Horizontal Gene Transfer HGT of a Catabolic Plasmid

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Horizontal Gene Transfer (HGT) of a Catabolic
Plasmid

• By Daniel Roeter
• November 2, 2005

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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

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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

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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

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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

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The Different Media

• PY Agar
• Abiotic Soil (autoclaved)
• Biotic Soil

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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

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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

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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

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

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• 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

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• 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

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• 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

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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

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Discussion of Results

• Non-sterile soil highlights biotic stresses

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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