Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

Abstract

Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. Type IV secretion systems (T4SS) in gram-negative bacteria mediate translocation of macromolecules out of the bacterial cell (14). The transmission of effector proteins and DNA into plant cells or other bacteria via cell-cell contact is one example of their function, and conjugation systems as well as the transferred DNA (T-DNA) delivery system of the phytopathogen Agrobacterium tumefaciens are prototypical of the T4SS family. Macromolecular translocation is achieved by a membranespanning protein machinery comprised of 12 gene products, VirB1 to VirB11 and an associated factor known as the coupling protein (VirD4) (66). The T4SS-associated coupling protein (T4CP) performs a crucial function in recognition of appropriate secretion substrates and governing entry of those molecules to the translocation pathway (7,8,10,30,41). In conjugation systems substrate recognition is applied to the relaxosome, a nucleoprotein complex of DNA transfer initiator proteins assembled specifically at the plasmid origin of transfer (oriT). In current models, initiation of the reactions that provide the single strand of plasmid (T-strand) DNA for secretion to recipient bacteria is expected to resemble the initiation of chromosomal replication (for reviews, see references 18, 54, and 81). Controlled opening of the DNA duplex is required to permit entry of the DNA processing machinery. The task of remodeling the conjugative oriT is generally ascribed to two or three relaxosome auxiliary factors, of host and plasmid origin, which occupy specific DNA binding sites at this locus. Intrinsic to the relaxosome is also a site-and strand-specific DNA transesterase activity that breaks the phosphodiester backbone at nic (5). Upon cleavage, the transesterase enzyme (also called relaxase) forms a reversible phosphotyrosyl linkage to the 5Ј end of the DNA. Duplex unwinding initiating from this site produces the single-stranded T strand to be exported. A wealth of information is available supporting the importance of DNA sequence recognition and binding by relaxosome components at oriT to the transesterase reaction in vitro and for effective conjugative transfer (for reviews, see references 18, 54, and 81). On the other hand, the mechanisms controlling release of the 3Ј-OH generated at nic and the subsequent DNA unwind...

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

“…Although a lot of information has been gathered over the years about the activity, oligomerization state and structure as well as the importance of VirD4 with respect to substrate recognition and processing, its location within the T4S system was unknown (Cascales & Christie, 2004; Mihajlovic et al , 2009; Lang et al , 2010; de Paz et al , 2010; Whitaker et al , 2016). This is the issue we set out to address in this study.…”

“…VirB11 has been reported as a hexamer in different systems (Machon et al , 2002; Savvides et al , 2003; Hare et al , 2006). VirD4 and VirB4, though expected to form hexamers, have been identified in monomeric and dimeric forms as well (Gomis‐Ruth et al , 2001; Schroder et al , 2002; Rabel et al , 2003; Arechaga et al , 2008; Mihajlovic et al , 2009; Durand et al , 2011; Pena et al , 2012; Wallden et al , 2012). All three ATPases interact with one another and these interactions likely direct the two processes in which T4S systems are involved: pilus biogenesis and substrate secretion (Atmakuri et al , 2004; Ripoll‐Rozada et al , 2013).…”

Mobility of Plasmids

“…The VirB4 tree showed a more scattered distribution of relaxase families, especially among MPF T plasmids. From a functional point of view, although T4CPs interact with components of both the relaxosome and the T4SS, the T4CP-relaxosome interactions seem to be more system specific (94,96,97,101). Besides, T4CP-encoding genes are usually adjacent to the genes coding for the relaxosome components (50,57), with gene order conservation being another sign of coevolution and functional relatedness.…”

“…This includes, besides the above-mentioned relaxosome components, the type IV coupling protein (T4CP) and the components of the mating channel that assemble a T4SS. The T4CP is involved in the connection between the relaxosome and the transport channel (11,41,94,101). It is also thought to energize the process of DNA transport (135,136).…”

Functional Dissection of the Conjugative Coupling Protein TrwB

“…In F-TraD, it was determined that its C terminus is essential for relaxosomal specificity, probably through an interaction with TraM (4,39,48). The cytoplasmic domain of the related TraD protein of plasmid R1 stimulates both transesterase and helicase activities of its cognate relaxase, TraI (41,51). A series of random mutations were shown to affect TraD oligomerization (23).…”