Post symbiotic analysis of the Bradyrhizobium japonicum-soybean symbiosis : proteomics and mutagenesis of protocatechuate 3, 4-dioxygenase paralogs

Proteins were isolated from the periplasm of Bradyrhizobium japonicum bacteroids by a modification of the method of Streeter and LeRudilier (1990) and compared to those isolated from the cytoplasm. The modification provides periplasmic protein preparations with minimal contamination of cytoplasmic proteins. The largest groups of identified proteins were in central intermediary metabolism and amino acid metabolism, whereas the smallest were in the categories of DNA replication, recombination, and repair, and purines, pyrimidines, and nucleosides. A theme in the carbon and amino acid metabolism groups of the periplasmic proteins was the production of succinate. A number of plant proteins were found which suggested contamination of the plant portion of the nodule during bacteroid isolation. However, antibodies to two of the identified plant proteins were applied as immunogold labels to soybean nodules sections. The labeling patterns suggested that these two proteins were specifically targeted to the bacteroids. Introduction The gram negative, soil bacterium Bradyrhizobium japonicum infects soybean (Glycine max) roots forming a new plant derived, nitrogen-fixing organ, the nodule. The free-living B. japonicum in the nodule tissue transform to a non-growing, non-motile form called a bacteroid that is retained within a plant-derived membrane, the peribacteroid or symbiosome membrane. Inside of the symbiosome membrane and