T-even-phage_mutations.html: 06_T01-T-even-phage_mutations.jpg
T4-cycle.html: 06_15-T4-cycle.jpg
1) The virus binds to the bacterial host cell surface by the tail fibers.
2) The tail sheath contracts and causes the central core to penetrate the cell wall.
3) The protein coat remains outside the host while viral DNA is injected;
host DNA is degraded.
4) Viral molecules are synthesized using host resources; assembly of progeny phages from components begins.
5) Viral assembly is completed, a phage enzyme (lysozyme) ruptures (lyses) the cell,
releasing progeny
phages.
T4.html: 06_14-T4.jpg
Bacteriophage T4 is one of a group of lytic (or virulent)
bacterial viruses called T-even phages.
The "head" is made up of an protein coat containing the DNA.
A "tail" contains a collar and a contractile sheath surrounding a central core;
tail fibers protruding from a base plate
contain binding sites that recognize the cell wall of the E. coli host.
T4_rII_map.html: 06_25-T4_rII_map.jpg
Benzer mapped 307 distinct sites after analyzing 20,000 mutations of the T4 rII locus.
Some areas, called hot spots, seem to be more susceptible to mutation than others.
U-tube.html: 06_04-U-tube.jpg
If F+ and F- cells are grown in a Davis U-tube which allows mixing of the medium
but not of the cells, no prototrophs are recovered.
Therefore physical contact is needed for conjugation and genetic recombination.
cistron.html: 06_22-cistron.jpg
Mixed infections of two rIIA mutants on E. coli B produce wild-type recombinants that can infect
the K12 strain (left).
The plate at right is a B strain control that reflect total phage progeny.
To calculate the frequency of recombination:
2 (recombinant phages) / total phages):
2 (4 * 103 / 8 * 109) = 2 (0.5 * 10-6) = 10-6
We need to multiply by 2 since only one of the two reciprocal recombinants is
detected.
complementation-no.html: 06_21b-complementation.jpg
Two mutations in the same cistron do not complement one another, and produce no
wild-type phenotype.
The mutants are classified into two complementation groups,
or cistrons, A or B.
Today we know that these cistrons represent two genes
in what was originally thought as a single rII locus.
complementation.html: 06_21a-complementation.jpg
Some rII mutants can complement each other without recombination.
In mixed infections, each mutant strain provided a function that the other lacked, restoring wild-type
phenotype,
though the phages themselves retain the mutant phenotype.
conjugation.html: 06_06-conjugation.jpg
Transfer of the Fertility (F) factor during conjugation.
1.
In the F+,
the two strands of the double helix of the F factor (a plasmid) separate.
2.
One of the two strands moves into the recipient (F-) cell.
3.
The other strand remains in the donor cell.
4.
Both strands are replicated, with clockwise rotation of the circles.
5.
Both the donor and the recipient cells are now F+.
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The F factor sometimes reverts from being integrated to a free plasmid,
this condition is called F'.
Often the F factor carries several adjacent bacterial genes
with it.
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Following conjugation with an F- cell,
the recipient cell is converted to an F+ donor cell and also
becomes partially diploid and is called a merozygote.
Merozygotes are useful for studying prokaryote gene regulation, described in chapter 16.
conjugation_integration.html: 06_10-conjugation_integration.jpg
Occasionally, an F+ cell is converted to an Hfr state by integration
of the F factor into the bacterial chromosome.
The point of integration determines the origin (O) of the
transfer.
conjugation_integration2.html: 06_10-conjugation_integration2.jpg
In a subsequent conjugation, an enzyme nicks the F factor,
now integrated into the host DNA, initiating DNA transfer there.
Conjugation is usually interrupted before transfer is complete:
only the A and B genes are transferred here,
and the F factor is not transferred, since it is last.
conjugation_map.html: 06_08-conjugation_map.jpg
A time map constructed from the oriented transfer of genes correlated with the length of time that
conjugation proceeded.
Minutes in bacterial mapping are equivalent to map units in eukaryotes.
The same type of experiment with other Hfr strains
to complete the E. coli gene map.
conjugation_order.html: 06_09-conjugation_order.jpg
Different Hfr strains yielded different points of the origin and
the direction in which gene transfer proceeded from that point.
This suggests that the E. coli chromosome is circular.
The origin is determined by the point of integration of the F factor
into the chromosome, and the direction is determined by the orientation of the F factor
as it integrates.
conjugation_transfer.html: 06_07-conjugation_transfer.jpg
Interrupted mating.
Wollman and Jacob
(Nobel 1965)
incubated mixtures of hfr bacteria and antibiotic-resistant F- strains with
several marker genes, and placed them in a blender at various times to interrupt
the conjugating process.
The cells are grown on antibiotic media so that only recipient cells were recovered.
Genetic recombination showed a linear progression with time.
thr and leu are always transferred and are used in the initial screen for recombinants.
After about 10 minutes, recombination of the genes started to be detected , in the order
aziR, tonS, lac+, and gal+.
This oriented transfer of genes can be used to construct a conjugation
map.
deletion_mapping.html: 06_24-deletion_mapping.jpg
If a point mutation tested against each deletion (dashed areas) in Series I
for the production of recombinant wild-type progeny shows the results at the right (+ or –),
the mutation must be in segment A5.
In Series II, the mutation is narrowed to segment A5c, and in Series III to segment A5c3.
deletion_testing.html: 06_23-deletion_testing.jpg
A point mutation can be quickly localized
within a deletion if it fails to
produce wild-type recombinants in complementation assays.
exponential_growth.html: 06_01-exponential_growth.jpg
Bacterial population growth.
Bacteria grown in liquid medium are started with an inoculum (a few cells).
The cells show an initial slow growth (lag phase) followed by a period of rapid exponential growth (log phase) when the doubling time can be as short as 20 minutes.
Nutrients eventually become limiting and cells enter the
stationary phase.
A prototroph
can synthesize all essential organic compounds and
can be grown on minimal medium
of carbohydrates and salts, while
an auxotroph has lost the ability to synthesize some compounds
which must be provided in the medium for the auxotroph to grow.
intragenic_recombination.html: 06_20-intragenic_recombination.jpg
Mixed infections of T4 phages with independent mutations at the rII (rapid lysis II)
locus that prevent the mutants from
infecting E. coli K12(λ) produced wild-type intragenic recombinants (right).
The other recombinant (left) cannot be detected.
These recombination frequencies allowed Benzer to produce a genetic map of this locus, proving that
a gene is not an indivisible particle.
mixed_infection.html: 06_T02-mixed_infection.jpg
phage_recombination.html: 06_19-phage_recombination.jpg
Hershey
and
Luria
(Nobel 1969)
discovered T2 mutations that affected plaque morphology and allow detection of genetic recombination.
Mixed infections of T2 phages with mutations at two loci, r (rapid lysis - large plaques)
and h (host range - dark center plaques when grown on 2 hosts),
resulted in intergenic recombination
that can be detected by morphology.
The relative distance between phage genes can be calculated by dividing the percentage of recombinant plaques by the total number of plaques:
recombination frequency = (((h+r+) + (hr)) / total plaques) x 100
pilus.html: 06_05-pilus.jpg
An F+ (contains "fertility factor", a small circular DNA molecule) E. coli cell
can "donate" parts of its chromosome to a recipient (F-) cell through a conjugation tube called a
sex pilus. This conjugation produces recombinant DNA in the recipient,
which also becomes F+.
plaque_assay.html: 06_16-plaque_assay.jpg
There are 23 phage plaques derived from a 0.1 ml aliquot (sample) of the 10–5 dilution.
plaque_dilution.html: 06_16-plaque_dilution.jpg
A plaque assay of a concentrated bacteriophage culture begins with serial
dilutions
of a viral culture. A small sample is then mixed with host bacteria and plated on nutrient agar.
After incubation, the bacteria grow to form a "lawn" of cells, with clear areas ("plaques") indicating
where one phage has initially infected one bacterium.
plasmid.html: 06_12b-plasmid.jpg
An R plasmid consists of a resistance transfer factor (RTF), which enables
conjugation, and one or more r-determinants: genes conferring resistance to antibiotics.
Plasmids are useful in recombinant DNA research, which is described in chapter 19.
The r-determinants in this diagram are
Tc, tetracycline; Kan, kanamycin; Sm, streptomycin; Su, sulfonamide; Amp, ampicillin; and Hg, mercury.
prototroph.html: 06_03-prototroph.jpg
Auxotroph A needs methionine (met) and biotin (bio) to grow;
auxotroph B needs threonine (thr), leucine (leu),
and thiamine (thi); both require supplemented media.
Lederberg
and
Tatum
(Nobel 1958) mixed the two strains and plated them on
minimal medium and recovered
wild-type prototrophs, indicating recombination had occurred.
serial_dilution.html: 06_02-serial_dilution.jpg
To estimate the population of a liquid bacterial culture, serial
dilutions
are made to reduce the cell density to a reasonable range.
A sample is then plated on semisolid medium in a petri dish.
Each bacterium grows into one visible colony after incubation,
so the original concentration can be found by multiplying the number of colonies by the dilution factor.
(Note: when high concentrations of cells are plated, the colonies can fuse into a
"lawn"
of cells.)
Each of the plates above is diluted by a factor of 10. The dish on the right contains 15 colonies.
transduction.html: 06_17-transduction.jpg
Zinder and Lederberg (Nobel 1958)
placed two auxotrophic strains of Salmonella
on opposite sides of a Davis U-tube.
Prototrophs were recovered from the side containing
LA-22 cells, but not from the side containing LA-2 cells.
Adding DNase, an enzyme that digests DNA, did not prevent recombination, so this is not transformation.
The filter prevented cell contact, so this is not conjugation.
LA-22 cells.
Rarely, some P22 enter the lytic phase,
exit the LA-22 cells,
and cross the filter to infect LA-2 cells.
When LA-2 cells are lysed, some LA-2 DNA may be packaged in P22 heads,
which can move back across the filter to infect LA-22 cells, enter lysogeny
and producing recombinants.
transduction_generalized.html: 06_18-transduction_generalized.jpg
Sometimes in a phage infection cycle, some host DNA is packaged in the phage head during assembly.
When that DNA is injected into the next host cell,
crossing over between the injected DNA and the homologous region of the bacterial chromosome results in
recombination, as in transformation.
Like transformation, transduction can be used to establish "linkage",
since bacterial genes that are close together have a high probability of cotransduction, and
relative mapping distances between linked genes can be determined.
transformation.html: 06_13-transformation.jpg
Transformation.
A piece of foreign DNA in the environment is taken up into a competent cell by active transport via a
receptor site on the surface of the cell.
transformation2.html: 06_13-transformation2.jpg
One of the two strands of the invading DNA molecule is digested by nucleases,
The surviving DNA strand aligns with a complementary region of the bacterial chromosome and replaces it,
producing a heteroduplex region where the two strands of DNA are not perfectly complementary.
Following DNA replication and cell division, one cell contains the original DNA sequence, while the other has been transformed to possess the foreign gene.
This process can be used to establish "linkage", since bacterial genes that are close together have a high probability of cotransformation, and relative mapping distances between linked genes can be determined.