Heme Biosynthesis in the Bradyrhizobium japonicum/Soybean Symbiosis

大豆根瘤菌是大豆中的细菌内共生菌，在一种被称为根瘤的特殊植物器官的细胞内起着固氮细胞器的作用。这两种共生体之间发生了许多相互作用，促进了结核的形成、发展和维持。这项拟议工作的广泛目标是了解豆科植物如何与它们的细菌伙伴沟通，以调节共生所必需的代谢过程。特别是，我们正在阐明日本双胞菌和大豆之间的相互作用事件，这些事件控制着根瘤中血红素的合成。一些数据支持这样的假设，即植物根瘤蛋白豆红蛋白的血红素修复基是细菌的起源，我的实验室的工作表明，细菌血红素残基的形成本身需要两种共生体的协同努力。针对上述两个假设，提出了三个目标。I)使用对温度敏感的突变体确定日本双胞菌血红素合成酶铁络合酶是否对大豆血红素的形成是必需的。Ii)确定日本芽孢杆菌对血红素前体--氨基乙酰丙酸(ALA)的摄取机制，并确定其在根瘤血红素合成中的作用。数据表明，细菌对植物来源的ALA的摄取是根瘤中组织间血红素途径的一个关键特征。Iii)构建并鉴定了日本血凝素合成途径的第二步丙氨酸脱水酶突变株，并研究了丙氨酸脱水酶基因的调控。突变体将使我们能够准确地确定有机体间途径中的哪些血红素合成步骤必须是细菌的。

Bradyrhizobium japonicum is the bacterial endosymbiont of soybean (Glycine max) that functions as a nitrogen-fixing organelle within cells of a specialized plant organ called a root nodule. Many interactions occur between the two symbionts for the initiation, development and maintenance of nodules. The broad objective of the proposed work is to understand how leguminous plants communicate with their bacterial partners to regulate metabolic processes essential for symbiosis. In particular, we are elucidating the interactive events between B. japonicum and soybean that control heme synthesis in root nodules. Some data support the hypothesis that the heme prosthetic group of the plant nodule protein leghemoglobin is bacterial in origin, and work from my laboratory indicates that formation of the bacterial heme moeity itself requires the coordinated effort of both symbionts. Three objectives are proposed which address the two aforesaid hypotheses. i) Determine whether the B. japonicum heme synthesis enzyme ferrochelatase is essential for soybean hemoglobin heme formation using a temperature-sensitive mutant. ii) Characterize the B. japonicum uptake mechanism of the heme precursor delta- aminolevulinic acid (ALA), and ascertain its role in nodule heme synthesis. Data indicate that bacterial uptake of plant-derived ALA is a key feature of an inter-organismic heme pathway in nodules. iii) Construct and characterize a B. japonicum mutant in ALA dehydratase, the second step of the heme synthetic pathway, and study the regulation of the ALA dehydratase gene. The mutant will allow us to determine exactly which heme synthesis steps in the inter-organismic pathway must be bacterial.