Graduate Group in Microbiology, University of California, Berkeley, California, USA.
Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, USA.
mBio. 2020 Aug 25;11(4):e01625-20. doi: 10.1128/mBio.01625-20.
The facultative intracellular pathogen , like many related , uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in , the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for -dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.
兼性细胞内病原体与许多相关病原体一样，利用核苷酸第二信使环二腺苷酸 (c-di-AMP) 来适应营养可用性、渗透压应激和细胞壁作用抗生素的变化。在丰富的培养基中，c-di-AMP 是必需的；然而，编码 c-di-AMP 结合蛋白 B 的 基因的突变抑制了必需性。在这项研究中，我们发现依赖的必需性的原因是通过 RelA 诱导严格反应。RelA 是一种双功能 RelA/SpoT 同源物 (RSH)，通过多种变构相互作用调节 (p)ppGpp 的水平，(p)ppGpp 是一种通过多个变构相互作用协调严格反应的二级信使。我们对缺乏 c-di-AMP 的细菌进行正向遗传抑制子筛选，以鉴定在 持续表达的同时挽救生长的基因组突变，并鉴定 RelA 合成酶结构域的突变。酵母 2 杂交筛选还鉴定了 RelA 合成酶结构域是 CbpB 的相互作用伙伴。生化分析证实，游离 CbpB 激活 RelA，而 c-di-AMP 抑制其激活。我们解决了与 c-di-AMP 结合和未结合的 CbpB 的晶体结构，并提供了有关 c-di-AMP 结合和 RelA 激活的重要区域的见解。这项研究的结果表明，CbpB 在 细菌中完成了 c-di-AMP 和 (p)ppGpp 之间的稳态调节回路，细菌必须有效地维持必需分子的内稳态以在环境中存活。我们发现，两种核苷酸第二信使，即 c-di-AMP 和 (p)ppGpp，在整个微生物世界中高度保守，在兼性细胞内病原体 中存在于一个稳态循环中。在这里，我们发现环二腺苷酸结合蛋白 B (CbpB) 作为 c-di-AMP 传感器起作用，当 c-di-AMP 水平较低时，通过与 RelA 结合促进 (p)ppGpp 的合成。添加 c-di-AMP 通过结合和隔离 CbpB 防止 RelA 激活。先前的研究表明，(p)ppGpp 结合并抑制 c-di-AMP 磷酸二酯酶，导致 c-di-AMP 增加。该途径通过直接酶促调节控制，并表明核糖体非依赖性严格激活的另一种机制。
