1 / 16

Horizontal Gene Transfer (HGT) of a Catabolic Plasmid

Horizontal Gene Transfer (HGT) of a Catabolic Plasmid. By Daniel Roeter November 2, 2005. My Research. Studying HGT of a catabolic gene that degrades parathion Models antibiotic resistances acquired by bacteria Much of this paper is very similar to Brandon’s and my research.

gigi
Download Presentation

Horizontal Gene Transfer (HGT) of a Catabolic Plasmid

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Horizontal Gene Transfer (HGT) of a Catabolic Plasmid By Daniel Roeter November 2, 2005

  2. My Research • Studying HGT of a catabolic gene that degrades parathion • Models antibiotic resistances acquired by bacteria • Much of this paper is very similar to Brandon’s and my research

  3. Why was this Paper Written? • Lack of study data on transfer of a catabolic plasmids • Differs from heavy metal/antibiotic resistance plasmids in size and copy number • Research on this subject could lead to bioaugmentation and/or bioremediation in soil

  4. Background Information • Genes can be horizontally transferred via conjugation, transformation, and transduction • Transfer had to be between 2 bacterial strains that could degrade 2,4-dichlorophenoxyacetic (2,4-D) acid • This requires chromosomally encoded maleylacetate reductase

  5. The Bacterial Strains • Donor: Alealigenes eutrophus JMP134, contains pJP4 plasmid, (2,4-D+ Hgr Kms) • Recipient: Variovorax paradoxus, (2,4-D- Hgs Kmr) • Plasmid Being Transferred: “pJP4” that contains (1) the catabolic enzyme that degrades 2,4-dichlorophenoxyacetic (2,4-D) acid and (2)resistance to Hg • All growth on media was done with both donor and recipient cells

  6. The Different Media • PY Agar • Abiotic Soil (autoclaved) • Biotic Soil

  7. The Different Selectors • PYM: PY + Mercury (Hg)  selects for donors and transconjugants (plasmid transferred) • PYK: PY + Kanamycin  selects for recipients and transconjugants • PYMK: PY + Hg + Kanamycin  selects for only transconjugants

  8. Confirmation of Transfer Procedure • The transconjugants from the PYMK media were grown in a solution where 2,4-D was the only carbon source (green to yellow) • Ability to grow in Hg • PCR of the Gene and visualization • Plasmid analysis and visualization

  9. Growth on Soil Media Grown on both sterile (abiotic) and non-sterile (biotic) soil Procedure for both soils was basically the same Samples were taken from the sterile soil immediately following addition to soil and at days 1,2,3, and 8 Non-sterile were taken immediately and at days 1 and 2 Suspected trasconjugants were confirmed in the same way as before

  10. In all experiments, the growth on solid agar was 1 transconjugant per 103 parents cells (1/103) 75 suspected transconjugants were confirmed as before

  11. Demonstrates that that during the transfer from media to plate for transconjugant analysis (done at 4°C) little or no HGT occurred to “spike” the numbers This is called plate mating, did not occur due to pre-incubation at 4°C

  12. Results (sterile) • Donor and recipient cells remained constant • Transconjugant number increased in the 1st day and remained fairly constant • Transfer of 1 transconjugant per 105 parents cells (1/105) Again transconjugants were confirmed

  13. Results (non-sterile) • Donor and recipient cells remained constant in day 1 and 2, but then declined • Transconjugant number were BDL until 6th day • Procedure was repeated with 2,4-D amended soil to increase the conc. of donors and transconjugants Change produced transconjugants within 2 days Transfer of 1 transconjugant per 106 parents cells (1/106) even though soil was amended Transconjugants were confirmed

  14. Discussion of Results • Transfer did occur and was confirmed by transconjugant analysis • Growth on agar media overestimates HGT frequency • Growth on sterile soil highlights abiotic stresses • Abiotic stresses are unique to the particular soil

  15. Discussion of Results • Non-sterile soil highlights biotic stresses

  16. The Bottom Line • Transfer of the large catabolic plasmid does occur, but its frequency is greatly reduced in soil • HGT effective over long period of time, but not effective with introduced donors

More Related