Hughes Research Presentation


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Hughes Research Presentation

  1. 1. Evidence for Styrene Oxidation in Pseudomonas sp. via a meta -fission Pathway Zhiguang Eric Zhang Dr. Anthony Hay Department of Microbiology Thursday, July 19, 2001
  2. 2. Overview <ul><li>Introduction </li></ul><ul><ul><li>Significance of styrene </li></ul></ul><ul><ul><li>Pathways for styrene degradation </li></ul></ul><ul><li>Methodology and experimental outline </li></ul><ul><ul><li>Isolation of organisms </li></ul></ul><ul><ul><li>Molecular characterization of organisms </li></ul></ul><ul><ul><li>Preliminary findings: evidence for involvement of the tod pathway / homologous styrene pathway </li></ul></ul><ul><li>Conclusions </li></ul><ul><li>Future work </li></ul>
  3. 3. What is styrene? <ul><ul><li>monosubstituted benzene (vinyl group) </li></ul></ul><ul><ul><li>strong odor, toxin, mutagen, and suspected carcinogen </li></ul></ul><ul><ul><li>starting material for industrial production of plastic and rubber </li></ul></ul><ul><ul><li>primarily released to the environment through wastewater, evaporation, and pyrolysis of polystyrene </li></ul></ul>
  4. 4. <ul><li>sinking of the Ievoli Sun off the coast of France - Oct. 31, 2000 </li></ul><ul><li>cargo included 4,000 tons of styrene </li></ul>Photo:Marine Nationalé
  5. 5. Pathways for styrene oxidation <ul><li>styABCD - monooxygenation of styrene and phenylacetate lower pathway </li></ul><ul><ul><li>Pseudomonas putida CA-3, Pseudomonas sp. VLB120 </li></ul></ul><ul><ul><li>Exophiala jeanselmei </li></ul></ul><ul><li>Dioxygenation of styrene and meta -fission </li></ul><ul><ul><li>Rhodococcus rhodocrous NCIMB 13259 </li></ul></ul><ul><ul><li>Pseudomonas sp. s7, s15? </li></ul></ul>
  6. 6. <ul><li>closely studied at the molecular level </li></ul><ul><li>regulatory genes stySR </li></ul><ul><li>metabolic end products: </li></ul>styABCD upper pathway lower pathway The University of Minnesota Biocatalysis/Biodegradation Database styAB styC styD fumarate acetoacetate
  7. 7. Styrene meta -fission upper pathway - direct aromatic attack through a catecholic intermediate <ul><ul><li>Enzymatic activity has been identified but there has been no molecular work done to characterize the genes or enzymes involved </li></ul></ul><ul><li>metabolic end products: </li></ul><ul><li>- acrylate </li></ul><ul><li>- acetaldehyde </li></ul><ul><li>- pyruvate </li></ul>The University of Minnesota Biocatalysis/Biodegradation Database meta- fission
  8. 8. Methods <ul><li>Identified a consortia that removes styrene from air via a biofilter system </li></ul><ul><li>Isolation and characterization of organisms </li></ul><ul><ul><li>grew consortia in enrichment media ( MSM+500ppm styrene) to select for styrene degraders </li></ul></ul><ul><ul><li>isolated several organisms capable of growing on styrene as the sole carbon source </li></ul></ul><ul><ul><li>characterized using RISA and BOXA1R genomic fingerprinting, 16s rRNA sequencing, and substrate utilization </li></ul></ul>
  9. 9. Characterization of isolates s7 and s15 <ul><li>Identical RISA and BOXA1R banding patterns </li></ul><ul><li>Same 16S rRNA sequence hit using BLAST search algorithm </li></ul><ul><li>Able to grow on the same substrates </li></ul><ul><li>s7 grows faster on liquid and solid media than s15 </li></ul><ul><li>s7 and s15 accumulate different colored metabolites when grown with styrene as the sole carbon source </li></ul><ul><li>One a derivative of the other? </li></ul>
  10. 10. Growth substrates utilized by s7 and s15
  11. 11. Same strain - different metabolite accumulation - the same Pseudomonas sp. s7 s15 MSM + Styrene atmosphere
  12. 12. How to explain different colored metabolites? <ul><li>Brown is characteristic of catecholic polymerization products </li></ul><ul><li>Yellow is characteristic of meta -fission products </li></ul><ul><li>Involvement of meta -fission pathway via a catecholic intermediate? </li></ul>
  13. 13. The University of Minnesota Biocatalysis/Biodegradation Database The styrene meta -fission pathway vs. the tod pathway for toluene oxidation todC1C2BA todE todD
  14. 14. Evidence for tod / homologous styrene pathway: <ul><li>PCR using degenerate aromatic dioxygenase primers gave a ~450bp fragment </li></ul><ul><li>BLAST results indicate region of sequence identity: </li></ul><ul><ul><li>todC1 : toluene dioxygenase iron-sulfur protein component - large subunit </li></ul></ul>506bp 396bp s15 s7 1% agarose
  15. 15. Evidence for meta -fission I: <ul><li>Yellow metabolite (s7) - meta -fission product </li></ul><ul><li>Yellow color is lost when acidified and is restored upon neutralization </li></ul><ul><li>Brown metabolite (s15) is characteristic of catecholic polymerization products </li></ul><ul><li>Possible structure of brown polymerization product (GC-MS) </li></ul>
  16. 16. Evidence for meta -fission II: <ul><li>- GC-MS metabolite derivitization: detection of the meta -fission intermediate 3-vinylcatechol </li></ul>Vinylcatechol (m/z=136)
  17. 17. Use of 3-fluorocatechol - a meta -fission inhibitor ? 3-vinylcatechol 3-fluorocatechol  2-hydroxy-6-oxo- octa-2,4,7-trienoate
  18. 18. Enzymatic activity? <ul><li>Resting cell assay </li></ul><ul><ul><li>indicates that s7 cells induced with styrene are capable of: </li></ul></ul><ul><ul><ul><li>using styrene </li></ul></ul></ul><ul><ul><ul><li>using toluene </li></ul></ul></ul><ul><ul><ul><li>using catechol </li></ul></ul></ul><ul><ul><li>...whereas cells that were grown on glucose were not </li></ul></ul><ul><li>This is evidence that… </li></ul><ul><ul><li>enzymes capable of utilizing styrene, toluene, and catechol as substrates were produced when s7 cells were induced with styrene </li></ul></ul>
  19. 19. Conclusions <ul><li>PCR, sequencing, resting cell assays, and GC-MS indicate that Pseudomonas sp. s7 and s15 utilize the upper part of the tod pathway or a homologous styrene pathway </li></ul><ul><li>s7 and s15 appear to differ only in catechol 2,3-dioxygenase ( todE ) activity - one a mutant of the other? </li></ul><ul><li>s7 and s15 provide a glimpse at an adaptation that has allowed for relaxed substrate specificity in the tod pathway </li></ul>
  20. 20. Future work <ul><li>todC1 knockout strains for s7 and s15 </li></ul><ul><li>todE knockout strains for s7 and s15 </li></ul><ul><li>Complementation of s15 todE knockout with s7 todE gene </li></ul><ul><ul><li>see accumulation of yellow meta -fission product instead of brown 3-vinylcatechol polymer? </li></ul></ul><ul><li>Sequence s7 and s15 todE genes - mutation? </li></ul>
  21. 21. Acknowledgements <ul><ul><li>Dr. Anthony Hay </li></ul></ul><ul><ul><li>Lauren Junker </li></ul></ul><ul><ul><li>Tammy Arbogast </li></ul></ul><ul><ul><li>Jeanne Kagel </li></ul></ul><ul><ul><li>Serena Mak </li></ul></ul><ul><ul><li>Paul Taylor at U. Melbourne for Ado primers </li></ul></ul><ul><ul><li>Lew Daly at Triad Technologies - Syracuse, NY </li></ul></ul><ul><ul><li>Cornell Center for Advanced Technology </li></ul></ul><ul><ul><li>Develop Your Own Internship Program </li></ul></ul><ul><ul><li>Laurel, Pam, and the Hughes Program </li></ul></ul>
  22. 22. Questions?