chaffeensis. The current study provides the first evidence suggesting that E. chaffeensis whole-cell protein lysates contain regulatory proteins which modulate transcription of p28-Omp14 and p28-Omp19 promoters Selleckchem Y 27632 in vitro. In support of further testing the hypothesis that E. chaffeensis whole-cell protein lysates contain proteins that bind to putative regulatory DNA sequences of these promoters, EMSA experiments were performed. A shift in mobility of DNA fragments was observed for several partial or complete DNA segments of the promoter regions of both p28-Omp14 and p28-Omp19 genes. These data suggest that the promoter region contained regulatory DNA

sequences that allowed binding of one or more E. chaffeensis proteins. The binding was specific as the addition of specific competitors considerably reduced the shift and the addition of a non-specific protein did not cause a shift. The binding of E. chaffeensis regulatory proteins to the DNA segments spanning putative DNA binding elements is consistent with previous studies on this organism [49] as well as in several other bacteria, including Anaplasma phagocytophilum [50–52], C. trachomatis [34, 35]and B. subtilis [53, 54]in which interaction of regulatory proteins with regulatory sequences have been demonstrated. The identity of DNA binding proteins and the location of protein binding sites remain

to be determined. Conclusions In this study, we developed in vitro transcription assays using a G-less cassette and described this website methods to isolate native

RNAP and the recombinant RNAP σ70 subunit of E. chaffeensis. The value of using these tools in evaluating the promoters of two differentially expressed genes has been demonstrated. The application of these tools to the study of E. chaffeensis is new and important for furthering our understanding of the regulation of gene expression in this pathogen. Specifically, the tools will be valuable in studies to map specific interactions of E. chaffeensis proteins in driving differential gene expression influenced by vertebrate and tick host cell environments. This is the first report of in vitro transcription using native E. chaffeensis RNAP and E. coli RNAP core enzyme reconstituted with the Acyl CoA dehydrogenase recombinant E. chaffeensis σ70 subunit. This study marks the beginning of a greater effort to broadly characterize the mechanisms that control the transcription in Anaplasmataceae pathogens in support of their growth in vertebrate and tick hosts. Methods PCR conditions PCRs for amplification of E. chaffeensis p28-Omp14 and p28-Omp19 promoters were carried out in a 25 μl reaction volume containing 0.2 μM of each primer, 250 ng of purified E. chaffeensis (Arkansas isolate) genomic DNA, 400 μM of each of the four deoxyribonucleoside triphosphates, 1.5 mM MgSO4, 1x native HiFi PCR buffer (60 mM Tris-SO4, 18 mM (NH4)2SO4), 2.