complexes, whose CECs were ⱕ 48 cmol(⫹) kg⫺1 (Table 2). In contrast, the amounts of DNA adsorbed on Al complexes were not related to the CEC (Fig. 2). In fact, the XRD patterns of the Al–M–HA complexes (A1–A5) showed relatively sharp and symmetrical peaks at 20⬚C, whereas the complexes F1 to

Fig. 2. (a) Amounts of DNA adsorbed on Al– or Fe–M–HA as a function of the cation-exchange capacity (CEC) of the complexes. Data were obtained by interpolating the best-fitting Langmuir, Freundlich, or linear equations, as reported in Table 3. (b) Percentage of DNA adsorbed on Al– or Fe–M–HA as a function of the organic C content of the complexes.

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CRECCHIO ET AL.: BINDING OF DNA FROM BACILLUS SUBTILIS

F5 showed broad and asymmetrical peaks with a shoulder at about 1.53 nm that rapidly collapsed at higher temperature (300–500⬚C), and which could be attributed to an irregular interstratification of the Fe hydrolytic products into the M (Table 2). The percentage of added DNA adsorbed on the Al and Fe complexes was also plotted as a function of their content of organic carbon (Fig. 2b). The percentages ranged from 60 to 85% in the absence of HA, similar to the 80% adsorption of DNA on Ca-M (Khanna and Stotzky, 1992) and to the 58 to 85% adsorption on HA (Crecchio and Stotzky, 1998). The differences in percentage adsorption between the Fe and Al complexes decreased as the organic C content increased. Adsorption decreased as the content of organic C increased, independently of other characteristics of the complexes, such as the type of cation, CEC, or SSA. When the Fe and Al polycations on the surface of the clay became complexed by the functional groups of HA, they were apparently less available to interact with the phosphate groups of DNA. In general, adsorption was greater at low pH and decreased as a function of the different characteristics of the complexes as the pH increased (Fig. 3). This was probably the result of the protonation at low pH of purines and pyrimidines that were then attracted with higher affinity by the net negatively charged M and on phosphate groups of DNA that did not expose negative charges and, as a consequence, showed a decreased repulsion toward the net negatively-charged M. At higher pH values, protonation decreased, reducing adsorption

Fig. 3. Effect of pH on the percentage of adsorption of DNA on Al– or Fe–M–HA complexes. Data expressed as the means ⫾ the standard errors of the means (SEMs). Where not reported, SEMs are within the dimensions of the symbols.

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on net negatively-charged M. The effects of pH on adsorption were greatest for complexes A1 and A2, whose adsorption decreased significantly from 85 to 90% at pH 3 to 40 to 45% at pH 10. The higher content of organic C of complexes A4 and A5, especially the greater amounts of functional acidic groups of the HA, apparently render adsorption independent of pH. The adsorption of DNA by complex A3 was also not particularly affected by pH. Although the content of organic C of sample A3 was lower than that of complexes A4 and A5 and comparable with complex A2 (Table 2), the modalities of preparation were different, as A3 was the only complex where the HA were added first to the Al hydroxypolymers and M was added 2 h later. The effects of pH on adsorption of DNA by the Fe complexes were similar to the Al complexes. Adsorption was generally higher at pH 3 and decreased as the pH increased. This trend was particularly apparent in complexes F1 and F2, whose adsorption as a function of pH was comparable with the corresponding Al complexes (A1 and A2), whereas the adsorption on complexes F3, F4, and F5 was almost independent of pH. This independence of pH indicated that adsorption of DNA occurs not only by cation exchange (after protonation of DNA molecules), but also at higher pH values, probably by hydrogen bridges between –COOAl or –COOFe groups of humic substances and the –RNH2 groups of DNA. When DNA adsorbed at equilibrium was eluted repeatedly (up to five washings) by washing the complexes for 3 h with ddH2O, 0.1 M NaCl, or 0.1 M Na4P2O7, essentially no desorption was observed (only 1% of added DNA). This strong binding of DNA on soil complexes was unusual, as significant, although variable, amounts of DNA were desorbed from HA (Crecchio and Stotzky, 1998), M (Khanna and Stotzky, 1992), soil particles (Blum et al., 1997), and sand (Romanowsky et al., 1991). No difference in desorption was observed between the two types of organomineral particles, and binding was independent of the pH at which adsorption occurred. Regardless of how they were constituted and prepared, Al– and Fe–M–HA particles strongly bound large amounts of chromosomal DNA from B. subtilis over a broad range of pH. This strong and constant binding could be explained by the presence of both positive and negative functional groups on the surface of organomineral soil particles, which are also differently charged at the broad range of pH investigated. The binding of DNA on the organomineral particles reduced the frequency of transformation (Fig. 4), as has also been observed with other particles (e.g., Khanna and Stotzky, 1992; Crecchio and Stotzky, 1998). Although only small amounts of DNA were desorbed, DNA–Al– or Fe–M–HA complexes were always first washed with ddH2O to eliminate the possibility that transformation was the result of desorbed free DNA. DNA bound on Fe–M–HA showed a lower capacity to transform B. subtilis than DNA bound on Al–M–HA. These differences in transforming capacity could have been the result more of differences in the coating of M by the hydroxypolymers and HA than of differences in interca-

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SOIL SCI. SOC. AM. J., VOL. 69, MAY–JUNE 2005

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CONCLUSIONS

Fig. 4. Transformation of Bacillus subtilis by DNA free or bound on Al– or Fe–M–HA and effects of DNase, free or bound on Al–M–HA or Fe–M–HA, on transformation. Data expressed as the means ⫾ SEs of the means.

lation of DNA within the clay layers, which was higher in the Al than in the Fe complexes. Furthermore, as only DNA is taken up by competent cells during transformation, these differences might have also been result of the different strengths of binding of DNA on the Al and Fe complexes. Bound DNA was protected against inactivation by DNase I more than free DNA. The number of transformants ␮g⫺1 of DNA bound on Al–M–HA was almost the same after incubation with 10 ng DNase than with no DNase, was slightly lower when DNA was bound on Fe–M–HA, but was significantly decreased when free DNA was hydrolyzed by DNase in solution (Fig. 4). The protection against inactivation was also evident when DNase was also bound on the organomineral particles to mimic environmental conditions, wherein soil enzymes presumably exist and catalyze primarily in a bound form rather than in the soil solution (e.g., Burns, 1982; Nannipieri et al., 2002) (Fig. 4). Probably as the result of the partial inactivation of its catalytic activity, 100 ng of bound DNase had essentially the same efficacy as 10 ng of free enzyme in decreasing the capacity of free DNA to transform competent cells, whereas bound as well as free DNase was essentially inactive against bound DNA. The apparent paradox between the resistance of bound DNA to DNase and the ability of bound DNA to transform can be reconciled according to Khanna et al. (1998), who hypothesized changes in electron distribution and/or conformation of clay bound DNA, which prevented DNase from recognizing or interacting with appropriate binding or cleavage sites on the DNA. In contrast, the extention of free ends of DNA from the edges of the soil particles enables the DNA to interact with receptor sites on competent cells and result in their transformation. During the uptake of naked DNA by competent cells, one end of the double-stranded DNA molecule penetrates the cell membrane at a receptor site, at the interior of which is a molecule of DNase, which splits the double-stranded DNA into two single-stranded molecules, one of which enters the cell (Lacks, 1962).

The adsorption of DNA on organomineral particles was strongly affected by the nature of the complexes, that is, the species of hydroxypolymers coprecipitated, the CEC of the particles, and, although not always significantly, the pH at which adsorption occurred. Relatively small quantities of the Fe hydrolytic species, compared with Al species and HA intercalated M, and these complexes showed different behavior in DNA adsorption. Therefore, knowledge of such characteristics is important to predict the adsorption capacity of a soil for DNA. Although binding protects the DNA from enzymatic degradation, it does not prevent transformation. Such protected cryptic genes (e.g., DNA from genetically modified bacteria) may persist undetected in soils but be subsequently expressed when a susceptible host comes into contact with soil particle-DNA complexes and transformation occurs. These results not only indicate the importance of assessing the risks and benefits associated with the release of genetically modified organisms, but they also emphasize the potential importance of HGT by transformation in soil in the evolution of bacteria. ACKNOWLEDGMENTS This research was in part supported by the Research Program of National Interest (PRIN), year 2001, contract 2001072713, by the Italian Ministry of Education and Research (MIUR).

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