Transfer of Potential pseudomonas stutzeri genes

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In this research of saxitoxin bacterial production bioinformatics will be utilized to identify sxt coding genes within pseudomonas stutzeri strain A1501. PCR will then be employed in the isolation of sxt genes from pseudomonas stutzeri. The isolated genes will be transformed into Ecoli cultures.

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Transfer of Potential pseudomonas stutzeri genes

  1. 1. Building Genetic Foundations in the saxitoxin Production of Pseudomonas stutzeri A Research Proposal Gabrielle Roberts
  2. 2. <ul><li>TABLE OF CONTENTS </li></ul><ul><li>ALGAE BLOOMS </li></ul><ul><li>BACTERIAL PRODUCTION OF SAXITOXIN: A CONTROVERISAL TOPIC </li></ul><ul><li>SAXITOXIN A NEUROTOXIN </li></ul><ul><li>CHEMICAL & MOLECULAR SIDES OF SAXITOXIN SYNTHESIS </li></ul><ul><li>METHODS </li></ul><ul><li>PRELIMINARY RESULTS </li></ul>
  3. 3. OVERVIEW OF BLOOMS <ul><li>Saxitoxin is a potent neurotoxin that can be found within Harmful Algae Blooms of cyanobacteria and dinoflagellates. </li></ul>
  4. 4. Harmful Algae Blooms <ul><li>Bloom: “population explosions” or large populations of algae within marine and fresh water environments. </li></ul>HAB “red tide” Blooms of toxic dinoflagellates in marine environment Green bloom of blue-green algae cyanobacteria in fresh water
  5. 5. <ul><li>Cause : </li></ul><ul><li>Eutrophication events : addition of nitrogen and phosphorus from agricultural run offs or sewage leakage can cause an increase in algae population number </li></ul><ul><li>Description of algae blooms : </li></ul><ul><li>Blooms can be toxic or nontoxic </li></ul><ul><li>Involve cyanobacteria, dinoflagellates </li></ul><ul><li>Have detrimental effects on environments and human population </li></ul>
  6. 6. Impacts of non-toxic/toxic blooms <ul><li>Large Populations of algae can prevent light from reaching lower depths </li></ul><ul><li>Submerged plants growing at the bottom don’t have access to light. Plants get rid of nitrogen/phosphorus pollutants. </li></ul><ul><li>Anoxia: blooms cause oxygen depletion of marine and fresh water environments </li></ul>Littoral zone: area where plants grow in pond or lakes http://www.dnr.state.mn.us/shorelandmgmt/apg/wheregrow.html
  7. 7. Impacts of toxic blooms “the food pyramid” <ul><li>Consumption of saxitoxin contaminated seafood can have devastating effects </li></ul>Humans SECONDARY CONSUMERS Fish PRIMARY CONSUMERS Filter feeders Shrimp “have natural saxitoxin resistance” PRIMARY PRODUCERS Photoautotrophs Dinoflagellates cyanobacteria
  8. 8. PSP (PARALYTIC SHELLFISH POISIONING) <ul><li>Saxitoxin effects respiratory muscles </li></ul><ul><li>Symptoms: shortness of breath, dizziness, numbness of extremities. If not treated it can cause respiratory failure </li></ul>
  9. 9. <ul><li>QUESTION: </li></ul><ul><li>How do dinoflagellates produce saxitoxin? </li></ul>Sxanning electron micrograph of dinoflagellate: Gymnodium DAB dinoflagellate asscoiated bacteria Phylum: Cytophage-Flavobacteroides and Proteobacteria Live in Alexandrium and Gymnodinium dinoflagellates
  10. 10. Bacterial saxitoxin production a controversial subject… <ul><li>Past 1995-1996 studies affermed bacterial paralytic saxitoxin production using bacteria isolated from Alexandrium lusitanicum and Gymnodium Catenatum </li></ul><ul><li>Current 2003 research findings have not found evidence linking bacteria to the production of paralytic saxitoxin </li></ul><ul><li>Incorrect identification of paralytic saxitoxin within dinoflagellate producing bacteria using methods such as HPLC. </li></ul>
  11. 11. Evidence of saxitoxin bacteria production <ul><li>Evidence: Horizontal gene transfers </li></ul><ul><li>Bacteria transferred saxitoxin gene into cyanobacteria. </li></ul><ul><li>Result: saxitoxin producing cyanobacteria </li></ul>Bacteria Bacteria cyanobacteria
  12. 12. Evidence of saxitoxin bacteria production… <ul><li>Sxt genes have been characterized within saxitoxin producing cyanobacteria. </li></ul><ul><li>Phylogenetic analysis show that some cyanobacteria stx genes have bacteria origins. </li></ul>Moustafa, A., Loram, J. E., Hackett, J. D., Anderson, D. M., Plumley, F. G., Bhattacharya, D. (2009). Origins of Saxitoxin Biosynthetic Genes in Cyanobacteria. Plos One , 4 (6). Retrieved from: www.plosone.org
  13. 13. <ul><li>SXTA: Polyketide synthase </li></ul><ul><li>First enzyme in biosynthesis. Has 4 domains. </li></ul><ul><li>Acyl-CoA/N-acyltransferase </li></ul><ul><li>Phosphopantetheine binding site </li></ul>Aminotransferase classI and II Proteobacterium Acintobacterium STX+ Cyanobacteria Moustafa, A., Loram, J. E., Hackett, J. D., Anderson, D. M., Plumley, F. G., Bhattacharya, D. (2009). Origins of Saxitoxin Biosynthetic Genes in Cyanobacteria. Plos One , 4 (6). Retrieved from: www.plosone.org SxtA
  14. 14. SAXITOXIN MODE OF ACTION <ul><li>Affects the sodium gated channels of nerve cells. </li></ul><ul><li>Prevents generation of neural impulses by inhibiting depolarization events . </li></ul>
  15. 15. Repolarization Rest State Passer, M.W. & Smith, R. E. (2004). Psychology the Science of Mind and Behavior: Second Edition. New York: McGraw Hill. Depolarization Repolarization
  16. 16. SAXITOXIN BINDING <ul><li>The sodium voltage gated channel </li></ul><ul><li>260kDa a-subunit and 33-36kDa beta subunit </li></ul>The a-subunit is made up of 4 transmembrane domains. Each domain has S1-S6 segments a-subunit Zimmer, R. K., & Ferrer, R. P. (2007). Neuroecology, Chemical Defense and the Keystone Species Concept. Biol. Bull., 213 , 208-225.
  17. 17. a-subunit Ion conducting pore The pore loop between the S5 and S6 segments 1. Na+ enter 2. Is the site where saxitoxin binds “ Inner ring pore loop”
  18. 18. SAXTITOXIN CHEMICAL STRUCTURE The guanidine, hydroxyl and carbamoyl groups of saxitoxin bind to the voltage gated sodium channel’s inner ring pore loop
  19. 19. SAXTITOXIN CHEMICAL SYNTHESIS <ul><li>Chemical synthesis of saxitoxin involves 9 steps </li></ul><ul><li>use several enzymes and co-factors </li></ul>
  20. 20. Transfer of guanidino group to intermediate Claisen condensation of arginine and acetate Kellman, R., Mihali, T. K., Jeon, Y. J., Pickford, R., Pomati, F., & Neilan, B. A. (2008). Biosynthetic Intermediate Analysis and Functional Homology Reveal a Saxitoxin Gene Cluster in Cyanobacteria. Applied and Environmental Microbiology , 74, No.13, 4044-4053. carbamoylation hydroxylation
  21. 21. SAXTITOXIN MOLECULAR SYNTHESIS SxtG Guanidino transfer SxtB SxtD Cyclization Methylation Epoxidation SxtS SxtS opening SxtU Reduction Carbamoylation SxtH/T SxtW SxtV Hydroxylation SxtI Claisen condensation: acetate to arginine
  22. 22. <ul><li>GOAL: TO PROVE THAT </li></ul><ul><li>PSEUDOMONAS STUTZERI CAN SYNTHESIZE SAXITOXIN </li></ul><ul><li>Increase our understanding of dinoflagellate saxitoxin synthesis </li></ul><ul><li>Develop methods to reduce the occurrence of Red Tide </li></ul><ul><li>Help fishing industries, reduce PSP fatalities </li></ul>
  23. 23. <ul><li>OBJECTIVE: </li></ul><ul><li>. </li></ul>To uncover evidence of the bacterial saxitoxin production within Pseudomonas stutzeri marine strain through the use of bioinformatics and molecular biology .
  24. 24. THE MODEL ORGANISMS <ul><li>Pseudomonas stutzeri strains </li></ul><ul><li>Pseudomonas stutzeri A1501 (sxt-) </li></ul><ul><li>Pseudomonas stutzeri marine strain (sxt+) </li></ul><ul><li>Cyanobacteria </li></ul><ul><li>Cylindrospermopsis raciborskii T3 (sxt+) </li></ul><ul><li>Saxitoxin genes have been sequenced </li></ul>
  25. 25. Question: Why use bioinformatics? <ul><li>Search for cyanobacteria saxitoxin genes within Pseudomonas stutzeri (sxt-) </li></ul><ul><li>Conduct BLAST search for cyanobacteria saxitoxin genes in Pseudomonas stutzeri (sxt-) </li></ul><ul><li>Phylogenetic analysis of saxitoxin producing enzymes in cyanobacteria and Pseudomonas stutzeri (sxt-) </li></ul><ul><li>Use CLUSTALW program to conduct a phylogenetic analysis of saxitoxin enzymes in Pseudomonas stutzeri A1501 and cyanobacteria Cylindrospermopsis raciborskii T3 </li></ul>
  26. 26. METHOD ONE <ul><li>Molecular approach </li></ul><ul><li>Design PCR primers to amplify the sxt genes in Pseudomonas stutzeri marine isolate . </li></ul>
  27. 27. METHOD ONE <ul><li>2. Insert the saxitoxin DNA within competent E. coli cells via bacterial transformation. </li></ul><ul><li>finding suitable vector that is able to express all amplified gene sequences </li></ul><ul><li>* BAC (bacterial artificial chromosome) </li></ul><ul><li>3. Confirm bacterial saxitoxin production using HPLC (high preformance liquid chromatography) </li></ul>Identify saxitoxin in E-coli cultures
  28. 28. Question: Why Pseudomonas stutzeri ? Pseudomonas stutzeri is a dinoflagellate associated bacteria that is commonly isolated from dinoflagellates Question: Why two strains of Pseudomonas stutzeri <ul><li>Pseudomonas stutzeri A1501 is a rice strain that does not make saxitoxin. But it is closely related to the Pseudomonas strutzeri marine strain </li></ul><ul><li>The genome of saxitoxin producing Pseudomonas stutzeri has not been sequenced </li></ul>
  29. 29. METHOD TWO <ul><li>Commercially sequence DNA isolated from Pseudomonas stutzeri marine strain </li></ul><ul><li>Conduct search for genes coding for saxitoxin enzymes </li></ul>Ambry Genetics
  30. 30. Preliminary Results
  31. 31. Preliminary Results <ul><li>Analysis of the P.sutzeri A1501 hypothetical enzymes show that the Pseudomonas stutzeri A1501 genes: </li></ul><ul><li>Do not form an open reading frame gene cluster </li></ul><ul><li>Are instead separated by great distances and some are on different strands. </li></ul>Positive strand Negative strand 13 million base pairs apart 3’ 5’ 5’ 3’ 5’ 3’ sxtH sxtU sxtI Where are the Pseudomonas stutzeri A1501 genes located?
  32. 32. Preliminary Results <ul><li>CLUSTAL W Phylogenetic Analysis </li></ul><ul><li>Cylindrospermopsis raciborskii T3 saxitoxin enzymes </li></ul><ul><li>Pseudomonas stutzeri A1501 BLAST results </li></ul>SxtU Short-chain dehydrogenase Cylindrospermopsis raciborskii T3 Pseudomonas stutzeri A1501 Common ancestor All C. Raciborskii T3 and Pseudomonas stutzeri A1501 enzymes are evolutionary related HYPOTHETICAL PHYLOGENETIC TREE
  33. 33. Preliminary Results <ul><li>E-value </li></ul><ul><li>Similarity between the genes </li></ul><ul><li>Lower E-value </li></ul><ul><li>Good match </li></ul>
  34. 34. Preliminary Results Good E-values Carbamoyl Transferase 7e-88 Short-chain dehydrogenase 3e-40 8-amino-7-oxononanoate 9e-44 Closest Blast Match in P. stutzeri A1501 STX Enzyme in C.raciborskii T3 sxtI sxtU sxtA
  35. 35. Carbamoyl transferase E-value: 7e-88 Exceptional E-value is evidence of horizontal gene transfer between Cylindrospermopsis raciborskii T3 and Pseudomonas stutzeri A1501 Pseudomonas stutzeri A1501
  36. 36. <ul><li>5 out of 12 P. stutzeri genes did not have a close BLAST match with cyanobacterial STX enzymes </li></ul>Preliminary Results
  37. 37. Preliminary Results Iron sulfur Protein 0.001 Sterol desaturase 0.037 Tyosine decarboxylase putative 0.063 Closest Blast Match in P. stutzeri A1501 STX Enzyme in C.raciborskii T3 sxtW sxtD sxtJ sxtB Hypothetical protein PST_2386 0.14 Chemotaxis Protein CheV sxtG 1.9 E-value
  38. 38. Preliminary Results <ul><li>Moderate </li></ul><ul><li>E-values </li></ul><ul><li>Proteins are found in all bacteria </li></ul><ul><li>Some have the same function as the cyanobacteria saxitoxin enzyme </li></ul>Short-chain dehydrogenase 3e-40 Protein involved in the biosynthesis of mitomycin antibiotics/polyketide fumonisin 2e-04 Closest Blast Match in P. stutzeri A1501 STX Enzyme in C.raciborskii T3 sxtU sxtS sxtH Benzoate dioxygenase, alpha subunit 2e-o4 8-amino-7-oxononanoate sxtA 9e-44 E-value 1e-o4 sxtI
  39. 39. <ul><li>we designed primers to match the Pseudomonas stutzeri A1501 saxitoxin enzymes that had a good-moderate </li></ul><ul><li>E-values </li></ul><ul><li>we designed primers for the cyanobacterial saxitoxin enzymes that had a bad </li></ul><ul><li>high-value </li></ul>
  40. 40. Currently… <ul><li>Use PCR to amplify P.stutzeri rice strain STX genes/ cyanobacterial STX genes from marine P.stutzeri strain. </li></ul><ul><li>Commercially sequence genes from the Pseudomonas stutzeri marine strain </li></ul><ul><li>using Ambry Genetics </li></ul>
  41. 41. ACKNOWLEDGEMENTS <ul><li>Special Thanks to: </li></ul><ul><li>Committee members </li></ul><ul><li>Dr. Marsh </li></ul><ul><li>Dr. Molina </li></ul><ul><li>Dr. Morin </li></ul><ul><li>Research funding: </li></ul><ul><li>ARCC program </li></ul>

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