1.5.2S
HOST-RESPONSIVE GENE EXPRESSION BY AGROBACTERIUM TUMEFACIENS
HOST-RESPONSIVE GENE EXPRESSION BY AGROBACTERIUM TUMEFACIENS SC WINANS Section of Microbiology, Cornell University, Ithaca, New York, USA 14853 Background and Objectives Agrobacterium tumefaciens offers a unique model to learn about host recognition by bacterial pathogens. A. tumefaciens causes crown gall tumors in a broad range of dicotyledenous plants by transferring one or more fragments of DNA from its large tumor-inducing (Ti) plasmid to the nuclei of wounded plant cells. This transferred DNA directs overproduction of mitogenic phytohormones as well as production of nutrient sources called opines. The genes that direct DNA transfer (vir genes) are transcriptionally induced by wound-released chemical stimuli, and three proteins, VirA, VirG, and ChvE, are required to transduce these signals to the level of gene expression. Later in colonization, opines provide a second class of host-released chemical signals. One such opine, octopine, activates transcription of the octopine catabolic operon. Induction of this operon requires OccR, a member of the LysR family. Directly downstream from the octopine catabolic genes is the traR gene, whose product activates the conjugal transfer regulon of the Ti plasmid. TraR is a member of the LuxR family of quorum-sensing regulators and functions in conjunction with the Tral protein (a Luxi homolog), which synthesizes the autoinducer 3-oxo-octanoyi-homoserine lactone (30C8-HL). Results and Conclusions VirA has a particularly complex structure, containing four modules: a periplasmic module, a cytoplasmic "linker" module, the kinase module, and a carboxyl terminal receiver module. We have assigned functions to each module by genetically ablating each one, by expressing certain modules as separate proteins, and by point mutagenesis [1]. The periplasmic module is required for perception of complexes containing host-released monosaccharides and ChvE, while the linker domain is required for perception of phenolic compounds. Both modules play stimulatory roles in the presence of inducing ligands, rather than inhibitory roles that are neutralized by ligands. The receiver module plays an inhibitory role that is abolished by phosphorylation. OccR, like many LysR-type proteins, binds to a rather long binding site and does so in the presence or absence of inducing ligands. Octopine must therefore cause a conformational change of the bound protein. We have demonstrated that octopine causes the OccR-protected region to shrink from five helical turns to four, and also to drastically reduce the angle of an OccR-induced DNA bend. We have obtained mutants of both OccR and its DNA binding site that perturb this conformational change, either abolishing it entirely, or altering the response to low concentrations of octopine. From these data, we have formulated a model about the roles of particular DNA sequences in ligandresponsive DNA bending [2]. As described above, OccR activates transcription of traR, whose product activates the tra regulon. We have shown that a second, truncated copy of traR (designated traS, originally identified by S.K. Farrand and colleagues, University of Illinois) encodes a protein that is both unable to activate this regulon and able to exert a dominant defective effect upon TraR. traS is expressed in response to another opine called mannopine, and mannopine prevents conjugation in a traS-dependent fashion [3]. In a separate study, we have found that a wild type strain of A. tumefaciens strongly discriminate between 30-C8-HL and similar compounds. Furthermore TraR activity is strongly inhibited by many autoinducer analogues. In contrast, overexpression of TraR drastically broadens its substrate specificity and abolishes antagonistic effects of all autoinducers. We propose that autoinducers act by oligomerizing TraR and that overexpression of TraR potentiates dimerization by mass action. References [1] Chang, C.-H., Zhu, J., and Winans, S. C. 1996. J. Bacteriol. 178:4710-4716. [2] Wang, L. and Winans, S. C. 1995. J. Mol. Biol. 253:691-702. [3] Zhu, J., and Winans, S. C. 1998. Mol. Microbiol. 27:289-297.