Transformation of Bartonella bacilliformis by electroporation

Abstract

Bartonella bacilliformis is a member of the order Rickettsiales, family Bartonellaceae. The bacterium is an intracellular parasite of human erythrocytes. To date, members of the family Bartonellaceae have not been transformed by standard chemical methods. We report the first successful transformation of a member of the Bartonellaceae family, B. bacilliformis, by the method of electroporation. The optimal conditions for electroporation of B. bacilliformis include a field strength of 12.5 kV/cm and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters and the cosmid pEST (RK2 replicon), a transformation efficiency of 7.8 × 10 5 was obtained. Transformants were readily cultured on medium containing kanamycin sulfate at concentrations ranging from 15 to 600 μg/mL. Bacterial survival was approximately 31% under optimal electroporation conditions, and the maximal number of transformants was obtained with 80 ng of pEST DNA. Bartonella bacilliformis was verified as the transformed organism by a comparison of transformant protein profiles with those of wild-type B. bacilliformis using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and detection of the exogenous plasmid in DNA from the transformed bacteria by DNA hybridization. Transformations using the plasmids pMK20, pML31, and pUCK18 (containing the replicons ColE1, F, and pMB1, respectively) were not successful.Key words: transformation, electroporation, Rickettsiales order.