Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/31/2005
Publication Date: 8/1/2005
Citation: Kim, W., Sun-Hyung, J., Park, R., Kim, K., Krishnan, H.B. 2005. Sinorhizobium fredii usda257 releases a 22 kda outer membrane protein (omp22) to the extra cellular milieu when grown in calcium-limiting conditions. Molecular Plant-Microbe Interactions. 18(8):808-818. Interpretive Summary: Biological nitrogen fixation enables soybean plants to grow in nitrogen-poor soils. Soil bacteria form nodules on the roots of soybean plants where atmospheric nitrogen is fixed by the bacterium, which in turn, is utilized by soybean plants for growth and development. Sinorhizobium fredii USDA257 is a nitrogen-fixing bacterium that forms nodules on primitive soybeans. This special bacterium secretes proteins into the soil near the roots of the soybean plant, the rhizosphere, where it comes into contact with chemicals that are excreted from the soybean root. Some of these proteins are involved in regulating the formation of nodules on soybean plants. Currently, very little is known about how these proteins are exported to the rhizosphere. Such information is required for designing strategies targeted toward improving biological nitrogen fixation. Deficiency of calcium adversely affects soybean nodulation. We have identified a protein of S. fredii USDA257 that is exported to the soybean rhizosphere when soybeans are grown where calcium is limited. Information obtained from this basic study will help to better understand the factors that limit the formation of nitrogen-fixing nodules on soybean cultivars. Such an understanding should enable scientists to manipulate biological nitrogen fixation so that farmers can increase the soybean yields with minimal use of nitrogen fertilizers.
Technical Abstract: Calcium, which regulates a wide variety of cellular functions, plays an important role in Rhizobium-legume interactions. We investigated the effect of calcium on the surface appendages of Sinorhizobium fredii USDA257. Cold field emission scanning electron microscopy observation of USDA257 grown in calcium-limiting conditions revealed cells with unusual shape and size. Transmission electron microscopy observation revealed intact flagella were present only when USDA257 cells were grown in calcium-sufficient conditions. SDS-PAGE analysis of the flagellar preparations from USDA257 cells grown in calcium-limiting conditions showed the presence of a 22 kDa protein, which was absent from cells grown in calcium-sufficient conditions. We have cloned and determined the nucleotide sequence of the gene encoding the 22 kDa protein. The deduced amino acid sequence is similar to that of several bacterial outer membrane proteins including S. meliloti, Agrobacterium tumefaciens, R. leguminosarum bv. viciae, and Brucella suis. The 22 kDa protein (Omp22) was successfully expressed in Escherichia coli and subsequently polyclonal antibodies were raised against the recombinant protein. Subcellular fractionation analysis demonstrated that the Omp22 was predominantly present in the extracellular fraction. Western blot analysis revealed the presence of immunologically related proteins from diverse rhizobia. Immunocytochemical localization of thin sections of USDA257 cells showed specific labeling of protein A-gold particles on protein inclusions found proximal to the cells. Accumulation of Omp22 was greatly reduced when USDA257 cells were grown in presence of increasing calcium. Northern blot analysis indicated that calcium, among the divalent cations examined, down-regulated the expression of omp22 gene. An omp22 mutant was able to grow in calcium-limiting conditions at a similar rate as the wild-type USDA257. The omp22 mutant initiated more nodules on soybean cultivar Peking than the wild-type when the plants were grown in calcium-limiting conditions.