RpoS and gene expression in Borrelia burgdorferi

Tulane University Of Louisiana, New Orleans LA

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Abstract

DESCRIPTION (provided by applicant): Borrelia burgdorferi, the etiologic agent of Lyme disease, cycles in nature between two evolutionarily diverse hosts, an invertebrate tick vector and a vertebrate host. The proliferation and maintenance of the spirochete in the two hosts is guided by several adaptive strategies involving the shuffling of proteins in response to changing environmental conditions. In this respect, molecular changes in the spirochete associated with tick feeding is of particular significance as they precede vertebrate infection. Tick feeding results in a dramatic burst in the spirochetal population, with accompanying changes in protein composition. These changes can be duplicated in vitro by shifting a spirochetal culture to a higher temperature and lower pH environment. One of the key proteins induced under these conditions is the transcription factor RpoS. Our long-term goal is to investigate the molecular events triggered in B. burgdorferi in response to changes in environmental temperature and pH. Our short-term objective is to exploit the in vitro model to develop a better understanding of this phenomenon. Specifically, the goals of this proposal are to (1) investigate the mechanism of regulation of RpoS, (2) identify chromosomal RpoS-regulated genes, (3) characterize the role of RpoS in the regulation of the chromosomal RpoS-regulated genes in vivo. The regulation of RpoS expression will be examined, as we and others have determined that its own expression is responsive to changes in temperature and pH. We have identified a putative DNA-binding protein by electromobility shift assay that specifically binds to the rpoS 5' sequence. Initially, a response regulator protein RRP-2 will be examined as a putative candidate, failing which the unknown protein will be screened for and identified. The role of this DNA-binding protein in the expression of RpoS will be defined in vitro. We will also identify new chromosomal RpoS-regulated proteins by an in vitro screening assay. This assay will involve in vitro transcription using recombinant B. burgdorferi RNA polymerases loaded with sigma D or sigma S. Finally, the regulation of expression of these newly identified genes by RpoS will be partially characterized in vivo. These studies will shed light on the mechanism guiding the flow of genetic information, and identify new effector proteins, in Lyme disease spirochetes poised to infect the vertebrate host.