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Title: Disruption of the Glycine Cleavage System Enables Sinorhizobium Fredii USDA257 to Form Nitrogen-fixing Nodules on Agonomically Improved North American Soybean Cultivars

Author
item LORIO, JULIO - University Of Missouri
item KIM, WON-SEOK - University Of Missouri
item KRISHNAN, AMMULU - University Of Missouri
item Krishnan, Hari

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/26/2010
Publication Date: 7/1/2010
Citation: Lorio, J.C., Kim, W., Krishnan, A.H., Krishnan, H.B. 2010. Disruption of the Glycine Cleavage System Enables Sinorhizobium Fredii USDA257 to Form Nitrogen-fixing Nodules on Agonomically Improved North American Soybean Cultivars. Applied and Environmental Microbiology. 76:4185-4193.

Interpretive Summary: Sinorhizobium fredii USDA257, a soil-bacterium, forms nodules on the roots of primitive 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. S. fredii USDA257, however, does not form nodules on most of the North American cultivars. In this basic study we provide evidence for the involvement of a new genetic locus (glycine cleavage system) that controls soybean cultivar specificity. Inactivation of glycine cleavage system of USDA257 enables this strain to nodulate several of the North American soybean cultivars. Information obtained from this basic study will enable manipulation of biological nitrogen fixation so that farmers can increase the yield of soybean with minimal use of nitrogen fertilizers.

Technical Abstract: The symbiosis between Sinorhizobium fredii USDA257 and soybean [Glycine max (L.) Merr.] exhibits a high degree of cultivar specificity. USDA257 nodulates primitive soybean cultivars but fails to nodulate agronomically improved cultivars such as McCall. In this study we provide evidence for the involvement of a new genetic locus that controls soybean cultivar specificity. This locus was identified in USDA257 by Tn5 transposon mutagenesis followed by nodulation screening on McCall soybean. We have cloned the region corresponding to the site of Tn5 insertion and found that it lies within a 1.5 kb EcoRI fragment. DNA sequence analysis of this fragment and an adjucent 4.4 kb region identified an operon made up of three open reading frames encoding proteins of deduced molecular weight of 41, 13 and 104 kDa, respectively. These proteins revealed significant amino acid homolgy to glycine cleavage (gcv) system T, H and P proteins of Escherichia coli and other organisms. Southern blot analysis revealed the presence of similar sequences in diverse rhizobia. Measurement of ß-galactosidase activity of USDA257 strain containing a transcrptional fusion of gcvT promoter sequences to lacZ gene revealed that USDA257 gcvTHP operon was inducible by glycine. Inactivation of either gcvT or gcvP of USDA257 enabled the mutant the ability to nodulate several agronomically improved north American soybean cultivars. These nodules revealed anatomical features typical of determinate nodules with numerous bacteroids within the infected cells. Unlike the previously characterized soybean cultivar specifcity locus, nolBTUVW, incativation of the gcv locus had no discernible effect on the secretion of nodulation outer proteins of USDA257.