Many important biological processes depend on corrinoid cofactors. Corrinoids are a family of cobalt-containing molecules that include vitamin B12. Corrinoids are utilized as enzymatic cofactors by animals, protists, and the majority of prokaryotes but are produced only by a subset of prokaryotes. Thus, corrinoids must be shared between corrinoid-producing and corrinoid-requiring organisms. More than 12 corrinoids with structural variability in the lower axial ligand have been observed. However, sequence-level understanding of specificity in corrinoid biosynthesis remains largely unexplored. This project seeks to identify the molecular factors that control the biosynthesis of structurally diverse corrinoids by examining specificity in the attachment of lower ligands to corrinoid precursors. Chemical analysis of corrinoids produced by five representative bacteria will be performed. Specificity in a lower ligand attachment enzyme will be examined both in vivo in a heterologous bacterial host and in vitro with purified enzymes. Based on these experimental results, bioinformatic predictions will be generated to identify amino acid residues that control specificity for attachment of particular lower ligands. These predictions will be tested by site-directed mutagenesis and further refined by additional sequence analysis and experimental validation.

The fundamental principles of specificity in corrinoid biosynthesis elucidated by this work will provide insights into the source and biological function of corrinoid structural variability. This research may form the foundation of a more general understanding of how small molecules are exchanged in microbial communities. Microbes in nearly all environments exist in complex communities containing numerous metabolically and phylogenetically diverse species. The complexity of organisms in microbial communities has made it difficult to analyze molecular interactions that occur between community members. Distinct combinations of structurally diverse corrinoids have been detected in five different microbial communities thus far. Given that half of sequenced bacteria are estimated to require corrinoids produced by other organisms, and that corrinoid function is influenced by the structure of the lower ligand, understanding specificity in lower ligand attachment will provide important insights into the molecular interactions that occur in microbial communities.

Broader impacts This research will provide insights into biological processes that require corrinoids and will contribute to the understanding of small molecule exchange in microbial communities. Understanding and manipulating corrinoid-dependent metabolism has diverse applications in bioremediation, bioenergy, and synthetic biology. This project will provide research training to a postdoctoral scholar, graduate student, and 2-3 undergraduate students. A major initiative to educate the public about microbial diversity will be created that will launch a new exhibit at UC Berkeley's annual open house, an event that draws approximately 35,000 visitors to the campus each year. A team of undergraduate and graduate students will design an interactive microbiology activity station to showcase the diversity of microbes in nature. The event will also include a "microbe art" station aimed at educating children about microorganisms. An outreach program will be created as an extension of this event in which undergraduate and graduate student volunteers will bring interactive components of the event to elementary school classes in surrounding communities. These educational initiatives have the potential to increase public awareness of the impact of microbial processes on the environment, to generate excitement about microbes among younger students, and to provide opportunities to engage university students in primary education.

National Science Foundation (NSF)
Division of Molecular and Cellular Biosciences (MCB)
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Jill Zeilstra-Ryalls
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University of California Berkeley
United States
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