Chapter 18

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Biology, Seventh EditionNeil Campbell and Jane Reece

Chapter 18Chapter 18

The Genetics of Viruses and Bacteria


Figure 18.1 T4 bacteriophage infecting an E. coli cell

0.5 m


Figure 18.2 Comparing the size of a virus, a bacterium, and an animal cell

0.25 m

Virus

Animalcell

Bacterium

Animal cell nucleus


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


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.


• 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.


Figure 18.3 Infection by tobacco mosaic virus (TMV)

Normal 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.


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


Figure 18.4 Viral structure

18 250 mm 70–90 nm (diameter) 80–200 nm (diameter) 80 225 nm

20 nm 50 nm 50 nm 50 nm

(a) Tobacco mosaic virus (b) Adenoviruses (c) Influenza viruses (d) Bacteriophage T4

RNA

RNACapsomereof capsid

DNACapsomere

Glycoprotein Glycoprotein

Membranousenvelope

CapsidDNA

Head

Tail fiber

Tail sheath


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.


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.


Figure 18.5 A simplified viral reproductive cycleVIRUS

Capsid proteins

mRNA

Viral DNA

HOST CELL

Viral DNA

Entry into cell anduncoating of DNA

Replication Transcription

DNA

Capsid

Self-assembly of new virus particles and their exit from cell


Figure 18.6 The lytic cycle of phage T4, a virulent phage

Attachment. The T4 phage usesits tail fibers to bind to specificreceptor sites on the outer surface of an E. coli cell.

Entry of phage DNA and degradation of host DNA.The sheath of the tail contracts,injecting the phage DNA intothe cell and leaving an emptycapsid outside. The cell’sDNA is hydrolyzed.

Synthesis of viral genomes and proteins. The phage DNAdirects production of phageproteins and copies of the phagegenome by host enzymes, usingcomponents within the cell.

Assembly. Three separate sets of proteinsself-assemble to form phage heads, tails,and tail fibers. The phage genome ispackaged inside the capsid as the head forms.

Release. The phage directs productionof an enzyme that damages the bacterialcell wall, allowing fluid to enter. The cellswells and finally bursts, releasing 100 to 200 phage particles.

12

4 3

5

Phage assembly

Head Tails Tail fibers


Figure 18.7 The lytic and lysogenic cycles of phage , a temperate phage

Many cell divisions produce a large population of bacteria infected with the prophage.

The bacterium reproducesnormally, copying the prophageand transmitting it to daughter cells.

Phage DNA integrates into the bacterial chromosome,becoming a prophage.

New phage DNA and proteins are synthesized and assembled into phages.

Occasionally, a prophage exits the bacterial chromosome, initiating a lytic cycle.

Certain factorsdetermine whether

The phage attaches to ahost cell and injects its DNA.

Phage DNAcircularizes

The cell lyses, releasing phages.Lytic cycleis induced

Lysogenic cycleis entered

Lysogenic cycleLytic cycle

or Prophage

Bacterialchromosome

Phage

PhageDNA


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.


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.


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.


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.


Table 18.1 Classes of Animal Viruses


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.


• 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.


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.


• 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.


Figure 18.8 The reproductive cycle of an enveloped RNA virus

Capsid and viral genomeenter cell2

The viral genome (red)functions as a template forsynthesis of complementary RNA strands (pink) by a viral enzyme.

3

New virus8

RNA

Capsid

Envelope (withglycoproteins)

Glycoproteins on the viral envelope bind to specific receptor molecules(not shown) on the host cell, promoting viral entry into the cell.

1

New copies of viralgenome RNA are madeusing complementary RNAstrands as templates.

4

Vesicles transportenvelope glycoproteins to

the plasma membrane.

6

A capsid assemblesaround each viral

genome molecule.

7

Complementary RNAstrands also function as mRNA,

which is translated into bothcapsid proteins (in the cytosol)and glycoproteins for the viral

envelope (in the ER).

5

HOST CELL

Viral genome (RNA)

Template

Capsidproteins

Glyco-proteins

mRNA

Copy ofgenome (RNA)

ER


Figure 18.9 The structure of HIV, the retrovirus that causes AIDS

Reversetranscriptase

Viral envelope

Capsid

Glycoprotein

RNA(two identicalstrands)


Figure 18.10 The reproductive cycle of HIV, a retrovirus

Vesicles transport theglycoproteins from the ER to the cell’s plasma membrane.

7

The viral proteins include capsid proteins and reverse transcriptase (made in the cytosol) and envelope

glycoproteins (made in the ER).

6

The double-stranded DNA is incorporatedas a provirus into the cell’s DNA.

4

Proviral genes are transcribed into RNA molecules, which serve as genomes for the next viral generation and as mRNAs for translation into viral proteins.

5

Reverse transcriptasecatalyzes the synthesis ofa second DNA strandcomplementary to the first.

3

Reverse transcriptasecatalyzes the synthesis of aDNA strand complementaryto the viral RNA.

2

New viruses bud off from the host cell.

9

Capsids areassembled aroundviral genomes and reverse transcriptase molecules.

8

mRNA

RNA genomefor the nextviral generation

Viral RNA

RNA-DNAhybrid

DNA

ChromosomalDNA

NUCLEUSProvirus

HOST CELL

Reverse transcriptase

New HIV leaving a cell

HIV entering a cell

0.25 µm

HIV Membrane of white blood cell

The virus fuses with thecell’s plasma membrane.The capsid proteins areremoved, releasing the viral proteins and RNA.

1


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.


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.


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.

(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.


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.


Figure 18.12 Viral infection of plants


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?


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?


• 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.


Figure 18.13 Model for how prions propagate

Prion

Normalprotein

Originalprion

Newprion

Many prions


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.


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.


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.


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


Figure 18.14 Replication of a bacterial chromosome

Replicationfork

Origin of replication

Termination of replication


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.


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.


Figure 18.15 Can a bacterial cell acquire genes from another bacterial cell?

Only the samples from the mixed culture, contained cells that gave rise to colonies on minimal medium, which lacks amino acids.

RESULTS

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.

Mutantstrain

arg+ trp–

Mutantstrain

arg trp+

Mixture


Mutantstrain

arg+ trp–

Mutantstrain

arg– trp+

No colonies(control)

No colonies(control)

Mixture

Coloniesgrew

Because only cells that can make both arginine and tryptophan (arg+ trp+ 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.

CONCLUSION


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.


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 18-16). 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.


• 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.


Figure 18.16 Generalized transduction

Phage DNA

Donorcell

Recipientcell

A+ B+

A+ B+

A+

A+ B–

A– B–

A+

Recombinant cell

Crossingover

Phage 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 bacterial DNA fragment (in this case a fragment withthe A+ allele) may be packaged in a phage capsid.

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).

The genotype of the resulting recombinant cell (A+B–) differs from the genotypes of both the donor (A+B+) and the recipient (A–B–).

1

2

3

4

5


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.


Figure 18.17 Bacterial conjugation

Sex pilus 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


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).


• 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.


Figure 18.18 Conjugation and recombination in E. coli (layer 4)

1 A cell carrying an F plasmid(an F+ cell) can form amating bridge with an F– celland transfer its F plasmid.

A single strand of the F plasmid breaks at a specific point (tip of blue arrowhead) and begins tomove into the recipient cell. As transfer continues, the donor plasmid rotates(red arrow).

2 DNA replication occurs inboth donor and recipientcells, using the single parental strands of the F plasmid as templates to synthesize complementary strands.

3 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+.

4

F Plasmid Bacterial chromosome

Bacterial chromosomeF– cell

F+ cell

F+ cell

F+ cell Hfr cell

F factorThe circular F plasmid in an F+ cellcan be integrated into the circularchromosome by a single crossoverevent (dotted line).

1The resulting cell is called an Hfr cell (for High frequency of recombination).

2

Since an Hfr cell has all the F-factor genes, it can form a mating bridge with an F– cell and transfer DNA.

3 A single strand of the F factorbreaks and begins to move through the bridge. DNA replication occurs in both donor and recipient cells, resulting in double-stranded DNA

4 The location and orientation 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.

5 The mating bridgeusually breaks well before the entire chromosome and the rest of the F factor are transferred.

6

Two crossovers can result in the exchange of similar (homologous) genes between the transferred chromosome fragment (brown) and the recipient cell’s chromosome (green).

7 The piece of DNA ending up outside thebacterial 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.

8

Temporarypartialdiploid

Recombinant F–

bacterium

Conjugation and transfer of an F plasmid from an F+ donor to an F– recipient

(a)

Conjugation and transfer of part of the bacterial chromosome from an Hfr donor to an F– recipient, resulting in recombination

(b)

A+B+ C+

D+

F– cell A–B–

C–

D–

A–B–

C–

D– D–

A–

C–B– D–

A–

C–

B–

A+

B+C+D+A+

B+C+D+A+B+

D+C+

A+

A+

B+

A–B–

C–

D–

A–B+

C–

D–

A+

B+ B–

A+

A+

B+

F+ cell

Mating bridge

Hfr cell


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.


• 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


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.


• 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.


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!!!!.


Figure 18.19 Transposable genetic elements in bacteria

(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.

(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.

Insertion sequence

Transposase geneInvertedrepeat

Invertedrepeat

Inverted repeats Transposase gene

Insertion sequence

Insertion sequence

Antibioticresistance gene

Transposon

3

5

3

5

5

3

5

3

A T C C G G T…

T A G G C C A …

A C C G G A T…

T G G C C T A …


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).


Figure 18.20 Regulation of a metabolic pathway

(a) Regulation of enzyme activity

Enzyme 1

Enzyme 2

Enzyme 3

Enzyme 4

Enzyme 5

Regulationof geneexpression

Feedbackinhibition

Tryptophan

Precursor

(b) Regulation of enzyme production

Gene 2

Gene 1

Gene 3

Gene 4

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.


Figure 18.21 The trp operon: regulated synthesis of repressible enzymes

(a) Tryptophan absent, repressor inactive, operon on. RNA polymerase attaches to the DNA at the promoter and transcribes the operon’s genes.

Genes of operon

Inactiverepressor

Protein

Operator

Polypeptides that make upenzymes for tryptophan synthesis

Promoter

Regulatorygene

RNA polymerase

Start codon Stop codon

Promoter

trp operon

5

3mRNA 5

trpDtrpE trpC trpB trpAtrpRDNA

mRNA

E D C B A


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.


• 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.


Figure 21b; action of tryptophan repressor

DNA

mRNA

Protein

Tryptophan(corepressor)

Active repressor

No RNA made

Tryptophan present, repressor active, operon off. As tryptophanaccumulates, it inhibits its own production by activating the repressor protein.

(b)


• 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.


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.


• 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.


Figure 18.22 The lac operon: regulated synthesis of inducible enzymes

DNA

mRNA

ProteinActiverepressor

RNApolymerase

NoRNAmade

lacZlacl

Regulatorygene

Operator

Promoter

Lactose absent, repressor active, operon off. The lac repressor is innately active, and inthe absence of lactose it switches off the operon by binding to the operator.

(a)

5

3


mRNA 5'

DNA

mRNA

Protein

Allolactose(inducer)

Inactiverepressor

lacl lacz lacY lacA

RNApolymerase

Permease Transacetylase-Galactosidase

5

3

(b) 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.

mRNA 5

lac operon


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.


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.


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


Figure 18.23 Positive control of the lac operon by catabolite activator protein (CAP)

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.

(a)

CAP-binding siteOperator

Promoter

RNApolymerasecan bindand transcribe

InactiveCAP

ActiveCAPcAMP

DNA

Inactive lacrepressor

lacl lacZ


(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.

Inactive lacrepressor

InactiveCAP

DNA

RNApolymerasecan’t bind

Operator

lacl lacZ

CAP-binding site

Promoter


The End

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Chapter 18

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