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...

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysenedegrading organism Sphingomonas sp. strain CHY-1 were investigated. [14 C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1 a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.

Understanding the prevalence and polymorphism of antibiotic resistance genes in soil bacteria and their potential to be transferred horizontally is required to evaluate the likelihood and ecological (and possibly clinical) consequences of the transfer of these genes from transgenic plants to soil bacteria. In this study, we combined culture-dependent and -independent approaches to study the prevalence and diversity of bla genes in soil bacteria and the potential impact that a 10-successive-year culture of the transgenic Bt176 corn, which has a blaTEM marker gene, could have had on the soil bacterial community. The bla gene encoding resistance to ampicillin belongs to the beta-lactam antibiotic family, which is widely used in medicine but is readily compromised by bacterial antibiotic resistance. Our results indicate that soil bacteria are naturally resistant to a broad spectrum of beta-lactam antibiotics, including the third cephalosporin generation, which has a slightly stronger discriminating effect on soil isolates than other cephalosporins. These high resistance levels for a wide range of antibiotics are partly due to the polymorphism of bla genes, which occur frequently among soil bacteria. The blaTEM116 gene of the transgenic corn Bt176 investigated here is among those frequently found, thus reducing any risk of introducing a new bacterial resistance trait from the transgenic material. In addition, no significant differences were observed in bacterial antibiotic-resistance levels between transgenic and nontransgenic corn fields, although the bacterial populations were different.antibiotic resistance ͉ GMO ͉ HGT

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Little information is available concerning the occurrence of natural transformation of bacteria in soil, the frequency of such events, and the actual role of this process on bacterial evolution. This is because few bacteria are known to possess the genes required to develop competence and because the tested bacteria are unable to reach this physiological state in situ. In this study we found that two soil bacteria, Agrobacterium tumefaciens and Pseudomonas fluorescens, can undergo transformation in soil microcosms without any specific physical or chemical treatment. Moreover, P. fluorescens produced transformants in both sterile and nonsterile soil microcosms but failed to do so in the various in vitro conditions we tested. A. tumefaciens could be transformed in vitro and in sterile soil samples. These results indicate that the number of transformable bacteria could be higher than previously thought and that these bacteria could find the conditions necessary for uptake of extracellular DNA in soil.

SummaryFor some crops, the only possible approach to gain a specific trait requires genome modification. The development of virus‐resistant transgenic plants based on the pathogen‐derived resistance strategy has been a success story for over three decades. However, potential risks associated with the technology, such as horizontal gene transfer (HGT) of any part of the transgene to an existing gene pool, have been raised. Here, we report no evidence of any undesirable impacts of genetically modified (GM) grapevine rootstock on its biotic environment. Using state of the art metagenomics, we analysed two compartments in depth, the targeted Grapevine fanleaf virus (GFLV) populations and nontargeted root‐associated microbiota. Our results reveal no statistically significant differences in the genetic diversity of bacteria that can be linked to the GM trait. In addition, no novel virus or bacteria recombinants of biosafety concern can be associated with transgenic grapevine rootstocks cultivated in commercial vineyard soil under greenhouse conditions for over 6 years.

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