Periplasmic expression of a restriction endonuclease in Escherichia coli and its effect on the antiviral activity of the host
One possible mechanism preventing phage infection of the bacterial cells is related to the presence of an effective restriction-modification system (R-M) which allows restriction of the invading DNA. However, there are some limitations to the absolute restriction of foreign DNA. Since there is a serious conflict between increase in the restriction-modification genes expression level and cell viability, we examined the antiviral effect of EcoRI restriction endonuclease after its translocation to the periplasmic space of the cell. We assumed that such reconstructed R-M system could be able to degrade foreign DNA at the stage of its passage through the cell envelope of Gram-negative bacteria, before its penetration into the bacterial cytoplasm. The Tat secretion pathway of Escherichia coli was used to export R.EcoRI fused to the TorA leader peptide across the cytoplasmic membrane. However, although we observed a huge accumulation of the TorAss-R.EcoRI pre-protein in the cytoplasm the Tat system did not provide an efficient transport across the cytoplasmic membrane. Moreover, our data strongly suggest that endonuclease cannot function under the conditions prevailing in periplasmic space, therefore, the transported endonuclease could not contribute to an increase in restriction properties of the host.
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