Abstract：
Phosphatidylcholine (PC) is critical for the nitrogen-fixing symbiosis between rhizobia and legumes. We characterized three PC biosynthesis pathways in Rhizobium leguminosarum and evaluated their impact on nitrogen fixation in clover nodules. In the presence of choline, a PC synthase catalyzes the condensation of cytidine diphosphate-diacylglycerol with choline to produce PC. In the presence of lyso-PC, acyltransferases acylate this mono-acylated phospholipid to PC. The third pathway relies on phospholipid N-methyltransferases (Pmts), which sequentially methylate phosphatidylethanolamine (PE) through three rounds of methylation, yielding PC via the intermediates monomethyl-PE and dimethyl-PE. In R. leguminosarum, at least three Pmts participate in this methylation cascade. To elucidate the functions of these enzymes, we recombinantly produced and biochemically characterized them. We moved on to determine the phospholipid profiles of R. leguminosarum mutant strains harboring single and combinatorial deletions of PC biosynthesis genes. The cumulative results show that PC production occurs through the combined action of multiple enzymes, each with distinct substrate and product specificities. The methylation pathway emerges as the dominant PC biosynthesis route, and we pinpoint PmtS2, which catalyzes all three methylation steps, as the enzyme responsible for providing adequate PC amounts for a functional nitrogen-fixing symbiosis with clover.
OBJECTIVE: Understanding the molecular mechanisms of symbiotic nitrogen fixation has important implications for sustainable agriculture. The presence of the phospholipid phosphatidylcholine (PC) in the membrane of rhizobia is critical for the establishment of productive nitrogen-fixing root nodules on legume plants. The reasons for the PC requirement are unknown. Here, we employed Rhizobium leguminosarum and clover as model system for a beneficial plant-microbe interaction. We found that R. leguminosarum produces PC by three distinct pathways. The relative contribution of these pathways to PC formation was determined in an array of single, double, and triple mutant strains. Several of the PC biosynthesis enzymes were purified and biochemically characterized. Most importantly, we demonstrated the essential role of PC formation by R. leguminosarum in nitrogen fixation and pinpointed a specific enzyme indispensable for plant-microbe interaction. Our study offers profound insights into bacterial PC biosynthesis and its pivotal role in biological nitrogen fixation.
OBJECTIVE: Understanding the molecular mechanisms of symbiotic nitrogen fixation has important implications for sustainable agriculture. The presence of the phospholipid phosphatidylcholine (PC) in the membrane of rhizobia is critical for the establishment of productive nitrogen-fixing root nodules on legume plants. The reasons for the PC requirement are unknown. Here, we employed Rhizobium leguminosarum and clover as model system for a beneficial plant-microbe interaction. We found that R. leguminosarum produces PC by three distinct pathways. The relative contribution of these pathways to PC formation was determined in an array of single, double, and triple mutant strains. Several of the PC biosynthesis enzymes were purified and biochemically characterized. Most importantly, we demonstrated the essential role of PC formation by R. leguminosarum in nitrogen fixation and pinpointed a specific enzyme indispensable for plant-microbe interaction. Our study offers profound insights into bacterial PC biosynthesis and its pivotal role in biological nitrogen fixation.
摘要：
磷脂酰胆碱（PC）对于根瘤菌和豆科植物之间的固氮共生至关重要。我们表征了豆科根瘤菌中的三种PC生物合成途径，并评估了它们对三叶草根瘤固氮的影响。在胆碱的存在下，PC合酶催化胞苷二磷酸-二酰甘油与胆碱的缩合以产生PC。在lyso-PC的存在下,酰基转移酶将这种单酰化磷脂酰化为PC。第三条途径依赖于磷脂N-甲基转移酶（Pmts），通过三轮甲基化依次甲基化磷脂酰乙醇胺（PE），通过中间体单甲基-PE和二甲基-PE产生PC。在豆科，至少有三个Pmts参与这个甲基化级联。为了阐明这些酶的功能，我们重组生产和生化特征。我们继续确定包含PC生物合成基因的单个和组合缺失的豆科R.leguminosarum突变菌株的磷脂谱。累积结果表明，PC的产生是通过多种酶的联合作用而发生的，每个都有不同的底物和产品特异性。甲基化途径成为PC生物合成的主要途径，我们确定了PmtS2，它催化所有三个甲基化步骤，作为负责为三叶草的功能性固氮共生提供足够PC量的酶。
目的：了解共生固氮的分子机制对可持续农业具有重要意义。根瘤菌膜中磷脂磷脂酰胆碱（PC）的存在对于在豆科植物上建立生产性固氮根瘤至关重要。PC要求的原因未知。这里,我们采用豆科根瘤菌和三叶草作为有益的植物-微生物相互作用的模型系统。我们发现豆科念珠菌通过三种不同的途径产生PC。这些途径对PC形成的相对贡献是在一系列单一的，双,和三重突变菌株。对几种PC生物合成酶进行了纯化和生化表征。最重要的是,我们证明了豆科动物形成PC在固氮中的重要作用，并指出了植物与微生物相互作用所必需的特定酶。我们的研究为细菌PC的生物合成及其在生物固氮中的关键作用提供了深刻的见解。
目的：了解共生固氮的分子机制对可持续农业具有重要意义。根瘤菌膜中磷脂磷脂酰胆碱（PC）的存在对于在豆科植物上建立生产性固氮根瘤至关重要。PC要求的原因未知。这里,我们采用豆科根瘤菌和三叶草作为有益的植物-微生物相互作用的模型系统。我们发现豆科念珠菌通过三种不同的途径产生PC。这些途径对PC形成的相对贡献是在一系列单一的，双,和三重突变菌株。对几种PC生物合成酶进行了纯化和生化表征。最重要的是,我们证明了豆科动物形成PC在固氮中的重要作用，并指出了植物与微生物相互作用所必需的特定酶。我们的研究为细菌PC的生物合成及其在生物固氮中的关键作用提供了深刻的见解。
