Sequence determinants spanning -35 motif and AT-rich spacer region impacting Ehrlichia chaffeensis Sigma 70-dependent promoter activity of two differentially expressed p28 outer membrane protein genes.

Abstract

Ehrlichia chaffeensis is an obligate intracellular tick-borne bacterium which causes the disease, human monocytic ehrlichiosis. Ehrlichia chaffeensis contains only two sigma factors, σ32 and σ70. It is difficult to study E. chaffeensis gene regulation due to lack of a transformation system. We developed an Escherichia coli-based transcription system to study E. chaffeensis transcriptional regulation. An E. coli strain with its σ70 repressed with trp promoter is used to express E. chaffeensis σ70. The E. coli system and our previously established in vitro transcription system were used to map transcriptional differences of two Ehrlichia genes encoding p28-outer membrane proteins 14 and 19. We mapped the -10 and -35 motifs and the AT rich spacers located between the two motifs by performing detailed mutational analysis. Mutations within the -35 motif of the genes impacted transcription differently, while -10 motif deletions had no impact. The AT-rich spacers also contributed to transcriptional differences. We further demonstrated that the domain 4.2 of E. chaffeensis σ70 is important for regulating promoter activity and the deletion of region 1.1 of E. chaffeensis σ70 causes enhancement of the promoter activity. This is the first study defining the promoters of two closely related E. chaffeensis genes.