The agent of Lyme disease or Lyme borreliosis, Borrelia burgdorferi, yields more arthropod-borne infections than any other pathogen in the United States with over 300,000 cases diagnosed each year. In areas where B. burgdorferi infections are prevalent, morbidity associated with this infection is high and is seen mostly in th form of cardiac, neurologic, or arthritic sequelae. Although B. burgdorferi has a simplistic genome relative to most living systems, it is adept at altering its gene regulatory pattern as the pathogen moves from the arthropod vector and mammalian species it can occupy during its lifecycle. Although we understand the role of several transcriptional regulators in this adaptive response, there are additional layers of regulation for some borrelial genes (e.g., bosR and dbpA) that have not been elucidated. Given their emergence as important regulatory effectors in many bacterial systems, including a linkage to virulence gene expression and protein production in a number of pathogens, we hypothesized that small, non-coding RNAs (sRNAs) were required for similar regulatory functions in B. burgdorferi. Using a collaborative approach, the B. burgdorferi Tn library coupled with Tn-seq, as well as RNA-seq technologies, identified 14 putative sRNA mutants that were attenuated following experimental infection. These same sites encoded sRNA previously identified via RNA- seq of total transcript profiles as well as libraries specific for sRNAs. Subsequent infections with individual Tn mutants at these sites indicated that 4 of the mutants were impaired in their ability to colonize mice. This proposal is designed to establish a link between the sRNAs and their role in regulating gene expression and/or protein production via alteration of target mRNAs. To address this we propose the following Specific Aims: (1) Link sRNA species to borrelial infectivity. Precise mutants, designed to eliminate specific sRNAs, will be tested to determine their role in borrelial infectivity in vivo in both a spatial and temporal manner; and (2) Determine the global regulatory effect of select borrelial sRNA depletion. The effect of the loss of specific sRNAs will be tracked using global transcript and protein profiles to determine how the sRNA affects transcription or translation of unlinked genes. The work outlined herein provides a link between sRNA regulatory pathways and B. burgdorferi infection. To date, this connection has not analyzed in depth and, as such, represents an opportunity to determine how sRNA modulate either transcriptional or post- transcriptional regulatory pathways that impact borrelial physiology and pathogenesis.