BIOL3060 #16 Bacterial Gene Exchange

Discovery of Genetic Exchange in Bacteria

Lederberg and Tatum’s 1946 Experument

• Before 1946, bacteria was believed to reproduce only by binary fission (dividing of organism by small polyp that breaks off and grows on its own), no genetic exchange was known

• Loshua Lederberg and Edward tatum challenged this idea using E. coli mutants

• Their experiment provided the first evidence of recombination in bacteria

Lederberg and Tatum: Experimental Design

• Auxotrophs: bacteria that cannot grow without certain nutrients because of gene mutations

• Prototrophs: can grow on minimal medium (make all nutrients themselves)

• Strains Used (auxotrophs for complementary mutations) DON’T NEED TO MEMORIZE STRAIN INFO

  • Y10: thr- thi- bio+ phe+ cys+

    • needs threonine, leucine, and thiamine added to grow

  • Y24: thr+ leu+ thi+ bio- phe- cys-

    • needs biotin, phenylalanine, and cysteine added to grow

• Hypothesis: if bacteria can exchange genes, mixing Y10 and Y24 might allow them to complement each other’s mutations, producing some offspring that can grow without supplements

Lederberg and Tatum: When Y10 and Y24 Were Mixed

• when mix ed and plated on minimal medium, a few colonies grew

• each strain alone produced no growth, therefore colonies could not have arisen by random mutation: too many simultaneous mutations would be required

• interpretation:

  • colonies were prototrophic recombinants with the genotype thr+ leu+ thi+ bio+ phe+ cys+

  • means that genetic exchange and recombination occurred between Y10 andY24

Does Gene Transfer Require Direct Contract?

• Bernard Davis (1950) designed a U-tube with a fine-pore filter separating two bacterial strains

  • Allowed liquid medium to pass

  • Blocked bacterial cells (to see if DNA would pass through filter if bacteria could not)

• Results:

  • After incubation, no colonies grew when plated on minimal medium

  • DNA did NOT pass through the filter

• Conclusion:

  • Gene transfer requires direct contact between bacterial cells

• Why did this not work?

  • cell has to be dead and break apart for DNA to be free-floating

  • this bacteria may just not be able to do transformation

The Fertility (F) Factor and Conjugation

• Conjugation depends on a plasmid present in the donor cell

  • In E. coli, plasmid is called the fertility (F) factor

• F factor carries genes to form a pilus, which allows transfer of DNA to another cell

• Cells with the F factor are called F+ (donor); cells lacking it are F- (recipient)

• F factor is an episome (can integrate into the chromosome bc it is a plasmid)

• Contains:

  • Origin of replication (oriV): site where plasmid replication begins

  • Origin of transfer (oriT): site where DNA transfer begins

  • Conjugation genes (tra genes): encode the proteins that form the pilus

• Once the entire F factor is transferred, the recipient (F-) becomes F+

  • both cells have full copy of F plasmid and can be donors in future conjugations

• Direct transfer is defined: the oriT site always enters the recipient first

• Only F plasmid genes are transferred in this type of conjugation - not chromosomal genes (yet)

  • could NOT explain the transfer of chromosomal genes discovered by Lederberg and Tatum

The Fertility (F) Factor and Conjugation: Steps

1. Contact: F+ cell forms a pilus that attaches to an F- cell

2. Connection: the pilus pulls the cells together, forming a cytoplasmic bridge

3. Nick and Transfer: One DNA strand of the F plasmic is nicked at oriT and begins to move into the recipient

4. Replication: as transfer occurs, rolling-circle replication replaces the transferred strand in the donor

5. Completion: The recipient replicates the incoming strand, forming a new double-stranded F plasmid

Note: now both cells can continue to pas on F+ (but chromosomal DNA is yet to be transferred)

Hfr Cells

Consequences and Frequency of Hfr Events

F’ Cells

Merozygotes

Cell Types (F factor)

Mapping Bacterial Genes with interrupted Conjugation

Transformation in Bacteria

Competence and DNA Uptake

Laboratory Techniques

Using Transformation for Gene Mapping

Horizontal Gene Transfer Definition

Horizontal Gene Transfer: Medical Importance