Pathogenic spirochetal bacteria cause serious illnesses—Syphilis, Leptospirosis, Periodontal disease, and Lyme disease—throughout the world. Despite these debilitating diseases, spirochetes remain poorly studied. Spirochete-specific research is crucial and urgently needed for the development of new treatment modalities. Spirochetes are well recognizable due to their distinctive spiral or wave-like morphology and cork-screw type motility. The organelles essential for the unique morphology and motility of the Lyme disease spirochete Borrelia burgdorferi are the periplasmic flagella, which are enclosed in the periplasmic space and are distinct in assembly, structure, and function from external flagella in the model organisms Escherichia coli and Salmonella enterica. Our goal is to understand the molecular mechanisms underlying the assembly and function of the unique periplasmic flagellar proteins and their specific roles in spirochetal motility. Since motility is crucial for pathogenic spirochetes to cause diseases in hosts, the objectives of the proposal are to, demonstrate novel mechanistic insights into how spirochete-specific collar proteins help recruit and stabilize sixteen stator complexes to generate high torque; discover and characterize novel flagellar proteins to gain a mechanistic understanding of the unique collar architecture and its impacts on the assembly of periplasmic flagella and motility of spirochetes; and to elucidate molecular mechanisms of how the collar proteins provide solid bearing support to the rotary periplasmic flagella, and how they impact the assembly and orientation of the flagella. These unique aspects of the periplasmic flagella are poorly studied. Three Aims are proposed to better understand those objectives in Lyme disease spirochete B. burgdorferi. Aim 1 is to elucidate how the flagellar collar proteins help recruit and stabilize the stator required for spirochetal motility. Aim 2 is to discover and characterize novel flagellar proteins in spirochetes and their impacts on the motility of spirochetes and Aim 3 is to elucidate molecular mechanisms of periplasmic flagellar orientation in Borrelia burgdorferi. State-of-the- art technologies including bioinformatics, genetic manipulations, microbiological, biochemical, and high resolution cryo-electron tomography will be utilized to accomplish the proposed aims. The project is expected to better understand the mechanisms of flagellar assembly and how they impact the very critical motility function of the spirochetes. Knowledge obtained from this project can directly be applied to other pathogenic spirochetes and diverse flagellated bacteria. Structure-based drug design targeting flagellar subunits present an effective prevention strategy because motility is critical for pathogenic spirochetes to produce disease.