LysR-Transcriptional Regulators (LTTRs) are the largest family of regulators in bacteria. They control diverse metabolic functions including amino acid biosynthesis, virulence, nitrate fixation, replication, and many other major processes. Despite their central role in allowing bacteria to respond to their environment, LTTRs are among the least studied types of regulators. The goal of this project is to expand our understanding of how these proteins function at an atomic level. Questions that will be addressed include: how do LTTRs bind to specific sequences of DNA in response to specific control molecules and what conformational changes take place when they bind their control molecules? New approaches to study LTTRs will be developed so that understanding these proteins and their potential beneficial applications can be advanced in the scientific community. The combined labs of the two PIs will focus on two proteins (BenM and CatM) from a soil bacterium, Acinetobacter baylyi. A wide range of methods will be used including mutant screens (genetics), DNA footprinting (molecular biology), and biochemical methods like tryptophan fluorescence, fluorescence polarization, and X-ray crystallography. The research will allow models to be developed to explain how diverse LTTRs function. Application of these studies may lead to novel biosensors and/or improved organisms for bioprocessing and bioremediation that can come from being able to modulate their functions. The research environment has a high educational impact. The project will allow high school, undergraduate and graduate students to have opportunities to experience experimental science and learn advanced methods in a highly collaborative environment that crosses multiple disciplines.

Project Report

Intellectual Merit: This research project was oriented toward understanding how regulatory proteins in bacteria, the LysR-type transcriptional regulators (LTTRs), work to control the synthesis of RNA. Our goals were to understand at a molecular level, how LTTRs interact with their promoters (where and how they bind to DNA), how small molecule regulatory molecules (effectors) influence the proteins, and how the LTTRs capture RNA polymerase (which makes RNA). Some of the highlights of the project included: The first report of a LTTR DNA binding domain bound to promoter DNA demonstrated that a common DNA binding mechanism is broadly shared among LTTRs. Analysis of full-length LTTRs (including our own structural contribution) demonstrated that the LTTRs use a very flexible domain arrangement to create very different overall structures, despite using conserved domains. New functions were genetically engineered into LTTRs to look at regulatory specificities of individual promoters. Effector binding was characterized in a variety of mutants to reveal that one ligand (benzoate) enhances binding of a second ligand (muconate) to achieve a synergistic effect. Solved the structures of several full-length LTTRs that regulate amino acid biosynthesis and other metabolic pathways. These studies of bacterial metabolism can lead to improved control of bacterial plant pathogens and new food preservation approaches. A motif was identified that may be used to create contacts with RNA polymerase control. Discovery of this motif may lead to the development of novel antibiotics for human health as well as new applications in biotechnology by controlling bacterial metabolism. Functions were assigned to several previously uncharacterized LTTRs and regulatory models were developed for several metabolic pathways. 6 atomic structures were submitted to the protein data base. Multiple additional structure determinations are complete or in progress. Broader Impacts of Work: This project supported diverse students in their intellectual development and transformation into future scientists. 10 high school students and 10 undergraduate students participated in various aspects of the research on LysR-type transcriptional regulators. All of the high school students moved into college degree programs, primarily STEM disciplines, and the undergraduates continued into graduate programs or professional schools. Several students are at prestigious institutions. 5 graduate students and 1 postdoctoral fellow worked on the project, 3 Ph.D.’s were granted and 1 student completed a M.S. degree. The impact on STEM training at the University of Georgia was significantly broadened through the integration of this research with a college laboratory course (MIBO4600L Experimental Microbiology Lab), taught by the Co-PI in 2012-2014. Students were taught to generate and experimentally test hypotheses about novel LTTRs. High school students cloned LTTRs as part of a week-long Summer Science Academy taught by the P.I.

National Science Foundation (NSF)
Division of Integrative Organismal Systems (IOS)
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Michael L. Mishkind
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University of Georgia
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