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Title: Y4XP, AN OPEN READING FRAME LOCATED IN TYPE III PROTEIN SECRETION SYSTEM LOCUS OF SINORHIZOBIUM FREDII USDA257, ENCODES CYSTEINE SYNTHASE

Author
item LORIO, JULIO - UNIVERSITY OF MISSOURI
item CHRONIS, DEMOSTHENIS - UNIVERSITY OF MISSOURI
item Krishnan, Hari

Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/1/2006
Publication Date: 6/1/2006
Citation: Lorio, J.C., Chronis, D., Krishnan, H.B. 2006. Y4xp, an open reading frame located in type iii protein secretion system locus of sinorhizobium fredii usda257, encodes cysteine synthase. Molecular Plant-Microbe Interactions. 19:635-643.

Interpretive Summary: Sinorhizobium fredii USDA257, a soil-bacterium, forms nodules on the roots of primitive soybean plants but not on advanced North American cultivars. The nodules are specialized structures in which atmospheric nitrogen is fixed by the bacterium, subsequently to be used by the soybean plants for growth and development. This process, termed biological nitrogen fixation, enables soybean plants to grow in nitrogen-poor soils. Soybean and the rhizobia communicate with each other by exchanging signal molecules. Sinorhizobium fredii USDA257 secretes proteins called Nops (nodulation outer proteins) into the rhizosphere when they come into contact with soybean root exudates. Some of these proteins are involved in regulating nodulation on soybean plants. In addition to Nops, there are other proteins encoded by the bacterial genes whose function has not been determined. In the present investigation, we have demonstrated that a rhizobium gene, named y4xP, encodes cysteine synthase, an enzyme important to the assimilation of sulfur. Inactivation of this enzyme by mutating the rhizobium gene resulted in reduced Nops production and enhanced the ability of this mutant rhizobium to nodulate the soybean cultivar ‘McCall’. Information obtained from this basic study will help to better understand the factors that limit the formation of nitrogen-fixing nodules on North American soybean cultivars. Such an understanding should enable scientists to manipulate biological nitrogen fixation thereby increasing crop productivity.

Technical Abstract: Sinorhizobium fredii USDA257, a soybean symbiont, exports several nodulation outer proteins (Nops) into the rhizosphere. These proteins, which are exported by a type III secretion system (TTSS), have a pivotal role in host-specific nodulation. The entire TTSS of S. fredii lay within a 31 kb region which includes conserved genes that code for secretion machinery proteins, Nops, and several open reading frames (ORFs) of unknown function. Identifying the functions of these ORFs is essential to understand fully the role of TTSS in nodulation. Here, we report the characterization of y4xP, an ORF of previously unknown function. Southern blot analysis revealed that USDA257 contains two copies of y4xP, while a sibling, USDA191, contains a single copy. The amino acid sequence of y4xP is homologous to both eukaryotic and prokaryotic cysteine synthase, a key enzyme in sulfur assimilation. The coding region of USDA257 y4xP under control of T7 promoter was expressed in Escherichia coli and the recombinant protein was purified by nickel-affinity chromatography. Antibodies generated against soybean cysteine synthase cross-reacted with the recombinant protein. A non-polar mutant of y4xP of USDA191 showed a marked reduction in cysteine synthase activity. Enzyme activity was completely restored when the mutant was complemented with a plasmid containing the y4xP sequence. A non-polar cysteine synthase mutant was able to export all the Nops to the rhizosphere, however, in reduced amounts compared to the wild-type. Interestingly, USDA191 cysteine synthase mutant was able to initiate nodules on McCall soybean more efficiently than the wild-type. Our results demonstrate that y4xP encodes a cysteine synthase and inactivation of this gene enhances the ability of USDA191 to form nodules on McCall soybean by regulating Nops production.