THE AGROBACTERIUM-TUMEFACIENS VIRB4 GENE-PRODUCT IS AN ESSENTIAL VIRULENCE PROTEIN REQUIRING AN INTACT NUCLEOSIDE TRIPHOSPHATE-BINDING DOMAIN

Products of the approximately 9.5-kb virB operon are proposed to direct the export of T-DNA/protein complexes across the Agrobacterium tumefaciens envelope en route to plant cells. The presence of conserved nucleoside triphosphate (NTP)-binding domains in VirB4 and VirB11 suggests that one or both proteins couple energy, via NTP hydrolysis, to T-complex transport. To assess the importance of VirB4 for virulence, a nonpolar virB4 null mutation was introduced into the pTiA6NC plasmid of strain A348. The 2.37-kb virB4 coding sequence was deleted precisely by oligonucleotide-directed mutagenesis in vitro. The resulting DELTAvirB4 mutation was exchanged for the wild-type allele by two sequential recombination events with the counterselectable Bacillus subtilis sacB gene. Two derivatives, A348DELTAB4.4 and A348DELTAB4.5, sustained a nonpolar deletion of the wild-type virB4 allele, as judged by Southern blot hybridization and immunoblot analyses with antibodies specific for VirB4, VirB5, VirB10, and VirB11. Transcription of wild-type virB4 from the lac promoter restored virulence to the nonpolar null mutants on a variety of dicotyledonous species, establishing virB4 as an essential virulence gene. A substitution of glutamine for Lys-439 and a deletion of Gly-438, Lys-439, and Thr-440 within the glycine-rich NTP-binding domain (Gly-Pro-Iso-Gly-Arg-Gly-Lys-Thr) abolished complementation of A348DELTAB4.4 or A348DELTAB4.5, demonstrating that an intact NTP-binding domain is critical for VirB4 function. Merodiploids expressing both the mutant and wild-type virB4 alleles exhibited lower virulence than A348, suggesting that VirB4, a cytoplasmic membrane protein, may contribute as a homo- or heteromultimer to A. tumefaciens virulence.