The sensor kinase QseC regulates the unlinked PmrA response regulator and downstream gene expression in Francisella

The facultative intracellular bacterial pathogen Francisella tularensis is the causative agent of tularemia in humans and animals. Gram negative bacteria utilize two-component regulatory systems (TCS) to sense and respond to their changing environment. No classical, tandemly arranged sensor kinase and response regulator TCS genes exist in the human virulent Francisella tularensis subsp. tularensis, but orphaned members are present. PmrA is an orphan response regulator responsible for intramacrophage growth and virulence; however, the regulation of PmrA activity is not understood. We and others have shown that PmrA represses the expression of priM, described to encode an anti-virulence determinant. By screening a mutant library for increased priM promoter activity, we identified the sensor kinase homolog QseC as an upstream regulator of priM expression, and this regulation is in part, dependent upon the aspartate phosphorylation site of PmrA (D51). PmrA directly binds to the promoter of priM and a PmrAD51A mutation decreases binding. Several examined environmental signals including epinephrine, which is reported to activate QseC in other bacteria, do not affect priM expression in a manner dependent on PmrA. Intramacrophage survival assays also question the finding that PriM is an anti-virulence factor. Thus, these data suggest that the PmrA-regulated gene priM is regulated by an uncoupled TCS QseC-PmrA (QseB) in Francisella. To identify the PmrA regulon in Francisella novicida, we compared transcriptional profiles between the wild type strain and its isogenic pmrA mutant. Total RNA from F. novicida and its isogenic mutant at the exponential phase of growth was extracted from four independent experiments and used in the RNAseq. A similar experiment design was performed to identify the PmrA regulon in Francisella tularensis LVS strain.