GENETIC AND SEQUENCE-ANALYSIS OF THE PTIC58 TRB LOCUS, ENCODING A MATING-PAIR FORMATION SYSTEM RELATED TO MEMBERS OF THE TYPE-IV SECRETION FAMILY

Conjugal transfer of pTiC58 requires two regions, tra which contains t he oriT and several genes involved in DNA processing and a region of u ndefined size and function that is: located at the 2-o'clock position of the plasmid, Using transposon mutagenesis with Tn3HoHo1 and a binar y transfer system, we delimited this second region, called trb, to an 11-kb interval between the loci for vegetative replication and nopalin e catabolism, DNA sequence analysis of this region identified 13 signi ficant open reading frames (ORFs) spanning 11,003 bp, The first, encod ing tr al, already has been described and is responsible fur the synth esis of Agrobacterium autoinducer (AAI) (I. Hwang, P.-L. Li, L. Zhang, K. R. Piper, D. M. Cook, M. E. Tate, and S. K. Farrand, Proc. Natl. A cad. Sci. USA 91:4639-4643, 1994). Translation products of the next 11 ORFs showed similarities to those of trbB, -C, -D, -E, -J, -K, -L, -F , -G, -H, and -I of the trb region of the octopine-type Ti plasmid pTi 15955 and of the tra2 core region of RP4, In RP4, these genes encode m ating-pair formation functions and are essential for the conjugal tran sfer of the IncP plasmid, Each of the trb gene homologues is oriented counterclockwise on the Ti plasmid, Expression of these gents, as meas ured by using the lacZ fusions formed by Tn3HoHo1, required the tra1 p romoter and the transcriptional activator TraR along with its coinduce r, AAI. While related to that of RP4, the trb system of pTiC58 did not allow propagation of the trb-specific bacteriophages PRD1, PRR1, and Pf3. The products of several trb genes of the Ti plasmid are similar t o those of other loci that encode DNA transfer or protein secretion sy stems, all of which are members of the type IV secretion family.