1. Triparental Mating!
13 June 2014

2. Classical Methods of Horizontal Gene
Transfer
● Transformation
● Transduction
● Conjugation

3. Furuya, et al. 2005.
Antimicrobial-resistant
bacteria in the
community setting.

4. Methods of Incorporating Foreign DNA into
the Cell
● Transformation
o Free DNA into “competent” recipient cell
o Natural competence is genetically determined
o Electroporation--high voltage makes cell envelope permeable
and facilitates transformation
o Transfection--transformation with DNA extracted from bacterial
virus
o Works “moderately well” in archaea by facilitating disassembly
of glycoprotein cell wall layer, also occasionally electroporation,
approaches are more diverse and organism-dependent

5. Methods of Incorporating Foreign DNA into
the Cell
● Transduction
o Bacteriophage transfers DNA from one cell to
another
o Generalized: donor genes cannot replicate
independently
o Specialized: bacterial DNA incorporated into viral
genome
o Extremely rare in archaea, only one archaeal virus
has been shown to transduce genes of its host

6. Methods of Incorporating Foreign DNA into
the Cell
● Conjugation
o Plasmid-mediated mechanism of transfer involving
cell to cell contact
o Process involves donor cell with conjugative plasmid
and recipient strain without
o Two types of conjugation known in Archaea, one
using plasmids--genes encoding conjugative
functions have little similarity with those in Bacteria

7. F-plasmid mediated
bacterial conjugation.
Wikimedia Commons
http://commons.wikime
dia.org/wiki/File:Conjug
ation.svg

8. (General) Mechanism of Conjugation
Four Components of Conjugative Apparatus
● origin of transfer (oriT) site
● gene for relaxase protein
● type IV coupling protein
● type IV secretion system

9. Self-transmissible or conjugative
plasmids code for the four
components of a conjugative
apparatus: an origin of transfer
(oriT) (violet), a relaxase (R) (red),
a type IV coupling protein (T4CP)
(green), and a type IV secretion
system (T4SS) (blue).
From Mobility of Plasmids, Smillie et al Microbiol.
Mol. Biol. Rev. September 2010 vol. 74 no. 3 434-
452

10. Plasmid Mobility “Types”
● Conjugative (self-transmissible),
● Mobilizable
● Nonmobilizable (most plasmids larger than
300kb)

11. (General) Mechanism of Conjugation
● All conjugative and mobilizable plasmids
carry a gene for relaxase
● Relaxase catalyzes nicking of oriT (origin of
transfer) site in donor plasmid and final
ligation of transported DNA in recipient
● DNA replication occurs by rolling circle
replication

12. Replication and transfer of plasmid DNA
during bacterial conjugation. From Brock
Biology of Microorganisms 12th Edition

13. (General) Mechanism of Conjugation
Mobilizable plasmids
● Only need oriT, relaxase gene, and nicking
auxiliary proteins (forms relaxosome)
● Will occasionally encode for type IV coupling
protein, which is involved in the connection
between the relaxosome and the transport
channel

14. (General) Mechanism of Conjugation
Self-Transmissible
● OriT, relaxase gene, and nicking auxiliary
proteins (forms relaxosome)
● Type IV coupling protein
● Type IV secretion system that forms
conjugative mating channel

15. Triparental Mating
● Donor strain: contains mobilizable plasmid of
interest
● Helper strain: has conjugative plasmid that
can mobilize desired plasmid into recipient
strain in trans
● Recipient strain

16. Helper Donor
Conjugation
Step 1: conjugation initiated by
self-mobilizable “helper
plasmid” (red); helper plasmid is
transconjugated into donor
strain that contains plasmid with
desired functions (green)
Donor

17. Donor
Conjugation
Step 2: Green donor plasmid is
mobilized in trans by helper,
transfers desired donor plasmid
into recipient cell
Recipient
Recipient

18. Strains/plasmids used in experiment
● Donor: E.coli with pHC60 plasmid encoding
constitutively expressed GFP, TetR constructed from
broad-host range plasmid pSW213, RP4 transfer genes
● Helper: E.coli with prk600 conjugative helper, CmR,
plasmid with RK2 transfer genes and ColE1 (narrow-
range) replication origin

19. Plasmid map of pHC60 encoding TetR ,
GFP, and an RP4 oriT site. From
http://www.biovisualtech.com/bvplasmid/pH
C60.jpg

20. Strains/plasmids used in experiment
● Recipient: putative endophyte strains from
Pseudomonadaceae, Xanthomonadaceae,
Paenibacillaceae, Comamonadaceae,
Enterobacteriaceae, Rhizobiaceae
● No known selectable traits

21. General Protocol
1. Mate all three strains on solid rich media
2. Scrape up mating mix and select for desired
transconjugant using antibiotic media, etc --
however, do not know any selectable
markers for recipient strains

22. Donor Selecting for TetR (encoded in
green plasmid) recovers both
donor and recipient strains
Recipient
Mating Mix
Plate on media + Tet

23. Donor
Can (ideally) select against
E.coli by plating on M9+sucrose
and select for desired
transconjugant with antibiotics
Recipient
Mating Mix

24. Spread plate of
phc60 x prk600
pairwise control,
plated on M9 +
Sucrose on 4 June
2014. Picture taken
11 June 2014.

25. Streak plate of
putative
transconjugant (6ST2)
for isolation. Plated 09
June, image taken 11
June 2014.

26. Donor
Conjugation
Step 2: Green donor plasmid is
mobilized in trans by helper,
transfers desired donor plasmid
into recipient cell
Recipient
Recipient
What happened to the helper
plasmid (with respect to the
recipient strain?)