Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens

Abstract

The VirB͞D4 type IV secretion system of Agrobacterium tumefaciens translocates virulence factors (VirE2, VirF, and the VirD2-T-DNA complex) to plant cells. The membrane-bound translocation machinery consists of 12 proteins (VirB1-11 and VirD4) required for substrate translocation. Protein-protein interactions in the membranes were analyzed after extraction with the mild detergent dodecyl-␤-D-maltoside followed by separation under native conditions. Incubation of the membranes with increasing concentrations of the detergent differentially extracted virulence proteins. Separation of the solubilized proteins by blue native electrophoresis revealed cofractionation between two classes of protein complexes containing VirB7. The first class, consisting of major T-pilus component VirB2 and associated proteins VirB5 and VirB7, comigrated in the low molecular mass portion of the gel of about 100 kDa. The second class contains putative translocation complex core components VirB8, VirB9, and VirB10 in the high molecular mass portion of the gel larger than 232 kDa, as well as VirB7. Solubilized proteins were characterized further by gel filtration chromatography. This procedure separated T-pilus-associated proteins VirB2, VirB5, and VirB7 in the low molecular mass range from the other components of the translocation machinery and the substrates VirE2 and VirD2. Fractionation of VirB7-containing complexes (VirB7-VirB7 homodimers and VirB7-VirB9 heterodimers) suggested that they may link the T-pilus components to the core of the translocation machinery. Based on previously described VirB protein interactions and biochemical analysis of C58 wild type as well as of virB5 and virB6 deletion mutants, a model of T-pilus assembly in A. tumefaciens is suggested.

The versatile bacterial type IV secretion systems

“…Further investigations exploring these stabilizing effects, as well as recent cell biology studies, have led to the development of a biogenesis pathway for this transfer apparatus 22,53 .A crucial intermediate in this pathway is a 'core' structure that is composed of VirB4, VirB7-VirB10 and, probably, VirB6. The existence of this structure is now supported by data from dihybrid screens and complementary biochemical assays [54][55][56][57][58][59] (Table 2). Additionally, some of the interactions required for assembly of the putative core are conserved in the B. pertussis Ptl system.…”

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

“…These data on A. tumefaciens VirB6 clearly demonstrate the importance of VirB6 as a central channel component engaged in a series of substrate-and Mpf-component interactions. The requirement of VirB6 for the stability or functionality of the minor pilus components VirB5 and VirB7 indicates that VirB6 may play an important role in pilus biogenesis or pilus elongation (Hapfelmeier et al, 2000;Jakubowski et al, 2003;Krall et al, 2002). The identiWcation of VirB6 insertion mutants that are deWcient in pilus production (Pil ¡ ) but proWcient in substrate transfer (Tra + ) (Jakubowski et al, 2003) lends further support to this suggestion.…”

“…VirB6 of A. tumefaciens localizes to the cell poles, and this localization is dependent on the presence of Wve other Mpf components, VirB7 through VirB11 (Judd et al, 2005). Conversely, the presence of VirB6 is required for stable expression of VirB3 and VirB5 and for formation of VirB7 homodimers and VirB7-VirB9 heterodimers (Hapfelmeier et al, 2000;Jakubowski et al, 2003;Krall et al, 2002). The domains of VirB6 mediating polar localization, interaction with the T-DNA substrate, substrate transfer to VirB8 or substrate transfer to VirB2 and VirB9 have been delimited (Jakubowski et al, 2004;Judd et al, 2005).…”

“…VirB9, in turn, is an outer-membrane component of the so-called core complex comprising VirB8, VirB9, and VirB10 (see below). Fractionation experiments of detergent-solubilized VirB7-containing protein complexes has identiWed two classes of complexes: one containing the pilus subunit VirB2 and the pilus-associated protein VirB5, and one containing the core complex components VirB8, VirB9, and VirB10 (Krall et al, 2002). Thus, VirB7 may play the role of a connector, attaching the exocellular pilus to the core complex.…”

“…In A. tumefaciens, it forms part of the core complex VirB8-VirB9-VirB10 Krall et al, 2002;Kumar and Das, 2001) and is needed for proper localization of VirB9 and VirB10 to discrete sites in the bacterial membrane probably representing the sites of Mpf pore complex (T complex) assembly (Kumar et al, 2000). Since VirB8, by itself, is able to localize at such discrete sites, VirB8 is thought to play the role of a nucleation centre, recruiting VirB9 and VirB10 to the sites of T complex assembly (Kumar et al, 2000).…”