Development of inducible promoter and CRISPRi plasmids functional in Rickettsia rickettsii

Rickettsia rickettsii is an obligate intracellular, tick-borne bacterium that causes Rocky Mountain spotted fever. The demanding nature of cultivating these bacteria within host cells and the labor involved in obtaining clonal isolates have severely limited progress regarding the development of compatible genetic tools to study this pathogen. Conditional expression of genes that might be toxic or have an otherwise undesirable effect is the next logical goal to expand upon the constitutive expression plasmids generated thus far. We describe the construction of an inducible promoter system based on the tet-On system, leveraging design elements from the anhydrotetracycline-inducible promoter system used for Borrelia burgdorferi and one of the few characterized rickettsial promoters for the outer membrane gene, rompB (sca5). The functionality of this promoter is demonstrated via fluorescence of induced mScarlet production and was then used to construct a generalized inducible expression vector for R. rickettsii. The development of a functional inducible promoter was then applied to the construction of a CRISPR interference plasmid as a means to reduce or essentially silence the transcription of targeted genes. We demonstrate the viability of a simplified, single vector CRISPRi system to disrupt gene expression in R. rickettsii targeting the type IV secreted effector rarP2 and autotransporter peptidase rapL as examples.

This work expands upon the genetic toolbox available for R. rickettsii. We describe both an inducible promoter and CRISPRi system compatible with Rickettsia, which may provide key instruments for the development of further tools. The development of an inducible promoter system allows for the overexpression of genes, which might be toxic when expressed constitutively. The CRISPRi system enables the ability to knock down genes with specificity, and critically, genes that may be essential and could not otherwise be knocked out. These developments may provide the foundation for unlocking genetic tools for other pathogens of the order Rickettsiales, such as the Anaplasma, Orientia, and Ehrlichia for which there are currently no inducible promoters or CRISPRi platforms.