Essential components of the Ti plasmid trb system, a type IV macromolecular transporter

The trb operon from pTiC58 is one of three loci that are required for conju gal transfer of this Ti plasmid, The operon, which probably codes for the m ating bridge responsible for pair formation and DNA transfer, contains 12 g enes, 11 of which are related to genes from other members of the type TV se cretion system family. The 12th gene, traI, codes for production of Agrobac terium autoinducer (AAI). Insertion mutations were constructed in each of t he 12 genes, contained on a full-length clone of the trb region, using anti biotic resistance cassettes or a newly constructed transposon, This transpo son, called mini-Tn5Ptrb, was designed to express genes downstream of the i nsertion site from a promoter regulated by TraR and AAI. Each mutation coul d trans complement downstream Tn3HoHo1 insertions in the trb operon of full -sized Ti plasmids, When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58 Delta accR mutations in trbB, -C, -D, -E, -L, -F, -G, and - H abolished conjugal transfer from strain UIA5, which lacks the 450-kb cata bolic plasmid pAtC58. However, these mutants retained residual conjugal tra nsfer activity when tested in strain NT1, which contains this large plasmid , The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbI mutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was res tored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation in traI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the m utant donor in the presence of AAI, We conclude that all of the trb genes e xcept trbI and trbK are essential for conjugal transfer of pTiC58, We also conclude that mutations in any one of the trb genes except trail and trbJ c an be complemented by functions coded for by pAtC58.