ABSTRACTBacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that theEscherichia colipKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning inAgrobacterium tumefaciens,Anaplasma phagocytophilum, orWolbachia pipientis. A chimeric receptor assembled fromA. tumefaciensVirD4 (VirD4At) mediated transfer of a MOBQ plasmid (pML122) andA. tumefacienseffector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4Atand the TraJ/VirD4Atchimera with their CTDs deleted supported pML122 transfer at wild-type levels, ΔCTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4At's CTD to the VirE2 effector, although other VirD4Atdomains bound this substratein vitro. We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains.IMPORTANCEBacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through theEscherichia colipKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.
Advances in the Assembly Model of Bacterial Type IVB Secretion Systems