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Title:Genetic and Biochemical Characterization of the Mechanisms Concerning the TraR/AAI/TraM Mediated Quorum Sensing That Regulates Ti Plasmid Conjugation
Author(s):Luo, Zhao-Qing
Doctoral Committee Chair(s):Farrand, Stephen K.
Department / Program:Crop Sciences
Discipline:Crop Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Microbiology
Abstract:Quorum sensing defines a regulatory strategy that allows bacteria to coordinate gene expression with their population density. Among Gram-negative bacteria, monitoring cell density often is achieved by producing and sensing an acyl-homoserine lactone molecule. In Agrobacterium tumefaciens , regulation of Ti plasmids conjugation is controlled in part by a quorum-sensing system consisting of four elements; the transcription activator TraR, the intercellular signal N-(3-oxo-octanoyl)-L-homoserine lactone, the cis-acting recognition site tra box located in the promoter region of the target gene and the antiactivator TraM that negatively modulates TraR activity. Using a combination of genetic and biochemical approaches, I have analyzed how these four elements interact with one the other to control population size-dependent expression of tra genes. By converting the activator TraR into a repressor, I established a system to measure interactions between TraR and the tra box. I demonstrated that TraR recognizes the tra box as its binding site, that binding of TraR to this site depends on AAI, and that the N-terminal half of the protein contains one or more domains that are required for activation. I identified a region of TraR spanning residues 39 to 140 important for binding the signal. I demonstrated that the antiactivator TraM directly binds to the C-terminal portion of TraR, a region important for DNA binding. Moreover, such binding could abolish TraR-mediated transcription. I also found that a functional rnd gene is required for proper induction of tra genes on the Ti plasmid. Further studies suggest that mutations in rnd affect expression of traR at the post-transcriptional level. Finally, I have cloned and characterized a tetracycline resistance gene unit present in the genome of the widely used A. tumefaciens strain C58. I found that tetracycline and 25 related compounds do not induce expression of the functional gene and that resistance to tetracycline often, if not always arises from mutations affecting regulation of the tetracycline resistance gene. Moreover, I have constructed a derivative of C58 with an internal deletion of tet C58 to facilitate the use of tetracycline as a selection marker in genetic analysis of this widely used A. tumefaciens strain.
Issue Date:2001
Description:417 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2001.
Other Identifier(s):(MiAaPQ)AAI9996657
Date Available in IDEALS:2015-09-25
Date Deposited:2001

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