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Horizontal Gene Transfer (HGT) of a Catabolic Plasmid

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Lack of study data on transfer of a catabolic plasmids ... HGT effective over long period of time, but not effective with introduced donors ... – PowerPoint PPT presentation

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Title: Horizontal Gene Transfer (HGT) of a Catabolic Plasmid


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 Brandons
    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 4C) little or no HGT occurred to spike the
    numbers
  • This is called plate mating, did not occur due
    to pre-incubation at 4C

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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