W&M ScholarWorks Undergraduate Honors Theses Theses, Dissertations, & Master Projects 7-2012 Transcriptional Regulation of the Acetone Carboxylase Operon via Two-Component Signal Transduction in Helicobacter pylori Samuel Emerson Harvey College of William and Mary Follow this and additional works at: https://scholarworks.wm.edu/honorstheses Part of the Biology Commons Recommended Citation Harvey, Samuel Emerson, "Transcriptional Regulation of the Acetone Carboxylase Operon via Two- Component Signal Transduction in Helicobacter pylori" (2012). Undergraduate Honors Theses. Paper 471. https://scholarworks.wm.edu/honorstheses/471 This Honors Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Undergraduate Honors Theses by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. Transcriptional Regulation of the Acetone Carboxylase Operon via Two- Component Signal Transduction in Helicobacter pylori A thesis submitted in partial fulfillment of the requirement for the degree of Bachelor of Science in Biology from The College of William & Mary By Samuel Emerson Harvey Accepted for ____________________________ (Honors) _______________________________________ Dr. Mark Forsyth, Chair _______________________________________ Dr. Oliver Kerscher _______________________________________ Dr. Kurt Williamson _______________________________________ Dr. Randolph Coleman Williamsburg, VA April 30, 2012 Abstract Helicobacter pylori is a gram negative gastric pathogen that infects the mucosal lining of the human stomach and is present is nearly half of the human population. H. pylori is the etiologic agent of peptic ulcer disease, and infection is highly associated with the development of gastric cancer. The H. pylori genome encodes three complete two- component signal transduction systems (TCSTs): ArsRS, CrdRS, and FlgRS. Each system regulates many genes in response to environmental stimuli. The genome also encodes an essential orphan response regulator, HP1021. Previous transcriptional profiling experiments indicate that each of these TCSTs regulates the expression of virulence genes. The acetone carboxylase operon, acxABC, is associated with virulence and is regulated by all H. pylori TCSTs and HP1021. We characterized the TCST- mediated transcriptional regulation of acxABC expression by examining the physical interaction of response regulators ArsR and HP1021, a repressor and activator of acxABC transcription, respectively, with the promoter region of acxA. Electrophoretic mobility shift assays suggest that both ArsR and HP1021 bind upstream and downstream of the -35 hexamer promoter element, with ArsR binding two distinct sites and HP1021 binding as many as six sites. All of the ArsR binding sites overlap with HP1021 binding sites, suggesting possible binding competition. Also, acxA expression was assayed via quantitative real-time PCR in H. pylori strains 26695 and J99 under both neutral and acidic conditions. Under neutral conditions, abrogation of arsS in H. pylori strain J99 resulted in a 4.4-fold increase in acxA transcription, however no significant change in transcription was observed in strain 26695. Grown under acidic conditions, the J99 arsS null mutant exhibited approximately a 2.6-fold increase in acxA transcription with no II significant differential regulation occurring in the 26695 arsS null mutant. Collectively, this study suggests that H. pylori uses multiple TCSTs to regulate the expression of the acxABC operon, forming an overlapping regulatory network that allows finely tuned control over transcriptional regulation. This multi-layered mechanism of regulation may apply to other H. pylori virulence genes and represents a unique way for H. pylori, a bacterium with a relative paucity of TCSTs, to maintain tight control over gene expression. Inter-strain variation in the ArsRS-mediated regulation of acxA occurs between H. pylori strains J99 and 26695, offering the acxABC operon as a model for understanding strain-specific variation in TCST-mediated regulation of virulence factors. III Table of Contents List of Figures and Tables ................................................................................................... V Chapters 1. Introduction ....................................................................................................................1 1.1. Helicobacter pylori ............................................................................................1 1.2. Pathogenicity in H. pylori ..................................................................................2 1.3. Two-Component Signal Transduction ...............................................................4 1.4. Two-Component Signal Transduction in Helicobacter pylori ..........................5 1.5. The Acid Response System (ArsRS) Two-Component Signal Transduction System ................................................................................................................8 1.6. The Copper Resistance Determinant (CrdRS) Two-Component Signal Transduction System ........................................................................................10 1.7. The Flagellar FlgRS Two-Component Signal Transduction System ..............10 1.8. The Orphan Response Regulator HP1021 .......................................................11 1.9. The Role of Two-Component Signal Transduction in Acetone Metabolism ..12 1.10. Research Goals and Experimental Approach ..................................................15 2. Methods........................................................................................................................16 2.1. Expression of ArsR and HP1021 .....................................................................16 2.2. Response Regulator Purification ......................................................................17 2.3. Electrophoretic Mobility Shift Assays .............................................................18 2.3.1. Probes.........................................................................................................18 2.3.2. Binding Reactions......................................................................................24 2.3.3. Electrophoresis...........................................................................................25 2.3.4. Membrane Blotting and Detection.............................................................25 2.4. Helicobacter pylori Liquid Culture and Acid Shock .......................................26 2.5. RNA Extraction ...............................................................................................26 2.6. Quantitative Real-Time PCR ...........................................................................27 3. Results and Discussion ................................................................................................28 3.1. Localizing ArsR Binding Sites via Electrophoretic Mobility Shift Assays .....28 3.2. Localizing HP1021 Binding Sites via Electrophoretic Mobility Shift Assays ..............................................................................................................33 3.3. Binding Specificity and Competition Assays ..................................................40 3.4. Modeling Regulation of acxA with ArsR and HP1021 ....................................42 3.5. Overlapping Binding Sites of ArsR and HP1021 ............................................45 3.6. Inter-strain Variation in acxA Expression ........................................................45 3.7. Future Considerations ......................................................................................46 3.8. Concluding Thoughts .......................................................................................49 4. References ....................................................................................................................51 IV List of Figures and Tables Figures 1. Conventional Paradigm for Two-Component Signal Transduction System ..................8 2. Sizes and locations of acxA EMSA probes ..................................................................23 3. ArsR EMSA with Up.1* - Up.4* probes .....................................................................29 4. ArsR EMSA with Up.5* - Up.8* probes .....................................................................30 5. ArsR EMSA with Down.1* - Down.3* probes ............................................................31 6. ArsR EMSA with Down.4* - Down.6* probes ............................................................32 7. HP1021 EMSA with Up.1* - Up.4* probes ................................................................35 8. HP1021 EMSA with Up.5* - Up.8* probes ................................................................36 9. HP1021 EMSA with Down.1* - Down.3* probes .......................................................37 10. HP1021 EMSA with Down.4* - Down.6* probes .......................................................38 11. ArsR EMSA with Up* and Up Competition Assays ...................................................42 12. Hypothetical model for the transcriptional regulation of acxA by ArsR .....................44 13. Hypothetical model for the transcriptional regulation of acxA by HP1021 .................44 14. qPCR of H. pylori strains grown at pH 7 .....................................................................48 15. qPCR of H. pylori strains