The replicator of the nopaline-type Ti plasmid pTiC58 is a member of the repABC family and is influenced by the TraR-dependent quorum-sensing: Regulatory system

The replicator (rep) of the nopaline-type Ti plasmid pTiC58 is located adja cent to the trb operon of this conjugal element. Previous genetic studies o f this region (D. R. Gallie, M. Hagiya, and C. I. Kado, J. Bacteriol. 161:1 034-1041, 1985) identified functions involved in partitioning, origin of re plication and incompatibility, and copy number control. In this study, we d etermined the nucleotide sequence of a 6,146-bp segment that encompasses th e rep locus of pTiC58. The region contained four full open reading frames ( ORFs) and one partial ORF. The first three ORFs, oriented divergently from the traI-trb operon, are closely related to the repA, repB, and repC genes of the octopine-type Ti plasmid pTiB6S3 as well as to other repA, -B, and - C genes from the Ri plasmid pRiA4b and three large plasmids from Rhizobium spp. The fourth ORF and the partial ORF are similar to y4CG and y4CF, respe ctively, of the Sym plasmid pNGR234a, The 363-bp intergenic region between traI and repA contained two copies of the ha box which is the cis promoter recognition site for TraR, the quorum-sensing activator of Ti plasmid conju gal transfer. Expression of the traI-trb operon from the tra box II-associa ted promoter mediated by TraR and its acyl-homoserine lactone ligand, AAI, was negatively influenced by an intact ha box III. On the other hand, the r egion containing the two ha boxes was required for maximal expression of re pA, and this expression was enhanced slightly by TraR and AAI, Copy number of a minimal rep plasmid increased five- to sevenfold in strains expressing traR but only when AAI also was provided, Consistent with this effect, con stitutive expression of the quorum-sensing system resulted in an apparent i ncrease in Ti plasmid copy number. We conclude that Ti plasmid copy number is influenced by the quorum-sensing system, suggesting a connection between conjugal transfer and vegetative replication of these virulence elements.