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Oligomerization of Two Membrane Proteins Critical for Bacterial Mating

Bacterial mating allows for the transfer of DNA through a DNA translocation channel. These large, multi‐protein complexes can be found in many bacteria and are known as Type IV secretion systems (T4SSs). Our work focuses on the T4SS of the integrative and conjugative element ICEBs1 found in the Gram‐positive bacterium Bacillus subtilis. Two crucial components of the ICEBs1 T4SS are ConE and ConB. ConE is a VirB4‐like peripheral membrane ATPase, likely providing the energy necessary to pump DNA though the cell membrane. ConB is a VirB8‐like integral membrane protein that recruits ConE to the membrane. Our objective was to purify ConB and to characterize its oligomerization state and potential interactions with ConE in vitro using blue native polyacrylamide gel electrophoresis (BN‐PAGE). We found that purified full‐length His6‐ConB is a mixture of oligomers, forming tetramers up to dodecamers. Truncated His6‐ConB lacking its N‐terminal transmembrane segment is monomeric. Confirming a prior study, we found that purified His6‐ConE and MBP‐His6‐ConE form dimers and a mixture of higher order oligomers. No interactions were detected between any of our purified ConE and ConB proteins using BN‐PAGE. Our results on ConB and ConE multimerization are consistent with previous findings on homologous T4SS proteins. We propose that VirB8‐like proteins function as dodecamers and VirB4‐like proteins function as hexamers in assembled T4SSs in vivo. ConB and ConE likely must interact with the membrane and/or other components of the T4SS to achieve the fully oligomerized state.