9404818 O'Brian 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 this 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. The demand for heme increases in both organisms as the respective cell types differentiate in nodules, and the corresponding heme proteins are an essential feature of symbiotic nitrogen fixation. Preliminary work indicates that the plant host is directly involved in bacterial heme synthesis in nodules, and other data have implicated B. japonicum as the source for soybean hemoglobin heme. The two aforesaid hypotheses are being tested by i) Constructing and characterizing B. japonicum mutants defective in uptake of the heme precursor delta-aminolevulinic acid (ALA). It is possible that bacterial heme can be synthesized from soybean-derived ALA in nodules, and the tenability of this mechanism will be tested by the acquisition and characterization of ALA uptake mutants. ii) Bacterial heme synthesis from plant ALA infers that B. japonicum ALA dehydratase is the first essential bacterial enzyme for heme formation in nodules, and analysis of mutants bears out that prediction. Therefore, the regulation of hemB. the gene encoding ALA dehydratase will be examined. iii) Determining whether B. japonicum can provide the heme prosthetic group of soybean leghemoglobin by construction and characterization of a temperature-sensitive ferrochelatase mutant. The mutant will allow normal nodule development at a permissive temperature, but will permit the inactivation of bacterial heme synt hesis in those nodules. %%% This work will increase our understanding of an important step in soybean and other legume growth. ***
