Interaction between the RP4 coupling protein TraG and the pBHR1 mobilization protein Mob

Abstract

SummaryIt is currently believed that interaction between the relaxosome of a mobilizable plasmid and the transfer machinery of the helper conjugative plasmid is mediated by a TraG family coupling protein. The coupling proteins appear as an essential determinant of mobilization specificity and efficiency. Using a two-hybrid system, we demonstrated for the first time the direct in vivo interaction between the coupling protein of a conjugative plasmid (the TraG protein of RP4) and the relaxase of a mobilizable plasmid (the Mob protein of pBHR1, a derivative of the broad host range plasmid pBBR1). This interaction was confirmed in vitro by an overlay assay and was shown to occur even in the absence of the transfer origin of pBHR1. We showed that, among 11 conjugative plasmids tested, pBHR1 is efficiently mobilized only by plasmids encoding an IncP-type transfer system. We also showed that the RP4 TraG coupling protein is essential for mobilization of a pBBR1 derivative and is the element that allows its mobilization by R388 plasmid (IncW) at a detectable frequency.

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

“…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). Also, accessory Dtr components such as TraM of the F plasmid (Disqué-Kochem and Dreiseikelmann, 1997), as well as the DNA itself are seen to interact with CPs (Moncalián et al, 1999;Panicker and Minkley, 1992;Schröder and Lanka, 2003;Schröder et al, 2002).…”

“…It can thus be assumed that the process of conjugative DNA transfer relies on secretion of the relaxase as a pilot protein that trails the covalently attached transfer strand. This view is further supported by the Wnding that the relaxase speciWcally interacts with the CP of the Mpf/CP secretion machinery (Llosa et al, 2003;Pansegrau and Lanka, 1996b;Schröder et al, 2002;Szpirer et al, 2000), an interaction that is probably essential for initiating the process of secretion. Finally, other type IV secretion systems, which are ancestrally related to the conjugative Mpf/CP machinery, are found to secrete protein substrates without any indication for concomitant DNA transfer (Cascales and Christie, 2003;Schröder et al, 2005).…”

Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium

“…Accumulating evidence suggests that it is the coupling protein that recruits both the protein substrates and the nucleoprotein complex to the T4SS (4,14,20,22,37,38), the latter by virtue of the relaxase protein that is covalently bound to the DNA. Our studies show that Cre::VirD2 chimeric proteins are translocated into host cells in the absence of T-DNA, strongly suggesting that the relaxase component VirD2 indeed provides the transport signal for transfer of the nucleoprotein complex.…”

Agrobacterium tumefaciens VirB6 Domains Direct the Ordered Export of a DNA Substrate Through a Type IV Secretion System

“…4 Next, a homomultimer of an integral membrane ATP-binding protein termed the coupling protein (T4CP) recruits the DNA substrate to the secretory apparatus, probably through recognition of C-terminal secretion signals carried by the relaxase and other relaxosome subunits. [5][6][7][8] Finally, the T4CP functions together with a trans-envelope secretion channel composed of the mating pair formation (Mpf) proteins to deliver the DNA transfer intermediate to the cell exterior. 9 The processing and recruitment reactions are biochemically well characterized for several conjugation systems, and recent structurefunction studies have been aided by available crystal structures for the TrwC 10,11 and TraI 12,13 relaxases of plasmids R388 and F, respectively, and TrwB T4CP 14 of plasmid R388.…”