AUTHOR=Liu Huitao , Ganta Roman R. 

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

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.01772

DOI=10.3389/fmicb.2019.01772

ISSN=1664-302X

ABSTRACT=<p><italic>Ehrlichia chaffeensis</italic> is an obligate intracellular tick-borne bacterium that causes human monocytic ehrlichiosis. Studying <italic>Ehrlichia</italic> gene regulation is challenge, as this and related rickettsiales lack natural plasmids and mutagenesis experiments are of a limited scope. <italic>E. chaffeensis</italic> contains only two sigma factors, σ<sup>32</sup> and σ<sup>70</sup>. We previously developed <italic>Escherichia coli</italic> surrogate system to study transcriptional regulation from RNA polymerase (RNAP) containing <italic>Ehrlichia</italic> σ<sup>32</sup> or σ<sup>70</sup>. We reported that RNAP binding motifs of <italic>E. chaffeensis</italic> genes recognized by σ<sup>32</sup> or σ<sup>70</sup> 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 <italic>E. chaffeensis dnaK</italic> gene promoter using the pathogen σ<sup>32</sup> expressed in <italic>E. coli</italic> lacking its native σ<sup>32</sup>. The <italic>E. coli</italic> surrogate system and our previously described <italic>in vitro</italic> transcription system aided in defining the unique −10 motif and spacer sequence of the <italic>dnaK</italic> promoter. We also mapped σ<sup>32</sup> amino acids/domains engaged in its promoter regulation in <italic>E. chaffeensis</italic>. The data reported in this study demonstrate that the −10 and −35 motifs and spacer sequence located between the two motifs of <italic>dnaK</italic> 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<sup>32</sup> from <italic>dnaK</italic> gene promoter. This is the first study in defining an <italic>E. chaffeensis</italic> σ<sup>32</sup>-dependent promoter and it offers insights about how this and other related rickettsial pathogens regulate stress response genes.</p>