Structural Mechanisms of Bacterial Extracellular DNA-recognition

University Of Nebraska-Lincoln, Lincoln NE

Investigators

Abstract

Bacteria interact with DNA from their environment to acquire novel genetic material and to stabilize multi-cellular structures called biofilms. Biofilms allow bacteria to persist in a variety of conditions where single bacterial cells could not. The proteins that bind to DNA outside of bacterial cells are often incorporated into fibers which extend out from the cell. Although the basic structure of these fibers is known, identification of the proteins that bind DNA and how the proteins and DNA are incorporated into the fibers are points of investigation for this study. The overall goal is to identify which proteins in the fibers bind DNA and how, to determine the specific molecular interactions critical to the process of assembling bacterial biofilms. As a result, this project will advance the field of bacteriology as well as train the next generation of graduate students in the use of quantitative and structural methods for microbiology. Educational materials centered on microbial fitness and communities will be developed and shared with local HS teachers and students through existing university partnerships, a workshop and a summer camp. This proposal will identify novel mechanisms through which extracellular DNA (eDNA) interacts with bacterial surface structures and provide a detailed molecular description of these interactions to understand how the eDNA is incorporated and how protein-DNA interactions stabilize the biofilms. The identification and characterization of diverse extracellular DNA receptors will highlight unique mechanisms of biofilm stabilization, potentially allowing for the development of interventions for specific bacteria. The project will identify and characterize novel DNA receptor subunits incorporated into bacterial surface appendages known to adhere to eDNA, including those from Gram-positive (Clostridioides) and Gram-negative (Acinetobacter) bacterial species. Goals of the work include purifying soluble protein constructs, measuring their DNA binding affinity and determining the structural mechanisms of recognition through X-ray crystallography. The goal for this aspect of the project is to create an atomistic description of DNA recognition by these novel receptors. Additionally, the project will investigate how these DNA receptors are incorporated into the protein fibers through isolation of natively-expressed fibers and reconstitution with soluble protein complexes. This project is jointly funded by the Molecular Biophysics Program of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate and the Established Program to Stimulate Competitive Research (EPSCoR). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.