ABSTRACT Spirochetes are a phylogenetically distinct group of bacteria that are of significant importance in human health as they cause major diseases such as syphilis (Treponema pallidum), Lyme disease (Borrelia burgdorferi), leptospirosis (Leptospira interrogans), and periodontitis (Treponema spp.). To infect and disseminate in mammalian hosts, spirochetes have evolved a unique morphology and motility that is highly effective at translocating through viscous media and tissue barriers. The organelles essential for spirochetal motility are periplasmic flagella, which reside in the bacterial periplasmic space and are distinct from the external flagella in the model systems Escherichia coli and Salmonella enterica. Given that flagella-driven motility is crucial for virulence of pathogenic spirochetes and many other bacteria, our long-term goal is to understand molecular mechanisms underlying flagellar assembly and function. During the previous funding period, we have demonstrated that the Lyme disease spirochete B. burgdorferi (Bb) is a great model system for characterizing periplasmic flagella in situ at an unprecedented resolution. In collaboration with Drs. Md Motaleb and Chunhao Li, we have generated and characterized a large Bb library including 60 different flagellar and chemotaxis mutants. Significant progress has been made in understanding the periplasmic flagella and their remarkable capacity in driving the unique spirochetal motility and morphology. The objective of this application is to illuminate three fundamental but challenging aspects of the periplasmic flagella: 1) the structure and function of the flagellar type III secretion apparatus; 2) the mechanism underlying the flagellar rotation driven by proton motive force across membrane; and 3) the mechanisms by which flagella switch rotational directions to control the motility and chemotaxis. Together with genetic and biochemical approaches, cryo-ET will be utilized to determine the structure/function relationship of the spirochetal flagellar motor in a native cellular environment at nanometer resolution.