HIGHLY EFFICIENT INTEGRATION OF FOREIGN DNA INTO THE GENOME OF THE GREEN SULFUR BACTERIUM, CHLOROBIUM-VIBRIOFORME BY HOMOLOGOUS RECOMBINATION

Highly efficient and reproducible transformation of Chlorobium vibrioforme with plasmid DNA has been achieved by electroporation. Specific parameters have been optimized for the electrotransformation procedure. The method was developed using a construct containing a full copy of the pscC gene encoding the cytochrome c(551) subunit of the photosynthetic reaction center complex and the aadA gene encoding streptomycin resistance as selectable marker. Southern blotting analysis showed that the tested colonies were true transformants with the plasmid integrated into the genome by single homologous recombination. No transformants were obtained using the vector without the pscC gene showing that this vector does not replicate in C. vibrioforme. Thus transformation is possible only by homologous recombination. When using constructs designed to inactivate the pscC gene by insertion no transformants were obtained, indicating that the gene is indispensable for growth. The vector pVS2 carrying genes for erythromycin and chloramphenicol resistance was shown to replicate in C. vibrioforme. The two transformations shown here, provide an important genetical tool in the further analysis of structure and function of the photosynthetic apparatus in green sulfur bacteria.