Triparental Mating!13 June 2014

Classical Methods of Horizontal Gene Transfer

● Transformation● Transduction● Conjugation

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

Methods of Incorporating Foreign DNA into the Cell

● Transformationo Free DNA into “competent” recipient cello Natural competence is genetically determinedo Electroporation--high voltage makes cell envelope permeable

and facilitates transformationo Transfection--transformation with DNA extracted from bacterial

viruso Works “moderately well” in archaea by facilitating disassembly

of glycoprotein cell wall layer, also occasionally electroporation, approaches are more diverse and organism-dependent

Methods of Incorporating Foreign DNA into the Cell

● Transductiono Bacteriophage transfers DNA from one cell to

anothero Generalized: donor genes cannot replicate

independentlyo Specialized: bacterial DNA incorporated into viral

genomeo Extremely rare in archaea, only one archaeal virus

has been shown to transduce genes of its host

Methods of Incorporating Foreign DNA into the Cell

● Conjugationo Plasmid-mediated mechanism of transfer involving

cell to cell contacto Process involves donor cell with conjugative plasmid

and recipient strain withouto Two types of conjugation known in Archaea, one

using plasmids--genes encoding conjugative functions have little similarity with those in Bacteria

F-plasmid mediated bacterial conjugation. Wikimedia Commons http://commons.wikimedia.org/wiki/File:Conjugation.svg

(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

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

Plasmid Mobility “Types”

● Conjugative (self-transmissible), ● Mobilizable● Nonmobilizable (most plasmids larger than

300kb)

(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

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

(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

(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

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

Helper DonorConjugation

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

DonorConjugation

Step 2: Green donor plasmid is mobilized in trans by helper, transfers desired donor plasmid into recipient cell

Recipient

Recipient

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

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

Strains/plasmids used in experiment

● Recipient: putative endophyte strains from Pseudomonadaceae, Xanthomonadaceae, Paenibacillaceae, Comamonadaceae, Enterobacteriaceae, Rhizobiaceae

● No known selectable traits

General Protocol

1. Mate all three strains on solid rich media2. Scrape up mating mix and select for desired

transconjugant using antibiotic media, etc -- however, do not know any selectable markers for recipient strains

Donor Selecting for TetR (encoded in green plasmid) recovers both donor and recipient strains

Recipient

Mating Mix

Plate on media + Tet

Donor

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

Recipient

Mating Mix

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

Streak plate of putative

transconjugant (6ST2) for isolation. Plated 09 June, image taken 11

June 2014.

DonorConjugation

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?)