Role of Agrobacterium VirB11 ATPase in T-Pilus Assembly and Substrate Selection

Abstract

The VirB11 ATPase is a subunit of the Agrobacterium tumefaciens transfer DNA (T-DNA) transfer system, a type IV secretion pathway required for delivery of T-DNA and effector proteins to plant cells during infection. In this study, we examined the effects of virB11 mutations on VirB protein accumulation, T-pilus production, and substrate translocation. Strains synthesizing VirB11 derivatives with mutations in the nucleoside triphosphate binding site (Walker A motif) accumulated wild-type levels of VirB proteins but failed to produce the T-pilus or export substrates at detectable levels, establishing the importance of nucleoside triphosphate binding or hydrolysis for T-pilus biogenesis. Similar findings were obtained for VirB4, a second ATPase of this transfer system. Analyses of strains expressing virB11 dominant alleles in general showed that T-pilus production is correlated with substrate translocation. Notably, strains expressing dominant alleles previously designated class II (dominant and nonfunctional) neither transferred T-DNA nor elaborated detectable levels of the T-pilus. By contrast, strains expressing most dominant alleles designated class III (dominant and functional) efficiently translocated T-DNA and synthesized abundant levels of T pilus. We did, however, identify four types of virB11 mutations or strain genotypes that selectively disrupted substrate translocation or T-pilus production: (i) virB11/virB11‫ء‬ merodiploid strains expressing all class II and III dominant alleles were strongly suppressed for T-DNA translocation but efficiently mobilized an IncQ plasmid to agrobacterial recipients and also elaborated abundant levels of T pilus; (ii) strains synthesizing two class III mutant proteins, VirB11, V258G and VirB11.I265T, efficiently transferred both DNA substrates but produced low and undetectable levels of T pilus, respectively; (iii) a strain synthesizing the class II mutant protein VirB11.I103T/M301L efficiently exported VirE2 but produced undetectable levels of T pilus; (iv) strains synthesizing three VirB11 derivatives with a four-residue (HMVD) insertion (L75.i4, C168.i4, and L302.i4) neither transferred T-DNA nor produced detectable levels of T pilus but efficiently transferred VirE2 to plants and the IncQ plasmid to agrobacterial recipient cells. Together, our findings support a model in which the VirB11 ATPase contributes at two levels to type IV secretion, T-pilus morphogenesis, and substrate selection. Furthermore, the contributions of VirB11 to machine assembly and substrate transfer can be uncoupled by mutagenesis.Agrobacterium tumefaciens VirB11 is a member of a family of ATPases widely distributed among members of the domains Bacteria and Archaea (38,48,67). Mutational studies have established the importance of VirB11 homologs for translocation of macromolecules across the cell envelope in association with type IV secretion (17, 29, 32, 44, 52) and competence (1) systems and type II protein secretion and pilus biogenesis systems (54). These proteins hydrolyze ATP, as demonstrat...

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

“…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. The Pil ¡ /Tra + phenotype has only been observed in mutants of the pilin (VirB2) and in mutants of VirB6 and VirB11, but not in mutants of any other Mpf/CP component (Jakubowski et al, 2003;Sagulenko et al, 2001a). According to the "stump" model discussed above, the observed Pil ¡ /Tra + phenotype suggests that it is pilus elongation, but not initial formation of a pilus stump, which is impaired in the identiWed VirB2, VirB6, and VirB11 mutants.…”

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

“…N-terminal lipoprotein processing of VirB7 is followed by VirB6-assisted formation of VirB7 homodimers, which may link the T-pilus to the core complex. Energy for T-pilus assembly may be provided by VirB11 (20), and it may be transduced to the pilus assembly complex in the outer membrane via VirB6 and VirB7. SPI and SPII, signal peptidases.…”

“…The core proteins stabilize other T4SS components, but so far, the nature of the channel for substrate translocation has not been revealed (18). Energy for substrate translocation and complex assembly may be provided by NTPases VirB4, VirB11, or VirD4 (17,(19)(20)(21). Crystal structure analyses of VirB11-like protein HP0525 from H. pylori and VirD4-like protein TrwB from plasmid R388 revealed central holes in the multimeric structures, suggesting a role as transporters (22,23).…”

The versatile bacterial type IV secretion systems

“…Intriguingly however, recent studies have identified mutations in the VirB11 and VirB6 subunits that 'uncouple' pilus biogenesis from substrate transfer. Some mutations do not affect assembly of a wild-type pilus, but block substrate transfer, whereas, conversely, others prevent pilus formation without affecting substrate transfer 59,69 . So, the formation of a conjugative pilus extending from the cell surface is not, in fact, an obligatory feature of conjugation machines of Gram-negative bacteria.…”