Key Laboratory of Systems Biology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China.
CAS Key Laboratory of Synthetic Biology, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China.
J Bacteriol. 2024 May 23;206(5):e0000324. doi: 10.1128/jb.00003-24. Epub 2024 Apr 12.
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 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 transcription was upregulated in the null mutant of . Next, we proved that the gene was essential for 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 promoter region from both and two other representative actinomycetes ( and ). Therefore, our data suggest that GlnR may repress 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 , 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.
在大多数放线菌中，GlnR 既控制氮代谢又控制非氮代谢（例如，碳、磷酸盐和次级代谢）。尽管 GlnR 已被认为是一种全局调节剂，但它在中心碳代谢（例如糖酵解、糖异生和三羧酸 (TCA) 循环）中的调节作用在很大程度上仍是未知的。在这项研究中，我们将 GlnR 鉴定为编码磷酸烯醇丙酮酸羧激酶的基因的直接转录抑制剂，该基因催化 TCA 循环中间产物草酰乙酸转化为磷酸烯醇丙酮酸，这是糖异生的关键步骤。通过转录组学和定量实时 PCR 分析，我们首先表明在缺失突变体中，基因的转录上调。接下来，我们证明当 TCA 循环中间产物用作唯一碳源时，基因对于是必需的。此外，通过电泳迁移率变动分析和 DNase I 足迹分析，我们揭示 GlnR 能够特异性结合来自和两种其他代表性放线菌（和）的基因启动子区域。因此，我们的数据表明 GlnR 可能在放线菌中抑制基因的转录，这突出了 GlnR 在应对环境营养胁迫时对氮和中心碳代谢的全局调节作用。
放线菌的 GlnR 调节剂控制氮代谢基因和许多其他涉及碳、磷酸盐和次级代谢的基因。目前，已知的 GlnR 调节的碳代谢基因涉及碳源的运输、短链脂肪酸的同化和 2-甲基柠檬酸循环，尽管对于 GlnR 与 TCA 循环和糖异生之间的关系知之甚少。在这里，基于生化和遗传结果，我们将 GlnR 鉴定为编码磷酸烯醇丙酮酸羧激酶的基因的直接转录抑制剂，该基因是糖异生的关键酶，从而强调了 GlnR 为动态协调放线菌中的细胞碳、氮和磷酸盐通量以及生物活性次级代谢物以适应不断变化的环境提供了核心和复杂的作用。
