PDF(Pubmed)
Abstract：
The stringent response enables bacteria to survive nutrient starvation, antibiotic challenge, and other threats to cellular survival. Two alarmone (magic spot) second messengers, guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp), which are synthesized by RelA/SpoT homologue (RSH) proteins, play central roles in the stringent response. The pathogenic oral spirochete bacterium Treponema denticola lacks a long-RSH homologue but encodes putative small alarmone synthetase (Tde-SAS, TDE1711) and small alarmone hydrolase (Tde-SAH, TDE1690) proteins. Here, we characterize the respective in vitro and in vivo activities of Tde-SAS and Tde-SAH, which respectively belong to the previously uncharacterized RSH families DsRel and ActSpo2. The tetrameric 410-amino acid (aa) Tde-SAS protein preferentially synthesizes ppGpp over pppGpp and a third alarmone, pGpp. Unlike RelQ homologues, alarmones do not allosterically stimulate the synthetic activities of Tde-SAS. The ~180 aa C-terminal tetratricopeptide repeat (TPR) domain of Tde-SAS acts as a brake on the alarmone synthesis activities of the ~220-aa N-terminal catalytic domain. Tde-SAS also synthesizes \"alarmone-like\" nucleotides such as adenosine tetraphosphate (ppApp), albeit at considerably lower rates. The 210-aa Tde-SAH protein efficiently hydrolyzes all guanosine and adenosine-based alarmones in a Mn(II) ion-dependent manner. Using a growth assays with a ΔrelAΔspoT strain of Escherichia coli that is deficient in pppGpp/ppGpp synthesis, we demonstrate that Tde-SAS can synthesize alarmones in vivo to restore growth in minimal media. Taken together, our results add to our holistic understanding of alarmone metabolism across diverse bacterial species. IMPORTANCE The spirochete bacterium Treponema denticola is a common component of the oral microbiota. However, it may play important pathological roles in multispecies oral infectious diseases such as periodontitis: a severe and destructive form of gum disease, which is a major cause of tooth loss in adults. The operation of the stringent response, a highly conserved survival mechanism, is known to help many bacterial species cause persistent or virulent infections. By characterizing the biochemical functions of the proteins putatively responsible for the stringent response in T. denticola, we may gain molecular insight into how this bacterium can survive within harsh oral environments and promote infection. Our results also expand our general understanding of proteins that synthesize nucleotide-based intracellular signaling molecules in bacteria.
摘要：
严格的反应使细菌能够在营养饥饿中生存，抗生素挑战，以及其他对细胞生存的威胁。两个Alarmone（魔术点）第二信使，五磷酸鸟苷（pppGpp）和四磷酸鸟苷（ppGpp），由RelA/SpoT同源（RSH）蛋白合成，在严格的应对措施中发挥核心作用。致病性口腔螺旋体细菌Denticola密螺旋体缺乏长RSH同源物，但编码推定的小alarmone合成酶（Tde-SAS，TDE1711)和小alarmone水解酶(Tde-SAH，TDE1690)蛋白质。这里,我们表征了Tde-SAS和Tde-SAH各自的体外和体内活性，其分别属于先前未表征的RSH家族DsRel和ActSpo2。四聚体410-氨基酸（aa）Tde-SAS蛋白优先合成ppGpp而不是pppGpp和第三种警报酮，pGpp.与RelQ同源物不同，alarmones不会变构刺激Tde-SAS的合成活性。Tde-SAS的〜180aaC末端四肽重复（TPR）结构域对〜220-aaN末端催化结构域的alarmone合成活性起制动作用。Tde-SAS还合成“Alarmone-like”核苷酸，例如四磷酸腺苷（ppApp），尽管利率相当低。210-aaTde-SAH蛋白以Mn（II）离子依赖性方式有效地水解所有基于鸟苷和腺苷的丙氨酸。使用缺乏pppGpp/ppGpp合成的大肠杆菌ΔrelAΔspoT菌株的生长测定法，我们证明Tde-SAS可以在体内合成alarmones以恢复在基本培养基中的生长。一起来看,我们的结果增加了我们对跨不同细菌物种的alarmone代谢的整体理解。重要性螺旋体细菌是口腔微生物群的常见成分。然而,它可能在多物种口腔感染性疾病中起重要的病理作用，如牙周炎:一种严重和破坏性的牙龈疾病，这是成年人牙齿脱落的主要原因。操作的严格响应，一种高度保守的生存机制，已知可以帮助许多细菌物种引起持续或致命的感染。通过表征推定负责T.denticola严格反应的蛋白质的生化功能，我们可以从分子上深入了解这种细菌如何在恶劣的口腔环境中存活并促进感染。我们的结果还扩展了我们对细菌中合成基于核苷酸的细胞内信号分子的蛋白质的一般理解。
