BadR directly represses the expression of the glycerol utilization operon in the Lyme disease pathogen.

Glycerol utilization as a carbohydrate source by , the Lyme disease spirochete, is critical for its successful colonization and persistence in the tick vector. The expression of the () operon, which encodes proteins for glycerol uptake/utilization, must be tightly regulated during the enzootic cycle of . Previous studies have established that the second messenger cyclic di-GMP (c-di-GMP) is required for the activation of expression, while an alternative sigma factor RpoS acts as a negative regulator for expression. In the present study, we report identification of a element within the 5´ untranslated region of that exerts negative regulation of expression. Further genetic screen of known and predicted DNA-binding proteins encoded in the genome of uncovered that overexpressing host adaptation regulator (BadR), a known global regulator, dramatically reduced expression. Similarly, the mutant significantly increased expression. Subsequent electrophoretic mobility shift assay analyses demonstrated that BadR directly binds to this element, thereby repressing independent of RpoS-mediated repression. The efficiency of BadR binding was further assessed in the presence of c-di-GMP and various carbohydrates. This finding highlights multi-layered positive and negative regulatory mechanisms employed by to synchronize expression throughout its enzootic cycle.IMPORTANCE, the Lyme disease pathogen, must modulate its gene expression differentially to adapt successfully to its two disparate hosts. Previous studies have demonstrated that the glycerol uptake and utilization operon, , plays a crucial role in spirochetal survival within ticks. However, the expression must be repressed when transitions to the mammalian host. In this study, we identified a specific element responsible for the repression of . We further pinpointed host adaptation regulator as the direct binding protein to this element, thereby repressing expression. This discovery paves the way for a deeper exploration of how zoonotic pathogens sense distinct hosts and switch their carbon source utilization during transmission.