TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388)

Abstract

TraG-like proteins are essential components of type IV secretion systems. During secretion, TraG is thought to translocate defined substrates through the inner cell membrane. The energy for this transport is presumably delivered by its potential nucleotide hydrolase (NTPase) activity. TraG of conjugative plasmid RP4 is a membrane-anchored oligomer that binds RP4 relaxase and DNA. TrwB (R388) is a hexameric TraG-like protein that binds ATP. Both proteins, however, lack NTPase activity under in vitro conditions. We charac- Type IV secretion is an energy-driven mechanism for delivery of effector molecules from bacterial donors to recipient cells. It mediates the transfer of toxic components into eukaryotic hosts by many pathogenic bacteria such as Helicobacter pylori, Legionella pneumophila, Brucella spp., and Bartonella henselae and enables DNA transfer in bacterial conjugation systems such as that encoded by plasmid RP4.

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

“…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). Whereas the interactions of the CP with the protein components of the Dtr system are speciWc, the binding to DNA is non-sequence-speciWc, with a preference for single-stranded (ss) DNA (Moncalián et al, 1999;Schröder and Lanka, 2003;Schröder et al, 2002).…”

“…Similarly to the situation encountered with VirB4, the initially postulated NTPase activity of the CP has not been detected in vitro (Moncalián et al, 1999;Schröder et al, 2002). However, CPs have been demonstrated to bind nucleotides (NTPs) and also nucleotide diphosphates (NDPs) in vitro (Gomis-Rüth et al, 2001;Moncalián et al, 1999;Schröder and Lanka, 2003). Binding and release of nucleotides, which is possibly triggered by Mg 2+ , may have a mechanistic function related to substrate translocation (Schröder and Lanka, 2003).…”

“…However, CPs have been demonstrated to bind nucleotides (NTPs) and also nucleotide diphosphates (NDPs) in vitro (Gomis-Rüth et al, 2001;Moncalián et al, 1999;Schröder and Lanka, 2003). Binding and release of nucleotides, which is possibly triggered by Mg 2+ , may have a mechanistic function related to substrate translocation (Schröder and Lanka, 2003). Mutational analysis of the putative nucleotide binding motifs (Walker A and B 'P-loop' motifs) indicates an essential function of this binding activity for the secretion process (Balzer et al, 1994;Kumar and Das, 2002;Moncalián et al, 1999).…”

“…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). Whereas the interactions of the CP with the protein components of the Dtr system are speciWc, the binding to DNA is non-sequence-speciWc, with a preference for single-stranded (ss) DNA (Moncalián et al, 1999;Schröder and Lanka, 2003;Schröder et al, 2002). This indicates that substrate recognition by the CP primarily occurs via interactions with protein components of the transfer-competent protein/DNA-intermediate.…”

“…The monomeric, cytoplasmic domain of the CP is suYcient for binding nucleotides as well as DNA (Moncalián et al, 1999;Schröder and Lanka, 2003). In contrast, binding to the relaxase substrate requires the presence of the N-terminal membrane anchor, probably because oligomerization of the CP is essential for relaxase binding (Schröder and Lanka, 2003). In A. tumefaciens, the CP (VirD4) localizes to the cellular poles.…”