Abstract

The VirB4 ATPase of Agrobacterium tumefaciens, a putative component of the T-complex transport apparatus, associates with the cytoplasmic membrane independently of other products of the Ti plasmid. VirB4 was resistant to extraction from membranes of wild-type strain A348 or a Ti-plasmidless strain expressing virB4 from an IncP replicon. To evaluate the membrane topology of VirB4, a nested deletion method was used to generate a high frequency of random fusions between virB4 and *phoA, which encodes a periplasmically active alkaline phosphatase (AP) deleted of its signal sequence. VirB4::PhoA hybrid proteins exhibiting AP activity in Escherichia coli and A. tumefaciens had junction sites that mapped to two regions, between residues 58 and 84 (region 1) and between residues 450 and 514 (region 2). Conversely, VirB4::b-galactosidase hybrid proteins with junction sites mapping to regions 1 and 2 exhibited low b-galactosidase activities and hybrid proteins with junction sites elsewhere exhibited high b-galactosidase activities. Enzymatically active VirB5::PhoA hybrid proteins had junction sites that were distributed throughout the length of the protein. Proteinase K treatment of A. tumefaciens spheroplasts resulted in the disappearance of the 87-kDa VirB4 protein and the concomitant appearance of two immunoreactive species of;35 and;45 kDa. Taken together, our data support a model in which VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains. Agrobacterium tumefaciens is a plant pathogen with the unique ability to deliver oncogenes to the genomes of suscep-tible plants cells (for reviews, see references 27 and 64). The translocation-competent form of the transferred DNA (T-DNA) has been proposed to consist of a single-stranded mol-ecule covalently associated at its 59 end with the VirD2 protein and noncovalently associated along its length with VirE2, a