Agrobacterium VirB10, an ATP energy sensor required for type IV secretion

Abstract

Bacteria use type IV secretion systems (T4SS) to translocate DNA and protein substrates to target cells of phylogenetically diverse taxa. Recently, by use of an assay termed transfer DNA immunoprecipitation (TrIP), we described the translocation route for a DNA substrate [T-DNA, portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] of the Agrobacterium tumefaciens VirB͞D4 T4SS in terms of a series of temporally and spatially ordered substrate contacts with subunits of the secretion channel. Here, we report that the bitopic inner membrane protein VirB10 undergoes a structural transition in response to ATP utilization by the VirD4 and VirB11 ATP-binding subunits, as monitored by protease susceptibility. VirB10 interacts with inner membrane VirD4 independently of cellular energetic status, whereas the energy-induced conformational change is required for VirB10 complex formation with an outer membrane-associated heterodimer of VirB7 lipoprotein and VirB9, as shown by coimmunoprecipitation. Under these conditions, the T-DNA substrate is delivered from the inner membrane channel components VirB6 and VirB8 to periplasmic and outer membrane-associated VirB2 pilin and VirB9. We propose that VirD4 and VirB11 coordinate the ATP-dependent formation of a VirB10 ''bridge'' between inner and outer membrane subassemblies of the VirB͞D4 T4SS, and that this morphogenetic event is required for T-DNA translocation across the A. tumefaciens cell envelope. conjugation ͉ energy coupling ͉ pathogenesis ͉ membrane complex ͉ translocation

Subcellular localization and functional domains of the coupling protein, TraG, from IncHI1 plasmid R27

“…The recently proposed 'shoot and pump' model suggests that the coupling protein secretes the relaxase into the conduit created by the T4SS and subsequently threads the DNA portion of the substrate through a channel that is formed by the hexameric coupling protein (Llosa & de la Cruz, 2005). Furthermore, the A. tumefaciens coupling protein, VirD4, has been found to require functional NTPbinding motifs for bridging the inner and outer subassemblies of VirB/D4 T4SS (Cascales & Christie, 2004a). Clearly, the coupling protein plays a vital role in the transfer of plasmid DNA from donor to recipient cells.…”

“…The coupling proteins TraG and TrwB from the R27 and R388 plasmids, respectively, were both independently determined to interact with the VirB10-like proteins, TrhB and TrwE, from R27 and R388 (Gilmour et al, 2003;Llosa et al, 2003). In the Ti plasmid of Agrobacterium tumefaciens, VirB10 is a well-conserved T4SS component and it has been recently proposed to be the ATP energy sensor of the T4SS (Cascales & Christie, 2004a).…”

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

“…Protease susceptibility experiments have shown that VirB10 can adopt two diVerent conformations, one of which requiring the presence of cellular ATP (Cascales and Christie, 2004a). Closer inspection has revealed that it is the ATP utilization by VirB11 and VirD4 (but not VirB4) that is responsible for the switch from the ATP-independent to the ATP-dependent "energized" conformation of VirB10 (Cascales and Christie, 2004a).…”

“…Closer inspection has revealed that it is the ATP utilization by VirB11 and VirD4 (but not VirB4) that is responsible for the switch from the ATP-independent to the ATP-dependent "energized" conformation of VirB10 (Cascales and Christie, 2004a). Only the energized conformation of VirB10 is able to form complexes with the outer-membrane component VirB9.…”

Structures of two core subunits of the bacterial type IV secretion system, VirB8 from Brucella suis and ComB10 from Helicobacter pylori

“…VirB10 is essential for the transfer of the substrate from the inner to the outer membrane but does not directly contact the substrate (16). Instead, it acts as an energy-sensing bridge between the inner and outer membranes (17). Interactions between VirB8 and many other T4SS proteins have been reported, including VirB10 (18,19), VirB9 (19), VirB1, VirB4, and VirB11 (20), as well as with itself (18)(19)(20).…”