ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria
ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria
复制标题
DOI：
10.1073/pnas.1522179113
复制
发表时间：
2016-03-22
影响因子：
11.1
通讯作者：
Gruendling, Angelika
中科院分区：
文献类型：
--
作者：
Corrigan, Rebecca M.;Bellows, Lauren E.;Gruendling, Angelika
严格反应是细菌用来应对压力的一种生存机制。它由核苷酸鸟苷四磷酸和五磷酸 [(p)ppGpp] 协调，与靶蛋白相互作用以促进细菌存活。尽管这种反应在变形菌中得到了很好的表征，但人们对革兰氏阳性物种中这种信号系统的效应器知之甚少。在这里，我们报告了革兰氏阳性细菌金黄色葡萄球菌中严格反应核苷酸的七种靶蛋白的鉴定。我们证明 GTP 合成酶 HprT 和 Gmk 以高亲和力结合，从而抑制 GTP 的产生。此外，我们还鉴定了五种假定的 GTPases-RsgA、RbgA、Era、HflX 和 ObgE-as (p) ppGpp 靶蛋白。我们表明，RsgA、RbgA、Era 和 HflX 是功能性 GTP 酶，并且它们的活性在核糖体存在下得到促进，但受到严格响应核苷酸的强烈抑制。通过表征 RsgA 在体内的功能，我们确定该蛋白参与核糖体组装，其中 rsgA 缺失菌株或 GTP 酶活性失活的菌株表现出生长下降、成熟 70S 核糖体数量减少以及对抗菌药物的耐受性增加。我们还证明，ppGpp 与细胞 GTP 酶的相互作用并非葡萄球菌所独有，因为来自枯草芽孢杆菌和粪肠球菌的同源物保留了这种能力。总而言之，这项研究揭示了核糖体失活是一种先前未识别的机制，革兰氏阳性细菌通过该机制发挥严格的反应作用。
The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Gram-positive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Gram-positive bacterium Staphylococcus aureus. We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases-RsgA, RbgA, Era, HflX, and ObgE-as (p) ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria.