Quorum sensing is a mechanism used by many proteobacteria to regulate expression of target genes in a population-dependent manner. The quorum sensing system of Vibrio fischeri activates the luminescence (lux) operon when the autoinducer signaling molecule (N-3-oxohexanoyl homoserine lactone) is recognized and bound by the activator protein LuxR. LuxR subsequently binds to the lux box centered at à 42.5 bp upstream of the transcription initiation site and activates transcription from the lux operon promoter, resulting in the emission of light at high cell densities. LuxR consists of 250 amino acids arranged into an N-terminal (regulatory) domain and a C-terminal (activation) domain, and is thought to function as an ambidextrous activator capable of making multiple contacts with the alpha and sigma subunits of RNA polymerase (RNAP). Published work describing the results of alanine scanning mutagenesis performed on the C-terminal domain of LuxR (residues 190-250) has identified residues (K198, W201 and I206) that appear to play a role in positive control of transcription initiation. Additional mutagenesis of residues 180-189 has been undertaken via a three-primer or four-primer PCR-based method in this study. Variants of LuxR were screened for their ability to activate luciferase production and to repress transcription from an artificial promoter, and production of full-length LuxR was measured, in an attempt to identify additional positive control variants. No additional positive control variants were found in this study. Work has also been undertaken to identify intergenic suppressors between positive control variants of LuxR and the RNAP alpha subunit, RpoA. Starting with a recombinant Escherichia coli strain encoding the lux operon and LuxR variant I206E, a random chemical mutagenesis was performed on a vector encoding RpoA. Following transformation of the mutated plasmids encoding RpoA, high throughput luminescence assays were used to identify isolates with phenotypes brighter than the control. Isolation of an intergenic suppressor will confirm the existence of protein-protein interactions between LuxR and RpoA within the transcription initiation complex. The ability of other LuxR family members to establish productive protein-protein interactions with RNAP necessary for transcription initiation was also examined. LuxR homologues EsaR of Pantoea stewarti ssp. stewartii, a repressor of known function, and ExpR of Erwinia carotovora subsp. carotovora were also analyzed for their ability to activate the lux operon, as well as to repress transcription from an artificial promoter containing the lux box. / Master of Science
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/31994 |
| Date | 05 May 2003 |
| Creators | Faini, Marie Annette |
| Contributors | Biology, Stevens, Ann M., Larson, Timothy J., Popham, David L. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | MasterThesis5_5_2003.PDF |
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