Our broad, long-term objective is to understand the molecular biology of Chlamydia with the goal of using acquired information for new therapeutic and prophylactic interventions and improved diagnostic and preventive strategies. Chlamydiae have been exceedingly difficult organisms to study from a biological or molecular genetic perspective. First, because they can only be grown within eukaryotic host cells, little is known concerning the basic biochemical and physiological pathways. Second, most chlamydial gene promoters are not efficiently recognized by E. coli transcriptional machinery. Third, there has not been a genetic system developed, wherein DNA can be stably introduced into these organisms. Moreover, even the selection of chlamydial mutants has not offered a general genetic approach because of their limited propagation in tissue culture, dependence upon intracellular growth and a single developmental form required for infection of mammalian cells. The lack of the ability to utilize direct genetic systems has effectively prevented an essential and powerful experimental approach from the repertoire of chlamydial research. The availability of the complete genome sequences for C. trachomatis and C. pneumoniae mitigate many of these technical research challenges because this information can be used to markedly facilitate experimental investigations. Our hypothesis is that the microbiologically unique and virulence-defining chlamydial developmental cycle is regulated at the level of transcription by signal transduction response regulators and other regulators of transcription. During the requested period of support we will test this hypothesis by: I. Map mRNA 5'-ends of developmentally regulated genes and operons. Based on transcriptional array data and prediction of operons, we will determine the 5' ends of mRNA for developmentally regulated genes or operons. This information will provide a database of chlamydial promoters and transcriptional regulatory regions required for analyzing mechanisms of developmental regulation. II. Identify and characterize genes regulated by two-component signal transduction response regulators and DNA-binding proteins. We identified only two signal transduction response regulators in the chlamydial genome, CtcB-CtcC, analogs of the NtrB-NtrC-family of response regulators and CpxR, an analog of OmpR. We will test which chlamydial genes are regulated by genome-wide array analysis for binding DNA of intergenic promoter regions. Mechanisms of gene regulation will be expanded by testing the intergenic arrays for proteins present in RB and EB that bind DNA and likely regulate transcription. III. Surrogate models of transcriptional regulation in E. coli. Based upon the information obtained from Aims 1 & 2, we will model chlamydial gene regulation in E. coli and test specific hypotheses of gene regulatory function. ? ?
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