Borrelia burgdorferi is a pathogenic spirochete and the etiological agent of Lyme disease. Spirochetes are defined by unique periplasmic flagella and distinctive flat wave morphology. B. burgdorferi’s flagellar synthesis and motility must be coordinated with cell division, growth, and cell-shape; however, little is known about the overlap and regulation of these cellular processes. We identified a new conserved family of helix-turn-helix containing proteins across Spirochaetes, Firmicutes, the PVC clade and other phyla, that coordinate motility and cell division in B. burgdorferi. Importantly, motility and cell division must also be tightly regulated during infection, such as when B. burgdorferi is transmitted to mammalian hosts through the bite of an infected Ixodes scapularis tick. Yet, low numbers of B. burgdorferi have been a barrier to characterizing gene expression during infection. We utilized the MATQ-seq workflow to amplify and sequence RNA from both bulk (culture pellet) and individual B. burgdorferi at log, stationary, and prolonged stationary phase. Collectively, our work has uncovered abundant transcriptional heterogeneity of B.burgdorferi grown in culture and provide a foundation to characterize gene expression of a single bacterium during infection.