Structural Mechanisms of Bacterial Extracellular DNA-recognition

细菌与环境中的DNA相互作用，获得新的遗传物质，并稳定称为生物膜的多细胞结构。生物膜允许细菌在各种条件下存活，而单个细菌细胞却不能。与细菌细胞外的DNA结合的蛋白质通常被合并成从细胞伸出的纤维。虽然这些纤维的基本结构是已知的，但鉴定结合DNA的蛋白质以及蛋白质和DNA如何结合到纤维中是本研究的调查要点。总体目标是确定纤维中哪些蛋白质与DNA结合以及如何结合，以确定对组装细菌生物膜过程至关重要的特定分子相互作用。因此，该项目将推动细菌学领域的发展，并培养下一代研究生使用微生物学的定量和结构方法。将开发以微生物适应性和群落为中心的教育材料，并通过现有的大学合作伙伴关系、研讨会和夏令营与当地HS教师和学生分享。该提案将确定细胞外DNA （eDNA）与细菌表面结构相互作用的新机制，并提供这些相互作用的详细分子描述，以了解eDNA如何被纳入以及蛋白质-DNA相互作用如何稳定生物膜。多种细胞外DNA受体的鉴定和表征将突出生物膜稳定的独特机制，潜在地允许开发针对特定细菌的干预措施。该项目将识别和表征新的DNA受体亚基，这些亚基结合到已知的附着在eDNA上的细菌表面附属物中，包括那些来自革兰氏阳性（梭状芽胞杆菌）和革兰氏阴性（不动杆菌）细菌物种。这项工作的目标包括纯化可溶性蛋白结构，测量它们的DNA结合亲和力，并通过x射线晶体学确定识别的结构机制。该项目的这一方面的目标是通过这些新的受体创建DNA识别的原子描述。此外，该项目将研究这些DNA受体如何通过分离天然表达的纤维和用可溶性蛋白质复合物重组来整合到蛋白质纤维中。该项目由生物科学理事会分子和细胞生物科学部的分子生物物理学计划和促进竞争性研究的既定计划（EPSCoR）共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.