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6 BACTERIAL AND PHAGE GENETICS
6 Bacteria interact with viruses called bacteriophages
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- A lytic phage is a bacteriophage whose life cycle
always involves the takeover of bacterial machinery for production of new phage
and lysis of the bacterial cell after production is complete.
- A lawn (Bacterial lawn) is a
uniform layer of bacteria on the surface of a growth medium.
- The titer is the measurement of the amount or
concentration of a substance in a solution.
- An infectious center is a bacterial cell that
has been infected by a bacteriophage, giving rise to a plaque.
- A temperate phage can form a lysogenic relationship
with its host bacterium by becoming integrated into the bacterial genome in the
form of a prophage.
- Lysogeny describes the ability of a phage to survive
in a bacterium as a stable prophage component of the bacterial genome.
- Prophage is a phage genome covalently integrated
as a linear part of the bacterial chromosome.
- Immunity in phages refers to the ability of a
prophage to prevent another phage of the same type from infecting a cell. It results
from the synthesis of phage repressor by the prophage genome.
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- Bacterial viruses called bacteriophages
can infect bacteria.
- All bacteriophage depend on the bacterial
host to provide functions for replication, transcription, and translation, modifying
the host functions as needed.
- Lytic bacteriophage infect a bacterial
cell, duplicate themselves, and then kill their host, releasing multiple copies
(a burst) of phage.
- Lysogenic bacteriophage have two
alternative lifestyles: lytic growth, with death of the host and release of a burst
of phage, and lysogenic growth, in which they form a prophage, a silent or nearly
silent component of the cell's genetic material.
- A prophage can be induced to return
to lytic growth; many prophages are induced by agents that damage DNA.
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Bacteriophages — "eating" (Greek: -phagos) bacteria — are bacterial
parasites, viruses that use the bacterial machinery to copy their own genetic material,
leading to the release of dozens to hundreds of copies of the invading bacteriophage.
Bacteriophages have been widely used as research tools both in the first decades
of molecular biology research (see 4230) and today. They have also become
important tools for biotechnology, as vectors and for phage display (plasmids and
phage are useful for carrying foreign pieces of DNA) and are being developed as
possible agents to attack bacterial pathogens.
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Figure 6.6
The steps shown in the figure can be completed within 20′, releasing a burst
of 100 phage per cell. Each of these phage may then infect a new bacterial cell.
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Like all viruses, bacteriophages are composed of a nucleic acid core, either DNA
or RNA, protected from the environment, generally by a protein coating (or head).
The nucleic acid encodes the essential elements for bacteriophage propagation. The
bacteriophage life cycle proceeds as shown in Figure 6.6. A phage attaches to a
sensitive host cell and injects its DNA (or RNA for RNA viruses). A lytic phage,
which always kills its host, quickly begins replication of its nucleic acid. This
step is followed by synthesis of the proteins necessary for packaging the nucleic
acid (heads), assembly of the heads and incorporation of the nucleic acid into the
phage heads, and attachment of any additional structures, such as the phage tails
that allow attachment to a new host. Finally, the phage induces lysis of the host.
The resulting progeny phages that are released are called a burst of phages; these
can go on to infect other sensitive bacteria (Lytic development is controlled by
a cascade).
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Figure 6.7
This photo shows phage λ plaques growing on a bacterial strain deleted for
the lac operon. Plaques with halos are produced by phage expressing the
lac genes, whose gene product, β-galactosidase
is detected by immunoenzymatic methods.
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When an infected bacterial cell is plated on a petri dish with an excess of uninfected
bacteria (enough to totally cover the petri dish without any individual colonies
being visible; this is called a lawn of bacteria), the phages released from the
infected cell will infect the neighbors, killing them and releasing a burst once
again, through many cycles. The result, after incubation of such a petri dish under
favorable conditions, will be the appearance of a circular area cleared of bacterial
growth by lysis; elsewhere the lawn of bacteria will make the plate opaque, as shown
in Figure 6.7. By counting these circles, called plaques, we can calculate how many
infected cells were present on the plate; thus, we can titer (count) bacteriophages
(and other viruses) by appropriate serial dilutions as we do for colonies. Each
infected cell that gives rise to a plaque is called an infectious center.
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Figure 6.8
The red line shows the phage counted (titered) from a culture at various times after
infection. The blue line shows the number titered after the culture is treated with
chloroform to lyse the cells.
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We can monitor the steps in an infection by taking samples at various times after
infection and assaying them for phage particles. As graphed in Figure 6.8, there
is an initial period called the eclipse period, when only very few phages can be
titered; in fact, what we are measuring here is infected cells that can form plaques.
This period corresponds to the part of the phage life cycle when the phage DNA is
inside the cell, replicating and starting to synthesize the components of the phage
head, but no new phages have yet assembled.
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As phages assemble but remain inside the cell, we still have only one infectious
center. However, if we artificially lyse the bacterial cells, we can release the
phages. This increase in phage particles is shown by the blue line in the figure.
Once the cells lyse naturally we detect the sudden increase in phage particles shown
in the graph (the burst of phage).
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Figure 6.9
During lysogenic growth, the expression of the lytic genes is repressed by a phage-encoded
repressor. Induction of the integrated, stable state (called a lysogen) requires
inactivation of the repressor.
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A group of phages called temperate phages have, in addition to
the lytic cycle, an alternative reproductive cycle called lysogeny, shown in Figure
6.9. E. coli phage λ is the most well-understood temperate phage.
Temperate phages can infect a bacterium, replicate, and lyse the host like lytic
phages, or they can establish a long-term quiescent state, in which their genome
is passed on to all daughter cells but phages are not released. This latter process,
called lysogenization, can be reversed, usually when the phage senses trouble in
its host, such as DNA damage. Then, the quiescent state ends and the phage resumes
its lytic life cycle. The phage is said to be induced when it switches from the
lysogenic to the lytic state (Lysogeny is maintained by repressor protein).
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In addition to the genetic information needed to carry out the lytic functions,
a lysogenic phage, such as bacteriophage λ, must carry the information for
its alternative, lysogenic lifestyle. During the lysogenic state, phage DNA replication,
packaging, and cell lysis are shut down. The lysogenic phage must also ensure that
the viral genome is passed down to daughter cells, which is often accomplished by
integrating its genome into the host chromosome, as seen in Figure 6.9. The integrated
phage genome is called a prophage. For another well-studied bacteriophage, P1, the
silenced DNA is maintained as a low copy number plasmid, and the phage has a mechanism
to ensure the distribution of this plasmid to daughter cells.
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Lysogenic phages also confer phage immunity — a way of preventing lysogenic
cells from being infected and killed by other phages of the same sort, by shutting
down the lytic functions of infecting phage. A plaque of a lysogenic phage will
not be totally clear because lysogenic bacteria will grow up within the plaque.
Instead, the plaque is referred to as turbid (see Repressor maintains an autogenous
circuit).
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The life cycle of most bacteriophages depends on many host functions, from DNA replication
and protein synthesis machinery through functions necessary for protein folding.
In fact, chaperones, proteins that aid in the folding of other proteins, were first
identified as functions essential for the growth of bacteriophage lambda (4210).
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There are two ways in which bacteriophages play a major role in genetic analyses.
As genetic systems, they are useful for selecting bacterial mutants, for comparing
the behavior of two different bacterial hosts with respect to the growth or behavior
of the bacteriophage, or for increasing our understanding of how cells function
by analyzing the ways in which the bacteriophage modifies and utilizes the cell
machinery. The history of molecular biology is based, in large part, on the use
of mutations in either the phage or bacterial host that changed their interaction
(see 4229). The second role of bacteriophages reflects their ability to
transfer genetic material from one cell to another and, in the case of lysogenic
phages, to stably maintain the carried material in the recipient cell.
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Bacteriophages also have enzymes with unique properties that are useful to the scientist.
The lytic phage T7 ensures that its own genes are transcribed by encoding a highly
efficient RNA polymerase. This polymerase recognizes the unique promoters of a set
of T7 genes, and not E. coli promoters. The structure of this polymerase
is different enough from that of the E. coli RNA polymerase to be resistant
to drugs that inhibit the E. coli enzyme. In the laboratory, the bacterial
cell transcription and translation machinery can be redirected to make large amounts
of a particular protein by using the specialized T7 promoter in front of a gene
of interest and expressing the T7 polymerase in a bacterial cell.
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What is the advantage to bacteria of interacting with the bacteriophage? As we have
mentioned and will discuss later (Bacteriophage can move bacterial genes from cell
to cell by transduction), phages can transfer DNA from one cell to another.
For example, lysogenic bacteriophages can carry genes contributing to pathogenesis,
such as the genes encoding the toxins of some pathogenic E. coli and V.
cholera (4211; 4212). Bacteriophages are released from
the cell as a particle with a protective protein coat around the enclosed DNA, free
to float through the environment until they find and infect a new cell. The transfer
of DNA by bacteriophages has had profound effects on the evolution of bacteria.
It has been suggested, for instance, that bacteriophage may be the major source
of many of the genes found in bacteria (4227).
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Last Revised on September 16, 2004
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- 4210 Polissi, A., Goffin, L., and Georgopoulos,
C. (1995). The Escherichia coli heat shock response and bacteriophage lambda
development. FEMS Microbiol Rev 17, 159-169. PubMed
- 4227 Ochman, H., Lawrence, J. G., and Groisman,
E. A. (2000). Lateral gene transfer and the nature of bacterial innovation.
Nature 405, 299-304. PubMed Journal
- 4229 J. H. Miller (1996). Discovering Molecular
Genetics (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).
- 4230 Cairns, J., Stent, G. S., Watson, J. D.,
(1992). Phage and the Origins of Molecular Biology, Expanded Edition (Cold
Spring Harbor, NY: Cold Spring Harbor Laboratory).
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- 4211 Waldor, M. K. and Mekalanos, J. J. (1996).
Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272,
1910-1914. PubMed
- 4212 O'Brien, A. D., Newland, J. W., Miller, S.
F., Holmes, R. K., Smith, H. W., and Formal, S. B. (1984). Shiga-like toxin-converting
phages from Escherichia coli strains that cause hemorrhagic colitis or infantile
diarrhea. Science 226, 694-696. PubMed
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©Jones and Bartlett Publishers (2007)
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