ABSTRACTThe Lyme disease spirocheteBorrelia burgdorferimigrates to distant sites in the tick vectors and mammalian hosts through robust motility and chemotaxis activities. FliH and FliI are two cytoplasmic proteins that play important roles in the type III secretion system (T3SS)-mediated export and assembly of flagellar structural proteins. However, detailed analyses of the roles of FliH and FliI inB. burgdorferihave not been reported. In this study,fliHandfliItransposon mutants were utilized to dissect the mechanism of theBorreliatype III secretion system. ThefliHandfliImutants exhibited rod-shaped or string-like morphology, greatly reduced motility, division defects (resulting in elongated organisms with incomplete division points), and noninfectivity in mice by needle inoculation. Mutants infliHandfliIwere incapable of translational motion in 1% methylcellulose or soft agar. Inactivation of eitherfliHorfliIresulted in the loss of the FliH-FliI complex from otherwise intact flagellar motors, as determined by cryo-electron tomography (cryo-ET). Flagellar assemblies were still present in the mutant cells, albeit in lower numbers than in wild-type cells and with truncated flagella. Genetic complementation offliHandfliImutants intransrestored their wild-type morphology, motility, and flagellar motor structure; however, full-length flagella and infectivity were not recovered in these complemented mutants. Based on these results, disruption of eitherfliHorfliIinB. burgdorferiresults in a severe defect in flagellar structure and function and cell division but does not completely block the export and assembly of flagellar hook and filament proteins.IMPORTANCEMany bacteria are able to rapidly transport themselves through their surroundings using specialized organelles called flagella. In spiral-shaped organisms called spirochetes, flagella act like inboard motors and give the bacteria the ability to bore their way through dense materials (such as human tissue) in a corkscrew manner. In this article, we studied how two proteins, called FliH and FliI, are important for the production of full-length flagella in the Lyme disease spirocheteBorrelia burgdorferi. Mutants with defective production of FliH and FliI have reduced flagellar length and motility; this deficiency in turn affects many aspects ofB. burgdorferi's biology, including the ability to undergo cell division and cause disease in mammals. Using a microscopic computed tomography (CT) scan approach called cryo-electron tomography, the structure that contains FliH and FliI was defined in the context of the flagellar motor, providing clues regarding how this amazing nanomachine is assembled and functions.