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J. Bacteriol. doi:10.1128/JB.01319-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Maintenance forced by a restriction-modification system can be modulated by a region in its modification enzyme not essential for the methyltransferase activity

Department of Medical Genome Sciences, Graduate School of Frontier Science and Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

^* To whom correspondence should be addressed. Email: ikobaya{at}ims.u-tokyo.ac.jp.

	Abstract

Several Type II restriction-modification gene complexes can force their maintenance on their host bacteria by killing cells that have lost them in a process called post-segregational killing or genetic addiction. It is likely to proceed by dilution of the modification enzyme molecule during rounds of cell division following the gene loss, which exposes unmethylated recognition sites on the newly replicated chromosomes to lethal attack by the remaining restriction enzyme molecules. This process is in apparent contrast to the process of the classical types of post-segregational killing systems, in which built-in metabolic instability of the antitoxin allows release of the toxin for lethal action after the gene loss. In the present study, we characterize a mutant form of the EcoRII gene complex that shows stronger capacity in such maintenance. This phenotype is conferred by L80P (T239C) mutation in the modification enzyme. This mutant enzyme showed decreased DNA methyltransferase activity at a higher temperature in vivo and in vitro, although a deletion mutant lacking N-terminal 83 amino acids did not lose the activity at both of the temperatures. Under a condition of inhibited protein synthesis, the activity of the L80P mutant was completely lost at a high temperature. In parallel, the L80P mutant protein disappeared more rapidly than the wild-type protein. These results demonstrate that the capability of a restriction-modification system in forcing maintenance on its host can be modulated by a region of its antitoxin, the modification enzyme, as in the classical post-segregational killing systems.