RNA分子伴侣Hfq是众多生物过程的全球调节因子,如碳/氮代谢和植物相关的重氮生物的环境适应;然而,其靶RNA和潜在的固氮机制在很大程度上仍然未知。这里,我们使用增强的UV交联免疫沉淀结合高通量测序来鉴定数百种可能参与固氮的Hfq结合RNA,碳基质利用,生物膜的形成,和其他功能。总的来说,这些过程使菌株A1501具有在竞争激烈的根际中茁壮成长的必要能力。我们的发现揭示了以前未知的Hfq靶基因。其中值得注意的是nifM,编码固氮酶还原酶溶解度所必需的异构酶;amtB,编码铵转运蛋白;oprB,编码碳水化合物孔蛋白;和cheZ,编码趋化蛋白。此外,我们鉴定了100多个功能未知的基因,这扩大了重氮生物中Hfq的潜在直接调控目标。我们的数据表明,Hfq直接与调节蛋白的mRNA相互作用(RsmA,阿尔古,和NifA),调节性ncRNARsmY,和其他潜在目标,从而揭示了固氮和其他代谢途径中的机制联系。
目的:许多实验方法在区分Hfq介导的调节的直接和间接作用方面经常面临挑战。基于高通量测序的新技术越来越深入地了解Hfq在基因表达中的全球调控。这里,增强的UV交联免疫沉淀与高通量测序相结合,用于鉴定根相关的Stutzeri假单胞菌A1501中的Hfq结合位点和潜在靶标,并鉴定数百种预测参与代谢的新型Hfq结合RNA。环境适应,和固氮。特别是,我们已经在转录后水平显示了Hfq与各种调节蛋白mRNA及其潜在靶标的相互作用。这项研究不仅增强了我们对Hfq调控的理解,重要的是,还提供了一个框架,用于解决根相关固氮的综合调控网络。
The RNA chaperone Hfq acts as a global regulator of numerous biological processes, such as carbon/nitrogen metabolism and environmental adaptation in plant-associated diazotrophs; however, its target RNAs and the mechanisms underlying nitrogen fixation remain largely unknown. Here, we used enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing to identify hundreds of Hfq-binding RNAs probably involved in nitrogen fixation, carbon substrate utilization, biofilm formation, and other functions. Collectively, these processes endow strain A1501 with the requisite capabilities to thrive in the highly competitive rhizosphere. Our findings revealed a previously uncharted landscape of Hfq target genes. Notable among these is nifM, encoding an isomerase necessary for nitrogenase reductase solubility; amtB, encoding an ammonium transporter; oprB, encoding a carbohydrate porin; and cheZ, encoding a chemotaxis protein. Furthermore, we identified more than 100 genes of unknown function, which expands the potential direct regulatory targets of Hfq in diazotrophs. Our data showed that Hfq directly interacts with the mRNA of regulatory proteins (RsmA, AlgU, and NifA), regulatory ncRNA RsmY, and other potential targets, thus revealing the mechanistic links in nitrogen fixation and other metabolic pathways.
OBJECTIVE: Numerous experimental approaches often face challenges in distinguishing between direct and indirect effects of Hfq-mediated regulation. New technologies based on high-throughput sequencing are increasingly providing insight into the global regulation of Hfq in gene expression. Here, enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing was employed to identify the Hfq-binding sites and potential targets in the root-associated Pseudomonas stutzeri A1501 and identify hundreds of novel Hfq-binding RNAs that are predicted to be involved in metabolism, environmental adaptation, and nitrogen fixation. In particular, we have shown Hfq interactions with various regulatory proteins\' mRNA and their potential targets at the posttranscriptional level. This study not only enhances our understanding of Hfq regulation but, importantly, also provides a framework for addressing integrated regulatory network underlying root-associated nitrogen fixation.