Ichige Asao, Kobayashi Ichizo
Department of Medical Genome Sciences, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
J Bacteriol. 2005 Oct;187(19):6612-21. doi: 10.1128/JB.187.19.6612-6621.2005.
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.
某些II型限制修饰基因系统在从宿主细胞中消除时会杀死宿主细胞。这种导致宿主细胞进行后分离杀伤的能力是细菌成瘾模块的特征，每个成瘾模块都由毒素和抗毒素基因组成。有了这些成瘾模块，毒素和抗毒素分子在细胞中的稳定性差异在执行后分离杀伤中起着至关重要的作用。我们在此研究了EcoRI限制酶（毒素）和修饰酶（抗毒素）在体内的稳定性，其基因系统先前已被证明会在大肠杆菌中导致后分离宿主杀伤。使用两种不同的方法，即定量蛋白质免疫印迹分析和脉冲追踪免疫沉淀分析，我们证明EcoRI限制酶和修饰酶都与大量细胞蛋白一样稳定，并且它们的稳定性没有明显差异。估计了宿主细胞在稳态生长期间存在的EcoRI限制酶和修饰酶分子的数量。我们监测了后分离杀伤过程中EcoRI限制酶和修饰酶的细胞水平变化。这些分析结果共同表明，EcoRI基因系统在执行后分离细胞杀伤时不依赖于毒素和抗毒素分子之间的稳定性差异。我们的结果为限制修饰系统的后分离杀伤机制提供了见解，这似乎与其他细菌成瘾模块的后分离杀伤机制不同。
