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Quorum Sensing in Vibrios and Cross-Species Activation Of University of Wisconsin Milwaukee UWM Digital Commons Theses and Dissertations May 2013 Quorum Sensing in Vibrios and Cross-Species Activation of Bioluminescence Lux Genes by Vibrio Harveyi LuxR in an Arabinose-Inducible Escherichia Coli Expression System Anne Marie Wannamaker University of Wisconsin-Milwaukee Follow this and additional works at: https://dc.uwm.edu/etd Part of the Evolution Commons, Microbiology Commons, and the Molecular Biology Commons Recommended Citation Wannamaker, Anne Marie, "Quorum Sensing in Vibrios and Cross-Species Activation of Bioluminescence Lux Genes by Vibrio Harveyi LuxR in an Arabinose-Inducible Escherichia Coli Expression System" (2013). Theses and Dissertations. 177. https://dc.uwm.edu/etd/177 This Thesis is brought to you for free and open access by UWM Digital Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UWM Digital Commons. For more information, please contact [email protected]. QUORUM SENSING IN VIBRIOS AND CROSS-SPECIES ACTIVATION OF BIOLUMINESCENCE LUX GENES BY VIBRIO HARVEYI LUXR IN AN ARABINOSE-INDUCIBLE ESCHERICHIA COLI EXPRESSION SYSTEM by Anne Marie Wannamaker A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Biological Sciences at The University of Wisconsin-Milwaukee May 2013 ABSTRACT QUORUM SENSING IN VIBRIOS AND CROSS-SPECIES ACTIVATION OF BIOLUMINESCENCE LUX GENES BY VIBRIO HARVEYI LUXR IN AN ARABINOSE-INDUCIBLE ESCHERICHIA EXPRESSION SYSTEM by Anne Marie Wannamaker The University of Wisconsin-Milwaukee, 2013 Under the Supervision of Dr. Charles Wimpee Bacterial bioluminescence is observed in over twenty known species, primarily in the family Vibrionaceae. However, only Vibrio fischeri and Vibrio harveyi bioluminescence expression mechanisms are well studied. In V. harveyi, expression of the lux operon is activated by the transcription factor LuxR (LuxRVH), resulting in bioluminescence. Homologs of LuxRVH in other Vibrio species have been shown to regulate transcription of a variety of genes. Three parallel quorum sensing pathways co-regulate the expression of LuxRVH. The first objective was to assess possible quorum sensing regulation of lux operon expression in V. cholerae, V. chagasii, V. orientalis, and V. vulnificus using V. harveyi as the control. Secondly, cross-species induction of bioluminescence by LuxRVH was tested on the aforementioned Vibrio lux operons using an Escherichia coli dual vector expression system. This was accomplished by first generating a plasmid with the V. harveyi luxR gene (luxRVH) driven by an arabinose promoter. Secondly, individual E. coli systems had one of the five Vibrio species-specific lux operons cloned on a separate plasmid. Luminescence was assayed qualitatively on plates, and quantitatively using a ii luminometer. Relative light per cell was calculated by dividing measured light by OD600. Relative light assays showed quorum sensing regulation of the lux operon in all five Vibrio species. Quantitative V. harveyi lux operon expression assays in the E. coli dual vector systems showed significant increase in light production in samples provided with arabinose. This demonstrated that induction of luxRVH by arabinose resulted in upregulation of lux genes. The level of LuxRVH activation of other Vibrio lux operons reflects the distance of evolutionary relationships to V. harveyi. The most induction was seen with the V. vulnificus lux operon followed by V. orientalis, V. chagasii, and finally V. cholerae. This implies conservation of the lux operon regulatory mechanism between closely related species and suggests that the studied Vibrios utilize LuxRVH-type transcription factors. The known LuxRVH homologs, SmcR (V. vulnificus) and HapR (V. cholerae) were suspected lux activators. In addition, other LuxRVH-type regulators are now implicated in the other Vibrio species. Furthermore, mechanistic conservation of these transcription factors implies regulation by V. harveyi-type quorum sensing. iii TABLE OF CONTENTS Abstract ..................................................................................................................... ii List of Figures ........................................................................................................... vii List of Tables .............................................................................................................. x List of Abbreviations .................................................................................................. xi Acknowlegdements ...................................................................................................xii Introduction ............................................................................................................... 1 Bioluminescence ............................................................................................................. 1 Bioluminescent Bacteria ................................................................................................. 2 The lux operon ................................................................................................................ 4 Biochemistry of bioluminescence reaction ..................................................................... 7 Quorum Sensing: Regulation of the lux operon .............................................................. 9 Vibrio fischeri Quorum Sensing .................................................................................... 9 Vibrio harveyi Quorum Sensing ................................................................................. 11 Vibrio cholerae Quorum Sensing ............................................................................... 15 LuxRVH Induction ............................................................................................................ 18 Homologs of LuxRVH ...................................................................................................... 19 Hypothesis ............................................................................................................... 22 Methods and Materials ............................................................................................ 23 Phylogenetic Analysis and Sequence Alignment........................................................... 23 iv Bacterial Strains ............................................................................................................. 23 Media ............................................................................................................................ 24 Gel Electrophoresis and Imaging ................................................................................... 25 Quorum Sensing Vibrio Assays ...................................................................................... 26 PCRs and Construct Generation .................................................................................... 27 Transformation .............................................................................................................. 30 E. coli Plate Assays ......................................................................................................... 32 E. coli Maximum Light Output Assays ........................................................................... 32 Results ..................................................................................................................... 34 Quorum Sensing Vibrio Assays ...................................................................................... 34 PCRs and Construct Generation .................................................................................... 41 Transformation .............................................................................................................. 47 E. coli Plate Assays ......................................................................................................... 50 E. coli Maximum Light Output Assays ........................................................................... 53 Sequence Analysis of LuxRVH Homologs ........................................................................ 58 Discussion ................................................................................................................ 61 Quorum Sensing Among Vibrios ................................................................................... 61 Dual vector system for arabinose-induced bioluminescence ....................................... 62 Cross Species Induction of Bioluminescence by V. harveyi LuxR .................................. 63 Conclusion ............................................................................................................... 68 References ............................................................................................................... 70 Appendix A: Relative Light Calculations for Vibrio Samples ....................................... 81 v Appendix B: Individual E. coli Growth & Light Curves ................................................ 86 Appendix C: Average Relative Light Calculations for E. coli Samples ........................ 110 vi LIST OF FIGURES FIGURE 1: PHYLOGENETIC ANALYSIS OF BIOLUMINESCENCE GENE LUXA.. ................... 3 THE LUXA TREE WAS CONSTRUCTED USING BAYESIAN MARKOV CHAIN MONTE CARLO STATISTICS WITH THE PARAMETERS OF GENERAL TIME REVERSIBLE NUCLEOTIDE SUBSTITUTION COST MATRIX (GENEIOUS). PHOTOBACTERIUM LEIOGNATHI IS SPECIFIED AS THE OUTGROUP. CONSENSUS SUPPORT
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