The Conjugation System of Ti Plasmids and its Response to Environmental Cues.

We are interested in the nature of the conjugal transfer system of Ti plasmids, and how the expression of the genes of this system is regulated by the conjugal opines. With respect to the first area, we have characterized the two regions of the nopaline/agrocinopine-type Ti plasmid pTiC58 that are essential for conjugal transfer. Our genetic and DNA sequence analyses indicate that the tra genes of the nopaline-type Ti plasmid are located in two clusters. One, called tra, contains the cis active oriT site at which conjugal transfer initiates, and also the genes that code for the DNA metabolism functions required for nicking the plasmid at the oriT site. The second region, called trb, is located approximately 100 kb away and contains 11 genes that code for the mating bridge which forms the conjugation junction between donor and recipient cells. Analysis of these genes and their products suggests that the conjugal transfer system of the Ti plasmid is chimeric, and contains elements that share ancestral relatedness with the conjugal transfer genes of several other plasmids including RP4, F, and RSF1010. In the course of this work, we developed a binary system for the genetic analysis of conjugal transfer. Using this system we conclusively showed that the tra system and the vir system of the Ti plasmid are completely independent and non interacting. Using this system we also showed that the tra and trb systems of the nopaline and the octopine/mannityl opine-type Ti plasmids are interchangeable and cross-functional.

With respect to regulation, we have identified the key opine-responsive master regulator of conjugation for the nopaline/agrocinopine-type Ti plasmid, pTiC58. This protein, called AccR, is a transcriptional repressor that responds to agrocinopines A and B, the conjugal opines for this Ti plasmid. AccR binds specifically to a DNA sequence located in the promoter region of the genes that are required for uptake and catabolism of agrocinopines A and B. This DNA binding activity is specifically inhibited by the two opines indicating that these metabolites act as the specific inducers. However, while AccR directly regulates expression of the genes for catabolism of the agrocinopine opines, it only indirectly regulates expression of the tra and trb operons required for conjugation. Expression of these two operons is controlled by a second signaling system involving the transcriptional activator, TraR and the cell-density sensing molecule Agrobacterium Autoinducer (AAI). AAI is N-(3-oxo octanoyl)-L-homoserine lactone and is made and secreted by the Agrobacterium donor cells themselves. It is a member of the family of N-acyl homoserine lactone signals used by many Gram-negative bacteria to regulate gene expression in response to their population density. These autoinducers function as self-made signals by which bacterial cells in a population communicate with each other. Our physiological and culture studies indicate that A. tumefaciens does indeed regulate Ti plasmid conjugal transfer in a cell density-dependent fashion. Thus, conjugation is dependent upon the donor cells reaching a critical population density, with TraR and AAI functioning as the quorum sensors.

How, then, does Agrobacterium integrate the requirement for the opine signal with the TraR/AAI quorum sensing signal system? Our genetic and DNA sequence analysis indicates that traR is itself located in an operon that is regulated by AccR. Thus, in the absence of the opine, AccR represses the operon and traR is not expressed. In the presence of the conjugal opine, repression is relieved resulting in expression of traR and induction of conjugal transfer. Interestingly, the operon in which traR is located does not, itself, play any role in conjugation. The genes within this operon appear to be associated with catabolism of some metabolite, perhaps related to agrocinopines A and B. This would account for why the operon is regulated by AccR. There is no apparent reason for traR to be associated with this operon. Thus, traR may be an intruder in this gene system. None the less, the effect is to place conjugation under the control of opines. A conceptually similar gene arrangement results in the regulation of traR by octopine in the octopine/mannityl opine-type Ti plasmids. However, the operon of the octopine-type Ti plasmid in which traR is located is not related to the operon of the nopaline-type Ti plasmid in which traR is located. In both cases, traR appears to be a relatively recent addition to the two operons. This means that the events that resulted in placing traR under control of the respective opine regulons occurred independently in the two Ti plasmid types. We now have found a third arrangement that places a traR gene under the control of another opine family; the mannityl opines which we study a in project described previously.

Recent publications relevant to this theme:

Cook, D.M., and Farrand, S.K. The oriTregion of the Agrobacterium tumefaciesTi plasmid pTiC58 shares DNA sequence identity with the T-region borders and the transfter origins of RSF1010, and RK2. J. Bacteriol. 174:6238-6246, 1992

Farrand, S.K. Conjugal Transfer of AgrobacteriumPlasmids. In: D.B. Clewell (ED.). Bacterial Conjugation., Plenum Publishing Corp. New York, N.Y. pp. 255-291. 1993

Piper, K. R., Beck von Bodman, S., and Farrand, S.K. Conjugation factor of Agrobacterium tumefaciensregulates Ti plasmid transfer by autoinduction. Nature (London) 362:448-450, 1993.

Hwang, I., Li., P.-L., Zhang, L., Piper, K., Cook, D.M., Tate, M.E., and Farrand, S.K. TraI, a LuxI homologue, is responsible for production of conjugaton factor, the Ti plasmid N-acyl-homoserine lactone autoinducer. Proc. Natl. Acad. Sci. (USA) 91:4639-4643, 1994

Hwang, I., Cook, D.M., and Farrand, S.K. A new element modulates homoserine lactone-mediated autoinduction of Ti plasmid conjugal transfer. J. Bacteriol. 177:449-458, 1995

Farrand. S.K., Hwang, I, and Cook, D.M. The tra region of the nopaline-type Ti Plasmid is a chimeric with elements related to the transfer systems of RSF1010, RP4, and F. J. Bacteriol. 178:4233-4247, 1996.

Alt-Morbe, J., Stryker, J.L., Fuqua, C., Farrand, S.K. and Winans, S.C. The conjugal transfer system of A.tumefaciensoctopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes. J. Bacteriol. 178:4248-4257, 1996.

Farrand, S. K., Piper, K. R., Sackett, R., Ping, G., Shaw, P. S., and Kim, K.-S. Homoserine Lactone-Mediated Microbial Signaling: A Communication System Common to Plant-Associated Bacteria. In: G. Stacey, B. Mullin and P. Gresshoff, (Eds.). Biology of Plant-Microbe Enteractions. Internatl. Soc., Molec. Plant-Microbe Interact., St. Paul, MN. pp. 173-179, 1996.

Cook, D.M., Li, P.-L., Ruchaud, F., Padden, S., and Farrand, S.K. Ti plasmid conjugation is independent of vir: Reconstitution of the tra functions from pTiC58 as binary system. J. Bacteriol. 179:1291-1297, 1997.

Shaw. P.D., Ping, G., Daly, S.L., Cha, C., Cronan, Jr., J.E., Rinehart, K.L., and Farrand, S.K. Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromotgraphy. Proc. Natl. Acad. Sci. (USA)94:6036-6041.

Oger, P., Kim, K.-S., Sackett, R. L., Piper, K. R., and Farrand, S. K. Octopine-type Ti plasmids code for a mannopine-induceble dominant-negative allele of traR, the quorum-sensing activator that regulates Ti plasmid conjugal transfer. Molec. Microbiol. 27:277-288, 1998.

Farrand, S. K. Conjugal Plasmids and Their Transfer. In: H. P. Spaink, A. Kondorosi, and P. J. J. Hooykaas, (Eds). The Rhizobiaceae. Kluwer Academic Publishing Co., Dorrecht, The Netherlands, In press, 1998.