(p)ppGpp-Dependent Persisters Increase the Fitness of Escherichia coli Bacteria Deficient in Isoaspartyl Protein Repair.
(p)ppGpp-Dependent Persisters Increase the Fitness of Escherichia coli Bacteria Deficient in Isoaspartyl Protein Repair.
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(p)ppGpp 依赖性持续存在可增强缺乏异天冬氨酰蛋白修复的大肠杆菌细菌的适应性。
DOI：
10.1128/aem.00623-16
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发表时间：
2016
影响因子：
4.4
通讯作者：
Visick,JonathanE
中科院分区：
文献类型：
--
作者：
VandenBerg,KelseyE;Ahn,Sarah;Visick,JonathanE
异天冬氨酸蛋白羧基甲基转移酶（PCM）可修复由天冬氨酸或天冬酰胺残基自发转化为异天冬氨酸所造成的蛋白质损伤，提高大肠杆菌在应激条件下的长期静止期存活率。在旨在检测代谢失活细胞中PCM功能的研究过程中，我们发现了pcmas基因，其突变影响氧氟沙星耐受持久性细胞的形成。具体而言，Δpcmmutant在固定阶段产生的持续细胞延长了一段时间，ΔglpDmutation在Δpcmbackground中显著增加了持续细胞，达到活细胞的23%。在长期稳定阶段，高持久性双突变体在与野生型的竞争中表现出比pcm突变体更高的竞争适应度，这表明持久性与减轻未修复的蛋白质损伤之间存在联系。我们假设，高持久性菌株的代谢降低可能会延缓蛋白质损伤，但与野生型或单突变菌株相比，代谢没有明显差异。然而，甲基乙二醛，其积累的单一pdmutants，也增加适合度，提示一个可能的机制。ΔpcmΔglpDmutant中高水平的持久性形成依赖于五磷酸鸟苷[(p)ppGpp]和多磷酸。相反，Δpcmmutant中的持久性形成与(p)ppGpp无关，因此可能通过不同的途径发生。我们还观察到在高持久性菌株中构象不稳定蛋白的增加，并讨论了这作为对未修复的蛋白质损伤的反应的持久性的可能触发因素。蛋白损伤是影响细胞和生物体生存和功能的重要因素。一种特殊形式的蛋白质损伤，即异常氨基酸异天冬氨酸的形成，可以通过一种几乎普遍保守的酶PCM来修复。pcm导向的修复与细菌、昆虫、蠕虫、植物、小鼠和人类的应激生存和长寿有关，但关于蛋白质损伤和修复的具体影响仍有待研究。本文确定了异天冬氨酸蛋白损伤与持久性之间的意外联系，细菌培养中的亚群显示出对抗生素的耐受性增加。在缺乏PCM的情况下，大肠杆菌的持久性种群增加，特别是当代谢基因eglpd也发生突变时。在竞争分析中，高水平的glpd双突变体与细菌适应度增加相关，适应度依赖于信号分子(p)ppGpp；这可能是对蛋白质损伤作出反应的另一种途径。
Thel-isoaspartyl protein carboxyl methyltransferase (PCM) repairs protein damage resulting from spontaneous conversion of aspartyl or asparaginyl residues to isoaspartate and increases long-term stationary-phase survival of Escherichia coli under stress. In the course of studies intended to examine PCM function in metabolically inactive cells, we identifiedpcmas a gene whose mutation influences the formation of ofloxacin-tolerant persisters. Specifically, a Δpcmmutant produced persisters for an extended period in stationary phase, and a ΔglpDmutation drastically increased persisters in a Δpcmbackground, reaching 23% of viable cells. The high-persister double mutant showed much higher competitive fitness than thepcmmutant in competition with wild type during long-term stationary phase, suggesting a link between persistence and the mitigation of unrepaired protein damage. We hypothesized that reduced metabolism in the high-persister strain might retard protein damage but observed no gross differences in metabolism relative to wild-type or single-mutant strains. However, methylglyoxal, which accumulates inglpDmutants, also increased fitness, suggesting a possible mechanism. High-level persister formation in the ΔpcmΔglpDmutant was dependent on guanosine pentaphosphate [(p)ppGpp] and polyphosphate. In contrast, persister formation in the Δpcmmutant was (p)ppGpp independent and thus may occur by a distinct pathway. We also observed an increase in conformationally unstable proteins in the high-persister strain and discuss this as a possible trigger for persistence as a response to unrepaired protein damage.IMPORTANCEProtein damage is an important factor in the survival and function of cells and organisms. One specific form of protein damage, the formation of the abnormal amino acid isoaspartate, can be repaired by a nearly universally conserved enzyme, PCM. PCM-directed repair is associated with stress survival and longevity in bacteria, insects, worms, plants, mice, and humans, but much remains to be learned about the specific effects of protein damage and repair. This paper identifies an unexpected connection between isoaspartyl protein damage and persisters, subpopulations in bacterial cultures showing increased tolerance to antibiotics. In the absence of PCM, the persister population in Escherichia coli bacteria increased, especially if the metabolic geneglpDwas also mutated. High levels of persisters inpcm glpDdouble mutants correlated with increased fitness of the bacteria in a competition assay, and the fitness was dependent on the signal molecule (p)ppGpp; this may represent an alternative pathway for responding to protein damage.