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Chapter 18
The Genetics of Viruses
and Bacteria
Biology, Seventh Edition
Neil Campbell and Jane Reece
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Figure 18.1 T4 bacteriophage infecting an E. coli cell
0.5 m
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Figure 18.2 Comparing the size of a virus,
a bacterium, and an animal cell
Virus
Bacterium
Animal
cell
Animal cell nucleus
0.25 m
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THE GENETICS OF VIRUSES
• How viruses were discovered?
• The story begins in 1883 with Adolf Mayer from
Germany who was studying the cause of tobacco
mosaic disease.
• Mayer discovered that the disease was contagious
• He tried to see any thing in the contagious sap
extracted form infected plants but could not seen any
thing.
• He concluded that the disease is caused by an
unusually small organism
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THE GENETICS OF VIRUSES
• Later in 1897 Martinus Beijerinck discovered that the
infectious agent can reproduce but only in the agent that
infects but NOT in the nutrient media unlike bacteria. In
addition the agent was not inactivated by alcohol which
inactivate bacteria.
• 1n 1935 the American Scientist Wendell Stanley
crystallized the infectious particle that is now know as
tobacco mosaic virus (TMV).
• Later with the aid of the electron microscopy, the virus was
seen.
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• So what is the virus?
• A virus is a genome enclosed in a protective coat
• The tiniest virus is only 20 nm in diameter that is smaller
than a ribosome.
• The largest virus can barely be resolved by light
microscope.
• Viruses are infectious particles consisting of nucleic acids
enclosed in a protein coat and some times a membranous
envelope.
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Figure 18.3 Infection by tobacco mosaic virus (TMV)
Normal
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Infected
Structure of Viruses
• Viruses are called DNA or RNA viruses based
on their genetic material which could consist of;
– double stranded DNA
– Single stranded DNA
– Double stranded RNA
– Single stranded RNA
• Smallest viruses have only 4 genes while the
largest have several hundred genes.
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Capsids and envelopes
• The protein shell that encloses the viral genome is called
the capsid which is made large number of protein subunits
called capsomeres.
• Tobacco mosaic virus has a structure that contains over a
thousand molecules of a single type of protein (helical)
• Adeno virus that causes respiratory infection in animals
made of 252 identical protein molecules (polyhydral).
• Influenza virus has a viral envelopes contain proteins,
glycoproteins and phospholipids
• The most complex capsids are found among viruses that
infect bacteria (bacteriophages). There are 7 types of
bacteriophages that infect E. coli called T1 –T7
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Figure 18.4 Viral structure
Capsomere
of capsid
RNA
Capsomere
Membranous
envelope
DNA
Head
Capsid Tail
sheath
RNA
DNA
Tail
fiber
Glycoprotein
18  250 mm
20 nm
(a) Tobacco mosaic virus
Glycoprotein
70–90 nm (diameter)
80–200 nm (diameter)
50 nm
50 nm
(b) Adenoviruses
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(c) Influenza viruses
80  225 nm
50 nm
(d) Bacteriophage T4
Viruses can reproduce only within a host cell
• Viruses are obligate intracellular parasites that is they reproduce
only within a host cell.
• Viruses have no enzymes for metabolism and have no ribosomes
or other equipement for making their own proteins.
• Each virus can infect only a limited range of hosts called the host
range.
• Viruses identify their hosts by a lock and key mechanism. However
some viruses have wider range than others such as swine flue virus
can infect both humans and hogs while rabies virus can infect a
number of mammalian species including raccoons, skunks, dogs
and humans.
• Viruses of eukaryotes are usually tissue specific such as human
cold virus that infects upper respiratory tract or AIDS virus that
attaches to CD4 cells of the immune system.
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How does a viral infection occur (Figure 18-5)
• A viral infection begins when a virus genome finds its way to a host
cell by the specific mechanism of injection used by the virus.
• Once inside, the viral genome can commandeer its host, reprogram
the cell to copy the viral nucleic acid and manufacture viral proteins
• Most viruses use DNA polymerase of the host cell to synthesize
new genomes along the template provided by viral DNA.
• With regard to RNA viruses they use special virus-encode
polymerase and use RNA as template.
• The host provide all the resources for nucleic acid synthesis such
as nucleotides (N), enzymes, ribosomes, tRNAs, amino acids, ATP
and other components needed for making proteins as dictated by
the viral genes.
• After the production of capsid proteins and the replication of viral
DNA their assembly of new viruses is spontaneous.
• The cycle completes after that hundreds or thousands emerging
from the infected cell causing the death of the cell and infecting
hundreds or thousands of other cells.
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Figure 18.5 A simplified viral reproductive cycle
Entry into cell and
uncoating of DNA
DNA
VIRUS
Capsid
Transcription
Replication
HOST CELL
Viral DNA
mRNA
Viral DNA
Capsid
proteins
Self-assembly of new
virus particles and
their exit from cell
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Figure 18.6 The lytic cycle of phage T4, a virulent phage
1 Attachment. The T4 phage uses
its tail fibers to bind to specific
receptor sites on the outer
surface of an E. coli cell.
5 Release. The phage directs production
of an enzyme that damages the bacterial
cell wall, allowing fluid to enter. The cell
swells and finally bursts, releasing 100
to 200 phage particles.
2 Entry of phage DNA
and degradation of host DNA.
The sheath of the tail contracts,
injecting the phage DNA into
the cell and leaving an empty
capsid outside. The cell’s
DNA is hydrolyzed.
Phage assembly
4 Assembly. Three separate sets of proteins
self-assemble to form phage heads, tails,
and tail fibers. The phage genome is
packaged inside the capsid as the head forms.
Head
Tails
Tail fibers
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3 Synthesis of viral genomes
and proteins. The phage DNA
directs production of phage
proteins and copies of the phage
genome by host enzymes, using
components within the cell.
Figure 18.7 The lytic and lysogenic cycles of phage
, a temperate phage
Phage
DNA
The phage attaches to a
host cell and injects its DNA.
Phage DNA
circularizes
Phage
Bacterial
chromosome
Lytic cycle
The cell lyses, releasing phages.
Occasionally, a prophage
exits the bacterial chromosome,
initiating a lytic cycle.
Lysogenic cycle
Certain factors
determine whether
Lytic cycle
Lysogenic cycle Prophage
or
is induced
is entered
New phage DNA and
proteins are synthesized
and assembled into phages.
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Many cell divisions
produce a large
population of bacteria
infected with the
prophage.
The bacterium reproduces
normally, copying the prophage
and transmitting it to daughter cells.
Phage DNA integrates into
the bacterial chromosome,
becoming a prophage.
With this mechanism how come that bacteriophage
have not exterminated all the bacteria ?
• Bacteria are not defense less.
• Natural selection favors bacterial mutants with receptor
sites that are no longer receptive for a particular
bacteriophage.
• When the virus inters several enzymes might break it
down, such enzymes are called restriction
endonucleasis.
• Bacterial DNA is chemically modified so that it can not be
destroyed by these restriction enzymes.
• Some phages can live inside the cell without lysing it
instead coexist in what is called lysogenic cycle.
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The Lysogenic cycle
• Unlike the lytic cycle that kills the cell, lysogenic cycle replicates the
phage genome without destroying the host.
• The phages that are capable of using both modes are called
temperate phages.
• An example of a temperate phage is called lamba (λ) and Figure
18-5 shows the Lysogenic and lytic reproductive cycles of phage λ
• During the Lysogenic cycle the viral genome behaves differently,
the λ DNA molecule is incorporated (by genetic recombination
called crossing over) into a specific site on the host cell’s
chromosome which is then known as prophage.
• One prophage gene codes for a protein that represses most of the
other prophage genes.
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The Lysogenic cycle…..cont.
• Now each time the bacteria divides it replicates
the phage DNA and passes that to the progeny
• Now once the phage genome is free in the cell,
and due to some environmental triggers such as
radiation, the cycle might go through the lytic path
instead of the Lysogenic
• The expression of certain prophage genes during
a Lysogenic cycle may alter the phenotype of
some bacteria.
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The Lysogenic cycle….cont.
• Example; bacteria that cause diphtheria, botulism
and scarlet fever become harmful due to
induction of certain prophage genome in the
bacteria to produce their toxins.
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Table 18.1 Classes of Animal Viruses
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Reproductive cycles of animal viruses
• Viral envelopes
• An animal virus equipped with an outer membrane or viral envelope
will use it to inter the host cell.
• The membrane is generally a lipid bylayer with glycoproteins
protruding from the outer surface
• These glycoprotein spikes bind to specific receptors on the surface
of the host cell
• Viral envelope then fuses with host’s plasma membrane
transporting the capsid and viral genome into the cell
• Cellular enzymes remove capsid
• Viral genome replicates and direct the synthesis of viral proteins by
the ER for new viral envelope
• The new virus buds from the cell like exocytosis wrapping it self in a
membrane and have the glycoprotein spikes on the surface.
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• Some viruses like herpes viruses have
envelopes that are derived from the nuclear
membrane of the host.
• Its genome is double stranded DNA which may
become integrated into the host genome as
provirus similar to the prophage.
• Once acquired this type of virus might stay in the
host for life as it will be in the nucleus however, will
cause and infection once the immune system is
weakened.
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RNA as Viral Genetic Material
• The broadest variety of RNA genomes is found among the
viruses that infects animals. There are three types of single
stranded RNAa genomes.
• Here the virus genome serves as a template for mRNA
synthesis.
• RNA viruses with most complicated reproductive cycles are
the retroviruses (backward) which refers to the reverse
direction in which genetic information flows for these
viruses.
• This group of viruses are equipped with an enzyme called
reverse transcriptase which transcribes DNA from RNA
template thus providing an RNA → DNA information flow.
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• The newly made DNA then integrates as a provirus into a
chromosome within the nucleus of the animal cell.
• The host RNA polymerase transcribes the viral DNA into
RNA molecules which can function both as mRNA for
protein synthesis and as a genome for the new virus
particles released from the cells.
• Example of this type of viruses is the HIV ( human
immunodeficiency virus) the virus that causes AIDS.
Figure 18-10 shows the structure of HIV and the
reproductive cycle of this virus.
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Figure 18.8 The reproductive cycle of an enveloped RNA virus
1 Glycoproteins on the viral envelope
bind to specific receptor molecules
(not shown) on the host cell,
promoting viral entry into the cell.
Capsid
RNA
Envelope (with
glycoproteins)
Capsid and viral genome
enter cell
2
HOST CELL
The viral genome (red)
functions as a template for
synthesis of complementary
RNA strands (pink) by a viral
enzyme.
3
Viral genome (RNA)
Template
5
Complementary RNA
strands also function as mRNA,
which is translated into both
capsid proteins (in the cytosol)
and glycoproteins for the viral
envelope (in the ER).
mRNA
Capsid
proteins
ER
Glycoproteins
New copies of viral
genome RNA are made
using complementary RNA
strands as templates.
4
Copy of
genome (RNA)
6
Vesicles transport
envelope glycoproteins to
the plasma membrane.
8
7
A capsid assembles
around each viral
genome molecule.
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New virus
Figure 18.9 The structure of HIV, the retrovirus that causes AIDS
Glycoprotein
Viral envelope
Capsid
Reverse
transcriptase
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RNA
(two identical
strands)
Figure 18.10 The reproductive cycle of HIV, a retrovirus
HIV
Membrane of
white blood cell
1 The virus fuses with the
cell’s plasma membrane.
The capsid proteins are
removed, releasing the
viral proteins and RNA.
2 Reverse transcriptase
catalyzes the synthesis of a
DNA strand complementary
to the viral RNA.
HOST CELL
3 Reverse transcriptase
catalyzes the synthesis of
a second DNA strand
complementary to the first.
Reverse
transcriptase
Viral RNA
0.25 µm
HIV entering a cell
RNA-DNA
hybrid
The double4
stranded
DNA is
incorporated
as a provirus into
the cell’s DNA.
DNA
NUCLEUS
Chromosomal
DNA
RNA genome
for the next
viral generation
New viruses
9
bud
off from the
New HIV leaving a cell
host cell.
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Provirus
mRNA
Capsids are
8
assembled around
viral genomes and
reverse transcriptase
molecules.
Proviral genes are
transcribed into RNA
5
molecules, which serve
as genomes for the
next viral generation
and as mRNAs for
translation into viral
proteins.
The viral proteins
include
capsid proteins and
6
reverse transcriptase (made
in the cytosol) and envelope
glycoproteins (made in the
ER).
Vesicles transport the
7
glycoproteins from the ER
to the cell’s plasma
membrane.
Causes and Prevention of Viral Diseases in Animals
• Viruses might cause the disease and its symptoms by
various mechanisms such as;
• Killing cells by release of hydrolytic enzymes
• Some viruses induce infected cells to produce toxins
that kills the cell itself.
• Some envelope proteins of some viruses are toxic and
cause the destruction.
• Now the extent of damage depends on the speed of
the regeneration of the infected tissue. Example we
completely recover from cold because the epithelial
tissue regenerated completely, while with polio virus,
the damage is permanent as the nerve tissue do not
regenerate at all.
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Emerging Viruses
• HIV, the AIDS virus seemed to make a sudden
appearance in 1980.
• In 1993 a dozen of people in southwestern USA died
from Hantavirus.
• 1976 the deadly virus Ebola horrified the people of
Africa.
• Nipah virus that appeared in 1999 in Malaysia and
killed 105 people and destroyed the pig industry.
• SARS virus that appeared in 2003 and killed several
hundred people in Hong Kong, China and elsewhere
in the world. It is source still unknown as of spring
2004.
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Figure 18.11 SARS (severe acute respiratory
syndrome), a recently emerging viral disease
(a) Young ballet students in Hong Kong
wear face masks to protect themselves
from the virus causing SARS.
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(b) The SARS-causing agent is a coronavirus
like this one (colorized TEM), so named for the
“corona” of glycoprotein spikes protruding from
the envelope.
The question is from where do these and other emerging viruses arise?
• Three processes contribute to the emerging viral
diseases;
• Mutation of existing viruses especially RNA viruses (high
rate mutations ) as the replication of their nucleic acids
does not have a proof reading mechanism.
• Spread of existing viruses from animals to humans or
form one host species to another. Example the spread
of hantavirus from dust contaminated with urine or feces
of infected rodents.
• The dissemination of a viral disease from a small
isolated population to the public as is the case in AIDS.
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Plant viruses are serious agricultural pests
• Plant viruses can stunt plant growth (dwarf the plant
growth) and diminishes crop yield.
• Most plant viruses discovered so far are RNA viruses
• Routes that plant viruses can spread;
– Horizontal transmission; a plant is infected from an
external source of virus due to a breakage in the tree
and the spread to other adjacent trees by wind or
insects.
– Vertical transmission; in this type a plant inherits a
disease form its parents.
• Is their any cure for these viruses? Not yet.
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Figure 18.12 Viral infection of plants
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Viroids and prions are infectious agents even simpler than viruses.
• Viroids are tinny molecules of naked circular RNA
that infect plants.
• Only several hundred nucleotide long, they do not
encode proteins but can replicate in the host cells.
Some how these tinny creatures can disrupt the
metabolism of plant cells and stunt the growth of
the whole plant.
• One viroid disease has killed 10 million coconut
palms in Philipines.
• Viroids are nucleic acids whose replication
mechanism is well known. But what about those
infectious proteins called prions?
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Prions
• Prions appears to cause a number of
degenerative brain diseases including but not
limited to;
– Scrapie in sheep
– Mad cow disease in cows
– Creutzfeldt-Jakob disease in humans
• How can a protein which cannot replicate itself
be a transmissible pathogen?
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• According to the leading hypothesis, a prion is a
misfolded from of protein normally found in the
brain.
• When a prion gets into a cell containing the
normal form, the prion coverts the normal one into
a misfolded protein. In this way the prions trigger a
chain reaction that increases their numbers.
(Figure 18-13).
• In 1997 Stanly Prusiner form Caltech in CA was
awarded Nobel Prize in Medicine for his work on
elucidating the mechanism of the disease.
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Figure 18.13 Model for how prions propagate
Prion
Original
prion
Many prions
Normal
protein
New
prion
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THE GENETICS OF BACTERIA
The short generation span of bacteria helps them adapt
to changing environments.
• The major component of the bacterial genome is one
double stranded, circular DNA molecule.
• In E. coli the chromosomal DNA consists of 4.6 million
nucleotides representing about 4300 genes.
• This is more than 100 times the DNA found in a typical
virus.
• If stretched out, it would be 500 times longer than the
cell itself, however, it is packed so tightly that it only
fills part of the cell.
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THE GENETICS OF BACTERIA …Cont.
• This dense region of DNA is called the nucleoid
which is not bound by membrane like eukaryotic
cells.
• In addition to the chromosome, many bacteria has
plasmids containing a small number of genes.
• Bacterial cells divide by binary fission proceeded
by chromosomal replication (Figure 18-14).
• Bacteria can proliferate very rapidly in a favorable
environment, e.g E. coli can divide every 20
minutes so that a culture started with a single cell
can reach 107 – 108 in just 12 hours.
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THE GENETICS OF BACTERIA ….Cont.
• In the human body E. coli reproduce so rapidly to
replace the 2 x 1010 bacteria lost every day in feces.
• Because the reproduction by binary fission is
asexual, the offspring are all identical to the parent
cell. Only due to mutations, some of the offspring
can differ slightly from the parents.
• For a given E. coli, the probability of a spontaneous
mutation is about 1x 10-7 per cell division i.e only 1
in 10 million. So in the 2x1010 that are replaced
every day their must be around 2000 bacteria that
have mutation in a gene.
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THE GENETICS OF BACTERIA ….Cont.
• With 4300 gene on average, multiply by 2000 = 9
million mutations per day per human host.
• This huge number of mutations predispose to the
vast genetic diversity observed in the bacterial
populations which is due to the short life span of
bacteria.
• In contrast, in humans mutations does not
contribute to the genetic diversity as the life span of
humans are much longer, instead the diversity in
humans is attributed to the sexual recombination of
existing alleles
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Figure 18.14 Replication of a bacterial chromosome
Replication
fork
Origin of
replication
Termination
of replication
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Genetic recombination produces new bacterial strains
• What is recombination?
• It is simply combining DNA from two individuals into
the genome of a single individual.
• How can we detect genetic recombination in bacteria?
• Consider two mutants of E. coli, one can not
synthesize arginine while the other can not
synthesize tryptophan. Due to the mutations they
can not reproduce on minimal media (glucose and
salt) so if we grow them separately on the minimal
media, no colonies will grow (Figure 18-15) while if we
mix them together some colonies will appear.
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How does that happen?
• By natural genetic recombination, i.e bacteria
acquired genes that are missing from the other
bacteria and thus were capable of producing
either arginine or tryptophan.
• In eukaryotic cells the sexual process of meiosis
and fertilization combines the DNA from two
individuals in a single zygote.
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Figure 18.15 Can a bacterial cell acquire genes
from another bacterial cell?
EXPERIMENT Researchers had two mutant strains, one that could make arginine but not tryptophan
(arg+ trp–) and one that could make tryptophan but not arginine (arg– trp+). Each mutant strain and a
mixture of both strains were grown in a liquid medium containing all the required amino acids. Samples
from each liquid culture were spread on plates containing a solution of glucose and inorganic salts (minimal
medium), solidified with agar.
Mixture
Mutant
strain
arg+ trp–
Mutant
strain
arg trp+
RESULTS Only the samples from the mixed culture, contained cells that gave rise to colonies on
minimal medium, which lacks amino acids.
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Mixture
Mutant
strain
arg+ trp–
Mutant
strain
arg– trp+
No
colonies
(control)
Colonies
grew
CONCLUSION
No
colonies
(control)
Because only cells that can make both arginine and tryptophan
cells) can grow into colonies on minimal medium, the lack of colonies on
the two control plates showed that no further mutations had occurred restoring this
ability to cells of the mutant strains. Thus, each cell from the mixture that formed a
colony on the minimal medium must have acquired one or more genes from a cell
of the other strain by genetic recombination.
(arg+
trp+
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Mechanisms of Gene Transfer and Genetic
Recombination in Bacteria
• Transformation
• Is the alteration of a bacterial cell’s genotype by the
uptake of naked foreign DNA from the surrounding
environment.
• Such process happened when a harmless bacterium takes
DNA (a pathogenic allele) from harmful one and the latter
become harmful by replacing one of its non-pathogenic
alleles with the newly acquired pathogenic one. A process
occurs by crossing over.
• Some bacteria posses proteins on their surface specialized
in taking naked DNA inside. In addition,high Ca+ stimulate
uptake of DNA into cells.
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Transduction
• In the DNA transfer process known as transduction,
phages carry bacterial genes from one host cell to
another. There are two forms of transduction;
• Generalized transduction, i.e random (Figure 1816). This part occurs when the phage infects a cell
which replicates the phage’s DNA, this DNA is
packaged within capsids to infect other cells.
• Occasionally, some of the host DNA is packaged in
the capsid, such a virus will be defected because it
lacks its own genetic material.
• This defected phage can infect other cells where some
of its DNA will combine with the hosts DNA to produce
a combination of DNA produced form two cells.
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• Specialized transduction; ( i.e only certain genes
located near the prophage).
• This type of transduction requires infection of a
temperate pahge. In the lysogenic cycle, the
genome of the temperate phage integrates as a
prophge in the host genome.
• Now when the phage genome is excised from the
chromosome, it sometimes take with it a small part
of the host DNA adjacent to the prophage. When
the phage infects other cells it introduces the
bacterial DNA along with the viral one.
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Figure 18.16 Generalized transduction
Phage DNA
1Phage
2
infects bacterial cell that has alleles A+ and B+
Host DNA (brown) is fragmented, and phage DNA
and proteins are made. This is the donor cell.
A+ B+
A+ B+
Donor
cell
3
A bacterial DNA fragment (in this case a fragment with
the A+ allele) may be packaged in a phage capsid.
A+
4
Phage with the A+ allele from the donor cell infects
a recipient A–B– cell, and crossing over (recombination)
between donor DNA (brown) and recipient DNA
(green) occurs at two places (dotted lines).
Crossing
over
A+
A– B–
Recipient
cell
5
The genotype of the resulting recombinant cell (A+B–)
differs from the genotypes of both the donor (A+B+) and
the recipient (A–B–).
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A+ B–
Recombinant cell
Conjugation and Plasmids
• Conjugation is the direct transfer of genetic
material between two bacterial cells that are
temporarily joined.
• In this process one cell (male) donates the DNA
and the other cell (female) receiving the DNA
(Figure 18-17).
• The sex pili hooks the female while a cytoplamsic
bridge forms to facilitate the DNA transfer. This
process results from the presence of a fertlitity
factor or F factor which can exist as a piece of
DNA or as a plasmid.
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Figure 18.17 Bacterial conjugation
Sex pilus
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1 m
Plasmids
• A plasmid is a small, circular, self-replicating DNA molecule
(not the chromosome). A genetic element that can exist as a
plasmid or as part of a chromosome is called episome.
• Temperate viruses such as phage λ qualify as episomes.
Because the genome of these viruses replicate
independently during lytic cycle and as a part of bacterial
chromosome during lysogenic cycle.
• Plasmids unlike viruses lack protein coats and do not
normally exist outside the cell. However, plasmids have
small number of genes which are NOT required for survival.
They help bacteria survive a stressful environment.
• Example, F plasmids (conjugation), antibiotic resistance
plasmids, heat or cold shock resistance protein-plasmids
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The F plasmid and conjugation
• Cells that contain F plasmid are denoted F+
(male) which is a heritable trait. This factor
replicates in synchrony with chromosomal DNA.
• Cells lack the F factor are called F- (females) and
thus are recipients of the DNA.
• How does conjugation occurs? Figure 18-18
summarizes these types;
• An F+ cell converts an F- cell to become F+ cell is
one type of conjugation (18-18a).
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• When the donor cell’s F factor integrated into the
chromosome the cell is called Hfr cell (high frequency
of recombination) (Figure 18-18b).
• Hfr cell acts like F+ cell, it initiates DNA replication at a
point on the F factor and starts to transfer the DNA
copy to its F- partner ( Figure 18-18b).
• If part of the newly acquired DNA ( as shown in the
last part of 18-18b), aligns with the homologous region
of the F- chromosome, segments of DNA can be
exchanged ( Figure 18-18b).
• Binary fission of this cell give rise to progeny with DNA
from two different cells.
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Figure 18.18 Conjugation and recombination in E. coli (layer 4)
F Plasmid
Bacterial chromosome
F+ cell
F+ cell
Mating
bridge
1
F+ cell
Bacterial
chromosome
F– cell
A cell carrying an F plasmid
(an F+ cell) can form a
mating bridge with an F– cell
and transfer its F plasmid.
2
3
A single strand of the F
plasmid breaks at a
specific point (tip of blue
arrowhead) and begins to
move into the recipient cell.
As transfer continues, the
donor plasmid rotates
(red arrow).
DNA replication occurs in 4
both donor and recipient
cells, using the single
parental strands of the
F plasmid as templates
to synthesize complementary
strands.
Hfr cell
A+
F factor
The circular F plasmid in an F+ cell
can be integrated into the circular
chromosome by a single crossover
event (dotted line).
B+
C+
(a) Conjugation and transfer of an
F plasmid from an F+ donor to
an F– recipient
Hfr cell
F+ cell
1
The plasmid in the
recipient cell
circularizes. Transfer
and replication result
in a compete F plasmid
in each cell. Thus, both
cells are now F+.
D+
A+
C+
B+
D+
2
The resulting cell is called an Hfr cell
(for High frequency of recombination).
D+ C+
B+
A+
D+ C+
B+
B+
A+
B–
A+
A+
F– cell
3
B–
B+
C– –
D
A–
B–
Since an Hfr cell has all 4
the F-factor genes, it can
form a mating bridge with
an F– cell and transfer DNA.
A single strand of the F factor 5
breaks and begins to move
through the bridge. DNA
replication occurs in both donor
and recipient cells, resulting in
double-stranded DNA
Temporary
partial
diploid
7
C– –
D
A–
B+
A+
B–
C– –
D
A–
Two crossovers can result
in the exchange of similar
(homologous) genes between
the transferred chromosome fragment
(brown) and the recipient cell’s
chromosome (green).
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A+
–
B– C D–
A–
The location and orientation
6
of the F factor in the donor
chromosome determine
the sequence of gene transfer
during conjugation. In this
example, the transfer sequence
for four genes is A-B-C-D.
B–
A+
B+
C– –
D
A–
C–
A–
D–
The mating bridge
usually breaks well
before the entire
chromosome and
the rest of the
F factor are transferred.
Recombinant F–
bacterium
8 The piece of DNA ending up outside the
bacterial chromosome will eventually be
degraded by the cell’s enzymes. The recipient
cell now contains a new combination of genes
but no F factor; it is a recombinant F – cell.
(b) Conjugation and transfer of part
of the bacterial chromosome from
an Hfr donor to an F– recipient,
resulting in recombination
R plasmids and antibiotic resistance
• Antibiotic resistance was first noticed in 1950s in
Japan when they noticed that some shigella
strains does not respond to certain antibiotics they
used to respond to before.
• What causes this resistance was a specific
gene(s) such as genes encode for enzymes that
destroy the antibiotic. These resistance genes
where found to exist in plasmids therefore, were
called R plasmids.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• An exposure of certain bacteria to an antibiotic will kill all
the sensitive bacteria but the ones have R plasmids will
survive.
• Like the F factors which move from one cell to another, the
R plasmids are moving from one strain to another thus
conferring resistance to the recipient cell.
• Some plasmids carry as many as 10 resistance genes, but
how come a single plasmid will carry this number of
resistance genes? Transposons!
• There are two types of transposons;
– Insertion sequences; simplest transposon
– Composite Transposons; more complex ones
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Transposons; insertion sequences
• They consist only of the DNA necessary for the
transposition
• Contain only one gene for transposase that
catalyzes the movement of the transposon from one
place to the other.
• Transposase gene is bracketed by a pair of what is
called inverted repeats that make the boundaries of
the transposon (Figure 18-19).
• DNA polymerase participate in the transposition by
creating identical regions of DNA called the direct
repeats.
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• Insertion sequences account for about 1.5% of E.
coli genome and cause the intrinsic mutations.
• Mutations occurred by this method occurs rarely at
a rate of a bout 1/107 generations.
• Composite Transposons
• They contain extra genes such as antibiotic
resistance genes that are taken with the
transposon for the free ride (Figure 18-19) and
help the bacteria adapt to tough environments.
• As the case in insertion transposons, there is a
direct repeat and an inverted repeat.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Discovery of Transposons
• Transposons are not unique to bacteria; they are
important components in the eukaryotic genome.
• This was postulated long time ago by Barbra
McClintock in 1940-1950s when she concluded
that the changes in the color of corn kernel can be
explained only by transposable elements that
move from one part of the genome to the genes of
the kernel color.
• At age 81 (30 years after she discovered the
transposons) she was awarded Noble Prize for
her discovery. Unfortunately she did not spend the
money!! some one else may be did!!!!.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Figure 18.19 Transposable genetic elements in bacteria
Insertion sequence
3
A T C C G G T…
A C C G G A T…
3
5
TAG G C CA…
TG G C CTA…
5
Transposase gene
Inverted
Inverted
repeat
repeat
(a) Insertion sequences, the simplest transposable elements in bacteria, contain a single gene that
encodes transposase, which catalyzes movement within the genome. The inverted repeats are
backward, upside-down versions of each other; only a portion is shown. The inverted repeat
sequence varies from one type of insertion sequence to another.
Transposon
Insertion
sequence
Antibiotic
resistance gene
Insertion
sequence
5
5
3
3
Transposase gene
Inverted repeats
(b) Transposons contain one or more genes in addition to the transposase gene. In the transposon
shown here, a gene for resistance to an antibiotic is located between twin insertion sequences.
The gene for antibiotic resistance is carried along as part of the transposon when the transposon
is inserted at a new site in the genome.
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The control of gene expression
• Metabolic control occurs in two levels;
• Regulation of enzyme production; Cells regulate
the expression of genes thus stop producing the
synthesis of the enzyme (Figure 18-20a).
• Regulation of enzyme activity; Cells can adjust the
activity of many enzymes to chemical cues that
increase or decrease their catabolic activity. In
this case the bacteria produce enough product so
that its accumulation sends a message, Feedback
inhibition, to stop the first enzyme in the series
(Figure 18-20b).
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Figure 18.20 Regulation of a metabolic pathway
(a) Regulation of enzyme
activity
Precursor
Feedback
inhibition
Enzyme 1
Enzyme 2
Enzyme 3
(b) Regulation of enzyme
production
Gene 1
Gene 2
Regulation
of gene
expression
Gene 3
–
Enzyme 4
Gene 4
–
Enzyme 5
Tryptophan
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Gene 5
Operons: the basic concept
• Consider the production of tryptophan by E. coli. And
consider that it needs 5 enzymes that are all needed
at once when tryptophan is needed. The switch for
those genes is a segment of DNA adjacent to the
promoter called the operator.
• This operator controls the access of RNA polymerase
to the genes.
• All together; promoter, operator, the 5 genes
necessary for tryptophan synthesis is called an
operon (Figure 18-21).
• The operator is always on, so the RNA polymerase
can bind to the promoter and start the synthesis.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Figure 18.21 The trp operon: regulated synthesis of
repressible enzymes
trp operon
Promoter
DNA
Promoter
Genes of operon
trpD
trpC
trpE
trpR
trpB
trpA
Operator
Regulatory
gene
mRNA
5
3
RNA
polymerase
Start codon
Stop codon
mRNA 5
E
Protein
Inactive
repressor
D
C
B
A
Polypeptides that make up
enzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on. RNA polymerase attaches to the DNA
at the promoter and transcribes the operon’s genes.
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Switching the operon off
• Then what makes it switches of? The operon can
be switched off by a protein called the repressor.
• The repressor binds to the operator and blocks
attachment of RNA polymerase to the promoter
preventing transcription of the genes. The
repressors are specific for certain operons.
• What happened is that, once tryptophan
accumulates it works as a co-repressor by binding
to an allosteric site in the repressor protein,
causing it to change its conformation thus
activating it.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• The active form of this repressor switches of the
operon by binding to the operator (reversibly) and
blocking access of RNA polymerase to the
promoter. This process is explained fully in Figure
18-22.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Figure 21b; action of tryptophan repressor
DNA
No RNA made
mRNA
Protein
Active
repressor
Tryptophan
(corepressor)
(b) Tryptophan present, repressor active, operon off. As tryptophan
accumulates, it inhibits its own production by activating the repressor protein.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Repressible versus inducible operons; two
types of negative gene regulation
– The tryptophan operon is said to be
repressible as it is inhibited by the production
of high amount of tryptophan.
– The other type of operons, inducible operons
are said to be stimulated.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
lac operon is an Example
The disaccharide lactose (milk sugar) is available to
intestinal E. coli if human drinks milk where the
bacteria can utilize it for energy and as a carbon
source for synthesizing other compounds.
• There are three enzymes involved in the utilization
of lactose and its metabolism all found in one
operon called the lac operon Figure 18-22b.
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• This entire transcription unit is under the control of
one promoter and one operator with a regulatory
gene lacI, that is located outside the operon.
• The regulatory gene codes for a repressor in the
same way as for the try repressor. However, the
trp repressor was innately inactive while the lac
repressor is innately active.
• In this case a specific molecule called the
inducer, inactivates the repressor.
• For the lac repressor the inducer is allolactose
(isormer of lactose) that is made from lactose
when it is available. Figure 18-22b.
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Figure 18.22 The lac operon: regulated synthesis of
inducible enzymes
Promoter
Regulatory
gene
DNA
Operator
lacl
lacZ
No
RNA
made
3
mRNA
Protein
RNA
polymerase
5
Active
repressor
(a) Lactose absent, repressor active, operon off. The lac repressor is innately active, and in
the absence of lactose it switches off the operon by binding to the operator.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
lac operon
DNA
lacl
lacz
3
mRNA
5
(b)
lacA
RNA
polymerase
mRNA 5'
5
mRNA
-Galactosidase
Protein
Allolactose
(inducer)
lacY
Permease
Transacetylase
Inactive
repressor
Lactose present, repressor inactive, operon on. Allolactose, an isomer of lactose, derepresses
the operon by inactivating the repressor. In this way, the production of enzymes for lactose utilization
is induced.
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Comparison between repressible and inducible
enzymes
• Repressible enzymes; accumulation of trp, the
end product of the anabolic pathway represses the
trp operon thus blocking synthesis of all enzymes
necessary for the pathway.
• Inducible enzymes: their synthesis is induced by
chemical signal with the enzyme produced when
the nutrient is available. they function in catabolic
pathways which break nutrient down to simpler
molecules. i.e breaking lactose to simple sugars
• Both systems are examples of negative control
genes because the operons are switched off by
the active form of the repressor protein.
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Positive gene regulation
• For the enzymes that breakdown lactose to be
synthesized in appreciable amount, it is not enough
that lactose be present in the bacterial cell, the
glucose also has to be in short supply.
• How does E. coli cell sense the glucose concentration
and how does that relate to the genome?
• The mechanism depends on the interaction of an
allosteric regulatory protein with a small organic
molecule cAMP that accumulates when the glucose is
scarce. The regulatory protein is the cAMP receptor
protein (CAP, catabolic activator protein) and it is an
activator of transcription.
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How does the regulation happen?
• When glucose is scarce, cAMP accumulates, so it bind to the CAP
thus activating it so that it bind to a specific site at the upstream end
of the lac promoter ( Figure 18-23a).
• The attachment of CAP bends the DNA facilitating binding of RNA
polymerase to the promoter i.e start transcription of the lactose
metabolism enzymes.
• Because CAP stimulates the gene expression, it is therefore, called
a positive regulation.
• If glucose amounts increase, every thing will be reversed, however,
transcription of the lac operon proceeds only at a very low level due
to other mechanisms that are controlled by the lac repressor.
• Thus the lac operon is therefore under dual control mechanisms;
–
Negative control by the lac repressor
–
Positive by the CRP control
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Figure 18.23 Positive control of the lac operon by
catabolite activator protein (CAP)
Promoter
DNA
lacl
lacZ
CAP-binding site
Active
CAP
cAMP
Inactive
CAP
RNA
polymerase
can bind
and transcribe
Operator
Inactive lac
repressor
(a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized.
If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces
large amounts of mRNA for the lactose pathway.
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Promoter
DNA
lacl
lacZ
CAP-binding site
Operator
RNA
polymerase
can’t bind
Inactive
CAP
Inactive lac
repressor
(b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized.
When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription.
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The End
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings