AN ESSENTIAL VIRULENCE PROTEIN OF AGROBACTERIUM-TUMEFACIENS, VIRB4, REQUIRES AN INTACT MONONUCLEOTIDE BINDING DOMAIN TO FUNCTION IN TRANSFER OF T-DNA

The 11 gene products of the Agrobacterium tumefaciens virB operon, tog ether with the VirD4 protein, are proposed to form a membrane complex which mediates the transfer of T-DNA to plant cells. This study examin ed one putative component of that complex, VirB4. A deletion of the vi rB4 gene on the Ti plasmid pTiA6NC was constructed by replacing the vi rB4 gene with the kanamycin resistance-conferring nptII gene. The virB 4 gene was found to be necessary for virulence on plants and for the t ransfer of IncQ plasmids to recipient cells of A. tumefaciens. Genetic complementation of the deletion strain by the virB4 gene under contro l of the virB promoter confirmed that the deletion was nonpolar on dow nstream virB genes. Genetic complementation was also achieved with the virB4 gene placed under control of the lac promoter, even though synt hesis of the VirB4 protein from this promoter is far below wild-type l evels. Having shown a role for the VirB4 protein in DNA transfer, lysi ne-439, found within the conserved mononucleotide binding domain of Vi rB4, was changed to a glutamic acid, methionine, or arginine by oligon ucleotide-directed mutagenesis. virB4 genes bearing these mutations we re unable to complement the virB4 deletion for either virulence or for IncQ transfer, showing that an intact mononucleotide binding site is necessary for the function of VirB4 in DNA transfer. The necessity of the VirB4 protein with an intact mononucleotide binding site for extra cellular complementation of vir2 mutants was also shown. In merodiploi d studies, lysine-439 mutations present in trans decreased IncQ plasmi d transfer frequencies, suggesting that VirB4 functions within a compl ex to facilitate DNA transfer.