The bacterium Borrelia burgdorferi causes Lyme disease. Transmission of B. burgdorferi from its tick vector to a mammalian host and infection of the mammal require changes in the expression of a suite of genes required for transmission and infection. The change in the program of gene expression is regulated in response to environmental signals associated with tick feeding, including increased temperature. The most dramatic change in gene expression is the reciprocal regulation of the major outer surface lipoproteins, including OspC, which is required for transmission and infection. The induction of ospC gene expression is dependent on the alternative sigma factor RpoS (C38 or CS). The central hypothesis of this application is that RpoS is the key regulator of the enzootic cycle and thus serves as the target of multiple signals through several regulatory mechanisms. The specific hypotheses are that RpoS and OspC syntheses are positively and negatively regulated through several trans-acting mechanisms, including translational control by a small regulatory RNA in response to temperature and post-translational degradation by a protease. The regulation is also hypothesized to be regulated through cis-acting DNA supercoiling in response to temperature. The long-term objective of this proposal is to understand the function and regulation of OspC in transmission of B. burgdorferi and pathogenesis of Lyme disease, which will lead to improved diagnostic, prevention, and treatment strategies because expression of OspC is required for B. burgdorferi to cause Lyme disease; this is relevant to the mission of the agency to pursue fundamental knowledge for the sake of alleviating human disease. The following specific aims are proposed toward achieving this objective: 1) identify and characterize factors that repress RpoS and/or OspC syntheses; 2) identify and characterize factors that induce RpoS and/or OspC syntheses; and 3) define the temporal requirement for OspC in transmission and determine the role of RpoS in regulating OspC during mammalian infection. Genetic, biochemical, molecular, transcriptomic, and proteomic approaches will be utilized to test these hypotheses. Specifically, genes encoding regulatory factors will be disrupted and/or fused to the new inducible promoter to assay function both in culture and in a tick-mouse model, reporter fusions will be constructed to quantitatively assay gene expression, the regulatory mechanism of the small RNA will be probed by RNA binding experiments, and the global effect of DNA supercoiling on gene expression will be determined by microarray and protein identification.