NSF-ANR MCB/PHY: The Bacteroidetes mechanism of gliding motility

For most organisms, motility provides key adaptive advantages to exploit available resources. Gliding is a smooth motion of individual bacterial cells on a solid surface that does not involve obvious appendages like flagella or pili, yet can rely on different mechanisms. In the model gliding bacteria Flavobacterium johnsoniae, gliding relies on highly dynamic molecular machineries composed of unique proteins. This project is to decipher the composition, architecture, and mechanism of the gliding machinery with a multidisciplinary approach. Graduate and postdoctoral fellows will actively participate in the project and will receive training in interdisciplinary research involving bacterial genetics, molecular biology, modern fluorescence microscopy and cryogenic electron microscopy. They will also receive training in academic writing and research presentations. The results of this research will be of interest to a large community of scientists studying cell motility and cell envelope processes and will be published in the best possible journals. The findings will also be reported by students and postdoctoral fellows at national and international conferences and in academic seminars. The results and data generated from this project will be incorporated into lectures for a cryogenic electron microscopy course designed for graduate students. The mechanism of the bacterial gliding motility is not fully understood. The goal of this project is to decipher the global composition, architecture and dynamics of the gliding machinery with a multidisciplinary approach heavily relying on cutting-edge cryo-electron tomography (Cryo-ET), single particle cryo-electron microscopy (Cryo-EM) and single-molecule fluorescence microscopy methods combined with protein biochemistry, bacterial genetics and bioinformatics. This project will address two specific aims: 1) Defining the gliding machinery and its dynamics in live cells. In this aim, a global picture of the gliding machinery will be determined by in situ APEX2-dependent proximity labeling. Then, the dynamics of the gliding machinery components will be determined by biochemistry and fluorescence microscopy approaches. 2) Mapping the architecture of the gliding machinery. The objective of this aim is to obtain the three-dimensional architecture of the gliding machinery by cryo-electron microscopy. Cryo-ET, an imaging technique to analyze the structure of biological assemblies in their native environments, will be used to determine the intact structure of the gliding machinery at nanometer resolution by imaging the bacterial cells. Single particle Cryo-EM is ideal for investigating purified protein complexes at near-atomic resolution, and this approach will be used to determine the structure of the purified subcomplex of gliding machinery. Completion of these aims will provide valuable insight on how proteins are organized to enable gliding motility. This collaborative US/France project is jointly supported by the Cellular Dynamics and Function program in the Division of Molecular and Cellular Biosciences and the Physics of Living Systems program in the Division of Physics at the US National Science Foundation and the French Agence Nationale de la Recherche, where NSF funds the US investigator and ANR funds the partners in France. 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.