VirB1* Promotes T-Pilus Formation in the vir -Type IV Secretion System of Agrobacterium tumefaciens

Abstract

The vir-type IV secretion system of Agrobacterium is assembled from 12 proteins encoded by the virB operon and virD4. VirB1 is one of the least-studied proteins encoded by the virB operon. Its N terminus is a lytic transglycosylase. The C-terminal third of the protein, VirB1*, is cleaved from VirB1 and secreted to the outside of the bacterial cell, suggesting an additional function. We show that both nopaline and octopine strains produce abundant amounts of VirB1* and perform detailed studies on nopaline VirB1*. Both domains are required for wild-type virulence. We show here that the nopaline type VirB1* is essential for the formation of the T pilus, a subassembly of the vir-T4SS composed of processed and cyclized VirB2 (major subunit) and VirB5 (minor subunit). A nopaline virB1 deletion strain does not produce T pili. Complementation with full-length VirB1 or C-terminal VirB1*, but not the N-terminal lytic transglycosylase domain, restores T pili containing VirB2 and VirB5. T-pilus preparations also contain extracellular VirB1*. Protein-protein interactions between VirB1* and VirB2 and VirB5 were detected in the yeast two-hybrid assay. We propose that VirB1 is a bifunctional protein required for virT4SS assembly. The N-terminal lytic transglycosylase domain provides localized lysis of the peptidoglycan cell wall to allow insertion of the T4SS. The C-terminal VirB1* promotes T-pilus assembly through protein-protein interactions with T-pilus subunits.

Secretome Analysis Uncovers an Hcp-Family Protein Secreted via a Type VI Secretion System inAgrobacterium tumefaciens

“…The identification of the known secretory protein VirB1* (1), which is required for T-pilus assembly (50), supports the conclusion that our proteomics approach was successful ( Table 2). We also identified ChvE, a sugar-binding protein previously detected in both the periplasm and extracellular space (4), in our secretome analysis; however, whether ChvE is actively secreted must still be determined.…”

The Bifunctional Cell Wall Hydrolase CwlT Is Needed for Conjugation of the Integrative and Conjugative Element ICEBs1in Bacillus subtilis and B. anthracis

“…That is, there can be redundancy, and the loss of one hydrolase is masked by the presence of others. For the conjugative elements, we suspect that the partial requirement for hydrolases in either Gram-positive or Gram-negative bacteria could be due to the activities of host hydrolases or hydrolases from other resident mobile elements (25,(45)(46)(47). For CwlT of ICEBs1 and the essential hydrolases from other mobile elements, it appears that the host hydrolases are not capable of providing sufficient function to allow any detectable conjugative transfer.…”

Agrobacterium-Mediated Plant Transformation: Biology and Applications

“…VirB1 is processed to a 73-residue, carboxy-terminal fragment termed VirB1*. The secreted VirB1* may play a direct role in T-pilus assembly, either by stabilizing VirB5 or by mediating VirB2-VirB5 interaction (Llosa et al, 2000;Zupan et al, 2007). VirB5 may directly interact with VirB2 or confer its effect by means of other proteins, e.g., VirB7.…”