TRANSFORMATION OF ROCHALIMAEA-QUINTANA, A MEMBER OF THE FAMILY RICKETTSIACEAE

Rochalimaea quintana is the only member of the family Rickettsiaceae that can be grown in vitro. Because of its relationship to the other members of this family, techniques developed to transform R. quintana might be applicable to the obligate intracellular bacteria of the Rickettsiaceae. These procedures are critical to understanding mechanisms of pathogenesis and the nature of obligate intracellular growth. A transformation procedure for R. quintana has been established by using electroporation techniques. Several cosmids or plasmids with replicons RK2 and RSF1010 have been successfully used to transform this organism. Transformants were obtained by selection for antibiotic resistance to chloramphenicol or kanamycin. Plasmid retention and replication has been verified by Southern blot analysis and chloramphenicol acetyltransferase assay. Experimentation with different voltage field strengths and pulse times indicate that 12.5 kV/cm at 10 ms (25 μF and 400 Ω) was optimal, giving a transformation frequency of ~ 0.3% and 3 x 105 transformants per μg of DNA.