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.