Submitted to: Bioscience Biotechnology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/21/2006
Publication Date: 12/7/2006
Citation: Yoshida, K., Kim, W., Kinehara, M., Mukai, R., Ashida, H., Ikeda, H., Fujita, Y., Krishnan, H.B. 2006. Identification of a functional 2-keto-myo-inositol dehydratase gene of Sinorhizobium fredii USDA191 required for myo-inositol utilization. Bioscience Biotechnology and Biochemistry. 70:2957-2964. Interpretive Summary: Sinorhizobium fredii USDA191, 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. Myo-inositol and its derivatives are abundant in soil and play an important role in plants, serving as signal transduction molecules, cell wall components, and as the major sink for phosphorus storage in seed. Bacterial myo-inositol catabolism has been shown to play an important role in rhizobium-legume symbiosis. In this study, we have identified the presence of 2-keto-myo-inositol dehydratase activity in free-living USDA191 cells. This observation indicates that a functional myo-inositol catabolic pathway is operational in S. fredii USDA191. 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 so that farmers can increase the soybean yields with minimal use of nitrogen fertilizers.
Technical Abstract: Sinorhizobium fredii USDA191 is a Gram-negative bacterium capable of forming nitrogen-fixing nodules on soybean roots. The USDA191 idhA gene encoding myo-inositol dehydrogenase, an enzyme necessary for myo-inositol utilization, is known to be involved in competitive nodulation and nitrogen fixation. In Bacillus subtilis, myo-inositol dehydrogenase catalyzes the first step of the myo-inositol catabolic pathway. Recently iolE was identified as the gene encoding 2-keto-myo-inositol dehydratase, which catalyzes the second-step in the pathway. Here, we report the presence of 2-keto-myo-inositol dehydratase activity in free-living USDA191 cells cultured in a medium containing myo-inositol, and an iolE ortholog was cloned from USDA191. USDA191 iolE was expressed in Escherichia coli as a His6-tag fusion and purified to exhibit 2-keto-myo-inositol dehydratase activity. Inactivation of USDA191 iolE led to defective myo-inositol utilization. USDA191 iolE partially complemented a B. subtilis iolE deficient mutant. These results suggested that S. fredii USDA191 could utilize a myo-inositol catabolic pathway, analogous to that of B. subtilis, involving at least idhA and iolE.