Sequence Determinants Spanning −10 Motif and Spacer Region Implicated in Unique Ehrlichia chaffeensis Sigma 32-Dependent Promoter Activity of dnaK Gene

Ehrlichia chaffeensis is an obligate intracellular tick-borne bacterium that causes human monocytic ehrlichiosis. Studying Ehrlichia gene regulation is challenge, as this and related rickettsiales lack natural plasmids and mutagenesis experiments are of a limited scope. E. chaffeensis contains only two sigma factors, σ(32) and σ(70). We previously developed Escherichia coli surrogate system to study transcriptional regulation from RNA polymerase (RNAP) containing Ehrlichia σ(32) or σ(70). We reported that RNAP binding motifs of E. chaffeensis genes recognized by σ(32) or σ(70) share extensive homology and that transcription may be initiated by either one of the sigma factors, although transcriptional efficiencies differ. In the current study, we investigated mapping the E. chaffeensis dnaK gene promoter using the pathogen σ(32) expressed in E. coli lacking its native σ(32). The E. coli surrogate system and our previously described in vitro transcription system aided in defining the unique −10 motif and spacer sequence of the dnaK promoter. We also mapped σ(32) amino acids/domains engaged in its promoter regulation in E. chaffeensis. The data reported in this study demonstrate that the −10 and −35 motifs and spacer sequence located between the two motifs of dnaK promoter are critical for the RNAP function. Further, we mapped the importance of all six nucleotide positions of the −10 motif and identified critical determinants within it. In addition, we reported that the lack of C-rich sequence upstream to the −10 motif is unique in driving the pathogen-specific transcription by its σ(32) from dnaK gene promoter. This is the first study in defining an E. chaffeensis σ(32)-dependent promoter and it offers insights about how this and other related rickettsial pathogens regulate stress response genes.