Transformation of Rhodococcus rhodnii, a symbiont of the Chagas disease vector Rhodnius prolixus, with integrative elements of the L1 mycobacteriophage.

Elimination of vector populations through the use of insecticides is the principal means of controlling Chagas disease. Because of the limitations of insecticide use, we have been developing a new potential method of control, to be used in conjunction with insecticide programs, a method which utilizes genetically modified symbiotic bacteria. These transformed bacteria can express anti-parasitic agents in the gut of the bug where the trypanosomes also are found. Previous studies have shown that it is possible to transform Rhodococcus rhodnii with a shuttle plasmid that contains the gene for cecropin A, an insect anti-microbial peptide. The bacteria expressed this peptide and reduced or eliminated the number of trypanosomes in the bug Rhodnius prolixus [Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 3274]. In an effort to improve efficacy and transformation stability, we have begun using plasmids that contain integrative elements from the L1 mycobacteriophage to insert DNA into the genome of the bacterium. The integrative plasmid pBP5 contains the attachment site (attP) and integrase gene (int) of the L1 mycobacteriophage, an antibiotic resistance gene and the lacZ gene. After transforming R. rhodnii with pBP5, nine positive clones were obtained and six different insertions sites were identified. In each clone, the integrative plasmid is inserted only once, the lacZ gene is expressed intensely and, all clones but one, remained stable for 100 generations of culture in the absence of antibiotic selection. In addition, the construct remains stable throughout the life cycle of the bug. These data demonstrate that L1 mycobacteriophage integrative plasmids are significantly more stable than episomally located plasmids used in previous studies and will be greatly beneficial for use in the transformation of symbiotic bacteria of Chagas disease vectors.