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Analysis of Oligomerization and Protein‐Protein Interactions within the Conjugation Machinery of Bacillus subtilis

Bacterial mating, or conjugation, is the transfer of DNA from one bacterial cell to another. Conjugation is significant to bacterial evolution and plays a large role in the spread of antibiotic resistance. The conjugation machinery of the Bacillus subtilis integrative and conjugative element ICEBs1 is a specialized DNA translocation channel known as a Type 4 Secretion System (T4SS). T4SSs are composed of multiple interacting oligomeric proteins that span the donor cell membrane. Previously, we found that the B. subtilis T4SS protein ConE (VirB4) interacts with itself and another T4SS protein, ConB (VirB8). These protein‐protein interactions were identified using a bacterial two hybrid method (BACTH) in which the proteins are fused to either the T18 or T25 domains of adenylate cyclase. Given that characterized VirB8 homologs oligomerize, we hypothesized that ConB interacts with itself, likely forming trimers or larger oligomers. To test this hypothesis, the conB gene was amplified by PCR and ligated into vectors that encode either the T18 or T25 domains. The plasmids were transformed into Escherichia coli and verified by sequencing. Subsequent BACTH assays in which the plasmids were co‐transformed into E. coli cells verified the interaction between T25‐ConB and T18‐ConB. We have performed site‐directed mutagenesis on the conB gene and intend to determine which domains of ConB are required for oligomerization and for interaction with ConE. Our work should provide greater insight into the structure and function of the T4SSs of B. subtilis and other Gram‐positive bacteria.