Quorum Sensing, Entry Exclusion And Replication Of The Ti Plasmid Of Agrobacterium Tumefaciens
Agrobacterium tumefaciens contains the tumor inducing (Ti) plasmid that stimulates tumor formation in wounded plant tissues. Replication, partitioning, conjugation and entry exclusion of this plasmid are under strict cellular control, and are regulated by quorum sensing. In Gram-negative bacteria, the quorum sensing circuitry is usually mediated by small diffusible signal molecules produced by LuxI homologues and signal receptor proteins homologous to LuxR. In A. tumefaciens, TraR is the LuxR-type quorum sensing receptor that is activated by the signal molecule N-3-oxooctanoyl-L-homoserine lactone (OOHL). We studied the TraR dimerization properties, the involvement of quorum sensing on entry exclusion, and replication of the Ti-plasmid. First we wanted to know whether TraR dimerization is required for protein stability. It is well established that transcriptional activation by TraR depends on the presence of the signal molecule OOHL, which is required for protein folding, protease resistance, and dimerization. My results suggest that dimerization of TraR enhances resistance to cellular proteases, further contributing to protein stability and function. In a second study it is shown that the Ti plasmid encodes for robust entry exclusion, which prevents conjugation between donor cells containing Ti plasmids. Entry exclusion of the Ti plasmid is tightly regulated by TraR and mediated by TrbK and TrbJ proteins. In the absence of OOHL, the Trb proteins are not expressed, and Agrobacterium cells harboring a Ti plasmid are efficient recipients. However, in the presence of OOHL, cells block the entry of Ti plasmids and instead become efficient conjugal donors. Finally, the replication properties of the Ti plasmid were analyzed and I show that the origin of replication resides within the repC gene. I also show that RepC protein binds to a site located at an AT-rich region within its own gene. The DNA binding domain of RepC was localized to a region at the N-terminus of the protein. RepC functions in cis to initiate replication, and this mode of action may have important implications for plasmid compatibility.
quorum sensing; entry exclusion; replication
Winans, Stephen C
Helmann, John D; Alani, Eric E
Ph.D. of Microbiology
Doctor of Philosophy
dissertation or thesis