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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.

frontiers in microbiology

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