Expression of Agrobacterium tumefaciens octopine Ti-plasmid virB8 gene is regulated by translational coupling.

Eleven proteins of the Agrobacterium tumefaciens virB operon are required for type IV secretion. All octopine Ti-plasmid pTiA6NC VirB proteins, except VirB8, could be expressed from a cloned monocistronic gene. Accumulation of VirB8 required translation of the upstream virB7 gene. Analysis of chimeric virB8 genes and a newly constructed virB7 deletion mutant Agrobacterium AD1275 showed that translation of virB7, and not the gene product, is required for VirB8 accumulation. Agrobacterium AD1275 accumulated VirB8 and other downstream virB gene products, and could be complemented with only virB7 in trans. In monocistronic virB8, sequences upstream of the virB8 ORF negatively controls virB8 expression possibly through the formation of a secondary structure that occludes both the ribosome binding site and translation start codon. Disruption of the structure through translation of the upstream gene ensures efficient translation of the virB8 mRNA in wild type bacteria. The pTiA6NC virB8 contains two potential translation start sites within the first eight codons. We show that the first AUG is used for virB8 translation initiation. The seven N-terminal residues resulting from translation initiation at the first AUG are required for both tumor formation and stabilization of VirB3. VirB8 and VirB4 are sufficient for the stabilization of VirB3, and VirB7 stabilizes VirB3 indirectly through its effect on virB8 expression.

Agrobacterium tumefaciens type IV secretion protein VirB3 is an inner membrane protein and requires VirB4, VirB7, and VirB8 for stabilization.

Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus and a T-pilus for secretion of DNA and proteins into plant cells. The pilin-like protein VirB3, a membrane protein of unknown topology, is required for the assembly of the T-pilus and for T-DNA secretion. Using PhoA and green fluorescent protein (GFP) as periplasmic and cytoplasmic reporters, respectively, we demonstrate that VirB3 contains two membrane-spanning domains and that both the N and C termini of the protein reside in the cytoplasm. Fusion proteins with GFP at the N or C terminus of VirB3 were fluorescent and, like VirB3, localized to a cell pole. Biochemical fractionation studies demonstrated that VirB3 proteins encoded by three Ti plasmids, the octopine Ti plasmid pTiA6NC, the supervirulent plasmid pTiBo542, and the nopaline Ti plasmid pTiC58, are inner membrane proteins and that VirB4 has no effect on membrane localization of pTiA6NC-encoded VirB3 (pTiA6NC VirB3). The pTiA6NC and pTiBo542 VirB2 pilins, like VirB3, localized to the inner membrane. The pTiC58 VirB4 protein was earlier found to be essential for stabilization of VirB3. Stabilization of pTiA6NC VirB3 requires not only VirB4 but also two additional VirB proteins, VirB7 and VirB8. A binary interaction between VirB3 and VirB4/VirB7/VirB8 is not sufficient for VirB3 stabilization. We hypothesize that bacteria use selective proteolysis as a mechanism to prevent assembly of unproductive precursor complexes under conditions that do not favor assembly of large macromolecular structures.