Investigation of protein oligomerization and assembly of a DNA translocation channel

Abstract

Mating, or conjugation, is the process by which a cell horizontally passes genetic information to another cell through a multi‐protein DNA translocation channel. ConE is a key ATPase of the mating machinery of ICEBs1, the Integrative and Conjugative element of Bacillus subtilis. Characterized homologs form a variety of oligomers in solution and crystallize as hexamers. Previously, we showed that purified His6‐ConE is a mixture of dimers, trimers, and higher‐order oligomers via Blue Native Polyacrylamide Gel Electrophoresis (BN‐PAGE). To more thoroughly understand the interactions between ConE and other players in the mating machinery, we have employed a bacterial two‐hybrid system. We investigated the interaction between ConE fused to the T18 and T25 domains of Bordetella pertussis adenylate cyclase. Using β‐galactosidase assay to indirectly report cyclic‐AMP levels, we found that T25‐ConE and T18‐ConE interact. We hope to use this technique to measure possible interactions between ConE and other ICEBs1 proteins that make up the DNA translocation channel. This research was funded by an NSF‐RUI grant to Melanie B. Berkmen.