ABSTRACT Stringent response is a physiological mechanism utilized by bacteria to adapt to nutrient deprivation and many other stresses. In Borrelia burgdorferi (Bb), the etiological agent of Lyme disease, stringent response is mediated by both the alarmone nucleotides (p)ppGpp and the RNA polymerase-binding protein DksA. Recently, we found that DksA plays an essential role in regulating Bb's virulence. First, dksA is highly expressed in infected animals and is required by Bb to infect the mammalian hosts. Second, DksA is critical for Bb's optimal growth. Third, DksA is required by Bb to cope with metabolic stress. Finally, in addition to function as a stringent response regulator, DksA promotes the transcription and/or translation of genes contributing to Bb's survival in the host. These combined data give rise to our hypothesis that DksA is a master regulator governing Bb's virulence gene expression. This hypothesis will be addressed in two Specific Aims. In Aim 1 of this proposal, we will determine the contribution of DksA to Bb's survival and persistence in ticks. In Aim 2, we will identify genes regulated by DksA under various in vitro and in vivo conditions. These combined studies will (i) define the entire DksA regulon; and (ii) provide a transformative understanding of the in vivo importance of DksA and the regulon. Resultant findings could lead to the development of new strategies to prevent and/or treat Lyme disease.