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Amino Acid Residues in LuxR Critical for its Mechanism of Transcriptional Activation during Quorum Sensing

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dc.contributor.authorTrott, Amy Elizabethen
dc.contributor.committeechairStevens, Ann M.en
dc.contributor.committeememberPopham, David L.en
dc.contributor.committeememberLarson, Timothy J.en
dc.contributor.departmentBiologyen
dc.date.accessioned2014-03-14T20:41:40Zen
dc.date.adate2000-07-21en
dc.date.available2014-03-14T20:41:40Zen
dc.date.issued2000-07-17en
dc.date.rdate2001-07-21en
dc.date.sdate2000-07-20en
dc.description.abstract<I>Vibrio fischeri</I>, a symbiotic bioluminescent bacterium, serves as one of the best understood model systems for a mechanism of cell-density dependent bacterial gene regulation known as quorum sensing. During quorum sensing in <I>V. fischeri</I>, an acylated homoserine chemical signal (autoinducer) is synthesized by the bacteria and used to sense their own species in a given environment. As the autoinducer levels rise, complexes form between the autoinducer and the N-terminal domain of a regulatory protein, LuxR. In response to autoinducer binding, LuxR is believed to undergo a conformational change that allows the C-terminal domain to activate transcription of the luminescence or <I>lux</I> operon. To further understand the mechanism of LuxR-dependent transcriptional activation of the <I>lux</I> operon, PCR-based site-directed mutagenesis procedures have been used to generate alanine-substitution mutants in the C-terminal forty-one amino acid residues of LuxR, a region that has been hypothesized to play a critical role in the activation process. An <I>in vivo</I> luminescence assay was first used to test the effects of the mutations on LuxR-dependent activation of the <I>lux</I> operon in recombinant <I>Escherichia coli</I>. Luciferase levels present in cell extracts obtained from these strains were also quantified and found to correlate with the luminescence results. Eight strains encoding altered forms of LuxR exhibited a "dark" phenotype with luminescence output less than 50% and luciferase levels less than 50% of the wildtype control strain. Western immunoblotting analysis with cell extracts from the luminescence and luciferase assays verified that the altered forms of LuxR were expressed at levels approximately equal to wildtype. Therefor, Low luminescence and luciferase levels could be the result of a mutation that either affects the ability of LuxR to recognize and bind its DNA target (the <I>lux</I> box) or to establish associations with RNA polymerase (RNAP) at the <I>lux</I> operon promoter necessary for transcriptional initiation. A third <I>in vivo </I>assay was used to test the ability of the altered forms of LuxR to bind to the <I>lux</I> box (DNA binding assay/repression). All of the LuxR variants exhibiting the "dark" phenotype in the luminescence and luciferase assay were also found to be unable to bind to the <I>lux</I> box in the<I> </I>DNA binding assay. Therefore, it can be concluded that the alanine substitutions made at these positions affect the ability of LuxR to bind to the <I>lux</I> box in the presence and absence of RNA polymerase. Another class of mutants exhibited wildtype phenotypes in the luminescence and luciferase assays but were unable to bind to the <I>lux</I> box in the DNA binding assay. The alanine substitutions made at these amino acid residues may be making contacts with RNAP that are important for maintaining the stability of the DNA binding region of LuxR. Alanine substitutions made at these positions have a defect in DNA binding at the promoter of the <I>lux</I> operon only in the absence of RNAP. None of the alanine substitutions made in the C-terminal forty-one amino acids of LuxR were found to affect activation of transcription of the <I>lux</I> operon without also affecting DNA binding. Taken together, these results support the conclusion that the C-terminal forty-one amino acids of LuxR are important for DNA recognition and binding of the <I>lux</I> box rather than positive control of the process of transcription initiation.en
dc.description.degreeMaster of Scienceen
dc.identifier.otheretd-07202000-10050050en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-07202000-10050050/en
dc.identifier.urihttp://hdl.handle.net/10919/34070en
dc.publisherVirginia Techen
dc.relation.haspartAET.thesis2.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectDNA bindingen
dc.subjecttranscriptional activationen
dc.subjectluminescenceen
dc.subjectautoinduceren
dc.subjectlux operonen
dc.subjectlux boxen
dc.subjectLuxRen
dc.subjectVibrio fischerien
dc.subjectluciferaseen
dc.titleAmino Acid Residues in LuxR Critical for its Mechanism of Transcriptional Activation during Quorum Sensingen
dc.typeThesisen
thesis.degree.disciplineBiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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