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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.

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Horizontal Gene Transfer (HGT) of a Catabolic Plasmid

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  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

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