Project Summary/Abstract Approximately 300,000 cases of Lyme disease are diagnosed each year in the United States. Lyme is caused by the spirochete Borrelia burgdorferi, a microorganism that cycles in nature between ticks and vertebrate hosts. Patterns of gene expression change dramatically as this bacteria navigates the different environments in its different hosts. Gene expression levels may be determined by the spatial arrangement of RNA transcripts in the bacterial cells. We propose to study this relationship in B. burgdorferi. B. burgdorferi cells are very long but incredibly narrow: less than ~200 nm in diameter. The narrow width limits the application of conventional fluorescence microscopy to study subcellular spatial arrangements of transcripts within these cells. The phenomenon of diffraction limits the resolution of a conventional microscope to approximately half the wavelength of light used, thereby obscuring biological structure and process occurring on length scales smaller than ~250 nm. Fortunately, the recent emergence of fluorescence nanoscopy techniques now provide near molecular-level resolution and allows the study of biological systems at the nanoscale. The overall aim of our work is to determine the spatial arrangement of a diverse set of RNA transcripts in B. burgdorferi cells. To test the hypothesize that RNA localization plays a role in the expression and turnover of transcripts in B. burgdorferi, including those that are involved in its virulence and pathogenicity, we propose to use fluorescence in situ hybridization (FISH) and STED nanoscopy to characterize the spatial arrangement of diverse transcripts encoding gene products that localize in different parts of the cell, have different turnover rates, and have diverse functions. Characterization of a set of diverse transcripts and advances to a custom built nanoscope to increase three dimensional resolution and multicolor imaging are proposed. Our elucidation of the localization patterns of these transcripts will increase the general understanding of the roles of RNA localization in gene expression and RNA turnover in bacteria and provide insight into the role RNA localization may play in the enzootic cycle of this pathogen.