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...

Our knowledge of Escherichia coli (E. coli) ecology in the field is very limited in the case of dairy alpine grassland soils. Here, our objective was to monitor field survival of E. coli in cow pats and underlying soils in four different alpine pasture units, and to determine whether the soil could constitute an environmental reservoir. E. coli was enumerated by MPN using a selective medium. E. coli survived well in cow pats (10(7) to 10(8) cells g(-1) dry pat), but cow pats disappeared within about 2 mo. In each pasture unit, constant levels of E. coli (10(3) to 10(4) cells g(-1) dry soil) were recovered from all topsoil (0-5 cm) samples regardless of the sampling date, that is, under the snow cover, immediately after snow melting, or during the pasture season (during and after the decomposition of pats). In deeper soil layers below the root zone (5-25 cm), E. coli persistence varied according to soil type, with higher numbers recovered in poorly-drained soils (10(3) to 10(4) cells g(-1) dry soil) than in well-drained soils (< 10(2) cells g(-1) dry soil). A preliminary analysis of 38 partial uidA sequences of E. coli from pat and soils highlighted a cluster containing sequences only found in this work. Overall, this study raises the possibility that fecal E. coli could have formed a naturalized (sub)population, which is now part of the indigenous soil community of alpine pasture grasslands, the soil thus representing an environmental reservoir of E. coli.

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This study is part of a European project focused on understanding the biotic and abiotic mechanisms involved in the retention and dissemination of transmissible spongiform encephalopathies (TSE) infectivity in soil in order to propose practical recommendations to limit environmental contamination. A 1-year field experiment was conducted with lamb carcasses buried in a pasture soil at three depths (25, 45, and 105 cm). Microbial community response to carcasses was monitored through the potential proteolytic activity and substrate induced respiration (SIR). Soil above carcasses and control soil exhibited low proteolytic capacity, whatever the depth of burial. Contrastingly, in soil beneath the carcasses, proteolysis was stimulated. Decomposing carcasses also stimulated SIR, i.e., microbial biomass, suggesting that proteolytic populations specifically developed on lixiviates from animal tissues. Decomposition of soft tissues occurred within 2 months at subsurface while it lasted at least 1 year at deeper depth where proteolytic activities were season-dependent. The ability of soil proteases to degrade the beta form of prion protein was shown in vitro and conditions of burial relevant to minimize the risk of prion protein dissemination are discussed.