Before we look at the methods for genetical analysis in bacteriophages
first I want to look at their infection cycles.
Many virulent bacteriophages can only conduct lytic cycles. These include
the T-even phages T2 & T4, and the T-odd phages T3 & T7. (Remember
the experiment of Hershey and Chase.) An infectious phage adsorbs to the
bacterial cell wall and injects its chromosome into the host. Three sets
of genes are then sequentially expressed:
At late stages of infection when rapid production of phages is in progress, the viral DNA replicates by the rolling circle mechanism.i) The early genes - products of the early genes inhibit host (E. coli) RNA transcription.
ii) The DNA metabolism genes - the products of these genes include enzymes to replicate the phage DNA and nucleases to digest the host chromosome so that free nucleotides are available for DNA synthesis. (Why is the phage genome not also degraded?)
iii) The late genes - their products include phage coat, tail and assembly proteins, and the assembly proteins to package the newly replicated chromosomes into the newly formed phage coats. A further late gene product is the enzyme lysozyme which digests the bacterial cell wall and gives rise to the burst or lytic release of the new phage particles.
Other bacteriophages can follow one of two delicately balanced developmental
pathways controlled by two sets of genes. One set of genes leads to the
lytic cycle as previously described, while the other set leads to the lysogenic
cycle which does not result in the immediate death of the host. Those phages
which do not necessarily immediately lyse the host are called temperate
phages, e.g. phage l and f80.
In the lysogenic cycle, the phage DNA is injected and dictates the synthesis
of a repressor molecule that inhibits
expression of the virulent, lytic genes. In most bacteriophages, the phage
DNA then becomes physically inserted into the host bacterial chromosome,
a process known as integration.
Fig 8b.3: Prophage formation
In this state the bacteriophage DNA is replicated along with the host
DNA for many generations and is called a prophage.
The bacterium carrying such a prophage is called a lysogen and it is the
act of repressor synthesis and prophage integration or stable maintenance
in a non-integrated state which is known as lysogeny.
An E. coli cell carrying a l prophage
is said to be lysogenic for l and is designated
E.
coli (l). Such a lysogen usually grows at
the same rate as a normal bacterium and very infrequently, about 10
– 4 divisions, the level of repressor falls below the threshold required
for lysogeny, whereupon the prophage excises itself from the host chromosome
and orchestrates a lytic cycle.
Lysogenic phages have an immense advantage over purely lytic phages.
Consider l bacteriophage: once the l
DNA has integrated as a prophage, the host is immune to further infection
(superinfection) and the l
DNA sequence is then transmitted to all descendants of this host with each
cell division. Consequently, millions of cells harbouring l
prophages are generated in a short period of time from a single lysogenic
cell. Transmission of phage information in space and time is thus assured
and not solely dependent on infectious meetings of virus with host.
Experimentally, phage particles are added at low dilution to a dense
suspension of E. coli cells and plated. The E. coli cells
give rise to a lawn of growth (a continuous covering of bacteria)
on the surface of the agar plate. In the majority of cases when wild type
l
infects E. coli, infection leads directly to the lytic cycle. After
a phage infects a single cell, the cell eventually bursts releasing about
250 progeny phages which infect nearby cells. This process is repeated
leading to a clear area of burst cells (a plaque)
resulting from a single original infection. In some cases, however, the
progeny phages will switch to the lysogenic cycle, some bacterial cells
in the plaque will survive and the plaques produced by the temperate l
phage growing on a lawn of E. coli will be opaque or turbid. In
contrast, lytic T2 phages produce totally clear plaques on a lawn of bacteria.
As phages are only visible under the e.m., morphological mutants are
not easily studied. However, many mutations affect the phage life cycle,
giving rise to differences in the appearances of plaques on a bacterial
lawn, e.g. minute mutant of l
where the plaques are much smaller than normal.
For phage crosses, mixed infections are performed as follows:
- incubate a dense suspension (108 ml– 1)
of sensitive E. coli cells with a denser suspension (109
ml– 1) of a 1:1 mixture of the phage strains (the multiplicity
of
infection
– m.o.i.– is 10, i.e. there are 10 phage particles per bacterial
cell).
- phage multiplication involving recombination (when two phage genomes
infect the same bacterial cell) can occur, the cells lyse and the lysates
are collected. (The suspension can be centrifuged and the bacterial cells
will pellet while the lysate containing the phages will be the supernatant.)
- the lysate is diluted to an appropriate concentration of phage particles
(to give a concentration such that about 100 phages are in the volume to
be plated, e.g. if 0.1 ml will be plated from a 10 ml suspension
of phages and bacteria, then the concentration of phage in the suspension
should be about 103 ml– 1). Or several dilutions
of lysate may be used.
- the diluted lysate is mixed with a suspension (> 107 ml– 1) of sensitive E. coli cells and plated.
- the E. coli cells produce a lawn of bacterial cells and at the appropriate dilution of phage particles discrete plaques are seen.
- plaques can then be examined for morphology and scored (remember, each plaque will be the result of infection by a single phage).
Consider a cross of a phage strain giving rise to minute plaques (mi) and the wild type. The mi and wild type strains are used in a mixed infection, giving rise to the following progeny:
Phenotype
number
+
2340
mi
2050
Total 4390
There were no other plaque phenotypes so the approximate 1:1 ratio implies
that mi is behaving as a single mutation at a single locus. i.e.,
a single gene mutation produces mi.
This was a one-factor cross but two-factor crosses can also be done. The assumption used is the same as in mapping eukaryotic genomes, namely, that crossovers occur randomly along the chromosome, so the recombination frequency represents the map distance (in map units) between the phage genes.
Consider a cross of mi with a clear (c) mutant strain (unable to achieve lysogeny, so plaques are clear) of l. In a mixed infection, then:
mi +
x
+ c
The following progeny were produced:
Plaque morphology Number scoredAs previously, identify the parental and recombinant phenotypes and establish recombination frequencies:mi + 1205
+ c 1213
+ + 84
mi c 75
Recombination frequency = 100% x No. of recombinants / Total number of progeny
= 100 % x (84 + 75) / (1205 + 1213 + 75 + 84)
= 100 % x (159 / 2577 ) = 6.17 % or 0.0617
Hence the loci are about 6 map units apart.
A map of the phage genome cen be built up in this way. Consider an additional
mutation, s, which leads to small plaques. A series of 2-factor
crosses in mixed infections were performed which allowed calculation of
recombination frequencies and map distances between the pairs of markers:
This gives the map:Markers Mapdistancess - mi 8.5 mu
s - c 2.8 mu
c - mi 6.2 mu
s --- 2.8 mu --- c -----6.2 mu -------- mi
ç------------8.5 mu ------------------è
Three-factor crosses can also be done which give the gene order and
recombination frequencies for three mutations in one cross. Examine the
single- and double-crossover classes and work out recombination frequencies.
You may attempt some of the problems at the end of chapter 15, remember that answers to odd-numbered problems are at the end of the text.
This template created by the Web Diner.
This
website is owned and maintained by Ronald Worrell.Copyright
© L.D. Waterman 1998
For
information regarding this web site and/or its contents please send mail
to