Motility and chemotaxis are essential components of pathogenesis for many infectious bacteria, including Borrelia burgdorferi, the causative agent of
Lyme disease. Motility and chemotaxis genes comprise 5 to 6% of the genome of B. burgdorferi, yet the functions of most of those genes remain uncharacterized, mainly due to the paucity of a nonpolar gene inactivation system. In this communication, we describe the development of a novel gene inactivation methodology to target B. burgdorferi fliL, a putative periplasmic flagellar gene located in a large motility operon and transcribed by RNA polymerase containing ?(70). Although the morphology of nonpolar fliL mutant cells was indistinguishable from that of wild-type cells, the mutant exhibited a defective-motility phenotype. Cryo-electron tomography (cryo-ET) of intact organisms revealed that the periplasmic flagella in the fliL mutant were frequently tilted toward the cell pole instead of their normal orientation toward the cell body. These defects were corrected when the mutant was complemented in cis. Moreover, a comparative analysis of flagellar motors from the wild type and the mutant provides the first structural evidence that FliL is localized between the stator and rotor. Our results suggest that FliL is likely involved in coordinating or regulating the orientation of periplasmic flagella in B. burgdorferi.