Conjugation is a major form of horizontal gene transfer, contributing to bacterial evolution and the acquisition of new traits. During conjugation, a donor cell transfers DNA to a recipient through a specialized DNA translocation channel classified as a type IV secretion system (T4SS). Here, we focused on the T4SS of ICEBs1, an integrative and conjugative element in Bacillus subtilis. ConE, encoded by ICEBs1, is a member of the VirB4 family of ATPases, the most conserved component of T4SSs. ConE is required for conjugation and localizes to the cell membrane, predominantly at the cell poles. In addition to Walker A and B boxes, VirB4 homologs have conserved ATPase motifs C, D, and E. Here, we created alanine substitutions in five conserved residues within or near ATPase motifs in ConE. Mutations in all five residues drastically decreased conjugation frequency but did not affect ConE protein levels or localization, indicating that an intact ATPase domain is critical for DNA transfer. Purified ConE is largely monomeric with some oligomers and lacks enzymatic activity, suggesting that ATP hydrolysis may be regulated or require special solution conditions. Finally, we investigated which ICEBs1 T4SS components interact with ConE using a bacterial two-hybrid assay. ConE interacts with itself, ConB, and ConQ, but these interactions are not required to stabilize ConE protein levels and largely do not depend on conserved residues within the ATPase motifs of ConE. The structure-function characterization of ConE provides more insight into this conserved component shared by all T4SSs. IMPORTANCE Conjugation is a major form of horizontal gene transfer and involves the transfer of DNA from one bacterium to another through the conjugation machinery. Conjugation contributes to bacterial evolution by disseminating genes involved in antibiotic resistance, metabolism, and virulence. Here, we characterized ConE, a protein component of the conjugation machinery of the conjugative element ICEBs1 of the bacterium Bacillus subtilis. We found that mutations in the conserved ATPase motifs of ConE disrupt mating but do not alter ConE localization, self-interaction, or levels. We also explored which conjugation proteins ConE interacts with and whether these interactions contribute to stabilizing ConE. Our work contributes to the understanding of the conjugative machinery of Gram-positive bacteria.
Keywords: Bacillus subtilis; ICE; T4SS; VirB4; conjugation; horizontal gene transfer; integrative and conjugative element; mating; type IV secretion system.