Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/26/2003
Publication Date: 6/1/2003
Citation: KRISHNAN, H.B., KIM, W., SUN-HYUNG, J., KIM, K.Y., JIANG, G. CITRATE SYNTHASE MUTANTS OF SINORHIZOBIUM FREDII USDA257 FORM INEFFECTIVE NODULES WITH ABERRANT ULTRASTRUCTURE. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 2003. v. 69. p. 3561-3568. Interpretive Summary: Sinorhizobium fredii USDA257, a soil-bacterium, forms nodules on the roots of soybean plants. The nodules are specialized structures where atmospheric nitrogen is fixed by the bacterium, which in turn, is utilized by soybean plants for growth and development. This process is termed biological nitrogen fixation and it enables soybean plants to grow in nitrogen-poor soils. Even though S. fredii USDA257 strain forms nitrogen-fixing nodules on soybean, the effectiveness of nitrogen fixation is poor. The tricarboxylic acid (TCA) cycle plays an important role in determining the effectiveness of biological nitrogen fixation. The TCA cycle provides the energy required by the S. fredii USDA257 to fix atmospheric nitrogen. Citrate synthase is the first enzyme that controls the entry of carbon into the TCA cycle. Currently, we know little about the TCA cycle enzymes in S. fredii USDA257. We have cloned the citrate synthase gene of S. fredii USDA257 and have shown that a functional citrate synthase gene is absolutely essential for efficient biological nitrogen fixation. Information obtained from this basic study will help to better understand the factors that limit the effectiveness of biological nitrogen fixation. Such an understanding should enable us to manipulate biological nitrogen fixation so that farmers can increase the yield of soybean with minimal use of nitrogen fertilizers.
Technical Abstract: The tricarboxylic acid (TCA) cycle plays an important role in generating energy required by the bacteroids to fix atmospheric nitrogen. Citrate synthase is the first enzyme that controls the entry of carbon into the TCA cycle. We have cloned and determined the nucleotide sequence of the gltA gene that encodes citrate synthase from Sinorhizobium fredii USDA257, a symbiont of soybeans (Glycine max [L.] Merr.) and several other legumes. The deduced citrate synthase protein has a molecular weight of 48,198 Da and shows sequence similarity to citrate synthase from several bacterial species, including S. meliloti and Rhizobium tropici. Southern blot analysis revealed that the fast growing S. fredii strains and Rhizobium sp. NGR234 contained a single copy of this gene located in the bacterial chromosome. Sinorhizobium fredii USDA257 gltA mutant HBK-CS1, which had no detectable citrate synthase activity, had a diminished nodulation capacity and produced ineffective nodules on soybean. Light and electron microscopy observations revealed that the nodules initiated by HBK-CS1 contained very few bacteroids. The infected cells contained large vacuoles and prominent starch grains. Within the vacuoles, membrane structures that appear to be reminiscent of disintegrating bacteroids were detected. The citrate synthase mutant had altered cell surface characteristics and produced three times more exopolysaccharides than the wild type. A plasmid carrying the USDA257 gltA gene, when introduced into HBK-CS1, was able to restore all aforementioned defects. Our results demonstrate that a functional citrate synthase gene of S. fredii USDA257 is essential for efficient soybean nodulation and nitrogen fixation.