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<p>Abstract</p> <p>Background</p> <p><it>Ehrlichia chaffeensis </it>is a tick-transmitted rickettsial pathogen responsible for an important emerging disease, human monocytic ehrlichiosis. To date how <it>E. chaffeen</it>sis and many related tick-borne rickettsial pathogens adapt and persist in vertebrate and tick hosts remain largely unknown. In recent studies, we demonstrated significant host-specific differences in protein expression in <it>E. chaffeensis </it>originating from its tick and vertebrate host cells. The adaptive response of the pathogen to different host environments entails switch of gene expression regulated at the level of transcription, possibly by altering RNA polymerase activity.</p> <p>Results</p> <p>In an effort to understand the molecular basis of pathogen gene expression differences, we isolated native <it>E. chaffeensis </it>RNA polymerase using a heparin-agarose purification method and developed an <it>in vitro </it>transcription system to map promoter regions of two differentially expressed genes of the p28 outer membrane protein locus, <it>p28-Omp14 </it>and <it>p28-Omp19</it>. We also prepared a recombinant protein of <it>E. chaffeensis </it>σ70 homologue and used it for <it>in vitro </it>promoter analysis studies. The possible role of one or more proteins presents in <it>E. chaffeensis </it>lysates in binding to the promoter segments and on the modulation of <it>in vitro </it>transcription was also assessed.</p> <p>Conclusions</p> <p>Our experiments demonstrated that both the native and recombinant proteins are functional and have similar enzyme properties in driving the transcription from <it>E. chaffeensis </it>promoters. This is the first report of the functional characterization of <it>E. chaffeensis </it>RNA polymerase and <it>in vitro </it>mapping of the pathogen promoters using the enzyme. This study marks the beginning to broadly characterize the mechanisms controlling the transcription by <it>Anaplasmataceae </it>pathogens.</p>