Fluorescence microscopy study of the effect of Esp1396I restriction-modification system proteins concentrations on protection against lambda phage.

Abstract

Restriction-modification systems are among wide-spread mechanisms protecting bacteria from foreign DNA, such as DNA of bacteriophages and plasmids. The action of such systems is based on the presence of two enzymes - methyltransferase and restriction endonuclease, first one protects host DNA from degradation by modification and second one cleaves foreign unmodified DNA. In some cases bacteriophage DNA is modified faster than being degraded by restriction endonuclease. This process, called breakdown, leads to formation of modified bacteriophage progeny which is not sensitive to the action of restriction endonuclease and can eliminate whole "protected" bacterial population. There is a hypothesis which assumes that the overcoming of the protective action of restriction-modification system by a bacteriophage depends on the relative concentrations of the restriction-modification system proteins. To check this assumption we decided to perform fluorescence microscopy of individual living E.coli cells. For this purposes we use fluorescently-labelled type II Esp1396I restriction-modification system and also create an artificial system which allows to regulate the ratio of restriction-modification system proteins through the additional restriction endonuclease expression. Preliminary results showed an increase in protection with an increase in the restriction endonuclease concentration in cells.