在大多数放线菌中,GlnR控制氮和非氮代谢(例如,碳,磷酸盐,和次生代谢)。尽管GlnR已被公认为全球监管机构,它在中心碳代谢中的调节作用[例如,糖酵解,糖异生,和三羧酸(TCA)循环]在很大程度上是未知的。在这项研究中,我们将GlnR描述为编码磷酸烯醇丙酮酸羧激酶的pckA基因的直接转录阻遏物,催化TCA循环中间体草酰乙酸转化为磷酸烯醇丙酮酸,糖异生的关键步骤.通过转录组学和定量实时PCR分析,我们首先表明,在麦田霉菌的glnRnull突变体中,pckA转录被上调。接下来,我们证明,当TCA循环中间体用作唯一的碳源时,pckA基因对于mediterranei糖异生至关重要。此外,采用电泳迁移率变化测定法和DNaseI足迹测定法,我们发现,GlnR能够特异性结合来自地中海曲霉和其他两种代表性放线菌(天黑链霉菌和耻垢分枝杆菌)的pckA启动子区域。因此,我们的数据表明GlnR可能抑制放线菌中的pckA转录,这突出了GlnR在响应环境营养胁迫的氮和中心碳代谢中的全球调节作用。
目的:放线菌的GlnR调节因子控制氮代谢基因和许多其他与碳,磷酸盐,和次级代谢。目前,碳代谢中已知的GlnR调节基因参与碳源的运输,短链脂肪酸的同化,和2-甲基柠檬酸盐循环,尽管对GlnR与TCA周期和糖异生之间的关系知之甚少。这里,根据生化和遗传结果,我们确定GlnR是pckA的直接转录抑制因子,编码磷酸烯醇丙酮酸羧激酶的基因,糖异生的关键酶,因此强调了GlnR在细胞碳的动态编排中起着核心和复杂的作用,氮,以及放线菌中的磷酸盐通量和生物活性次级代谢产物,以适应不断变化的环境。
In most actinomycetes, GlnR governs both nitrogen and non-nitrogen metabolisms (e.g., carbon, phosphate, and secondary metabolisms). Although GlnR has been recognized as a global regulator, its regulatory role in central carbon metabolism [e.g., glycolysis,
gluconeogenesis, and the tricarboxylic acid (TCA) cycle] is largely unknown. In this study, we characterized GlnR as a direct transcriptional repressor of the pckA gene that encodes phosphoenolpyruvate carboxykinase, catalyzing the conversion of the TCA cycle intermediate oxaloacetate to phosphoenolpyruvate, a key step in
gluconeogenesis. Through the transcriptomic and quantitative real-time PCR analyses, we first showed that the pckA transcription was upregulated in the glnR null mutant of Amycolatopsis mediterranei. Next, we proved that the pckA gene was essential for A. mediterranei
gluconeogenesis when the TCA cycle intermediate was used as a sole carbon source. Furthermore, with the employment of the electrophoretic mobility shift assay and DNase I footprinting assay, we revealed that GlnR was able to specifically bind to the pckA promoter region from both A. mediterranei and two other representative actinomycetes (Streptomyces coelicolor and Mycobacterium smegmatis). Therefore, our data suggest that GlnR may repress pckA transcription in actinomycetes, which highlights the global regulatory role of GlnR in both nitrogen and central carbon metabolisms in response to environmental nutrient stresses.
The GlnR regulator of actinomycetes controls nitrogen metabolism genes and many other genes involved in carbon, phosphate, and secondary metabolisms. Currently, the known GlnR-regulated genes in carbon metabolism are involved in the transport of carbon sources, the assimilation of short-chain fatty acid, and the 2-methylcitrate cycle, although little is known about the relationship between GlnR and the TCA cycle and
gluconeogenesis. Here, based on the biochemical and genetic results, we identified GlnR as a direct transcriptional repressor of pckA, the gene that encodes phosphoenolpyruvate carboxykinase, a key enzyme for
gluconeogenesis, thus highlighting that GlnR plays a central and complex role for dynamic orchestration of cellular carbon, nitrogen, and phosphate fluxes and bioactive secondary metabolites in actinomycetes to adapt to changing surroundings.