CONSTRUCTION OF TRANSPOSON TN3PHOA - ITS APPLICATION IN DEFINING THE MEMBRANE TOPOLOGY OF THE AGROBACTERIUM-TUMEFACIENS DNA TRANSFER PROTEINS

Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane proteins. To study the topology of the Agrobacterium T-DNA transfer proteins, we co nstructed a transposon, Tn3phoA. The transposon mobilizes into plasmid s at a high frequency, is stable after transposition, can produce phoA translational fusions and can be used for the analysis of protein top ology directly in the bacterium of interest. For studies on the DNA tr ansfer proteins, an Agrobacterium strain deficient in phoA under our e xperimental conditions was constructed by chemical mutagenesis. A plas mid containing virB and virD4 was used as a target for mutagenesis. Tw enty-eight unique phoA-positive clones that mapped to eight virB genes were isolated. Multiple insertions throughout VirB1, VirB5, VirB7, Vi rB9 and VirB10 indicated that these proteins primarily face the peripl asm. Insertions in VirB2, VirB6 and VirB8 allowed the identification o f their periplasmic domains. No insertions were found in VirB3, VirB4 and VirB11. These proteins either lack or have a short periplasmic dom ain. No insertions mapped to VirD4 either. To study VirD4 topology, ta rgeted phoA fusions and random lacZ fusions were constructed. Analysis of the fusion proteins indicated that VirD4 contains a single peripla smic domain near the N-terminus, and most of the protein lies in the c ytoplasm. A hypothetical model for the T-DNA transport pore is present ed.