TI PLASMID CONJUGATION IS INDEPENDENT OF VIR - RECONSTITUTION OF THE TRA FUNCTIONS FROM PTIC58 AS A BINARY-SYSTEM

Two regions of the nopaline-type Ti plasmid pTiC58 are important for c onjugal transfer of this element to recipient bacteria, These two regi ons were cloned into two independent replicons to produce a binary tra nsfer system, For one region, oriT/tra, we constructed two derivatives , pFRtra and pDCtra-5. Each contains the oriT site and the two flankin g, divergently transcribed tra operons that encode the DNA processing functions associated with the relaxosome. These two plasmids also carr y traR, which encodes the transtriptional activator necessary for expr ession of transfer genes, The two plasmids differ by the amounts of tr aB sequence or sequence downstream of traG present in the construct, T he second replicon, pPLE2, carries the traI/trb region, The tral gene confers production of the Agrobacterium tumefaciens N-acyl homoserine lactone autoinducer, while the remaining genes in the trb operon encod e components of the mating bridge. Donors harboring the two plasmids m obilized the transfer of the plasmid carrying the oriT/tra region to a n A. tumefaciens recipient at frequencies similar to that at which the intact Ti plasmid transferred, Plasmid pFRtra, which encodes most of traB, was mobilized at a frequency almost 10-fold higher than was pDCt ra-5, which lacks most of the gene, A. tumefaciens donors also mobiliz ed pFRtra to Escherichia coli and Pseudomonas fluorescens recipients a t frequencies similar to those observed with A. tumefaciens recipients , Rhizobium meliloti harboring the binary system also transferred the oriT/tra component to these recipients. However, E. coli or P. fluores cens donors harboring the binary system did not transfer pFRtra to any of the recipients, Furthermore, while the A. tumefaciens and R. melil oti donors produced high levels of the autoinducer, the P. fluorescens and E. coli donors produced only trace amounts of this signal molecul e, These results indicate that the tra system of pTiC58 is fully conta ined within the characterized tra and trb regions of the Ti plasmid, t hat conjugation does not require functions encoded by the vir system f or maximal activity, and that while the Ti plasmid tra system recogniz es diverse gram-negative bacteria as recipients, of the hosts tested, it functions only in members of the family Rhizobiaceae.