Bio3400
Chapter 6
Genetic Analysis and Mapping in Bacteria and Bacteriophages
Bacterial growth can be divided into three phases:
phase,
phase (
growth), and
phase.
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. wmplayer 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.
Cells grown in liquid medium can be quantified by the
serial
technique.
To estimate the population of a liquid bacterial culture, serial dilutions are made. A sample is then plated on semisolid medium in a petri dish. Each bacterium grows into one visible colony, 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.
If these grew from 1 ml of a total dilution of 10^5, the original concentration of bacteria is:
15 x 10^5 / ml, or + 1.5 x 10^6 / ml.
Bacteria can undergo
, in which genetic information from one bacterium is
transferred
to another,
resulting
in
in the
latter.
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^+.
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.
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.
F
+
cells contain a
(F)
factor
that has genes for sex
formation and allows the host to donate
DNA
during
. Recipient cells are usually converted to F
+
.
1. In the F^+, the two strands of the double helix of the Fertility (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^+ after conjugation.
An
(high-frequency recombination)
strain
has an
F
factor,
and can donate
DNA
in an
fashion
to an F
-
cell.
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.
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.
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.
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.
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.
Sometimes, an F factor is
from the chromosome of an Hfr cell, producing an
cell.
Transfer of an F' to an F
-
cell
results
in a
.
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.
Following conjugation with an F^- cell, the recipient cell becomes partially diploid and is called a merozygote. The recipient also behaves as an F^+ donor cell.
such as
Factors
contain one or more genes and replicate independently of the bacterial chromosome.
plasmids confer antibiotic
resistance.
An R plasmid consists of a resistance transfer factor (RTF), which enables conjugation, and one or more r-determinants: genes conferring resistance to antibiotics. The r-determinants in this diagram are Tc, tetracycline; Kan, kanamycin; Sm, streptomycin; Su, sulfonamide; Amp, ampicillin; and Hg, mercury.
Bacteria can also undergo genetic recombination by
, in which pieces of extracellular
DNA
are taken up by a cell and
into the
chromosome.
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.
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.
(phages) such as T4 are viruses that
infect
a host bacterium and use the host enzymes to
reproduce.
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.
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) A phage enzyme (lysozyme) ruptures (lyses) the cell, releasing progeny phages and completing the lytic cycle.
The number of phages produced following the infection of bacteria can be
determined
by the
assay.
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.
To estimate the population of a liquid bacterial culture, serial dilutions are made. A sample is then plated on semisolid medium in a petri dish. Each bacterium grows into one visible colony, 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.
If these grew from 1 ml of a total dilution of 10^5, the original concentration of bacteria is:
15 x 10^5 / ml, or + 1.5 x 10^6 / ml.
There are 23 phage plaques derived from a 0.1 ml aliquot (sample) of the 10^5 dilution.
To calculate the initial undiluted viral density (particles / ml):[7;11H+ (23 * 10^5 ) / 0.1 ml = 23 * 10^6 / ml, or[8;11H+ 2.3 * 10^7 / ml
Sometimes a
phage integrates its DNA into the bacterial chromosome in a
process
called
, and the viral DNA (
) is replicated with the host DNA.
The lytic and lysogenic cycles of l, a temperate phage. After entering the host cell and circularizing, the l (lambda) DNA can enter the lytic cycle or integrate into the bacterial chromosome (lysogenic cycle) as a prophage, where it may be carried in the host DNA for many generations. Review:
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Bacteriophages can mediate genetic
in their bacterial hosts in a
process
called
.
Transduction. 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.
Recombination stopped when the pore size of the Davis U-tube was reduced below the size of phages.
This transduction was mediated by phage P22, starting as a prophage in 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
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In
transduction,
DNA instead of
DNA is packaged in a phage particle and transferred to a recipient
host.
Transduction. 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.
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.
mapping in bacteriophages can be done by examining phage
mutations
that affect plaque
.
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