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Chapter 18 : Cell-Cell Signaling within Crown Gall Tumors
Category: Microbial Genetics and Molecular Biology; Bacterial Pathogenesis
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It has been 100 years since Agrobacterium tumefaciens was demonstrated to cause crown gall tumors at plant wound sites. This chapter first presents background information relevant to quorum sensing in A. tumefaciens and then focuses on our current knowledge of the molecular biology of the TraR-TraI system. Regulation of TraR activity is complex and occurs at the levels of transcription, protein folding, resistance to proteolysis, and the formation of quaternary complexes with other TraR subunits or with two different antiactivators of TraR. Control of traR expression by opines therefore has evolved independently in these two types of Ti plasmids. The genes of the arc operon and occ operon are not similar, except for traR. LysR and AccR are also dissimilar, as OccR is a LysR-type transcriptional activator that binds to promoter DNA both in the presence and absence of the inducing signal. Overproduction of TraR can fully overcome inhibition, suggesting that TraM acts by making stoichiometric contacts with TraR. Direct interactions were confirmed by yeast two-hybrid assays and by far Western immunoblots. The author recently used gene arrays to profile the TraR transcriptome and found that all induced genes were Ti plasmid-encoded. These genes include the tra and trb genes, which are involved in conjugal transfer; the rep genes, which are required for vegetative replication and plasmid partitioning into daughter cells; and traM.
A model of the quorum-sensing system in octopine-type Ti plasmids. TraR is expressed in response to the tumor-released nutrient octopine, whereas the TraR antiactivator TrlR is expressed in response to mannopine, which is also released from tumors. Apo-TraR is rapidly degraded by proteases but is rescued from proteolysis by binding OOHL, the quorum-sensing signal that is produced by TraI. TraR-OOHL dimers activate transcription of traM and the tra, trb, and rep operons. TraR-OOHL complexes can be inactivated through direct interactions with TraM or TrlR. OOHL can be destroyed by the BlcC protein (formerly AttM).
A comparison of traR regulation via opines on different types of Ti plasmids. On octopine-type Ti plasmids, traR is activated by OccR in response to octopine. The TraR antiactivator TrlR is expressed in response to mannopine, probably via inactivation of the MocR repressor. On nopaline-type Ti plasmids, traR is expressed when AccR repression is relieved by agrocinopines A and B. Regulation of traR on the chrysopine-type plasmid is similar, except the inducing opines are agrocinopines C and D. There are two copies of traR on pAtK84b, one thought to be activated by NocR in response to nopaline, while the other is activated in response to agrocinopines A and B via derepression of AccR.
(A) Induction of the tra regulon using full-genome microarrays. Black circles indicate genes located on the circular or linear chromosome or on the cryptic Ti plasmid pAtC58, while gray circles indicate genes located on the octopine-type Ti plasmid pTiR10. The x axis shows the range of expression in the absence of TraR, while the y axis shows the range of expression when TraR is overproduced, which suffices to induce the entire regulon ( 22 ). Note that virtually all Ti plasmid genes are expressed more strongly in the presence than in the absence of TraR, as TraR activates the repABC operon, increasing Ti plasmid copy number. (B) Induction of the vir regulon using Ti plasmid microarrays. Open symbols represent non-Ti plasmid genes, while filled symbols represent Ti plasmid genes. The x axis represents the range of gene expression in the absence of acetosyringone (AS), while the y axis represents the range of expression in the presence of 100 μM AS. Note that all Ti plasmid genes are slightly induced by AS, as AS acts through VirG to activate the repABC operon, increasing the Ti plasmid copy number.
Interactions between TraR and other regulators. (A) TraR and VirG both regulate the repABC operon and therefore influence the Ti plasmid copy number. TraROOHL complexes bind to tra boxes II and III (tbII and tbIII) to activate promoters P1, P2, and P3, as well as the divergent promoter PtraI. P~VirG bind to a vir box (vb) to activate promoter P4. Of these, P4 is active in the absence of either protein, causing a basal level of gene expression sufficient for low-level Ti plasmid replication. (B) RepA and RepB form a complex that binds to an operator directly downstream of promoter P4 and also binds to a site between repA and repB (not shown). Binding represses all four promoters of the repABC operon. RepC is negatively regulated at the transcript elongation or translational level by a small antisense RNA encoded by the repE gene. (C) The divergent traCDG and traAFGB operons are activated by TraR-OOHL complexes bound to the tra box I (tbI). Both promoters are also repressed by the TraA protein bound to the origin of conjugal transfer (oriT). TraA also processes one DNA strand at oriT in a step that is essential for conjugation. TraC and TraD assist TraA in repression, in DNA processing, and in conjugation but are not essential for any of these events.