Spirochetes are a group of poorly studied medically significant bacteria. The motility of spirochetes is driven by periplasmic flagella, which reside and rotate within the periplasmic space. These bacterial motility is crucial for virulence by all pathogenic spirochetes studied to-date including Borrelia burgdorferi. While motility plays such a vital role, we know very little about the periplasmic flagellar assembly, operation or organization in any spirochete. Although many components of the periplasmic flagellum have highly conserved counterparts in the external flagella from the model organisms Salmonella enterica and Escherichia coli, some unique components of the periplasmic flagella clearly distinguish them from external flagella. Most importantly, our recent studies have provided the first evidence that the novel spirochete-specific component known as the periplasmic collar is essential for flagellar assembly and orientation, the distinctive morphology and motility of these bacteria. However, very little is known about the genes encoding the periplasmic collar or their function in any spirochete. Based on our preliminary data, we hypothesize that the periplasmic collar is comprised of multiple novel spirochete-specific proteins, and that each of these proteins plays a distinct role in flagellar assembly, spirochetes distinctive morphology and motility. To address this hypothesis, we propose two specific aims. Aim 1 is to identify the proteins that make-up the large collar complex, determine their native cellular structure and function in B. burgdorferi. Moreover, to extend the relevance of B. burgdorferi periplasmic collar studies, we propose to determine if these novel flagellar proteins or their function is conserved in other spirochetes. Aim 1 is expected to be accomplished by using bioinformatics, genetics, various biochemical assays and cryo- electron tomography. We expect to identify the proteins encoding the collar complex structure, their function, location, structure, and assembly in the spirochetes. Aim 2 is proposed to determine the sequential assembly of the periplasmic collar proteins in the cell envelope and to understand the interactions of novel collar proteins with other prominent flagellar proteins and their impacts on increased torque required for the periplasmic flagella to rotate in viscous or complex medium such as the mammalian tissues. We plan to accomplish this aim using various mutational, biochemical, and cryo-electron tomography. We expect to understand how the periplasmic flagella are assembled in the spirochete and their overall impacts in B. burgdorferi. The knowledge gained from this project is fundamental to understand the biosynthesis, assembly, and function of the novel flagellar proteins not only in B. burgdorferi and Leptospira but also in other medically significant yet uncultivable spirochetes such as Treponema pallidum. These studies can also lead to applications in structure- based drug design to di...