Agrobacterium tumefaciensVirB6 Protein Participates in Formation of VirB7 and VirB9 Complexes Required for Type IV Secretion

Abstract

This study characterized the contribution of Agrobacterium tumefaciens VirB6, a polytopic inner membrane protein, to the formation of outer membrane VirB7 lipoprotein and VirB9 protein multimers required for type IV secretion. VirB7 assembles as a disulfide cross-linked homodimer that associates with the T pilus and a VirB7-VirB9 heterodimer that stabilizes other VirB proteins during biogenesis of the secretion machine. Two presumptive VirB protein complexes, composed of VirB6, VirB7, and VirB9 and of VirB7, VirB9, and VirB10, were isolated by immunoprecipitation or glutathione S-transferase pulldown assays from detergent-solubilized membrane extracts of wild-type A348 and a strain producing only VirB6 through VirB10 among the VirB proteins. To examine the biological importance of VirB6 complex formation for type IV secretion, we monitored the effects of nonstoichiometric VirB6 production and the synthesis of VirB6 derivatives with 4-residue insertions (VirB6.i4) on VirB7 and VirB9 multimerization, T-pilus assembly, and substrate transfer. A virB6 gene deletion mutant accumulated VirB7 dimers at diminished steady-state levels, whereas complementation with a plasmid bearing wild-type virB6 partially restored accumulation of the dimers. VirB6 overproduction was correlated with formation of higher-order VirB9 complexes or aggregates and also blocked substrate transfer without a detectable disruption of T-pilus production; these phenotypes were displayed by cells grown at 28°C, a temperature that favors VirB protein turnover, but not by cells grown at 20°C. Strains producing several VirB6.i4 mutant proteins assembled novel VirB7 and VirB9 complexes detectable by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two strains producing the D60.i4 and L191.i4 mutant proteins translocated IncQ plasmid and VirE2 effector protein substrates in the absence of a detectable T pilus. Our findings support a model that VirB6 mediates formation of VirB7 and VirB9 complexes required for biogenesis of the T pilus and the secretion channel.

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.…”

“…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.…”

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.…”

“…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. 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).…”

“…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).…”