Green Fluorescent Protein as a Marker in Rickettsia typhi Transformation

Transformation of rickettsiae is a recent accomplishment, but utility of this technique is limited due to the paucity of selectable markers suitable for use in this intracellular pathogen. We chose a green fluorescent protein variant optimized for fluorescence under UV lights (GFP(UV)) as a fluorometric marker and transformed Rickettsia typhi with an rpoB-GFP(UV) fusion construct. The rickettsiae were subsequently grown in Vero cells, and cultures were screened by PCR and restriction fragment length polymorphism (RFLP) to confirm incorporation of the rpoB-GFP(UV) construct. Cultures were then analyzed by flow cytometry for detection of GFP(UV) expression, and transformed R. typhi were isolated in a fluorescence-activated cell sorter. This is the first report of transformation of rickettsiae with a nonrickettsial (GFP(UV)) gene.