Fluorescence Localization of the Conjugation Machinery of Bacillus subtilis

Abstract

Conjugation, or mating, is the transfer of DNA from a donor to a recipient cell through conjugation machinery. Conjugation contributes to bacterial genome innovation and the spread of virulence and antibiotic resistance genes between bacteria. DNA is transferred during conjugation through a multi‐protein DNA translocation channel, classified as a Type IV secretion system (T4SS), embedded in the membrane. Our work focuses on characterizing the conjugation machinery of the integrative and conjugative element ICEBs1 of Bacillus subtilis. Previously, we identified several protein components of the ICEBs1 conjugation machinery, including the bitopic membrane protein ConB and the peripheral membrane ATPase ConE. We have used fluorescence microscopy to observe the locations of ConB and ConE inside cells. A fusion of ConE to green fluorescent protein (GFP) localizes to the membrane, predominantly at the cell poles. Here, we show that a fusion of ConB to the red fluorescent protein mCHERRY localizes similarly to ConE‐GFP. Consistent with ConE lacking predicted transmembrane segments, ConE‐GFP’s localization to the membrane requires ConB. In contrast, localization of ConB‐mCHERRY to the membrane does not depend on other conjugation proteins. Current studies are focused on which domains of ConB are required for its own localization as well as the localization of ConE.Support or Funding InformationThis research was funded by Suffolk University and an NSF‐RUI grant to M. Berkmen.