Interactions between proteins and clays perturb biological activity in ecosystems, particularly soil extracellular enzyme activity. The pH dependence of hydrophobic, hydrophilic, and electrostatic interactions on the adsorption of bovine serum albumin (BSA) is studied. BSA secondary structures and hydration are revealed from computation of the Amide I and II FTIR absorption profiles. The influence of ionization of Asp, Glu, and His side chains on the adsorption processes is deduced from correlation between p 2 H dependent carboxylic/carboxylate ratio and Amide band profiles. We quantify p 2 H dependent internal and external structural unfolding for BSA adsorbed on montmorillonite, which is an electronegative phyllosilicate. Adsorption on talc, a hydrophobic surface, is less denaturing. The results emphasize the importance of electrostatic interactions in both adsorption processes. In the first case, charged side chains directly influence BSA adsorption that generate the structural transition. In the second case, the forces that attract hydrophobic side chains toward the protein-clay interface are large enough to distort peripheral amphiphilic helical domains. The resulting local unfolding displaces enough internal ionized side chains to prevent them from establishing salt bridges as for BSA native structure in solution. On montmorillonite, a particular feature is a higher protonation of the Asp and Glu side chains of the adsorbed BSA than in solution, which decreases coulombic repulsion. C 2000 Academic Press

In order to determine the mechanisms involved in the persistence of extracellular DNA in soils and to monitor whether bacterial transformation could occur in such an environment, we developed artificial models composed of plasmid DNA adsorbed on clay particles. We determined that clay-bound DNA submitted to an increasing range of nuclease concentrations was physically protected. The protection mechanism was mainly related to the adsorption of the nuclease on the clay mineral. The biological potential of the resulting DNA was monitored by transforming the naturally competent proteobacterium Acinetobacter sp. strain BD413, allowing us to demonstrate that adsorbed DNA was only partially available for transformation. This part of the clay-bound DNA which was available for bacteria, was also accessible to nucleases, while the remaining fraction escaped both transformation and degradation. Finally, transformation efficiency was related to the perpetuation mechanism, with homologous recombination being less sensitive to nucleases than autonomous replication, which requires intact molecules.In the environment, three mechanisms are thought to be involved in gene uptake by bacteria (31), namely, conjugation, transformation, and transduction. Natural bacterial genetic transformation is the mechanism by which a bacterium acquires naked DNA. Such a mechanism is thought to have been involved in gene transfers during evolution and particularly in transfers among unrelated organisms such as plants and bacteria (1,8,21). However, numerous reports indicate that gene transfer events may be very rare in the environment (14,18,33). This could be due to the numerous steps that are required to achieve transformation. DNA released by organisms must persist under adverse conditions such as those encountered in soils. Naked DNA must then encounter competent recipient bacteria. Moreover, the incorporated DNA will only be perpetuated if its nucleotide sequences exhibit sufficient similarity to the recipient genome to allow recombination, unless the sequences possess a replicon which is operational in the new host (14,16,33).Nevertheless, there is a general agreement that natural transformation may occur in complex media such as soils. Indeed, large amounts of naked DNA, which is the preliminary key factor for transformation, can be detected in soils (7,35,40). Moreover, there is much evidence that extracellular DNA can persist for periods of time up to several months or years (8,18,25,27,28,38). Adsorption of DNA on soil components, particularly on clay minerals such as montmorillonite, illite, and kaolinite, is thought to be involved in protection of nucleic acids against nucleases, and could explain the high content of DNA in soils (2, 10, 32). However, soil or microcosm-based experiments have indicated that the adsorption-related protection process has only limited impact (3,17,25,27). In fact, very little is known about the protection mechanism itself, and the influence of parameters such as clay type, the size of DNA or its conformati...

Soils have a large solid surface area and high adsorptive capacities. To determine if structural and solvation changes induced by adsorption on clays are related to changes in enzyme activity, alpha-chymotrypsin adsorbed on a phyllosilicate with an electronegative surface (montmorillonite) has been studied by transmission FTIR spectroscopy. A comparison of the pH-dependent structural changes for the solution and adsorbed states probes the electrostatic origin of the adsorption. In the pD range 4.5-10, adsorption only perturbs some peripheral domains of the protein compared to the solution. Secondary structure unfolding affects about 15-20 peptide units. Parts of these domains become hydrated and others entail some self-association. However, the inactivation of the catalytic activity of the adsorbed enzyme in the 5-7 pD range is due less to these structural changes than to steric hindrance when three essential imino/amino functions, located close to the entrance of the catalytic cavity (His-40 and -57 residues and Ala-149 end chain residue), are oriented toward the negatively charged mineral surface. When these functions lose their positive charge, the orientation of the adsorbed enzyme is changed and an activity similar to that in solution at equivalent pH is recovered. This result is of fundamental interest in all fields of research where enzymatic activity is monitored using reversible adsorption procedures. Copyright 1999 Academic Press.

The interaction of extracellular enzymes with the solid phase of the soil affects their mobility and their catalytic properties. In particular, adsorption on clay minerals is known to shift the optimum pH of the catalytic activity towards alkaline values. Two conflicting interpretations of this phenomenon have been proposed: a surface pH effect and a pH-dependent modification of the conformation of the adsorbed enzyme. To assess the two mechanisms, we studied the adsorption on montmorillonite of two extracellular acid phosphatases of the ectomycorrhizal fungus Hebeloma cylindrosporum, and its consequences on catalytic activities. The results obtained are better interpreted by a pH-dependent modification of enzyme conformation due mainly to electrostatic interactions with the clay surface. At low pH, the two positively charged enzymes unfold on the negatively charged montmorillonite surface. At high pH, both the enzymes and the clay are negatively charged, and adsorption decreases. Adsorption and modification of conformation are largely irreversible, which should be taken into account when considering the fate of enzymes in soil. Finally, the comparison with the effect of clays on the catalytic activities of intracellular enzymes raises the hypothesis of a selection pressure of the soil solid phase leading to more stable extracellular enzymes.ActivitC enzymatique extracellulaire dans Ie sol: effet du pH et de la force ionique sur l'interaction avec la montmorillonite de deux phosphates acides sCcrdteds par le champignon ectomycorhizien Hebeloma cylindrosporum RCsumC L'interaction des enzymes extracellulaires avec la phase solide du sol modifie leur mobilitt et leur proprittts catalytiques. En particulier, I'adsorption sur les minCraux argileux provoque une augmentation du pH optimal de I'activitt catalytique. Deux interprktations opposkes de ce phtnombne ont Ctt propostes: un effet de pH de surface et une modification de conformation de I'enzyme adsorbte qui dtpend du pH. De faGon ? I tvaluer ces deux mtcanismes, I'adsorption sur la montmorillonite de deux phosphatases acides extracellulaires du champignon ectomycorhizien Hebeloma cylindrosporum a Ct C CtudiCe, ainsi que les constquences de I'adsorption sur leurs activitts catalytiques. Les rtsultats obtenus s'interprbtent mieux par une modification de conformation des enzymes dtpendant du pH et principalement due aux interactions klectrostatiques avec la surface d'argile. A bas pH, les deux enzymes positivement chargtes se dtnaturent sur la surface de montmorillonite chargke ntgativement. A pH tlevt, les enzymes et I'argile sont ntgativement chargtes, et ]'adsorption diminue. L'adsorption et la modification de conformation sont largement irrkversibles, ceci devrait Ctre prise en compte dans I'ttude de I'activitk enzymatique du sol. Enfin, la comparaison avec I'effet des argiles sur I'activitt catalytique d'enzymes intracellulaires soulBve I'hypothbse d'une pression de stlection de la phase solide du sol conduisant A des enzymes extracellulaires plus stables.

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Prions, the infectious agents thought to be responsible for transmissible spongiform encephalopathies, may contaminate soils and have been reported to persist there for years. We have studied the adsorption and desorption of a model recombinant prion protein on montmorillonite and natural soil samples in order to elucidate mechanisms of prion retention in soils. Clay minerals, such as montmorillonite, are known to be strong adsorbents for organic molecules, including proteins. Montmorillonite was found to have a large and selective adsorption capacity for both the normal and the aggregated prion protein. Adsorption occurred mainly via the N-terminal domain of the protein. Incubation with standard buffers and detergents did not desorb the full length protein from montmorillonite, emphasizing the largely irreversible trapping of prion protein by this soil constituent. An original electroelution method was developed to extract prion protein from both montmorillonite and natural soil samples, allowing quantification when coupled with rapid prion detection tests. This easy-to-perform method produced concentrated prion protein extracts and allowed detection of protein at levels as low as 0.2 ppb in natural soils.

This paper discusses the enzymatic hydrolysis of organic phosphorus with reference to the classical inorganic and organic compartmental analysis of phosphorus compounds in ecosystems. Also discussed are: the potential use of soil organic phosphorus by plants and microorganisms; properties of phosphohydrolases; inhibitors and activators; phosphohydrolase activity in soil; rhizosphere and mycorrhizal aspects of phosphohydrolase activity; and the regulation of the phosphohydrolase activity in the rhizosphere.

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