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Stability of EcoRI Restriction-Modification Enzymes In Vivo Differentiates the EcoRI Restriction-Modification System from Other Postsegregational Cell Killing Systems

Asao Ichige^1, and Ichizo Kobayashi^1,2,3*

Department of Medical Genome Sciences, Graduate School of Frontier Science,¹ Institute of Medical Science,² Graduate Program in Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan³

Received 31 March 2005/ Accepted 11 July 2005

Certain type II restriction modification gene systems can kill host cells when these gene systems are eliminated from the host cells. Such ability to cause postsegregational killing of host cells is the feature of bacterial addiction modules, each of which consists of toxin and antitoxin genes. With these addiction modules, the differential stability of toxin and antitoxin molecules in cells plays an essential role in the execution of postsegregational killing. We here examined in vivo stability of the EcoRI restriction enzyme (toxin) and modification enzyme (antitoxin), the gene system of which has previously been shown to cause postsegregational host killing in Escherichia coli. Using two different methods, namely, quantitative Western blot analysis and pulse-chase immunoprecipitation analysis, we demonstrated that both the EcoRI restriction enzyme and modification enzyme are as stable as bulk cellular proteins and that there is no marked difference in their stability. The numbers of EcoRI restriction and modification enzyme molecules present in a host cell during the steady-state growth were estimated. We monitored changes in cellular levels of the EcoRI restriction and modification enzymes during the postsegregational killing. Results from these analyses together suggest that the EcoRI gene system does not rely on differential stability between the toxin and the antitoxin molecules for execution of postsegregational cell killing. Our results provide insights into the mechanism of postsegregational killing by restriction-modification systems, which seems to be distinct from mechanisms of postsegregational killing by other bacterial addiction modules.

Present address: Department of Life Science, Rikkyo University, 3-34-1 Nishi-ikebukuro, Toshima-ku, Tokyo 171-8501, Japan.

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