Conjugation is the transfer of DNA from one bacterial cell to another. Conjugation is a major form of horizontal gene transfer, resulting in the spread of genes that play roles in antibiotic resistance, virulence, symbiosis and metabolism. ICEBs1 is an integrative and conjugative element of Bacillus subtilis. The conjugation machinery that transfers DNA is a Type 4 Secretion System (T4SS). The ICEBs1 T4SS includes the proteins ConB, ConC, ConD, ConE, ConG, ConQ, and CwlT. The focus of our work is the ConE peripheral membrane protein, which belongs to the VirB4 family of ATPases. We analyzed whether ConE interacts with any of the other ICEBs1 T4SS proteins using bacterial two hybrid (BACTH). We found that ConE interacts with itself, ConB, and ConQ. We used site-directed mutagenesis of the conE gene to make alanine substitutions within five conserved residues within ATPase motifs of the ConE protein that have previously been shown to be critical for mating. We used BACTH to determine if these mutations affect ConE’s protein interactions. We discovered that interaction of ConE with itself or ConB is unaffected by mutations in any of the ATPase residues tested, but that the ConE-ConQ interaction depends on two conserved residues within the ATPase motifs. Our research sheds new light on the role of conserved residues within the ATPase motifs of ConE.
This research was funded by a National Science Foundation Research at Undergraduate Institutions grant.
