Regulation of the opposing (p)ppGpp synthetase and hydrolase activities in a bifunctional RelA/SpoT homologue from Staphylococcus aureus.
Regulation of the opposing (p)ppGpp synthetase and hydrolase activities in a bifunctional RelA/SpoT homologue from Staphylococcus aureus.
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DOI：
10.1371/journal.pgen.1007514
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发表时间：
2018-07
期刊：
影响因子：
4.5
通讯作者：
Wolz C
中科院分区：
文献类型：
--
作者：
Gratani FL;Horvatek P;Geiger T;Borisova M;Mayer C;Grin I;Wagner S;Steinchen W;Bange G;Velic A;Maček B;Wolz C
严格反应的特点是 (p)ppGpp 合成导致翻译抑制和转录组重编程。在金黄色葡萄球菌中，(p)ppGpp 由长 RSH（RelA/SpoT 同源物）酶 RelSau 或两种短合成酶（RelP、RelQ）之一合成。 RSH 酶的特点是 N 端酶结构域带有 (p)ppGpp 合成酶或水解酶活性的不同基序，C 端调节结构域 (CTD) 包含保守基序（TGS、DC 和 ACT）。 RelSau 合成酶和水解酶活性之间的分子内转换对于金黄色葡萄球菌适应应激（严格）或非应激（松弛）条件至关重要。我们阐明了 CTD 在 RelSau 酶活性中的作用。生长模式、转录分析和体外测定得出以下结果：i)在体内、在松弛条件下以及在体外，CTD抑制合成酶活性，但不是水解酶活性所必需的； ii) 在严格条件下，CTD 对于 (p)ppGpp 合成至关重要； iii)缺乏CTD的RelSau在金黄色葡萄球菌中表达时表现出净水解酶活性，但在大肠杆菌中表达时表现出净(p)ppGpp合成酶活性； iv) CTD 内的 TGS 和 DC 基序对于正确的严格响应是必需的，而 ACT 基序是可有可无的，v) 免疫共沉淀表明 CTD 与核糖体相互作用，这在很大程度上依赖于 TGS 基序。总之，RelSau 主要以合成酶关闭/水解酶开启状态存在，CTD 内的 TGS 基序需要在严格条件下激活 (p)ppGpp 合成。严格反应是一种普遍的应激反应，它使细菌能够在营养有限的条件下生存并更好地耐受抗生素治疗。在人类病原体金黄色葡萄球菌中，严格的反应对于毒力、吞噬体逃逸和抗生素耐受性起着重要作用。该反应由核苷酸衍生物 (p)ppGpp 的合成引发，进而导致生长停滞和基因表达重编程。然而，这些生长抑制分子的快速和受控失活对于生物体同样重要。 (p)ppGpp 合成和水解是通过双功能 RelA/SpoT 同源物 RelSau 完成的，RelSau 具有不同的合成酶、水解酶和感觉域。我们阐明了 RelSau 的 C 端感觉结构域如何控制金黄色葡萄球菌中水解酶和合成酶活性之间的分子间转换。这种转换对于金黄色葡萄球菌适应感染过程中不断变化的环境的适当反应至关重要。
The stringent response is characterized by (p)ppGpp synthesis resulting in repression of translation and reprogramming of the transcriptome. In Staphylococcus aureus, (p)ppGpp is synthesized by the long RSH (RelA/SpoT homolog) enzyme, RelSau or by one of the two short synthetases (RelP, RelQ). RSH enzymes are characterized by an N-terminal enzymatic domain bearing distinct motifs for (p)ppGpp synthetase or hydrolase activity and a C-terminal regulatory domain (CTD) containing conserved motifs (TGS, DC and ACT). The intramolecular switch between synthetase and hydrolase activity of RelSau is crucial for the adaption of S. aureus to stress (stringent) or non-stress (relaxed) conditions. We elucidated the role of the CTD in the enzymatic activities of RelSau. Growth pattern, transcriptional analyses and in vitro assays yielded the following results: i) in vivo, under relaxed conditions, as well as in vitro, the CTD inhibits synthetase activity but is not required for hydrolase activity; ii) under stringent conditions, the CTD is essential for (p)ppGpp synthesis; iii) RelSau lacking the CTD exhibits net hydrolase activity when expressed in S. aureus but net (p)ppGpp synthetase activity when expressed in E. coli; iv) the TGS and DC motifs within the CTD are required for correct stringent response, whereas the ACT motif is dispensable, v) Co-immunoprecipitation indicated that the CTD interacts with the ribosome, which is largely dependent on the TGS motif. In conclusion, RelSau primarily exists in a synthetase-OFF/hydrolase-ON state, the TGS motif within the CTD is required to activate (p)ppGpp synthesis under stringent conditions. The stringent response is a general stress response, which allows bacteria to survive nutrient limited conditions and to better tolerate antibiotic treatment. In the human pathogen, Staphylococcus aureus, the stringent response plays an important role for virulence, phagosomal escape and antibiotic tolerance. The response is initiated by the synthesis of the nucleotide derivative (p)ppGpp which in turn leads to growth arrest and reprogramming of gene expression. However, a rapid and controlled inactivation of these growth inhibitory molecules is equally important for the organism. (p)ppGpp synthesis as well as hydrolysis is accomplished by a bi-functional RelA/SpoT homolog, RelSau bearing distinct synthetase, hydrolase and sensory domains. We elucidated how the C-terminal sensory domain of RelSau controls the intermolecular switch between hydrolase and synthetase activities in S. aureus. The switch is crucial for the appropriate response of S. aureus to adapt to changing environment encountered during infection.
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影响因子：
14.9
作者：
Arenz S;Abdelshahid M;Sohmen D;Payoe R;Starosta AL;Berninghausen O;Hauryliuk V;Beckmann R;Wilson DN
通讯作者：
Wilson DN
严格控制核糖体依赖性激活。
影响因子：
64.8
作者：
Brown A;Fernández IS;Gordiyenko Y;Ramakrishnan V
通讯作者：
Ramakrishnan V
影响因子：
3.7
作者：
Atkinson GC;Tenson T;Hauryliuk V
通讯作者：
Hauryliuk V
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
4.4
作者：
Cox, Juergen;Neuhauser, Nadin;Mann, Matthias
通讯作者：
Mann, Matthias