Data_Sheet_1_Rhamnolipid Enhances the Nitrogen Fixation Activity of Azotobacter chroococcum by Influencing Lysine Succinylation.docx

The enhancement of nitrogen fixation activity of diazotrophs is essential for safe crop production. Lysine succinylation (KSuc) is widely present in eukaryotes and prokaryotes and regulates various biological process. However, knowledge of the extent of KSuc in nitrogen fixation of Azotobacter chroococcum is scarce. In this study, we found that 250 mg/l of rhamnolipid (RL) significantly increased the nitrogen fixation activity of A. chroococcum by 39%, as compared with the control. Real-time quantitative reverse transcription PCR (qRT-PCR) confirmed that RL could remarkably increase the transcript levels of nifA and nifHDK genes. In addition, a global KSuc of A. chroococcum was profiled using a 4D label-free quantitative proteomic approach. In total, 5,008 KSuc sites were identified on 1,376 succinylated proteins. Bioinformatics analysis showed that the addition of RL influence on the KSuc level, and the succinylated proteins were involved in various metabolic processes, particularly enriched in oxidative phosphorylation, tricarboxylic acid cycle (TCA) cycle, and nitrogen metabolism. Meanwhile, multiple succinylation sites on MoFe protein (NifDK) may influence nitrogenase activity. These results would provide an experimental basis for the regulation of biological nitrogen fixation with KSuc and shed new light on the mechanistic study of nitrogen fixation.

提升固氮微生物（diazotrophs）的固氮活性，对于作物安全生产至关重要。赖氨酸琥珀酰化（lysine succinylation, KSuc）广泛存在于真核生物与原核生物中，可调控多种生物学过程。然而，目前关于褐球固氮菌（Azotobacter chroococcum）固氮过程中KSuc修饰的相关认知仍较为匮乏。本研究发现，250 mg/L的鼠李糖脂（rhamnolipid, RL）可使褐球固氮菌的固氮活性较对照组显著提升39%。实时定量反转录PCR（real-time quantitative reverse transcription PCR, qRT-PCR）验证结果显示，RL可显著上调nifA与nifHDK基因的转录水平。此外，本研究采用4D无标记定量蛋白质组学技术，对褐球固氮菌的全局KSuc修饰谱进行了分析。最终在1376个琥珀酰化蛋白质上鉴定到5008个KSuc修饰位点。生物信息学分析表明，RL处理可显著影响KSuc修饰水平，相关琥珀酰化蛋白质参与多种代谢过程，尤其富集于氧化磷酸化、三羧酸循环（tricarboxylic acid cycle, TCA循环）以及氮代谢通路。同时，钼铁蛋白（MoFe protein, NifDK）上的多个琥珀酰化位点可能会影响固氮酶活性。本研究结果可为基于KSuc修饰调控生物固氮提供实验依据，并为固氮机制的深入研究提供新的思路。