Agrobacterium tumefaciens uses a type IV or ‘adapted conjugation’ system to export oncogenic nucleoprotein particles as well as the VirE2 single-stranded DNA-binding protein (SSB) to plant cells. The 7.5-kDa cytoplasmic protein, VirE1, is a putative chaperone required for export of VirE2. In this study, we examined the requirements for VirE1/VirE2 complex formation. A nonpolar virE1 null mutant accumulated low levels of VirE2, and the trans expression of virE1 in this mutant partially restored VirE2 abundance. The virE1 deletion did not affect transcription but decreased the translation of virE2, as shown by analysis of lacZ transcriptional and translational fusions. VirE2 exhibited half-lives in excess of 2 hr when expressed in cis or trans with virE1, and less than 10 min when expressed in the absence of virE1, as shown by pulse-chase experiments. Previous studies showed that VirE1 interacts with an internal domain of VirE2. Analyses of amino- and carboxy-terminal VirE2 truncation mutants synthesized in the presence and absence of virE1 in A. tumefaciens supplied evidence for a stabilizing interaction between VirE1 and a domain near the N-terminus of VirE2. VirE1 self-association was demonstrated by use of the bacteriophage cI repressor fusion assay, native polyacrylamide gel electrophoresis, and gel filtration chromatography. Results of gel filtration chromatography studies further provided evidence that VirE2 aggregated in A. tumefaciens mutants deleted of virE1 and assembled as a VirE1/VirE2 complex with a molecular size of approximately 75-kDa when synthesized in the wide-type strain A348. Our findings support a model in which virE1 contributes to assembly of export-competent VirE2 at two levels, by promoting efficient translation of virE2 and posttranslationally whereby the VirE1 chaperone stabilizes VirE2 in vivo, and by assembling as a complex composed of a dimer of VirE1 chaperone bound to a monomer of VirE2 SSB. Finally a novel method was developed for identifying and characterizing protein-protein interactions in bacteria.
Studies on the Role of the VirE1 Chaperone for Export of the VirE2 Single-Stranded DNA-Binding Protein Across the Agrobacterium Cell Envelope