Nojirimycins (NJs) are nitrogen analogs of common sugars, in which the nitrogen atom is found in the ring, in place of the endocyclic ring oxygen. The NJs are found in some soil dwelling bacteria (Bacilli and Streptomyces) and in some plants. The initial and notable observation about NJ and various homologs are that these compounds often exhibit selective and potent inhibition of glycosyl hydrolases and glycosyltransferases. Despite over 40 years of recognition, the biosynthesis of these important natural products remains undefined to date, apart from knowledge that glucose is the biosynthetic precursor. Further, the reason or reasons for the NJ's occurrence in Nature remains unknown, but their function as inhibitors of glycoside hydrolase/transferase enzymes suggests that these compounds may serve as part of a complex chemical ecology of soil bacteria, plant host and other interacting species via perturbation of glycosylation states. The studies set forth in this project seek to identify the complete set of genes coding for the enzymes responsible for the synthesis of nojirimycin and deoxynojirimycin (DNJ) in the bacteria B. amyloliquefaciens and B. atrophaeus. The genes will be cloned and expressed in E. coli for production of recombinant biosynthetic enzymes. The enzymes will be characterized in terms of their substrate specificity and other steady state kinetic parameters. Finally, knockout strains of B.amyloliquefaciens will be produced and compared to wild type strains in microcosm experiments with pea seedlings and plant pathogens and perturbations in the populations as a function of the presence of DNJ will be identified. A correlation between DNJ production and changes in the microbial ecology of the rhizosphere would provide new evidence for the natural function of the nojirimycins.
Broader Impacts
The research project will involve undergraduate and graduate students, and provide them with a multidisciplinary research training experience. An important part of the project will include outreach to secondary schools. Floridian secondary school science teachers will be recruited to participate in summer research. The teachers will be provided the opportunity to develop learning/teaching modules based on their research experience that can be brought back to the classroom. The science developed in this research project may have broad impact in terms of providing new catalysts for "green" synthetic reactions, and provide new insight into manipulating interactions among members of microbial communities.
