Type II restriction-modification (R-M) systems serve to protect bacteria against incoming foreign DNA. Enzymes being part of these systems [restriction endonucleases (R), and DNA methyltransferases (M)] apart being indispensable tools of genetic engineering constitute exemplary models to study DNA-protein interactions. Genes of numerous type-II R-M systems are carried by mobile genetic elements, such as: (i) natural plasmids, e.g.: Bsp6I; EcoRI; EcoRII, EcoRV, PvuII; (ii) bacteriophages, e.g.: LlaI; (iii) defective prophages, e.g.: EcoO109I, EcoT38I, HindIII. In case of plasmids it has been shown that their stable maintenance inside bacterial cells is influenced by the presence of genes coding for R-M systems. In our laboratory we have found that derivatives of plasmid pEC156 carrying EcoVIII R-M system were stably maintained in E. coli after growth for 100 generation in the absence of selection. It is a common belief that localization of genes encoding R-M systems on mobile genetic elements can facilitate the spread of these systems among bacteria by horizontal gene transfer. Our intention is to verify hypothesis according to which R-M systems can be freely exchanged between bacteria by the means of horizonatal gene transfer.

As a model for our studies we have choosen a natural ColE1-type plasmid (pEC156) isolated from Escherichia coli E1585-68 which carries type II R-M system EcoVIII consisting of restriction endonuclease and DNA methyltransferase. Computational analysis of pEC156 revealed the presence of a DNA region that has high homology to the cer locus of ColE1 plasmids. This analyzed region contains binding sites for XerC and XerD recombinases, which are responsible for the maintaining of plasmids in the monomeric form in E. coli cells, as well as an ArgR-like binding site located upstream of the XerC-XerD region. These sequences are almost identical to those of the plasmid ColE1 cer locus.

To investigate a role of particular genetic elements affecting pEC156 stability we have constructed several derivatives of this plasmid. Each of them was deffcient either in plasmid resolution system (cer locus) or elements of the EcoVIII R-M system. Our experiments revealed that, a gene encoding R.EcoVIII is responsible for stable maintenance of pEC156. We have found the presence of the cer locus increases the plasmid stability. Moreover, we have shown that derivatives which lacked cer locus and gene encoding R.EcoVIII as well as a gene for M.EcoVIII were highly unstable in E. coli and in bacteria belonging to the Enterobacteriaceae family (Klebsiella oxytoca, Salmonella enteritidis, Serratia marcescens, Enterobacter cloaceae, Proteus vulgaris) after growth for 100 generations in the abscence of selection.

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1. On relaxed specific sequence recognition of the EcoVIII restriction endonuclease and cognate DNA methyltransferase.
2. Characterization of a cryptic plasmid from Moraxella bovis strain ATCC 10900.