Isolation and characterization of Ehrlichia chaffeensis RNA polymerase and its use in evaluating p28 outer membrane protein gene promoters

In an effort to understand the molecular basis of pathogen gene expression differences, we isolated native E. chaffeensis RNA polymerase using a heparin-agarose purification method and developed an in vitro transcription system to map promoter regions of two differentially expressed genes of the p28 outer membrane protein locus, p28-Omp14 and p28-Omp19. We also prepared a recombinant protein of E. chaffeensis σ70 homologue and used it for in vitro promoter analysis studies. The possible role of one or more proteins presents in E. chaffeensis lysates in binding to the promoter segments and on the modulation of in vitro transcription was also assessed.Our experiments demonstrated that both the native and recombinant proteins are functional and have similar enzyme properties in driving the transcription from E. chaffeensis promoters. This is the first report of the functional characterization of E. chaffeensis RNA polymerase and in vitro mapping of the pathogen promoters using the enzyme. This study marks the beginning to broadly characterize the mechanisms controlling the transcription by Anaplasmataceae pathogens.Ehrlichia chaffeensis is an obligate intracellular rickettsial pathogen and the causative agent of an important emerging zoonotic disease, human monocytic ehrlichiosis [1-4]. This Amblyomma americanum tick-transmitted pathogen causes infections in susceptible hosts (humans), host reservoirs (white-tailed deer), and less well described hosts such as the dog, goat and coyote [5-10]. E. chaffeensis has an unusual developmental cycle that requires growth and replication within eukaryotic cells of vertebrate and tick hosts [11]. During its developmental cycle, there is conversion between two distinct morphological forms, the elementary bodies (EBs) and reticulate bodies (RBs) [12,13]. The EBs are the infectious form and upon entry into a host cell, they differentiate into metabolically active reticulate bodies (RBs), which are larger compared to EBs and divide by