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Transformation ofrickettsiae 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 (GFPUV ) as a fluorometric marker and transformedRickettsia typhi with anrpoB -GFPUV 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 therpoB -GFPUV construct. Cultures were then analyzed by flow cytometry for detection of GFPUV expression, and transformedR. typhi were isolated in a fluorescence-activated cell sorter. This is the first report of transformation of rickettsiae with a nonrickettsial (GFPUV ) gene.

Green Fluorescent Protein as a Marker in Rickettsia typhi Transformation