RESUMEN
Bacterial defence systems are tightly regulated to avoid autoimmunity. In Type I restriction-modification (R-M) systems, a specific mechanism called restriction alleviation (RA) controls the activity of the restriction module. In the case of the Escherichia coli Type I R-M system EcoKI, RA proceeds through ClpXP-mediated proteolysis of restriction complexes bound to non-methylated sites that appear after replication or reparation of host DNA. Here, we show that RA is also induced in the presence of plasmids carrying EcoKI recognition sites, a phenomenon we refer to as plasmid-induced RA. Further, we show that the anti-restriction behavior of plasmid-borne non-conjugative transposons such as Tn5053, previously attributed to their ardD loci, is due to plasmid-induced RA. Plasmids carrying both EcoKI and Chi sites induce RA in RecA- and RecBCD-dependent manner. However, inactivation of both RecA and RecBCD restores RA, indicating that there exists an alternative, RecA-independent, homologous recombination pathway that is blocked in the presence of RecBCD. Indeed, plasmid-induced RA in a RecBCD-deficient background does not depend on the presence of Chi sites. We propose that processing of random dsDNA breaks in plasmid DNA via homologous recombination generates non-methylated EcoKI sites, which attract EcoKI restriction complexes channeling them for ClpXP-mediated proteolysis.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Plásmidos , Rec A Recombinasas , Plásmidos/genética , Escherichia coli/genética , Rec A Recombinasas/metabolismo , Rec A Recombinasas/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Recombinación Genética , Desoxirribonucleasas de Localización Especificada Tipo I/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo I/genética , Endopeptidasa Clp/metabolismo , Endopeptidasa Clp/genética , Exodesoxirribonucleasa V/metabolismo , Exodesoxirribonucleasa V/genética , ADN Bacteriano/metabolismo , Elementos Transponibles de ADN/genética , Enzimas de Restricción del ADN , Proteínas de Unión al ADNRESUMEN
Metagenomics is a powerful tool to study marine microbial communities. However, obtaining high-quality environmental DNA suitable for downstream sequencing applications is a challenging task. The quality and quantity of isolated DNA heavily depend on the choice of purification procedure and the type of sample. Selection of an appropriate DNA isolation method for a new type of material often entails a lengthy trial and error process. Further, each DNA purification approach introduces biases and thus affects the composition of the studied community. To account for these problems and biases, we systematically investigated efficiency of DNA purification from three types of samples (water, sea sediment, and digestive tract of a model invertebrate Magallana gigas) with eight commercially available DNA isolation kits. For each kit-sample combination we measured the quantity of purified DNA, extent of DNA fragmentation, the presence of PCR-inhibiting contaminants, admixture of eukaryotic DNA, alpha-diversity, and reproducibility of the resulting community composition based on 16S rRNA amplicons sequencing. Additionally, we determined a "kitome", e.g., a set of contaminating taxa inherent for each type of purification kit used. The resulting matrix of evaluated parameters allows one to select the best DNA purification procedure for a given type of sample.