Identification of subcomplexes and protein-protein interactions in the DNA transporter of Thermus thermophilus HB27

Abstract

The natural transformation system of the thermophilic bacterium Thermus thermophilus comprises at least 16 competence proteins. Recently we found that the outer membrane (OM) competence protein PilW interacts with the secretin channel, which guides type IV pili (T4P) and potential DNA transporter pseudopili through the OM. Here we have used biochemical techniques to study the interactions of cytoplasmic, inner membrane (IM) and OM components of the DNA transporter in T. thermophilus. We report that PilW is part of a heteropolymeric complex comprising of the cytoplasmic PilM protein, IM proteins PilN, PilO, PilC and the secretin PilQ. Co-purification studies revealed that PilO directly interacts with PilW. In vitro affinity co-purification studies using His-tagged PilC led to the detection of PilC-, PilW-, PilN- and PilO-containing complexes. PilO was identified as direct interaction partner of the polytopic IM protein PilC. PilC was also found to directly interact with the cytoplasmic T4P disassembly ATPase PilT1 thereby triggering PilT1 ATPase activity. This, together with the detection of heteropolymeric PilC complexes which contain PilT1 and the pilins PilA2, PilA4 and PilA5 is in line with the hypothesis that PilC connects the depolymerization ATPase to the base of the pili possibly allowing energy transduction for disassembly of the pilins.