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 (GFPUV) as a fluorometric marker and transformed Rickettsia typhi with an rpoB-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 the rpoB-GFPUV construct. Cultures were then analyzed by flow cytometry for detection of GFPUV 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 (GFPUV) gene.
Troyer
J M
JM
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
Radulovic
S
S
Azad
A F
AF
D015415:Biomarkers; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D012286:Rickettsia typhi / Q000235:genetics*; D014169:Transformation, Bacterial
D015415:Biomarkers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D012321:DNA-Directed RNA Polymerases