Conjugation, or mating, plays a profound role in bacterial evolution by spreading genes that allow bacteria to adapt to and colonize new niches. ICEBs1, an integrative and conjugative element of Bacillus subtilis, can transfer itself and mobilize resident plasmids. DNA transfer is mediated by a type IV secretion system (T4SS). Characterized components of the ICEBs1 T4SS include the conserved VirB4-like ATPase ConE, the bifunctional cell wall hydrolase CwlT, and the presumed VirD4-like coupling protein ConQ. A fusion of ConE to green fluorescent protein (GFP) localizes to the membrane preferentially at the cell poles. One or more ICEBs1 proteins are required for ConE's localization at the membrane, as ConE lacks predicted transmembrane segments and ConE-GFP is found dispersed throughout the cytoplasm in cells lacking ICEBs1. Here, we analyzed five ICEBs1 genes to determine if they are required for DNA transfer and/or ConE-GFP localization. We found that conB, conC, conD, and conG, but not yddF, are required for both ICEBs1 transfer and plasmid mobilization. All four required genes encode predicted integral membrane proteins. conB and, to some extent, conD were required for localization of ConE-GFP to the membrane. Using an adenylate cyclase-based bacterial two-hybrid system, we found that ConE interacts with ConB. We propose a model in which the ICEBs1 conjugation machinery is composed of ConB, ConC, ConD, ConE, ConG, CwlT, ConQ, and possibly other ICEBs1 proteins, and that ConB interacts with ConE, helping to recruit and/or maintain ConE at the membrane. IMPORTANCEConjugation is a major form of horizontal gene transfer and has played a profound role in bacterial evolution by moving genes, including those involved in antibiotic resistance, metabolism, symbiosis, and infectious disease. During conjugation, DNA is transferred from cell to cell through the conjugation machinery, a type of secretion system. Relatively little is known about the conjugation machinery of Gram-positive bacteria. Here, we analyzed five genes of the integrative and conjugative element ICEBs1 of Bacillus subtilis. Our research identifies four new components of the ICEBs1 conjugation machinery (ConB, ConC, ConD, and ConG) and shows an interaction between ConB and ConE that is required for ConE to associate with the cell membrane. Conjugation is a major form of horizontal gene transfer and plays a profound role in bacterial evolution and the acquisition of new traits (1-3). Conjugation can spread antibiotic resistance and disseminate genes involved in symbiosis, degradation of pollutants, metabolism, and pathogenesis. Conjugative elements encode specialized DNA translocation channels classified as type IV secretion systems (T4SSs) (4-7). T4SSs are composed of many interacting proteins that span the envelope of the donor cell. In addition to transferring the conjugative DNA element, the conjugation machinery also can mobilize resident plasmids or other DNA elements that do not encode their own machinery.There is a rich body of mechanis...

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Mating, or conjugation, is the process by which a cell transfers DNA to another cell through a multi‐protein DNA translocation channel (Type IV Secretion System) at the membrane. Our research focuses on identifying and characterizing the components of the mating machinery of ICEBs1, a conjugative element of Bacillus subtilis. Our previous work indicates that the ICEBs1 protein ConE is a critical energetics component of the channel. ConE is essential for mating, localizes to the cell membrane, and belongs to the HerA/FtsK superfamily of ATPases. Here, we show that ConE likely functions as an oligomer. His6‐ConE forms dimers, trimers, and higher order oligomers in vitro and ConE self‐interacts in vivo. We hypothesize that the ICEBs1 proteins ConB and ConD interact with and recruit ConE to the cell membrane, as ConE‐GFP is dispersed throughout the cytoplasm in strains lacking these proteins. Since ConB and ConD are conserved membrane proteins and are required for DNA transfer, we hypothesize that they encode critical components of the ICEBs1 DNA translocation channel. This research was supported by an NSF‐RUI grant.