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首页> 外文期刊>Applied Microbiology >Gpp-Dependent Persisters Increase the Fitness of Escherichia coli Bacteria Deficient in Isoaspartyl Protein Repair
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Gpp-Dependent Persisters Increase the Fitness of Escherichia coli Bacteria Deficient in Isoaspartyl Protein Repair

机译：Gpp依赖的持久性有机污染物增加缺乏异黄酮蛋白修复的大肠杆菌细菌的适应性。

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The l-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 identified pcm as a gene whose mutation influences the formation of ofloxacin-tolerant persisters. Specifically, a Δ pcm mutant produced persisters for an extended period in stationary phase, and a Δ glpD mutation drastically increased persisters in a Δ pcm background, reaching 23% of viable cells. The high-persister double mutant showed much higher competitive fitness than the pcm mutant 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 in glpD mutants, also increased fitness, suggesting a possible mechanism. High-level persister formation in the Δ pcm Δ glpD mutant was dependent on guanosine pentaphosphate [(p)ppGpp] and polyphosphate. In contrast, persister formation in the Δ pcm mutant 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.IMPORTANCE Protein 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 gene glpD was also mutated. High levels of persisters in pcm glpD double 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.
机译：l-异天冬氨酰蛋白质羧基甲基转移酶（PCM）修复了由于天冬氨酰或天冬酰胺基残基自发转化为异天冬氨酸而导致的蛋白质损伤，并增加了大肠杆菌在压力下的长期固定期存活率。在旨在检查代谢不活跃细胞中PCM功能的研究过程中，我们确定pcm为基因，其突变会影响耐氧氟沙星的持久性分子的形成。具体来说，Δpcm突变体在固定相中会产生持久的持久性，而ΔglpD突变会在Δpcm背景中急剧增加持久性，达到存活细胞的23％。在长期静止期与野生型竞争中，高持久性双突变体显示出比pcm突变体高得多的竞争适应性，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新陈代谢降低可能会延迟蛋白质的破坏，但与野生型或单突变菌株相比，新陈代谢没有明显的差异。但是，在glpD突变体中积累的甲基乙二醛也会增加适应性，提示可能的机制。 ΔpcmΔglpD突变体中的高水平持久性形成取决于五磷酸鸟苷[（p）ppGpp]和多磷酸盐。相反，Δpcm突变体中的持久性形成不依赖（p）ppGpp，因此可能通过不同的途径发生。我们还观察到高持久性菌株中构象不稳定蛋白的增加，并讨论了这可能是持久性的持续触发，是对未修复的蛋白损伤的反应。重要信息蛋白损伤是细胞和生物体存活和功能的重要因素。蛋白质损伤的一种特定形式，即异常氨基酸异天冬氨酸的形成，可以通过几乎普遍保守的酶PCM来修复。 PCM定向修复与细菌，昆虫，蠕虫，植物，小鼠和人类中的应力存活和寿命有关，但是关于蛋白质损伤和修复的特定作用尚有待研究。本文确定了异天冬氨酰蛋白损伤与持久性之间的意外联系，持久性是细菌培养物中的亚群，显示出对抗生素的耐受性增加。在没有PCM的情况下，大肠杆菌细菌的持久性种群增加，尤其是如果代谢基因glpD也发生突变时。 pcm glpD双重突变体中高水平的持久性与竞争分析中细菌的适应性增强相关，适应性取决于信号分子（p）ppGpp。这可能代表了应对蛋白质损伤的另一种途径。

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