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.
