TraG from RP4 and TraG and VirD4 from Ti Plasmids Confer Relaxosome Specificity to the Conjugal Transfer System of pTiC58

Abstract

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG RP4 ) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG RP4 . A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG RP4 was expressed in the donors. Mutations in traG RP4 with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG RP4 nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG RP4 mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG RP4 -mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG RP4 and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.Plasmid conjugation conceptually can be divided into two functions. In the first, the DNA is processed by a complex of proteins, one of which introduces a single-strand nick at the nic site within the oriT recognition sequence. Called the relaxosome, the proteins of this complex are coded for by genes of the Dtr (DNA transfer and replication) component of the transfer system. In the second, the nucleoprotein transfer intermediate comprised of the nicked strand covalently linked at the 5Ј end to the relaxase is secreted from the donor directly into the recipient via a bridge that forms between the mating pair. This translocation apparatus is a complex membraneassociated structure coded for by the Mpf (mating pair formation) genes.The relaxosome of one conjugal plasmid may or may not be transferrable by the Mpf system of another. Specificity is conferred, in part, by a...

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…In conjugation experiments with chimeric conjugation systems, composed of Mpf and Dtr systems originating from two diVerent plasmids, the CP is the factor that determines whether the chimeric machinery is functional or not. By exchanging the CP, the speciWcity of a given Mpf system for transport of diVerent mobilizable plasmids can be switched, implying that CPs represent an interface between the Mpf complex and the DNA substrate (Cabezón et al, 1994Hamilton et al, 2000;Lessl et al, 1993). This conclusion has been validated by biochemical data: CPs of conjugation systems have indeed been detected to directly interact with the relaxase, which is the Dtr component that covalently attaches to the DNA substrate during conjugative transfer (Llosa et al, 2003;Pansegrau and Lanka, 1996b;Schröder et al, 2002;Szpirer et al, 2000).…”

“…Remarkably, the VirB10-CP interaction is not system-restricted: VirB10-like proteins can interact, although with lower aYnity, with CPs of heterologous T4SS, as suggested by data of a twohybrid interaction analysis (Llosa et al, 2003). The versatility of the VirB10-CP interaction largely explains the functionality of chimeric bacterial conjugation systems containing an Mpf system of one plasmid and a Dtr system of a diVerent plasmid (Bolland et al, 1990;Cabezón et al, 1997;Hamilton et al, 2000).…”

Polar location and functional domains of the Agrobacterium tumefaciens DNA transfer protein VirD4

“…Like VirD4, its homologues in the E. coli plasmids F, RP4 and R388 are essential for conjugal transfer of plasmid DNA (Balzer et al, 1994). These proteins can functionally substitute for one another in the transfer of only a subset of conjugal plasmids (Cabezon et al, 1994;Hamilton et al, 2000). Two regions of the VirD4 homologues that correspond to motifs A and B of the Walker box have the highest sequence conservation.…”

Extensive Horizontal Gene Transfer in Cheese-Associated Bacteria

“…Integrative and conjugative elements (ICEs) can in part be identified by the presence of signature proteins associated with core functions of integration into and excision from the host genome (recombinase), replication as an extrachromosomal element (polymerase), and conjugation from the host to recipient cell (conjugation) (Ghinet et al, 2011). Analysis of the Glutamicibacter JB182 RUSTI region revealed homologs of each of these protein classes ( Figure 3A): a recombinase of the site-specific tyrosine recombinase XerD family (Ga0099663_102762) (Subramanya et al, 1997), a hexameric ATPase conjugation protein of the VirD4/TraG/TraD family (Ga0099663_102784) (Hamilton et al, 2000), and a homolog of the bi-functional primase-polymerase DNA replication protein family (Ga0099663_102766). Interestingly, Actinobacterial ICE systems typically use conjugation apparatus belonging to the SpoIIE/FtsK family, which allows transfer of double-stranded DNA (te Poele et al, 2008;Bordeleau et al, 2012).…”