Download Conjugation

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Time line for bacterial genetics
1946 Lederberg and Tatum - demonstrated conjugation in E. coli
1950 Cavalli-Sforza - created an E. coli Hfr strain
1952 Lederberg and Zinder - phage and Salmonella, transduction (“filterable agent”)
1953 Hayes - plated exconjugants on strep -> one way transfer during conjugation
1957 Wollman and Jacob - bacterial gene mapping with Hfr strains, interrupted mating
1958 Lederberg wins the Nobel prize
1959 Adelberg - Hfr -> F’ factor
1961 Jacob and Monod develop the operon model
1965 Jacob, L’Woff, Monod win the Nobel prize
1966 Gilbert, Muller-Hill isolate the Lac repressor
1969 Delbruck, Hershey, Luria - Nobel prize for bacteriophage genetics
1996 Lewis and Lu determine crystal structure of the Lac repressor
Bacterial Genetics, key concepts
3 mechanisms for recombination in bacteria: transformation, conjugation, and
transduction. All three involve the unidirectional transfer of genetic information to a
recipient.
Conjugation (Lederberg and Tatum, 1946)
The experiment:
Strain A, is met- and bio-, cannot grow on minimal medium
Strain B, is thr-, leu-, and thi-, cannot grow on minimal medium
A mix A and B is allowed to grow for a few cell divisions in complete medium
and then plated on minimal medium
1/10,000,000 cells grow into colonies; these are prototrophs, therefore, a
recombinational process is taking place.
1. The F factor is a plasmid that replicates episomally which allows it to be maintained in
the cytoplasm of the F+ cell. It contains ~100,000 base pairs and 19 genes that encode
for proteins involved in pili synthesis, DNA transfer, replication, and other functions.
2. Only F+ cells produce a pilus which allows the cell to attach to an F- cell. They are
called “donor” or “male” bacteria
3. One strand of the F factor is transferred to the F- cell (recipient or “female”) where the
complementary strand is synthesized. A copy of the F factor remains in the F+ cell.
Both exconjugate cells now contain the circular F factor (plasmid)
4. Recombination of F with recipient (exconjugant) chromosome does not occur
5. The F factor occasionally integrates randomly into the E. coli chromosome creating an
Hfr (high frequency of recombination) cell. Hfr strains have the F factor integrated in a
specific location with polarity (direction). Insertion sequences on the bacterial
chromosome are required for insertion of the F factor into the chromosome.
6. Hfr cells transfer bacterial genes in a linear fashion into an F- cell. The F factor is
transferred last. Conjugation rarely occurs long enough for the F factor to be
transferred. Therefore, recipient cells remain F-.
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7. The interrupted mating technique involves conjugation for specific times and then
plating exconjugants on media containing streptomycin to select for F- exconjugants.
The use of minimal media +/- nutritional additives is used to determine which genes
have been transferred and the order of gene transfer.
The longer the cells conjugate, the more of the donor bacterial chromosome moves into the recipient Fcell. The pilus is broken at various times by placing the bacteria in a blender
The F- cell does not become F+ or Hfr because the F factor does not transfer
The F factor can be inserted at different positions in different bacterial chromosomes, the genes move
over in the same order but from different starting points in different strains.
The F factor can be present in the reverse orientation, so the order with which the genes would move
over would be reversed in these strains
8. There may be homologous recombination between the bacterial genes transferred and
the recipient cell’s chromosome.
An Hfr cell can revert to an F+ cell and the F+ may clip out bacterial genes. The F plasmid is now referred to as an F’ (F prime) factor.
An F’ factor can carry many bacterial genes.
A mating of a F' cell with an F- bacterium results in a merodiploid cell - two copies of the transferred bacterial genes. One copy is on
the F’ factor and the other is on the recipient bacterial chromosome.
Transformation
1. A plasmid or other piece of DNA is enters a competent bacterium via receptors on the
bacterial cell
2. In the lab, bacterial cells can be made "competent" by treatment with calcium chloride.
A brief heat shock facilitates uptake of DNA into the bacterial cell
3. The plasmid is maintained extra chromosomally because is has an origin of replication.
It can integrate into the bacterial chromosome. The genes on the plasmid may confer
new traits to the bacterium such as antibiotic resistance.
4. In nature, transformation is a rare event
A recA- bacterium will not integrate the plasmid into the chromosomal DNA. This type of bacteria is used in laboratory transformation
to make sure the plasmid does not integrate into the chromosome. Then the plasmid can be recovered using standard laboratory
techniques (plasmid prep)
Transduction (Zinder, Lederberg, 1952, S. typhimurium)
1. U-tube experiment showed that bacterial contact is not necessary because transduction
is virus-mediated. The virus is not sensitive to DNAse
2. A bacteriophage infects bacteria (P22 phage and Salmonella) and begins the lytic cycle
(adsorption, injection of DNA, repression of bacterial genes, synthesis of phage
components, packaging of phage, lysis of cell)
3. During new phage packaging, pieces of chromosomal DNA (bacterial genes) may be
incorporated into the phage head = faulty head-stuffing. Up to 1% of the bacterial
genome may be incorporated (about 50,000 bp). All bacterial DNA has an equal
probability of being packaged. This is generalized transduction.
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4. When the host cell lyses, the new viral particles are released and can infect new
bacterial cells. Because infection is a property of the phage protein coat, the virus can
adsorb to cells and packaged bacterial genes will be injected.
5. The injected DNA may integrate into the host DNA via homologous recombination.
6. In lysogeny, the phage genome integrated into the bacterial genome creating a
prophage.
7.
8.
 phage integrates at a specific location, the  att site, between gal and bio genes in E. coli.
The existence of the integrated prophage prevents superinfection by additional phage
9. In response to bacterial damage or stress, the prophage enters the lytic cycle (genes for
phage assembly, packaging, cell lysis are expressed)
10. When the phage excises, it may also clip bacterial genes. These genes are then packaged into new phage
particles. The new phage infects new cells and carries the bacterial genes with it. This is referred to as
specialized transduction.
The Lac operon
Operon – a set of genes that is coordinately expressed
1. The I gene encodes the repressor which binds to the operator in the absence of lactose.
When the repressor is bound to the operator, RNA polymerase, which normally binds
the promoter, cannot transcribe the structural genes, Z,Y,A
2. Lactose binds repressor molecules which undergo a conformational change. The change
in shape prevents repressor binding to the operator. Transcription of the structural
genes, Z, Y, A, is now derepressed. Lactose is referred to an "inducer" of the operon
3. Structural gene Z encodes the enzyme, betagalactosidase, which cleaves lactose into
glucose + galactose. Structural genes Y and A encode enzymes also involved in lactose
metabolism
4. Various lac operon mutants have been used to elucidate control of operon expression
5. A constitutive mutant is one in which the gene product is produced continually, that is
there is no control over its expression. I- is constitutive because the repressor is not
produced, the operon is always on. Oc is constitutive because the operator cannot bind
the repressor.
6. Conjugation with F (plasmid) cells bearing certain operon genes also demonstrates
operon regulation. If a bacterium is I- and the plasmid is I+, the repressor can be
synthesized and function normally. The operon would then be inducible with lactose.
7. Operons may be positively or negatively regulated, there are others besides the lactose
operon