Center for Oral Biology and Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA.
mBio. 2013 Sep 24;4(5):e00646-13. doi: 10.1128/mBio.00646-13.
The stringent response (SR), mediated by the alarmone (p)ppGpp, is a conserved bacterial adaptation system controlling broad metabolic alterations necessary for survival under adverse conditions. In Enterococcus faecalis, production of (p)ppGpp is controlled by the bifunctional protein RSH (for "Rel SpoT homologue"; also known as RelA) and by the monofunctional synthetase RelQ. Previous characterization of E. faecalis strains lacking rsh, relQ, or both revealed that RSH is responsible for activation of the SR and that alterations in (p)ppGpp production negatively impact bacterial stress survival and virulence. Despite its well-characterized role as the effector of the SR, the significance of (p)ppGpp during balanced growth remains poorly understood. Microarrays of E. faecalis strains producing different basal amounts of (p)ppGpp identified several genes and pathways regulated by modest changes in (p)ppGpp. Notably, expression of numerous genes involved in energy generation were induced in the rsh relQ [(p)ppGpp(0)] strain, suggesting that a lack of basal (p)ppGpp places the cell in a "transcriptionally relaxed" state. Alterations in the fermentation profile and increased production of H2O2 in the (p)ppGpp(0) strain substantiate the observed transcriptional changes. We confirm that, similar to what is seen in Bacillus subtilis, (p)ppGpp directly inhibits the activity of enzymes involved in GTP biosynthesis, and complete loss of (p)ppGpp leads to dysregulation of GTP homeostasis. Finally, we show that the association of (p)ppGpp with antibiotic survival does not relate to the SR but rather relates to basal (p)ppGpp pools. Collectively, this study highlights the critical but still underappreciated role of basal (p)ppGpp pools under balanced growth conditions.
Drug-resistant bacterial infections continue to pose a significant public health threat by limiting therapeutic options available to care providers. The stringent response (SR), mediated by the accumulation of two modified guanine nucleotides collectively known as (p)ppGpp, is a highly conserved stress response that broadly remodels bacterial physiology to a survival state. Given the strong correlation of the SR with the ability of bacteria to survive antibiotic treatment and the direct association of (p)ppGpp production with bacterial infectivity, understanding how bacteria produce and utilize (p)ppGpp may reveal potential targets for the development of new antimicrobial therapies. Using the multidrug-resistant pathogen Enterococcus faecalis as a model, we show that small alterations to (p)ppGpp levels, well below concentrations needed to trigger the SR, severely affected bacterial metabolism and antibiotic survival. Our findings highlight the often-underappreciated contribution of basal (p)ppGpp levels to metabolic balance and stress tolerance in bacteria.
严格反应（SR）由警报素（p）ppGpp 介导，是一种保守的细菌适应系统，控制着在不利条件下生存所需的广泛代谢改变。在粪肠球菌中，（p）ppGpp 的产生受双功能蛋白 RSH（“Rel SpoT 同源物”的缩写；也称为 RelA）和单功能合成酶 RelQ 控制。以前对缺乏 rsh、relQ 或两者的粪肠球菌菌株的表征表明，RSH 负责激活 SR，并且（p）ppGpp 产生的改变对细菌应激存活和毒力有负面影响。尽管它作为 SR 的效应物具有很好的特征，但在平衡生长期间（p）ppGpp 的意义仍知之甚少。对产生不同基础水平（p）ppGpp 的粪肠球菌菌株进行微阵列分析，确定了受（p）ppGpp 适度变化调节的几个基因和途径。值得注意的是，在 rsh relQ [（p）ppGpp（0）] 菌株中，许多参与能量产生的基因的表达被诱导，这表明缺乏基础（p）ppGpp 使细胞处于“转录松弛”状态。发酵谱的改变和（p）ppGpp（0）菌株中 H2O2 产量的增加证实了观察到的转录变化。我们证实，与枯草芽孢杆菌中所见类似，（p）ppGpp 直接抑制参与 GTP 生物合成的酶的活性，并且（p）ppGpp 的完全缺失导致 GTP 动态平衡的失调。最后，我们表明，（p）ppGpp 与抗生素存活的关联与 SR 无关，而是与基础（p）ppGpp 池有关。总的来说，这项研究强调了在平衡生长条件下基础（p）ppGpp 池的关键但仍未被充分认识的作用。
耐药细菌感染继续对公共卫生构成重大威胁，因为这限制了医疗保健提供者可获得的治疗选择。严格反应（SR）由两种称为（p）ppGpp 的修饰鸟嘌呤核苷酸的积累介导，是一种高度保守的应激反应，广泛重塑细菌生理学以达到存活状态。鉴于 SR 与细菌对抗生素治疗的存活能力之间的强烈相关性，以及（p）ppGpp 产生与细菌感染性之间的直接关联，了解细菌如何产生和利用（p）ppGpp 可能会揭示开发新的抗菌疗法的潜在目标。我们使用多药耐药病原体粪肠球菌作为模型，表明（p）ppGpp 水平的微小变化，远低于触发 SR 所需的浓度，严重影响了细菌的新陈代谢和抗生素的存活。我们的研究结果强调了基础（p）ppGpp 水平对细菌代谢平衡和应激耐受的经常被低估的贡献。
