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Role of bacteria in the adsorption and binding of DNA on soil colloids and minerals
Colloids and Surfaces B: Biointerfaces 69 (2009) 26–30
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Role of bacteria in the adsorption and binding of DNA on soil colloids and minerals Peng Cai a , Jun Zhu b , Qiaoyun Huang a,b,∗ , Linchun Fang b , Wei Liang b , Wenli Chen a,∗∗ a b
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China Key Laboratory of Subtropical Agricultural Resources and Environment, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China
a r t i c l e
i n f o
Article history: Received 4 September 2008 Received in revised form 20 October 2008 Accepted 20 October 2008 Available online 30 October 2008 Keywords: Adsorption Desorption DNA Bacteria Composite Soil colloid Mineral
a b s t r a c t Adsorption and desorption of salmon sperm DNA on bacteria (Bacillus thuringiensis, Pseudomonas putida), two different colloidal fractions (organic and inorganic clay) from an Alfisol, minerals (montmorillonite, kaolinite and goethite) and colloid–bacteria composites were studied. Similar adsorption capacity and affinity of DNA were observed on two bacterial cells. However, the two bacterial strains played different roles in affecting the adsorption of DNA on the composites of soil colloidal particles with bacteria. The introduction of B. thuringiensis in soil colloids and minerals systems dramatically promoted DNA adsorption on colloidal particles especially organic clay, while P. putida decreased the adsorption of DNA on kaolinite and goethite. Electrostatic force and ligand exchange are regarded to be the major driving forces involved in the adsorption of DNA on bacterial cells, montmorillonite, soil colloids and goethite. Presence of bacteria enhanced the proportion of DNA adsorption on soil colloidal particles by electrostatic force and depressed that by ligand exchange process. Information obtained in this study is of fundamental significance for the understanding of the ultimate fate of extracellular DNA in soil systems. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In soil environments, the genetic material of organisms (DNA molecules) is liberated by excretion from microorganisms, plants, and animals or by lysis of dying cells [1]. These extracellular molecules, physically or chemically adsorbed on surface active particles in soils are partially protected against degradation by nucleases and retain the ability to transform competent bacterial cells [2,3]. This adsorbed DNA, which has been termed “environmentally cryptic genes”, has a crucial role in biological activity and diversity, as well as in the transfer of genetic information among bacteria [4,5]. In natural soil environments, soil inorganic components are often associated with the microorganisms and these interactions have profound impacts on microbial activity, mineral weathering, formation of aggregates and the mobility of a wide variety of contaminants [6–9]. Meanwhile, the surface properties of the composites of minerals with microorganisms can differ dramatically
∗ Corresponding author at: Key Laboratory of Subtropical Agricultural Resources and Environment, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China. Tel.: +86 27 87671033; fax: +86 27 87280670. ∗∗ Corresponding author. Tel.: +86 27 87671033; fax: +86 27 87280670. E-mail addresses: [email protected], [email protected] (Q. Huang), [email protected] (W. Chen). 0927-7765/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2008.10.008
from pure bacterial or mineral surfaces, particularly in terms of surface charge and numbers of reactive sites [10,11]. The ultimate fate of DNA released from organisms in soils depends largely on the interactions of DNA molecules with organic and inorganic surfaces. A number of investigations have been devoted to the adsorption and binding of DNA on various soil components such as sand, clay minerals, humic substances and soils. It was found that this process is affected by the ionic strength, pH of the medium, type and content of clay as well as the characteristics and configurations of DNA molecules [12–17]. An increase in the concentration of cations and/or a decrease in the pH favored DNA adsorption on soil colloids and clay minerals [12–14]. Organic matter played an inhibitory role in DNA adsorption on permanent-charge soil colloids. Montmorillonite clay dominates the amount of DNA adsorption and kaolinite probably plays a key role in the extent of DNA binding in permanent-charge soil [14]. The amount of supercoiled plasmid DNA adsorbed by sand was slightly less than that of linearized or open circular plasmid DNA [15]. The lower molecular mass DNA from Bacillus subtilis showed higher adsorption capacity by Ca-montmorillonite and Ca-kaolinite than the higher molecular mass DNA [16]. Recently, Pietramellara et al. [17] reported that the presence of constitutional organic components (coc) and cellular wall debris (cwd) improved the adsorption of the DNA molecule on montmorillonite and kaolinite, whereas only the cwd had a positive effect on the binding of DNA on clays. In B. subtilis and Streptococcus pneumoniae, double-stranded DNA associates rapidly with
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 69 (2009) 26–30
competent cells through non-covalent bonds to form a complex that is resistant to gentle washing or to replacement by excess DNA [18]. In competition studies, it was found that double-stranded DNA of any source adsorbs to the cell surface and is taken up, indicating no specificity of this process for homologous DNA [19]. Long DNA ( DNA, 48.5 kb) was adsorbed to Escherichia coli surfaces preferentially versus short DNA (herring sperm DNA, 23 kb) [20]. However, little information is available on DNA adsorption on the composites of bacteria with soil colloidal particles. Therefore, the objectives of the present study were to study the adsorption behavior of DNA on two bacterial strains (Bacillus thuringiensis, Pseudomonas putida) and their composites with clay fractions from an Alfisol and minerals including kaolinite, montmorillonite and goethite. The release of DNA molecules from their colloidal complexes was also examined by sequential desorption with neutral salt and phosphate solutions. 2. Materials and methods 2.1. DNA Salmon sperm DNA was purchased from Sigma Chemical Co., St. Louis, MO. 2.2. Preparation of bacteria, soil colloids and minerals The cells of B. thuringiensis and P. putida were initially cultured in 5 ml of beef-extracted peptone medium at 28 ◦ C for 7 h. Two ml of bacterial suspension was then transferred to 200 ml of the same medium for another 18 h. Cells at the stationary growth phase were harvested by centrifugation at 10,000 × g for 10 min, and washed twice by deionized distilled water (ddH2 O), and finally resuspended in ddH2 O. An aliquot of the cell suspension was centrifuged and dried overnight at 60 ◦ C to determine the dry weight of the cells in the stock suspension. The specific surface area (SSA), point of zero charge (PZC) and zeta potential of bacteria were measured by the methylene blue adsorption method [21], acid–base titration (Automatic Potentiometric Titrator, Electronics Manufacturing Corporation, Japan), and Zeta Potential Analyzer (ZetaPlus 90, Brookhaven Instruments Corporation, USA), respectively. The properties of bacteria are listed in Table 1. The preparation of organic clay (organo-mineral complex), inorganic clay (H2 O2 -treated clay) from a clay loamy Brown soil (Alfisol), goethite and less than 2 m fractions from kaolinite and montmorillonite as well as their properties was described previously [22].
Two grams of soil colloid or mineral were suspended into 100 ml ddH2 O and dispersed by sonication. To explore the adsorption of DNA in mixtures of bacteria and soil colloid or mineral, we used mixtures containing 5 mg soil colloid or mineral and 0.5 mg bacteria. The mixture was shaken for 30 min. Different amount of DNA (25–250 g) was added and the volume was brought to 2.5 ml with Tris buffer (pH 7.0). The mixture was shaken at 25 ◦ C for 2 h and centrifuged at 20,000 × g for 20 min. The concentration of DNA in the supernatant was measured at 260 nm. The adsorption of DNA on 5 mg of soil colloid or mineral was also determined as described above. 2.4. Desorption of DNA Two hundred and fifty ml of soil colloid or mineral suspension and 0.5 mg of bacteria were mixed and shaken at 25 ◦ C for 30 min in the centrifuge tube. Two ml of buffer containing 200 g of DNA was added into the mixture, and shaken for another 2 h and centrifuged at 20,000 × g for 20 min. The concentration of DNA was determined at 260 nm and the amount of DNA adsorbed was calculated by the difference between the amount of DNA added and that in the supernatant. The mixture–DNA complexes formed after equilibrium adsorption were washed with 1 ml of 100 mM NaCl (dissolved in 10 mM Tris buffer) and 100 mM phosphate at pH 7.0 sequentially. Each washing was repeated until no more DNA was detected in the supernatant. The percentage of DNA desorbed was calculated according to the following expression: the percentage of DNA desorption (%) = the total amount of DNA desorbed/the amount of DNA adsorbed. The desorption of DNA adsorbed on soil colloid or mineral or bacteria was also examined as described above. 3. Results and discussion 3.1. Equilibrium adsorption of DNA on bacterial cells The adsorption isotherms of DNA on B. thuringiensis and P. putida are shown in Fig. 1. DNA adsorbed by the bacterial cells fitted Langmuir equation X = Xm KC/(1 + KC), where X is the amount of DNA adsorbed per unit mass of bacterial cells (g mg−1 ), Xm is the maximum amount of DNA adsorbed (g mg−1 ), K is a constant related to the adsorption energy (ml g−1 ) and C stands for the concentration of DNA in the equilibrium solution (g ml−1 ). The adsorption of DNA by bacteria increased steadily as the increment of DNA concentration in solution. As presented in Table 2, the maximum amount of DNA adsorption on B. thuringiensis and P. putida were 7.79 and
2.3. Adsorption of DNA In the 10 ml centrifuge tube, 250 l (0.5 mg) of B. thuringiensis or P. putida were mixed with 2250 l of 10 mM Tris buffer (pH 7.0) containing 25–250 g of DNA. The mixture was gently shaken at 25 ◦ C for 2 h and centrifuged at 20,000 × g for 20 min. DNA in the supernatant was determined by spectrophotometry at 260 nm. The amount of DNA adsorbed by bacterial cells was calculated by the difference between the amount of DNA added and that remaining in the supernatant. Table 1 Selected properties of bacteria. Bacteria
SSA (m2 g−1 )
PZC
Zeta potential (mV)
B. thuringiensis P. putida
192.4 242.7
2.66 2.90
−16.12 −48.12
SSA and PZC stand for specific surface area and point of zero charge.
27
Fig. 1. Adsorption isotherms of DNA on B. thuringiensis and P. putida at pH 7.0.
28
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Table 2 Langmuir parameters for the adsorption of DNA on bacteria, soil colloidal particles and their composites. Bacteria, soil colloidal particles and their composites
Xm (g mg−1 )
K (ml g−1 )
r
B. thuringiensis P. putida Organic clay Organic clay + B. thuringiensis Organic clay + P. putida Inorganic clay Inorganic clay + B. thuringiensis Inorganic clay + P. putida Montmorillonite Montmorillonite + B. thuringiensis Montmorillonite + P. putida Kaolinite Kaolinite + B. thuringiensis Kaolinite + P. putida Goethite Goethite + B. thuringiensis Goethite + P. putida
7.79 7.29 1.81 11.01 3.41 2.68 7.89 5.11 6.90 11.24 15.39 4.82 7.78 4.69 3.86 7.09 2.30
0.04 0.05 0.02 0.01 0.01 0.14 0.03 0.59 0.01 0.01 0.01 0.05 0.07 0.02 0.01 0.01 0.03
0.98 0.99 0.99 0.99 0.99 0.99 0.97 0.98 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99
7.29 g mg−1 , respectively. The K values for the adsorption of DNA on B. thuringiensis and P. putida were 0.04 and 0.05 ml g−1 , respectively. The greater the K value, the higher the affinity between bacterial cells and DNA molecules. These results suggest that the two bacterial cells have not significant differences in DNA adsorption capacity and affinity although they have different physical and chemical properties (Table 1). 3.2. Equilibrium adsorption of DNA on bacteria composites with soil colloidal particles The adsorption isotherms of DNA on soil colloids, minerals and their corresponding bacterial composites are shown in Figs. 2 and 3. The adsorption data over the range of DNA concentrations were also conformed to the Langmuir equation and the simulation parameters are listed in Table 2. As for individual components, the order of DNA adsorption capacity was montmorillonite (6.90 g mg−1 ) > kaolinite (4.82 g mg−1 ) > goethite (3.86 g mg−1 ) > inorganic clay (2.68 g mg−1 ) > organic clay (1.81 g mg−1 ). The unit layers of montmorillonite, a 2:1 lattice aluminosilicate, are held together by weak van der Waals forces and have hydrated interlayer cations (Ca2+ , Mg2+ , and Na+ ) between unit layers in the packets. However, the unit layers of kaolinte are tightly stacked by strong hydrogen bonds and goethite is an iron hydroxide which possesses only one type of functional group (hydroxyl) [23]. As a result, in comparison with kaolinite and goethie, montmorillonite has a greater specific surface area which provides more adsorption sites for DNA. Greaves and Wilson [24] also reported that purified montmorillonite may adsorb more than its own weight in DNA. A maximum of 59.4% of 50 g chromosomal DNA from B. subtilis was adsorbed by montmorillonite, versus 31.4% by kaolinite [16]. As for soil colloids, the amount of DNA adsorbed by organic clay was lesser than that by inorganic clay, indicating that DNA adsorption is depressed by organic matter in permanent-charge soil. As compared with the pure soil colloids systems, the adsorption of DNA on organic clay and inorganic clay in the presence of B. thuringiensis increased by 5.1- and 1.9-times, respectively. The presence of B. thuringiensis also enhanced the adsorption capacity of DNA on minerals by 61.4–83.7%. These results suggest that new interfacial sites may be produced at the adhesion zones between the cell walls of B. thuringiensis and the colloidal particles especially organic clay. However, the effect of P. putida on DNA adsorption by soil colloidal particles was different from that of B. thuringien-
Fig. 2. Adsorption isotherms of DNA on soil colloids and their composites with bacteria at pH 7.0.
sis. There was only an increase of DNA adsorption by 88.4–123.0% on soil colloids and montmorillonite–P. putida composites as compared with the pure colloids systems. The adsorption of DNA on kaolinite and goethite in the presence of P. putida was 2.7% and 40.4% lower than that in the absence of bacteria. These different behaviors between B. thuringiensis and P. putida on the adsorption of DNA by their composites with soil colloidal particles may be related to the discrepancies in their cell wall structures which result in varying interactions with soil colloids and minerals. Grampositive cell walls are constructed of peptidoglycan polymers which are thicker (25 nm), rich in carboxyl groups and covalently linked together as they assemble around the cell. The secondary polymers (teichoic or teichuronic acids) are also bonded into the peptidoglycan framework [25]. Gram-negative cell walls have a more complex structure format. These walls contain a thin layer of peptidoglycan (7.5 nm) and a second membrane (outer membrane) within the cellular envelope which is porous structure, rich in protein, lipid and lipopolysaccharide [11,26]. Approximately 45% of the surface of Gram-negative bacteria is covered by lipopolysaccharides [27], which are anchored with their lipid part in the outer membrane and can be adsorbed mainly by hydrogen bonds on mineral oxides such as TiO2 , Al2 O3 , and SiO2 [28]. Rong et al. [29] reported that hydrogen bonding and electrostatic interaction were the main forces governing the adsorption of P. putida on kaolinite and montmorillonite by using isothermal titration calorimetry (ITC) and Fourier transform infrared spectra (FTIR). Therefore, the lesser amount of DNA adsorption on P. putida composites with soil colloidal particles
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 69 (2009) 26–30
29
Fig. 4. Desorption of DNA from bacteria, organic clay (OC), inorganic clay (IC) and their composites with bacteria by NaCl and phosphate sequentially at pH 7.0.
order of goethite > kaolinite > montmorillonite. Scanning electron microscopy (SEM) images showed that kaolinite has a tendency to aggregate and form large particles with P. putida whereas fewer bacterial cells coupled with the aggregation of montmorillonite particles [29]. The tighter associations of kaolinite or goethite with P. putida than montmorillonite may mask the reactive adsorption sites that are normally available to DNA molecules. These results indicate that Gram-positive bacteria play a more important role than Gram-negative bacteria in promoting DNA adsorption on soil colloids and minerals. 3.3. Desorption of DNA from bacteria, soil colloids and their composites Desorption was conducted after the adsorption of DNA on bacteria, soil colloids and their bacterial composites. Figs. 4 and 5 illustrate that the percent desorption of DNA by NaCl from B. thuringiensis, P. putida, organic clay and montmorillonite was in the range of 45.9–70.0% whereas that from inorganic clay, kaolinite and goethite was less than 13.2%. The desorption ratio of DNA molecules from their complexes with B. thuringiensis, P. putida, organic clays and montmorillonite by phosphate was 7.3–24.3%, while that from inorganic clays and goethite was 36.0–53.7%. Only 5.3% of DNA
Fig. 3. Adsorption isotherms of DNA on minerals and their composites with bacteria at pH 7.0.
than B. thuringiensis composites may be attributed to the competition of more macromolecules (especially lipopolysaccharide) with DNA on mineral surface for adsorptive sites. It is noteworthy to mention that P. putida played different roles in the adsorption of DNA for the three examined clay minerals systems. The possible reason for the decreased adsorption of DNA on kaolinite and goethite in the presence of P. putida as compared to individual components is the alteration of disperse state and aggregate structure of mineral particles after the introduction of bacteria. It was reported by Jiang et al. [30] that the affinity and the amount of P. putida adsorption on clay minerals and iron oxide followed the
Fig. 5. Desorption of DNA from montmorillonite (M), kaolinite (K), goethite (G) and their composites with bacteria by NaCl and phosphate sequentially at pH 7.0.
30
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 69 (2009) 26–30
on kaolinite was released by phosphate. Nucleic acids in solution resemble a polyelectrolyte and several mechanisms have been proposed to be responsible for the adsorption of salmon sperm DNA on soil colloids and minerals such as van der Waals force, electrostatic force, hydrogen bonding, ligand exchange and hydrophobic force [14,31]. DNA molecules desorbed by NaCl were usually regarded as exchangeable, while those removed by phosphate were considered as specifically adsorbed (ligand exchange form). The data obtained in this experiment imply that more than 50% of DNA was adsorbed by electrostatic force on bacteria, organic clay and montmorillonite. The proportion of DNA adsorbed by ligand exchange on inorganic clays and goethite was 36.0% and 53.7%, respectively. Only 21.9% of DNA molecules are adsorbed on kaolinite by electrostatic force and ligand exchange, suggesting that other interaction forces such as hydrophobic force and hydrogen bonding may play more important roles in the adsorption of DNA. As compared with pure organic clay system, the release of DNA by NaCl decreased by 73.5% from its composite with B. thuringiensis. For kaolinite–B. thuringiensis composite, DNA desorbed by phosphate was 64.0% higher than pure kaolinite. Except for organic clay- and kaolinite–B. thuringiensis composites, the desorption rate of DNA by NaCl increased while that by phosphate decreased in soil colloidal particles–bacteria composites as compared to pure colloids. It suggests that the introduction of bacteria in colloidal particles systems increased the proportion of DNA adsorbed electrostatically and decreased that by ligand exchange. 4. Conclusions The interactions between microorganisms and minerals occur widely in soils, sediments and groundwater and have profound impacts on the mobility of a wide variety of contaminants such as heavy metals. To our knowledge, this is the first paper reporting the adsorption and desorption of extracellular biomacromolecules (DNA) in the mixtures of bacteria and soil colloidal particles. Our results suggest that the introduction of B. thuringiensis in soil colloids and minerals systems promoted DNA adsorption on colloidal particles especially organic clay, while P. putida inhibited the adsorption of DNA on kaolinite and goethite. Moreover, the addition of bacteria in colloidal particles systems increased the proportion of DNA adsorbed electrostatically and decreased that by ligand exchange. These results indicate that bacteria play an important role in regulating the fate of DNA molecules in soil environments.
Acknowledgement The authors are grateful to the National Natural Science Foundation of China for the financial support of the research (40801095). References [1] D.C. Reanney, P.C. Gowland, J.H. Slater, Genetic interactions among microbial communities, in: J.H. Salter, R. Whittenbury, J.W.T. Wimpenny (Eds.), Microbes in their Natural Environments, Cambridge University Press, Cambridge, 1983. [2] E. Paget, P. Simonet, FEMS Microbiol. Ecol. 15 (1994) 109. [3] G. Stotzky, J. Environ. Qual. 29 (2000) 691. [4] M.G. Lorenz, W. Wackernagel, Microbiol. Rev. 58 (1994) 563. [5] P. Cai, Q.Y. Huang, X.W. Zhang, H. Chen, Pedosphere 15 (2005) 16. [6] P.M. Huang, J.M. Bollag, N. Senesi, Interactions between Soil Particles and Microorganisms: Impact on the Terrestrial Ecosystem, vol. 8: IUPACSeries on Analytical and Physical Chemistry of Environmental Systems, Wiley, Chichester, UK, 2002, p. 566. [7] J.B. Fein, C.J. Daughney, N. Yee, T. Davis, Geochim. Cosmochim. Acta 61 (1997) 3319. [8] C.J. Daughney, J.B. Fein, Environ. Sci. Technol. 32 (1998) 749. [9] N. Yee, J.B. Fein, Geochim. Cosmochim. Acta 65 (2001) 2037. [10] A.S. Temleton, T.P. Trainor, A.M. Spormann, S.J. Traina, G.E. Brown Jr., Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 11897. [11] Q.Y. Huang, W.L. Chen, L.H. Xu, Geomicrobiol. J. 22 (2005) 227. [12] M. Khanna, G. Stotzky, Appl. Environ. Microbiol. 58 (1992) 1930. [13] C. Crecchio, P. Ruggiero, M. Curei, C. Colombo, G. Palumbo, G. Stotzky, Soil Sci. Soc. Am. J. 69 (2005) 834. [14] P. Cai, Q.Y. Huang, X.W. Zhang, H. Chen, Soil Biol. Biochem. 38 (2006) 471. [15] G. Romanowski, M.G. Lorenz, W. Wackernagel, Appl. Environ. Microbiol. 57 (1991) 1057. [16] G. Pietramellara, M. Franchi, E. Gallori, P. Nannipieri, Biol. Fertil. Soils 33 (2001) 402. [17] G. Pietramellara, J. Ascher, M.T. Ceccherini, P. Nannipieri, D. Wenderoth, Biol. Fertil. Soils 43 (2007) 731. [18] H.O. Smith, D.B. Danner, R.A. Deich, Annu. Rev. Biochem. 50 (1981) 41. [19] D. Dubnau, Microbiol. Rev. 55 (1991) 395. [20] H.H. Liu, Y.R. Yang, X.C. Shen, Z.L. Zhang, P. Shen, Z.X. Xie, Curr. Microbiol. 57 (2008) 139. [21] L.M. He, B.M. Tebo, Appl. Environ. Microbiol. 64 (1998) 1123. [22] P. Cai, Q.Y. Huang, J. Zhu, D.H. Jiang, X.Y. Zhou, X.M. Rong, W. Liang, Colloids Surf. B 54 (2007) 53. [23] G. Stotzky, Mechanisms of adhesion to clays, with reference to soil systems, in: D.C. Savage, M. Fletcher (Eds.), Bacterial Adhesion, Plenum Publishing, Corp., New York, 1985. [24] M.P. Greaves, M.J. Wilson, Soil Biol. Biochem. 1 (1969) 317. [25] T.J. Beveridge, Int. Rev. Cytol. 72 (1981) 229. [26] S. Schultze-Lam, D. Fortin, B.S. Davis, T.J. Beveridge, Chem. Geol. 132 (1996) 171. [27] J.M. Dirienzo, K. Nakamura, I. Masayori, Annu. Rev. Microbiol. 47 (1978) 481. [28] B.A. Jucker, H. Harms, S.J. Hug, A.J.B. Zehnder, Colloids Surf. B 9 (1997) 331. [29] X.M. Rong, Q.Y. Huang, X.M. He, H. Chen, P. Cai, W. Liang, Colloids Surf. B 64 (2007) 49. [30] D.H. Jiang, Q.Y. Huang, P. Cai, X.M. Rong, W.L. Chen, Colloids Surf. B 54 (2007) 217. [31] K.A. Melzak, C.S. Sherwood, R.F.B. Turner, C.A. Haynes, J. Colloid Interface Sci. 181 (1996) 635.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Role of bacteria in the adsorption and binding of DNA on soil colloids and minerals Peng Cai a , Jun Zhu b , Qiaoyun Huang a,b,∗ , Linchun Fang b , Wei Liang b , Wenli Chen a,∗∗ a b
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China Key Laboratory of Subtropical Agricultural Resources and Environment, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China
a r t i c l e
i n f o
Article history: Received 4 September 2008 Received in revised form 20 October 2008 Accepted 20 October 2008 Available online 30 October 2008 Keywords: Adsorption Desorption DNA Bacteria Composite Soil colloid Mineral
a b s t r a c t Adsorption and desorption of salmon sperm DNA on bacteria (Bacillus thuringiensis, Pseudomonas putida), two different colloidal fractions (organic and inorganic clay) from an Alfisol, minerals (montmorillonite, kaolinite and goethite) and colloid–bacteria composites were studied. Similar adsorption capacity and affinity of DNA were observed on two bacterial cells. However, the two bacterial strains played different roles in affecting the adsorption of DNA on the composites of soil colloidal particles with bacteria. The introduction of B. thuringiensis in soil colloids and minerals systems dramatically promoted DNA adsorption on colloidal particles especially organic clay, while P. putida decreased the adsorption of DNA on kaolinite and goethite. Electrostatic force and ligand exchange are regarded to be the major driving forces involved in the adsorption of DNA on bacterial cells, montmorillonite, soil colloids and goethite. Presence of bacteria enhanced the proportion of DNA adsorption on soil colloidal particles by electrostatic force and depressed that by ligand exchange process. Information obtained in this study is of fundamental significance for the understanding of the ultimate fate of extracellular DNA in soil systems. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In soil environments, the genetic material of organisms (DNA molecules) is liberated by excretion from microorganisms, plants, and animals or by lysis of dying cells [1]. These extracellular molecules, physically or chemically adsorbed on surface active particles in soils are partially protected against degradation by nucleases and retain the ability to transform competent bacterial cells [2,3]. This adsorbed DNA, which has been termed “environmentally cryptic genes”, has a crucial role in biological activity and diversity, as well as in the transfer of genetic information among bacteria [4,5]. In natural soil environments, soil inorganic components are often associated with the microorganisms and these interactions have profound impacts on microbial activity, mineral weathering, formation of aggregates and the mobility of a wide variety of contaminants [6–9]. Meanwhile, the surface properties of the composites of minerals with microorganisms can differ dramatically
∗ Corresponding author at: Key Laboratory of Subtropical Agricultural Resources and Environment, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China. Tel.: +86 27 87671033; fax: +86 27 87280670. ∗∗ Corresponding author. Tel.: +86 27 87671033; fax: +86 27 87280670. E-mail addresses: [email protected], [email protected] (Q. Huang), [email protected] (W. Chen). 0927-7765/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2008.10.008
from pure bacterial or mineral surfaces, particularly in terms of surface charge and numbers of reactive sites [10,11]. The ultimate fate of DNA released from organisms in soils depends largely on the interactions of DNA molecules with organic and inorganic surfaces. A number of investigations have been devoted to the adsorption and binding of DNA on various soil components such as sand, clay minerals, humic substances and soils. It was found that this process is affected by the ionic strength, pH of the medium, type and content of clay as well as the characteristics and configurations of DNA molecules [12–17]. An increase in the concentration of cations and/or a decrease in the pH favored DNA adsorption on soil colloids and clay minerals [12–14]. Organic matter played an inhibitory role in DNA adsorption on permanent-charge soil colloids. Montmorillonite clay dominates the amount of DNA adsorption and kaolinite probably plays a key role in the extent of DNA binding in permanent-charge soil [14]. The amount of supercoiled plasmid DNA adsorbed by sand was slightly less than that of linearized or open circular plasmid DNA [15]. The lower molecular mass DNA from Bacillus subtilis showed higher adsorption capacity by Ca-montmorillonite and Ca-kaolinite than the higher molecular mass DNA [16]. Recently, Pietramellara et al. [17] reported that the presence of constitutional organic components (coc) and cellular wall debris (cwd) improved the adsorption of the DNA molecule on montmorillonite and kaolinite, whereas only the cwd had a positive effect on the binding of DNA on clays. In B. subtilis and Streptococcus pneumoniae, double-stranded DNA associates rapidly with
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 69 (2009) 26–30
competent cells through non-covalent bonds to form a complex that is resistant to gentle washing or to replacement by excess DNA [18]. In competition studies, it was found that double-stranded DNA of any source adsorbs to the cell surface and is taken up, indicating no specificity of this process for homologous DNA [19]. Long DNA ( DNA, 48.5 kb) was adsorbed to Escherichia coli surfaces preferentially versus short DNA (herring sperm DNA, 23 kb) [20]. However, little information is available on DNA adsorption on the composites of bacteria with soil colloidal particles. Therefore, the objectives of the present study were to study the adsorption behavior of DNA on two bacterial strains (Bacillus thuringiensis, Pseudomonas putida) and their composites with clay fractions from an Alfisol and minerals including kaolinite, montmorillonite and goethite. The release of DNA molecules from their colloidal complexes was also examined by sequential desorption with neutral salt and phosphate solutions. 2. Materials and methods 2.1. DNA Salmon sperm DNA was purchased from Sigma Chemical Co., St. Louis, MO. 2.2. Preparation of bacteria, soil colloids and minerals The cells of B. thuringiensis and P. putida were initially cultured in 5 ml of beef-extracted peptone medium at 28 ◦ C for 7 h. Two ml of bacterial suspension was then transferred to 200 ml of the same medium for another 18 h. Cells at the stationary growth phase were harvested by centrifugation at 10,000 × g for 10 min, and washed twice by deionized distilled water (ddH2 O), and finally resuspended in ddH2 O. An aliquot of the cell suspension was centrifuged and dried overnight at 60 ◦ C to determine the dry weight of the cells in the stock suspension. The specific surface area (SSA), point of zero charge (PZC) and zeta potential of bacteria were measured by the methylene blue adsorption method [21], acid–base titration (Automatic Potentiometric Titrator, Electronics Manufacturing Corporation, Japan), and Zeta Potential Analyzer (ZetaPlus 90, Brookhaven Instruments Corporation, USA), respectively. The properties of bacteria are listed in Table 1. The preparation of organic clay (organo-mineral complex), inorganic clay (H2 O2 -treated clay) from a clay loamy Brown soil (Alfisol), goethite and less than 2 m fractions from kaolinite and montmorillonite as well as their properties was described previously [22].
Two grams of soil colloid or mineral were suspended into 100 ml ddH2 O and dispersed by sonication. To explore the adsorption of DNA in mixtures of bacteria and soil colloid or mineral, we used mixtures containing 5 mg soil colloid or mineral and 0.5 mg bacteria. The mixture was shaken for 30 min. Different amount of DNA (25–250 g) was added and the volume was brought to 2.5 ml with Tris buffer (pH 7.0). The mixture was shaken at 25 ◦ C for 2 h and centrifuged at 20,000 × g for 20 min. The concentration of DNA in the supernatant was measured at 260 nm. The adsorption of DNA on 5 mg of soil colloid or mineral was also determined as described above. 2.4. Desorption of DNA Two hundred and fifty ml of soil colloid or mineral suspension and 0.5 mg of bacteria were mixed and shaken at 25 ◦ C for 30 min in the centrifuge tube. Two ml of buffer containing 200 g of DNA was added into the mixture, and shaken for another 2 h and centrifuged at 20,000 × g for 20 min. The concentration of DNA was determined at 260 nm and the amount of DNA adsorbed was calculated by the difference between the amount of DNA added and that in the supernatant. The mixture–DNA complexes formed after equilibrium adsorption were washed with 1 ml of 100 mM NaCl (dissolved in 10 mM Tris buffer) and 100 mM phosphate at pH 7.0 sequentially. Each washing was repeated until no more DNA was detected in the supernatant. The percentage of DNA desorbed was calculated according to the following expression: the percentage of DNA desorption (%) = the total amount of DNA desorbed/the amount of DNA adsorbed. The desorption of DNA adsorbed on soil colloid or mineral or bacteria was also examined as described above. 3. Results and discussion 3.1. Equilibrium adsorption of DNA on bacterial cells The adsorption isotherms of DNA on B. thuringiensis and P. putida are shown in Fig. 1. DNA adsorbed by the bacterial cells fitted Langmuir equation X = Xm KC/(1 + KC), where X is the amount of DNA adsorbed per unit mass of bacterial cells (g mg−1 ), Xm is the maximum amount of DNA adsorbed (g mg−1 ), K is a constant related to the adsorption energy (ml g−1 ) and C stands for the concentration of DNA in the equilibrium solution (g ml−1 ). The adsorption of DNA by bacteria increased steadily as the increment of DNA concentration in solution. As presented in Table 2, the maximum amount of DNA adsorption on B. thuringiensis and P. putida were 7.79 and
2.3. Adsorption of DNA In the 10 ml centrifuge tube, 250 l (0.5 mg) of B. thuringiensis or P. putida were mixed with 2250 l of 10 mM Tris buffer (pH 7.0) containing 25–250 g of DNA. The mixture was gently shaken at 25 ◦ C for 2 h and centrifuged at 20,000 × g for 20 min. DNA in the supernatant was determined by spectrophotometry at 260 nm. The amount of DNA adsorbed by bacterial cells was calculated by the difference between the amount of DNA added and that remaining in the supernatant. Table 1 Selected properties of bacteria. Bacteria
SSA (m2 g−1 )
PZC
Zeta potential (mV)
B. thuringiensis P. putida
192.4 242.7
2.66 2.90
−16.12 −48.12
SSA and PZC stand for specific surface area and point of zero charge.
27
Fig. 1. Adsorption isotherms of DNA on B. thuringiensis and P. putida at pH 7.0.
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Table 2 Langmuir parameters for the adsorption of DNA on bacteria, soil colloidal particles and their composites. Bacteria, soil colloidal particles and their composites
Xm (g mg−1 )
K (ml g−1 )
r
B. thuringiensis P. putida Organic clay Organic clay + B. thuringiensis Organic clay + P. putida Inorganic clay Inorganic clay + B. thuringiensis Inorganic clay + P. putida Montmorillonite Montmorillonite + B. thuringiensis Montmorillonite + P. putida Kaolinite Kaolinite + B. thuringiensis Kaolinite + P. putida Goethite Goethite + B. thuringiensis Goethite + P. putida
7.79 7.29 1.81 11.01 3.41 2.68 7.89 5.11 6.90 11.24 15.39 4.82 7.78 4.69 3.86 7.09 2.30
0.04 0.05 0.02 0.01 0.01 0.14 0.03 0.59 0.01 0.01 0.01 0.05 0.07 0.02 0.01 0.01 0.03
0.98 0.99 0.99 0.99 0.99 0.99 0.97 0.98 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99
7.29 g mg−1 , respectively. The K values for the adsorption of DNA on B. thuringiensis and P. putida were 0.04 and 0.05 ml g−1 , respectively. The greater the K value, the higher the affinity between bacterial cells and DNA molecules. These results suggest that the two bacterial cells have not significant differences in DNA adsorption capacity and affinity although they have different physical and chemical properties (Table 1). 3.2. Equilibrium adsorption of DNA on bacteria composites with soil colloidal particles The adsorption isotherms of DNA on soil colloids, minerals and their corresponding bacterial composites are shown in Figs. 2 and 3. The adsorption data over the range of DNA concentrations were also conformed to the Langmuir equation and the simulation parameters are listed in Table 2. As for individual components, the order of DNA adsorption capacity was montmorillonite (6.90 g mg−1 ) > kaolinite (4.82 g mg−1 ) > goethite (3.86 g mg−1 ) > inorganic clay (2.68 g mg−1 ) > organic clay (1.81 g mg−1 ). The unit layers of montmorillonite, a 2:1 lattice aluminosilicate, are held together by weak van der Waals forces and have hydrated interlayer cations (Ca2+ , Mg2+ , and Na+ ) between unit layers in the packets. However, the unit layers of kaolinte are tightly stacked by strong hydrogen bonds and goethite is an iron hydroxide which possesses only one type of functional group (hydroxyl) [23]. As a result, in comparison with kaolinite and goethie, montmorillonite has a greater specific surface area which provides more adsorption sites for DNA. Greaves and Wilson [24] also reported that purified montmorillonite may adsorb more than its own weight in DNA. A maximum of 59.4% of 50 g chromosomal DNA from B. subtilis was adsorbed by montmorillonite, versus 31.4% by kaolinite [16]. As for soil colloids, the amount of DNA adsorbed by organic clay was lesser than that by inorganic clay, indicating that DNA adsorption is depressed by organic matter in permanent-charge soil. As compared with the pure soil colloids systems, the adsorption of DNA on organic clay and inorganic clay in the presence of B. thuringiensis increased by 5.1- and 1.9-times, respectively. The presence of B. thuringiensis also enhanced the adsorption capacity of DNA on minerals by 61.4–83.7%. These results suggest that new interfacial sites may be produced at the adhesion zones between the cell walls of B. thuringiensis and the colloidal particles especially organic clay. However, the effect of P. putida on DNA adsorption by soil colloidal particles was different from that of B. thuringien-
Fig. 2. Adsorption isotherms of DNA on soil colloids and their composites with bacteria at pH 7.0.
sis. There was only an increase of DNA adsorption by 88.4–123.0% on soil colloids and montmorillonite–P. putida composites as compared with the pure colloids systems. The adsorption of DNA on kaolinite and goethite in the presence of P. putida was 2.7% and 40.4% lower than that in the absence of bacteria. These different behaviors between B. thuringiensis and P. putida on the adsorption of DNA by their composites with soil colloidal particles may be related to the discrepancies in their cell wall structures which result in varying interactions with soil colloids and minerals. Grampositive cell walls are constructed of peptidoglycan polymers which are thicker (25 nm), rich in carboxyl groups and covalently linked together as they assemble around the cell. The secondary polymers (teichoic or teichuronic acids) are also bonded into the peptidoglycan framework [25]. Gram-negative cell walls have a more complex structure format. These walls contain a thin layer of peptidoglycan (7.5 nm) and a second membrane (outer membrane) within the cellular envelope which is porous structure, rich in protein, lipid and lipopolysaccharide [11,26]. Approximately 45% of the surface of Gram-negative bacteria is covered by lipopolysaccharides [27], which are anchored with their lipid part in the outer membrane and can be adsorbed mainly by hydrogen bonds on mineral oxides such as TiO2 , Al2 O3 , and SiO2 [28]. Rong et al. [29] reported that hydrogen bonding and electrostatic interaction were the main forces governing the adsorption of P. putida on kaolinite and montmorillonite by using isothermal titration calorimetry (ITC) and Fourier transform infrared spectra (FTIR). Therefore, the lesser amount of DNA adsorption on P. putida composites with soil colloidal particles
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29
Fig. 4. Desorption of DNA from bacteria, organic clay (OC), inorganic clay (IC) and their composites with bacteria by NaCl and phosphate sequentially at pH 7.0.
order of goethite > kaolinite > montmorillonite. Scanning electron microscopy (SEM) images showed that kaolinite has a tendency to aggregate and form large particles with P. putida whereas fewer bacterial cells coupled with the aggregation of montmorillonite particles [29]. The tighter associations of kaolinite or goethite with P. putida than montmorillonite may mask the reactive adsorption sites that are normally available to DNA molecules. These results indicate that Gram-positive bacteria play a more important role than Gram-negative bacteria in promoting DNA adsorption on soil colloids and minerals. 3.3. Desorption of DNA from bacteria, soil colloids and their composites Desorption was conducted after the adsorption of DNA on bacteria, soil colloids and their bacterial composites. Figs. 4 and 5 illustrate that the percent desorption of DNA by NaCl from B. thuringiensis, P. putida, organic clay and montmorillonite was in the range of 45.9–70.0% whereas that from inorganic clay, kaolinite and goethite was less than 13.2%. The desorption ratio of DNA molecules from their complexes with B. thuringiensis, P. putida, organic clays and montmorillonite by phosphate was 7.3–24.3%, while that from inorganic clays and goethite was 36.0–53.7%. Only 5.3% of DNA
Fig. 3. Adsorption isotherms of DNA on minerals and their composites with bacteria at pH 7.0.
than B. thuringiensis composites may be attributed to the competition of more macromolecules (especially lipopolysaccharide) with DNA on mineral surface for adsorptive sites. It is noteworthy to mention that P. putida played different roles in the adsorption of DNA for the three examined clay minerals systems. The possible reason for the decreased adsorption of DNA on kaolinite and goethite in the presence of P. putida as compared to individual components is the alteration of disperse state and aggregate structure of mineral particles after the introduction of bacteria. It was reported by Jiang et al. [30] that the affinity and the amount of P. putida adsorption on clay minerals and iron oxide followed the
Fig. 5. Desorption of DNA from montmorillonite (M), kaolinite (K), goethite (G) and their composites with bacteria by NaCl and phosphate sequentially at pH 7.0.
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on kaolinite was released by phosphate. Nucleic acids in solution resemble a polyelectrolyte and several mechanisms have been proposed to be responsible for the adsorption of salmon sperm DNA on soil colloids and minerals such as van der Waals force, electrostatic force, hydrogen bonding, ligand exchange and hydrophobic force [14,31]. DNA molecules desorbed by NaCl were usually regarded as exchangeable, while those removed by phosphate were considered as specifically adsorbed (ligand exchange form). The data obtained in this experiment imply that more than 50% of DNA was adsorbed by electrostatic force on bacteria, organic clay and montmorillonite. The proportion of DNA adsorbed by ligand exchange on inorganic clays and goethite was 36.0% and 53.7%, respectively. Only 21.9% of DNA molecules are adsorbed on kaolinite by electrostatic force and ligand exchange, suggesting that other interaction forces such as hydrophobic force and hydrogen bonding may play more important roles in the adsorption of DNA. As compared with pure organic clay system, the release of DNA by NaCl decreased by 73.5% from its composite with B. thuringiensis. For kaolinite–B. thuringiensis composite, DNA desorbed by phosphate was 64.0% higher than pure kaolinite. Except for organic clay- and kaolinite–B. thuringiensis composites, the desorption rate of DNA by NaCl increased while that by phosphate decreased in soil colloidal particles–bacteria composites as compared to pure colloids. It suggests that the introduction of bacteria in colloidal particles systems increased the proportion of DNA adsorbed electrostatically and decreased that by ligand exchange. 4. Conclusions The interactions between microorganisms and minerals occur widely in soils, sediments and groundwater and have profound impacts on the mobility of a wide variety of contaminants such as heavy metals. To our knowledge, this is the first paper reporting the adsorption and desorption of extracellular biomacromolecules (DNA) in the mixtures of bacteria and soil colloidal particles. Our results suggest that the introduction of B. thuringiensis in soil colloids and minerals systems promoted DNA adsorption on colloidal particles especially organic clay, while P. putida inhibited the adsorption of DNA on kaolinite and goethite. Moreover, the addition of bacteria in colloidal particles systems increased the proportion of DNA adsorbed electrostatically and decreased that by ligand exchange. These results indicate that bacteria play an important role in regulating the fate of DNA molecules in soil environments.
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