Green fluorescent protein as a marker in Rickettsia typhi transformation

Transformation of rickettsiae is a recent accomplishment, but utility of th is technique is limited due to the paucity of selectable markers suitable f or use in this intracellular pathogen, We chose a green fluorescent protein variant optimized for fluorescence under UV lights (GFP(UV)) as a fluorome tric marker and transformed Rickettsia typhi with an rpoB-GFP(UV), fusion c onstruct. The rickettsiae were subsequently grown in Vero cells, and cultur es 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 trans formed R, typhi were isolated in a fluorescence-activated cell sorter, This is the first report of transformation of rickettsiae with a nonrickettsial (CGF(UV)) gene.