Structural and biochemical characterization of the relaxosome auxiliary proteins encoded on the Bacillus subtilis...

Bacterial conjugation is an important route for horizontal gene transfer. The initial step in this process involves a macromolecular protein-DNA complex called the relaxosome, which in plasmids consists of the origin of transfer (oriT) and several proteins that prepare the transfer. The relaxosome protein named relaxase introduces a nick in one of the strands of the oriT to initiate the process. Additional relaxosome proteins can exist. Recently, several relaxosome proteins encoded on the Bacillus subtilis plasmid pLS20 were identified, including the relaxase, named Rel_pLS20, and two auxiliary DNA-binding factors, named Aux1_pLS20 and Aux2_pLS20. Here, we extend this characterization in order to define their function. We present the low-resolution SAXS envelope of the Aux1_pLS20 and the atomic X-ray structure of the C-terminal domain of Aux2_pLS20. We also study the interactions between the auxiliary proteins and the full-length Rel_pLS20, as well as its separate domains. The results show that the quaternary structure of the auxiliary protein Aux1_pLS20 involves a tetramer, as previously determined. The crystal structure of the C-terminal domain of Aux2_pLS20 shows that it forms a tetramer and suggests that it is an analog of TraM_pF of plasmid F. This is the first evidence of the existence of a TraM_pF analog in gram positive conjugative systems, although, unlike other TraM_pF analogs, Aux2_pLS20 does not interact with the relaxase. Aux1_pLS20 interacts with the C-terminal domain, but not the N-terminal domain, of the relaxase Rel_pLS20. Thus, the pLS20 relaxosome exhibits some unique features despite the apparent similarity to some well-studied G- conjugation systems.