title: Stability of EcoRI restriction-modification enzymes in vivo differentiates the EcoRI restriction-modification system from other postsegregational cell killing systems.
abstract: 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.
authors.affiliation: Department of Medical Genome Sciences, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Asao
authors.initials: A
authors.lastname: Ichige
authors.forename: Ichizo
authors.initials: I
authors.lastname: Kobayashi
mesh_terms: D015246:Deoxyribonuclease EcoRI / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D010957:Plasmids / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D015246:Deoxyribonuclease EcoRI