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 self- aggregation 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 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 bacteria 10 with efficiencies depending on whether the species of recipient bacteria can integrate the genetic information via a homologous recombination process 2 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 ho- moionic to Ca frequently encountered in soils at mid- latitudes 12 was determined. Ca is known to act as cationic bridge between anionic groups of organic molecules and negative charges on the clay 13 and favors DNA adsorption. The adsorption isotherm technique was used to provide * 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, 1930- 1939. (5) Lorenz, M. G.; Wackernagel, W. In Gauthier, M. J., Ed.; Springer- Verlag: 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. (8) Widmer, F.; Seidler, R. J.; Watrud, L. S. Mol. Ecol. 1996, 5, 603- 613. (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, 563- 602. (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. 1233 Langmuir 2000, 16, 1233-1238 10.1021/la990506z CCC: $19.00 © 2000 American Chemical Society Published on Web 11/27/1999