Localization of two interacting protein components of the conjugation machinery of Bacillus subtilis

Abstract

Conjugation, or mating, is a major form of horizontal gene transfer, contributing to the spread of antibiotic resistance and virulence genes in bacterial populations. During conjugation, DNA is transferred from donor to recipient cell through a large multi-protein DNA translocation channel produced in the donor cell. We are interested in understanding the localization of the conjugation machinery of the conjugative element ICEBs1 of Bacillus subtilis. We previously identified several proteins that make up the ICEBs1 DNA translocation channel, including the peripheral membrane protein ConE and the transmembrane protein ConB. ConE fused to green fluorescent protein (GFP) localizes to the cell membrane, predominantly at the poles. ConE interacts with the bitopic membrane protein ConB. This interaction is functionally important as ConE-GFP mislocalizes to the cytoplasm in a strain deleted for conB. Here, we discovered that a fusion of ConB to the red fluorescent protein mCHERRY also localizes to the cell membrane at the poles, similarly to ConE-GFP. We also show that ConB-mCHERRY localization is independent of other conjugation proteins, but dependent on its own transmembrane segment. Finally, we discovered which domains of ConB are required for ConE-GFP localization. Our work provides new insights into the subcellular localization of two conserved interacting proteins that are critical for mediating horizontal gene transfer.