Kasai Koji, Usami Syoji, Yamada Takashi, Endo Yaeta, Ochi Kozo, Tozawa Yuzuru
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
Nucleic Acids Res. 2002 Nov 15;30(22):4985-92. doi: 10.1093/nar/gkf628.
A gene encoding a putative guanosine 3',5'-bispyrophosphate (ppGpp) synthase-degradase, designated Cr-RSH, was identified in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii. The encoded Cr-RSH protein possesses a putative chloroplast-targeting signal at its NH2-terminus, and translocation of Cr-RSH into chloroplasts isolated from C.reinhardtii was demonstrated in vitro. The predicted mature region of Cr-RSH exhibits marked similarity to eubacterial members of the RelA-SpoT family of proteins. Expression of an NH2-terminal portion of Cr-RSH containing the putative ppGpp synthase domain in a relA, spoT double mutant of Escherichia coli complemented the growth deficits of the mutant cells. Chromatographic analysis of 32P-labeled cellular mononucleotides also revealed that expression of Cr-RSH in the mutant bacterial cells resulted in the synthesis of ppGpp. SpoT, which catalyzes (p)ppGpp degradation, is dispensable in E.coli only if cells also lack RelA, which possesses (p)ppGpp synthase activity. The complementation analysis thus indicated that Cr-RSH possesses both ppGpp synthase and degradase activities. These results represent the first demonstration of ppGpp synthase-degradase activities in a eukaryotic organism, and they suggest that eubacterial stringent control mediated by ppGpp has been conserved during evolution of the chloroplast from a photosynthetic bacterial symbiont.
在单细胞光合真核生物莱茵衣藻中鉴定出一个编码假定的鸟苷3',5'-双焦磷酸（ppGpp）合成酶-降解酶的基因，命名为Cr-RSH。编码的Cr-RSH蛋白在其NH2末端具有一个假定的叶绿体靶向信号，并且在体外证明了Cr-RSH可转运到从莱茵衣藻分离的叶绿体中。预测的Cr-RSH成熟区域与RelA-SpoT家族蛋白的真细菌成员具有显著相似性。在大肠杆菌的relA、spoT双突变体中表达含有假定ppGpp合成酶结构域的Cr-RSH的NH2末端部分，可弥补突变细胞的生长缺陷。对32P标记的细胞单核苷酸的色谱分析还表明，在突变细菌细胞中表达Cr-RSH会导致ppGpp的合成。仅当细胞也缺乏具有（p）ppGpp合成酶活性的RelA时，催化（p）ppGpp降解的SpoT在大肠杆菌中才是可有可无的。因此，互补分析表明Cr-RSH同时具有ppGpp合成酶和降解酶活性。这些结果首次证明了真核生物中存在ppGpp合成酶-降解酶活性，并且表明由ppGpp介导的真细菌严谨反应在叶绿体从光合细菌共生体进化的过程中得以保留。
