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.