The bacterial stringent stress response, mediated by the signaling molecule guanosine tetraphosphate, ppGpp, has recently gained attention as being important during normal cellular growth and as a potential new therapeutic target, which warrants detailed mechanistic understanding. Here, we used intracellular protein tracking in Pseudomonas aeruginosa PAO1, which indicated that RelA was bound to the ribosome, while SpoT localized at the cell poles. Transcriptome sequencing (RNA-Seq) was used to investigate the transcriptome of a ppGpp-deficient strain under nonstressful, nutrient-rich broth conditions where the mutant grew at the same rate as the parent strain. In the exponential growth phase, the lack of ppGpp led to >1,600 transcriptional changes (fold change cutoff of ±1.5), providing further novel insights into the normal physiological role of ppGpp. The stringent response was linked to gene expression of various proteases and secretion systems, including aprA, PA0277, impA, and clpP2. The previously observed reduction in cytotoxicity toward red blood cells in a stringent response mutant appeared to be due to aprA. Investigation of an aprA mutant in a murine skin infection model showed increased survival rates of mice infected with the aprA mutant, consistent with previous observations that stringent response mutants have reduced virulence. In addition, the overexpression of relA, but not induction of ppGpp with serine hydroxamate, dysregulated global transcriptional regulators as well as >30% of the regulatory networks controlled by AlgR, OxyR, LasR, and AmrZ. Together, these data expand our knowledge about ppGpp and its regulatory network and role in environmental adaptation. It also confirms its important role throughout the normal growth cycle of bacteria.

中文翻译：

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铜绿假单胞菌在最佳生长条件下的严格应激反应控制蛋白酶和全局调节剂。

最近，由信号分子四磷酸鸟苷（ppGpp）介导的细菌严格的应激反应已引起注意，因为它在正常细胞生长过程中很重要，并且作为潜在的新治疗靶标，需要进行详细的机理理解。在这里，我们在铜绿假单胞菌中使用了细胞内蛋白追踪PAO1，表明RelA绑定到核糖体，而SpoT定位在细胞极。转录组测序（RNA-Seq）用于研究在无压力，营养丰富的肉汤条件下ppGpp缺陷菌株的转录组，其中突变体以与亲本菌株相同的速率生长。在指数生长期，缺乏ppGpp导致> 1,600的转录变化（倍数变化截止值为±1.5），为ppGpp的正常生理作用提供了新的见解。严格的反应与包括aprA，PA0277，impA和clpP2在内的各种蛋白酶和分泌系统的基因表达有关。。先前观察到的在严格应答突变体中对红细胞的细胞毒性降低似乎是由于aprA引起的。在鼠类皮肤感染模型中对aprA突变体的研究表明，感染了aprA突变体的小鼠的存活率提高了，这与以前观察到的严格应答突变体降低了毒力的观察结果一致。另外，relA的过表达，而不是用丝氨酸异羟肟酸酯诱导ppGpp，失调的全局转录调节子以及AlgR，OxyR，LasR和AmrZ控制的调节网络中的> 30％。这些数据一起扩展了我们对ppGpp及其监管网络的知识以及在环境适应中的作用。它还证实了其在细菌正常生长周期中的重要作用。