集胞藻PCC6803中relA/spoT同源基因syn-rsh(slr1325)的鉴定

Characterization of the relA/spoT homologue slr1325 (syn-rsh) of the cyanobacterium Synechocystis sp. PCC6803

【目的】四磷酸或五磷酸鸟苷(Guanosine 3',5'-bispyrophosphate,(p)ppGpp)是细菌在遭遇环境胁迫时细胞产生应激反应的信号分子,(p)ppGpp由其合成酶RelA或具有合成酶或水解酶双重催化功能的RelA/SpoT合成。本文证明了集胞藻PCC6803(Synechocystis sp.)中唯一编码RelA/SpoT同源蛋白(命名为Syn-RSH)的基因slr1325(syn-rsh)的功能。【方法】通过互补试验证明syn-rsh表达产物的生物学功能;以纤维素薄层层析检测不同条件下Escherichia coli(p)ppGpp合成缺陷突变株及集胞藻PCC6803细胞中的(p)ppGpp。【结果】诱导Syn-RSH表达可使(p)ppGpp合成酶和水解酶基因缺失的E.coli突变株回复野生型表型,并在细胞中积累一定水平的ppGpp;在实验室培养条件下,集胞藻PCC6803细胞中可检测到低水平的ppGpp,氨基酸饥饿可诱导ppGpp水平升高并维持在相应水平。【结论】Syn-RSH具有(p)ppGpp合成酶和水解酶的双重功能,(p)ppGpp是集胞藻PCC6803在实验室生长条件下细胞生长所必需的。

[Objective] Nucleotide guanosine-3′,5′-（bis）pyrophosphate（ppGpp） synthesized by（ppGpp） synthesase RelA or bifunctional ppGpp synthase/degradase RelA/SpoT,mediates bacterial stringent response to various stressful conditions.Here we characterized the slr1325（syn-rsh） gene encoding a RelA/SpoT homolog（Syn-RSH） of the cyanobacterium Synechocystis sp.PCC6803.[Methods] We performed phenotypic complement test using Escherichia coli strain with（p）ppGpp-synthesis defect to determine Syn-RSH function（s）,and employed chromatographic analysis of32P-labeled cellular mononucleotides to detect the accumulation of ppGpp in Escherichia coli strains expressing Syn-RSH and in Synechocystis sp.PCC6803.[Results] Syn-RSH expression in E.coli relA/spoT double mutant was able to restore the cell growth arrest;Chromatographic analysis of 32P-labeled cellular mononucleotides revealed that Syn-RSH expression resulted in the synthesis of ppGpp in E.coli strain with relA and spoT mutant mutation.Additionally,Synechocystis cells accumulated a low level of ppGpp under laboratory growth conditions.[Conclusion] Syn-RSH possesses ppGpp synthase/degradase activities,and ppGpp is required for Synechocystis cell viability under normal growth conditions.