Biology 2

CHAPTER 17 Notes

MICROBIAL MODELS: THE GENETICS OF VIRUSES AND BACTERIA

Scientists discovered the role of DNA in heredity by studying the simplest of biological systems - viruses and bacteria. Most of the molecular principles discovered through microbe research applies to higher organisms, but viruses and bacteria also have unique genetic features.

· Knowledge of these unique genetic features has helped scientist understand how viruses and bacteria cause disease.

· Techniques for gene manipulation emerged from studying genetic peculiarities of microorganisms.

I. Researchers discovered viruses by studying a plant disease: science as a process

The discovery of viruses resulted from the search for the infection agent causing tobacco mosaic disease. This disease stunts the growth of tobacco plants and gives their leaves a mosaic coloration.

1883: A. Mayer, a scientist demonstrated that the disease was contagious and proposed that the infectious agent was an unusually small bacterium that could not be seen with a microscope.

· He successfully transmitted the disease by spraying sap from infected plants onto the healthy ones.

· Using a microscope, he examined the sap and was unable to identify a microbe.

1890's: D. Ivanowsky, a Russian scientist proposed that tobacco moszaic disease was caused by a bacterium that was either to small to be trapped by filter or that produced a filterable toxin.

· To remove bacteria, he filtered sap from infected leaves.

· Filtered sap still transmitted disease to healty plants.

1897: Martinus Beijerinck, a Dutch microbiologist proposed that the disease was caused by a reproducing particle much smaller and simpler than a bacterium.

· He ruled out the theory that a filtrable toxin caused the disease by demonstrating that the infectious agent filtered sap could reproduce.

Plants were sprayed with filtered sap from diseased plant.

Sprayed plants developed tobacco mosaic disease.

Sap from newly infected plants was used to infect others.

· This experiment was repeated for several generations. He concluded that the pathogen must be reproducing because its ability to infect was undiluted by transfers from plant to plant.

· He also noted that unlike bacteria, the pathogen:

* Reproduced only within the host it infected.

* Could not be cultured on media.

* Could not be killed by alcohol.

1935: Wendell M. Stanley, and American biologist, rystallized the infections particle now kow as tobacco mosaic virus (TMV).

II. Most viruses consist of a genome enclosed in a protein shell

In the 1950's, TMV and other viruses were finally observed with electron microscopes. Viral structure appeared to be unique from the simplest of cells.

· The smallest viruses are only 20 nm in diameter.

· The virus particle, or virion, is just nucleic acid enclosed by a protein coat.

A. Viral Genomes

Depending upon the virus, viral genomes:

· May be double-stranded DNA, single-stranded DNA, double-stranded RNA or single-stranded RNA.

· Are organized as single nucleic acid moleculed that are either lineat or circular.

· May have as few as four genes or as many as several hundred.

B. Capsids and Envelopes

Capsid = Protein coat that enclosed the viral genome.

· Its structure may be rod-shaped, polyhedral or complex.

· Composed of many capsomeres - protein subunits made from only one or a few types of protein.

Envelope = Membrane that cloaks some viral capsids.

· Helps viruses infect their host.

· Derived from host cell membrane which is usually virus-modified and contains proteins and glycoproteins of viral origin.

The most complex capsids are found among bacteriophages or bacterial viruses.

· Of the first phages studied, seven infected E. coli. these were named types 1-7 (T1, T2, T3...T7)

· The T-even phages- T2, T4 and T6 - are structurally very similar.

* The icosohedral head encloses the genetic material.

* The protein tailpiece with tail fibers attaches the pahge to its bacterial host and injects its DNA into the bacterium.

III. Viruses can only reproduce within a host cell

Viral reproduction differs markedly from cellular reproduction, because viruses are obligate intracellular parasites, which can express their genes and reproduce only within a living cell. Each virus has a specific host range.

Host range = Limited number or range of host cells that a parasite can infect.

· Viruses recognize host cells by a complementary fit between external viral proteins and specific cell surface receptor sites.

· Some viruses have broad host ranges which may include several species (e.g. swine flu and rabies).

· Some viruses have host ranges so narrow that they can:

* infect on;y one species (e.g. phages of E.coli)

* infect only one a single tissue type of one species (e.g. human cold virus infects only cells of the upper respiratory tract; AIDS virus binds only to specific receptors on certain white blood cells).

There are many patterns of viral life cycles, but they all generally include:

· Infecting the host cell with viral genome.

· Coopting host cell's resources to:

*replicte the viral genome.

* manufacture capsid protein.

· Assembling newly produced viral nucleic acid and capsomeres into the next generation of viruses.

There are several mechanisms used to infect with viral DNA.

· For example, T-even phages use an elaborate tailpiece to inject DNA into the host cell.

· Once the viral genome is inside its host cell, it commandeers the host's resourced and reprograms the cell to copy the viral genes and manufacture capsid protein.

There are three possible patterns of viral genome replication:

1. DNA - DNA. If viral DNA is double-stranded, DNA replication resembles that of cellular DNA, and the virus uses DNA polymerase produced by the host.

2. RNA- RNA. Since host cells lack the enzyme to copy RNA, most RNA viruses contain a gene that codes for RNA replicase, an enzyme that uses viral RNA as a template to produce complementary RNA.

3. RNA - DNA - RNA. Some RNA viruses encode reverse transcriptase, an enzyme that transcribes DNA from an RNA template.

Viral genomic RNA

reverse transcribes

Viral DNA

transcibes transcribes

messenger RNA genomic RNA

for new virions

Regardless of how viral genomes replicate, all viruses divert host cell resources for viral production.

· Viral genes use the host cell's enzymes, ribosomes, tRNAs, amino acids, ATP and other resources to make copies of the viral genome and produce viral capsid proteins.

· These viral components - nucleic acid and capsids - are assembled into hundreds or thousands of virions, which leave to parasitize new hosts.

Viral nucleic acid and capsid proteins assemble spontaneously into new virus particles, a process called self-assembly.

· Since most viral components are held together by weak bonds (e.g. hydrogen bonds and Van der Waals forces), enzymes are not usually necessary for assembly.

· For examples, TMV can be dissembled in the laboratory. When mixed together, the RNA and capsids spontaneously reassemble to form complete TMV virions.

IV. Phages exhibit two reproductive cycles, the lytic and lysogenic cycles

Bacteriophages are the best understood of all viruses, and many of the important discoveries in molecular biology have come from bacteriophage studies.

· In the 1940s, scientists determined how the T phages reproduce within a bacterium; this research.

* Demonstrated that DNA is the genetic material.

* Established the phage-bacterium system as an imortant experimental tool.

· Studies on lambda (l) phage of E. coli showed that double-stranded DNA viruses reproduce by two alternative mechanisms: the lytic cycle and the lysogenic cycle.

A. The Lytic Cycle

Virulent bacteriophages reproduce by a lytic replication cycle.

Virulent phages = Phages that lyse their host cells.

Lytic Cycle = A viral replication cycle that results in the death or lysis of the host cell.

The lytic cycle of phage T4 illustrates this type of replication cycle:

1.Phage attaches to cell surface.

· T4 recognizes a host cell by a complementary fir between proteins on the virion's tail fibers and specific receptor sites on the outer surface of an E. coli cell.

1.Phage contracts sheath and injects DNA.

· ATP stored in the phage tailpiece is the energy source for the phage to: a) pierce the E. coli wall and membrane, b) contract its tail sheath, and c) inject its DNA.

· The genome separates from the capsid leaving a capsid "ghost" outside the cell.

1.Hydrolytic enzymes destroy host cell's DNA.

· The E. coli host cell begins to transcribe and translate the viral genome.

· One of the first viral proteins produced is an enzyme that degrades host DNA. The phage's own DNA is protected, because it contains modified cytosine not recognized by the enzyme.

1.Phage genome directs the host cell to produce phage components: DNA and capsid proteins.

·Using nucleotides from its own degraded DNA, the host cell makes many copies of the phage genome.

·The host cell also produces three sets of capsid proteins and assembles them into phage tails, tail fibers and polyhedral heads.

·Phage components spontaneously assemble into virions.

2.Cell lyses and releases phage particles.

· Lysozymes specified by the viral genome digest the bacterial cell wall.

· Osmotic swelling lyses the cell which releases hundreds of phages from their host cell.

· Released virions can infect nearby cells.

· Lytic cycle takes only 20-30 minutes at 37°C. In that period, a T4 population can increase a hundredfold.

Bacteria have several defenses against destruction by phage infection.

· Bacterial mutations can change receptor sites used by phages for recognition, and thus avoid infection.

· Bacterial restriction enzymes recognize and cut up foreign DNA, including certain phage DNA. Bacterial DNA is chemically altered, so it is not destroyed by the cell's own restriction enzymes.

Restriction Enzymes = Naturally occurring bacterial enzymes that protect bacterua against intruding DNA from other organisms; catalyze restriction – the process of cutting foreign DNA into small segments.

Bacterial hosts and their viral parasites are continually coevolving.

· Most successful bacteria have effective mechanisms for preventing phage entry or reproduction.

· Most successful phages have evolved ways around bacterial defenses.

· Many phages check their own destructive tendencies and may coexist with their hosts.

B. The Lysogenic Cycle

Some viruses can coexist with their hosts by incorporating their genome into the host's genome.

Temperate viruses = Viruses that can integrate their genome into a host chromosome and remain latent until they initiate a lytic cycle.

· They have two possible modes of reproduction, the lytic cycle and the lysogenic cycle.

· An example is phage l, discovered by E. Lederberg in 1951.

Lysogenic cycle = A viral replication cycle that involves the incorporation of the viral genome into the host cell genome.

Details of the lysogenic cycle were discovered through studies of phage l life cycle:

1. Phage l binds to the surface of an E. coli cell.

2.Phage l injects its DNA into the bacterial host cell.

3.l DNA forms a circle and either begins a lytic cycle or a lysogenic cycle.

4.During a lysogenic cycle, l DNA inserts by genetic recombination into a specific site on the bacterial chromosome and becomes a prophage.

Prophage = A phage genome that is incorporated into a specific site on the bacterial chromosome.

· Most prophage genes are inactive.

· One active prophage gene codes for the production of repressor protein which switches off most other prophage genes.

· Prophage genes are copied along with cellular DNA when the host cell reproduces. As the cell divides, both prophage and cellular DNA are passes on the daughter cells.

· A prophage may be carried in the host cell's chromosomes for many generations.

Occasionally, a prophage may leave the bacterial chromosome.

· This may be spontaneous or caused by environmental factors (e.g. radiation).

· The excision process may begin the phage lytic reproductive cycle.

· Virions produced during the lytic cycle may begin either a lytic or lysogenic cycle in their new hosts cells.

Lysogenic cell = Host cell carrying a prophage in its chromosome.

· It is called lysogenic because it has the potential to lyse.

· Some prophage genes in a lysogenic cell may be expressed and change the cell's phenotype in a process called lysogenic conversion.

· Lysogenic conversion occurs in bacteria that cause diphtheria, botulism and scarlet fever. Pathogenicity results from toxins coded for by prophage genes.

V. Animal viruses are diverse in their modes of infection and mechanisms of replication.

A. Reproductive Cycles of animal Viruses

Replication cycles of animal viruses may show some interesting variations from those of other viruses. Two examples are the replication cycles of: 1) viruses with envelopes and 2) viruses with RNA genomes.

1.Viruses with Envelopes

Some animal viruses are surrounded by a memebaranous envelope, which is unique to several groups of animal viruses. This envelope is:

· Outside the capsid and helps the virus enter host cells.

· A lipid bilayer with g;ycoprotein spikes protuding from the outer surface.

Envelope viruses have replication cycles characterised by:

a.Attachment. Clycoprotein spikes protruding from the viral envelope attach to receptor sites on the host's plasma membrane.

b.Entry. As the envelope fuses with the plasma membrane, the entire virus (capsid and genome) is transported into the cytoplasm by receptor-mediated endocytosis

c.Uncoating. Cellular enzymes uncoat the genome by removing the protein capsid from viral RNA.

d.Viral RNA and Protein Synthesis. Viral enzymes are required to replicate the RNA genome and to transcribe mRNA.

· Some viral RNA polymerase is packaged in the virion.

· Viral mRNA polymerase (transcriptase) replicates the viral genome and transcribes viral mRNA. Note that the viral genome is a strand complementary to mRNA.

· Viral mRNA is translated into viral proteins including:

· Capsid proteins synthesized in the cytoplasm by free ribosomes.

· Viral-envelope glycoproteins synthesized by a ribosomes bound to rough ER. Glycoprotiens produced in the host's ER are sent to the Golgi apparatus for further processing. Golgi vesicles transport the glycoproteins to the plasma memebrane, where they cluster at exit sites for the virus.

a.Assembly and Release. New capsids surround viral genomes. Once assembled, the virions envelop with host plasma membrane as they bud off from the cell's surface. The viral envelope is derived from:

· Host cell's plasma membrane lipid.

· Virus-specific glycoprotein.

Some viral envelopes are not derived from host plasma membrane. For example herpesviruses are double-stranded DNA viruses which:

· Contain envelopes derived from the host cell's nuclear envelope rather than from the plasma membrane.

· Reproduce within the host cell's nucleus.

· Use both viral and cellular enzymes to replicate and transcribe their genomic DNA.

· May integrate their DNA into the cell's genome as provirus. Evidence comes from the nature of herpes infections, which tend to recur. After a period of latency, physical or emotional stress may cause the proviruses to begin a productive cycle again.

Provirus – Viral DNA that inserts into a host cell chromosome.

1.RNA Viruses

All possible types of viral genomes are represented among animal viruses. Since mRNA is common to all types, DNA and RNA viruses are classified according to the relationship of their mRNA to the genome. In this classification:

· MRNA or the strand that corresponds to mRNA is the plus(+) strand; it has the nucleotide sequence that codes for proteins.

· The minus (-) strand is a tempkate for synthesis of a plus strand; it is complementary to the sense strand of mRNA.

Animal RNA viruses are classified as following:

· Class III RNA viruses. Double-stranded RNA genome; the minus strand is the template for mRNA. (Reoviruses)

· Class IV RNA viruses. Single plus strand genome; the plus strand can function directly as mRNA, but also is a template for synthesis of minus RNA. ( Minus RNA is a template for synthesis of additional plus strands.) Viral enzymes are required for RNA synthesis form RNA templates. (Picornavirus, Togavirus)

· Class V RNA viruses. Single minus strand genome; mRNA is transcribed directly form this genomic RNA. (Rhabdovirus, Paramyxovirus, Orthomyxovirus)

· Class VI RNA viruses. Single plus strand genome; the plus strand is a template for complementary DNA synthesis. Reverse transcriptase catalyzes this reverse transcription from RNA to DNA. mRNA is then trnascribed from a DNA template. (retroviruses)

Retrovirus = (retro = backward) RNA virus that uses reverse transcriptase to transcribe DNA from viral RNA genome.

· Reverse transcriptase is a type of DNA polymerase that transcribes DNA from ab\n RNA template.

· HIV (human dimmunodeficiency virus), the virus that causes AIDS 9 \acquired immuni\odefiency syndrome) is a retrovirus.

RNA viruses with the most complicated reproductive cycles are the retroviruses, because retroviruses must first carry out reverse transcription:

Attachment and entry of the virion.

Enters host cell cytoplam.

Uncoating of single-stranded RNA genome.

Capsid proteins are enzymetically removed

Reverse transcription.

Viral RNA is the template to produce minus strand DNA - the

template for complementary DNA strand.

Integration.

Newly produced double-stranded viral DNA enters the nucleus.

Viral DNA insertsinto chromosomal DNA and becomes a

provirus.

Viral RNA and protein synthesis

Proviral DNA is transcribed into mRNA and is translated into

proteins. Transcribed RNA may provide genomes for next viral

generation.

Expression of provirus genes may:

Produce Cause expression of

new virons oncogenes, if present

Capsid assembly and Transformation of host

release of new virions cell into a cancerous state

B. important Viral Disease in Animals

It is often unclear how certain viruses cause disease symptoms. Viruses may:

· Damage or kill cells. In response to a viral infection, lysomes may release hydrolytic enzymes.

· Be toxic themselves or cause infected cells to produce toxins.

· Cause varying degrees of cell damage depending upon regenerative ability of the infected cell. We recover from colds because infected cells of the upper respiratory tract can regenerate by cell division. Poliovirus, however, causes permanent cell damage because the virus attacks nerve cells which cannot divide.

· Be inderectly responsible for disease symptoms. Fever, aches and inflammation may result from activities of the immune system.

Medial weapons used to fight viral infections include vaccines and antiviral drugs.

Vaccines = Harmless variants or derivatives of pathogenic microbes that mobilize a host's immune mechanism against the pathogen.

· Edward Jenner developed the first vaccine (against smallpoc) in 1796. According to the WHO, a vaccine has almsot completely eradicated smallpox.

· Effective vaccines now exist for polio, rubella, measles, mumps and many other viral diseases.

While vaccines can prevent some viral illnesses, little can be done to cure a viral disease once it occurs. Some antiviral drugs have recently been developed.

· Severl are analogs of purine mucleosides taht interfere with viral nucleic acid synthesis (e.g. adenine arabinoside and acyclovir).

C. Emerging Viruses

Emerging viruses are viruses that make an apparent sudden apperance. in reality, they are not likely to be new viruses, but rather existing ones that have expanded their host territory. Emerging viral disease can arise if an existing virus:

1. Evolves and thus causes disease in individuals who hace immunity onely to the ancestral virus (e.g. influenza virus).

2. Spreads from one host species to another.

· fore example, monkeypox virus spread from African to Asian monkeys in the laboratory (1950's); and, in Zaire (1970's), spread to humans from other mammals that harbored the virus.

3. Disseminates from a small population to become more widespread.

· For example, the 1993 hantavirus outbreak in New Mexico was the result of a populaion explosion in deer mice taht are the viral reservoirs. Humand became infected by inhaling airborne hantavirus that came from the excreta of deer mice.

· AIDS, once a rare disease, had become a global epidemic. Technological and social factors influenced the spread of AIDS virus.

4. Enviromental disturn\bances can increase the viral traffic responsible for emerging disease. For example:

· traffic on newly cut roads thrpough remote areas can spread viruses among previously isolated hu,an populations.

· Deforestaion activities brings humans into contact with animals that my host viruses capable of infecting humans.

D. Viruses and Cnacer

Some tumor viruses cause cance in animals.

· When animal cells grown in tissue culture are infected with tumor viruses, they transform to a cancerous state.

· Examples are members of the retrovirus, papovavirus, adenovirus and herpesvirus groups.

· Certain viruses are implicated in human cancers:

Tumor viruses transform cells by inserting viral nucleic aids into host cell DNA.

· This inserion is permanent as the provirus never excises.

· Insertion for DNA tumor viruses is straightforward.

Several viral genes have been identified ads oncogenes.

Oncogenes = Genes found in viruses or as part of the normal eukaruoti c genome, that trigger transformation of a cell to a cancerous state.

· Code for cellular growth factors or for proteins invloved in the function of growth factors.

· Are not unique to tumor viruses, but are found in the normal cells of many species. In fact, some tumor viruses transform cells by activating cellulat oncogenes.

More than one oncogene must usually be activated to completely transform a cell.

· Indications are that tumor viruses are effective only in combination with other events such as exposure to carcinogens.

· Carcinogens probably also act by turning on cellular oncogenes.

VI. Plant viruses are seroious agricultaral pests

As serious agricultural pests, many of the plant viruses:

· Stunt plant growth and diminish vrop yields.

· Are RNA viruses.

· Have rod-shaped capsids with capsomeres arranged in a spiral.

Capsomere = Complex capsid subunit consisting of several identical or differet protein molecules.

Plant viruses spread from plant to plant by two major routes: horizontal transmission and vertical transmission.

Horizontal transmission = Route of viral transmission in which an organism receives the virus from an external source.

· Plants are more susceptible to viral infection if their protective epidermal layer is damaged.

· Insects may be vectors that trasmit viruses from plant to plant and can ingect the virus directly into the cytoplasm.

· By using contaminated tools, gardeners and farmers may transmit plant viruses.

Vertical transmission = Route of viral transmission in which an organism inherits a viral infection from its parent.

· Can occur in asexual propagation of infected plants (e.g. by taking cuttings).

· Can occur in sexual reproduction via infected seeds.

Once a plant is infected, viruses reproduce and spread from cell to cell by passing through plasmadesmata.

Most plant viral diseases have no cure, so current efforts focus on reducing viral propagation and breeding resistant plant varieties.

VII. Viroids and prions are infectious agents even simpler than viruses

Another class of plant pathogens called viroids are smaller and simpler than viruses.

· They are small naked RNA molecules with only several hundred nucleotides.

· It is likely that viroids disrupt normal plant metabolism, development and growth by causing errors in regulatoru systems that control gene expression.

· Viroid disease affect many commercially important plants such as coconut palms, chrysanthemums, potaotes and tomatoes.

Some scientists believe that viroids originated as escaped introns.

· Nucleotide sequences of viroid RNA are similar to self-splicing introns found within some normal eukaryotic genes, including rRNA genes.

· An alternative hypothesis is that viroids and self-splicing introns share a common ancestral molecule.

As nucleic acids, viroids self-direct their replication and thus are not diluted during transmission from host to host. Molecules other than nucleic acids can be infectious agents even though they cannot self-replicate.

· For example, prions are pathogens that are proteins.

* Cause scrapie in sheep.

* May cause degenerative disease of the nervous system in humans.

· How can a protein which cannot replicate itself, be an infectious pathogen? According to one hypotehsis:

* Prions are defectice versions of normally occuring cellular proteins.

* When prions infect normal cells, they catalyze conversion of noraml protein to the prion

version.

* Prions could thus trigger chain reaction that increase their numbers and allow them to

spread through a host population without dilution.

VII. Viruses may have evolved from other mobile genetic elements

Viruses do not fit our usual definitions of livign organisms. They cannot reproduce independently, yet they:

· Have a genome with the same genetic code as living organisms.

· Can mutate and evolve.

Viruses probably evolved after the first cells, from fragments of cellular nucleic acid that were mobile genetic elements. Evidence to support this includes:

· Genetic material of different viral families is more similar ro host genomes than to that of other viral families.

· Some viral genes are identical to cellular genes (e.g. oncognes in retroviruses).

· Viruses of eukaryotes are more similar in genomic structure to their cellular hosts thatn to bacterial viruses.

· Viral genomes are similar to certain cellular genetic elements such as plasmids and transposons; they are all mobile genetic elements.

IX. The short generation span of bacteria facilitates their evolutionary adaptation to changing environments.

The average bacterial genome is larger than viral genome, but much smaller than a typical eukayotic genome.

· Through prokaryotes contain only about 1/1000 the DNA of eukaryotes, prokaryotic chromosomes still contain a large amount of DNA relaice to the small prokaryotic cell.

· Bacterial chromosomes, consequently, are highly folded and packed within the cell.

Most DNA in a bacterium is found in a single circular bacterial chromosome (genophore) that is:

· Composed of double-stranded DNA.

· Structurally simpler and has fewer associated proteins than a eukaryotic chromosome.

· Structurally sumpler and has fewer associated proteins than a eukaryotic chromosome.

· Found in the nucleotid region. Since this region is not seperated from the rest of the cell (by a membrane), transcription and translation can occur simultaneously.

Many bacteria also contain extrachromosomal DNA in plasmids.

Plasmid = A small double-stranded ring of DNA that carries extrachromosoal genes in some bacteria.

Most bacteria can rapidly reproduce by binary fission, which is preseded by DNA replication.

· Semi-conservative replication of the bacterial chromosme begins at a single origin of replication.

· The two replicaion forks move bidirectionally until they meet and replication is complete.

· under optimal conditions, some bacteria can divide in twenty minutes. Because of this rapid reproductive rate, bacteria are useful for genetic studies.

Binary fission is asexual reproduction that produces clones - daughter cells that are genetically identical to the parent.

· Though mutauton are rare evnets, they can impat genetic diversity in bacteria because of their rapid reproductive rate.

· Though mutation can be a major source of genetic variation in bacteriam it is not a major source in more slowly reproducing organisms (e.g. humans). In most higher organism, genetic recombinatio from sexual reproduciton is responsible for most of the genetic diversity within populations.

X. genetic reconbination and transposition produce new bacterial strains

There are three natural processes of genetic recombination in bacteria: transformation, transduction and conjugation. These mechanisms of gene transfer occur seperately from bacterial reproduction; and in additonf to mutatiom, are anotehr major source of genetic variation in bacterial populations.

A. Transformation

Transformation = Process of gene transfer during which a bacterial cell assimilates foreign DNA from the surroundings.

· Some bacteria can take up naked DNA from the surroundings.

· Assimilated foreign DNA may be integrated into the bacterial chromosome by recombination (crossing over).

· Progeny of the recipient bacterium will carry a new combination of genes.

Many bacteria have surface proteins that recognize and import naked DNA from closely related bacterial species.

• Though lacking such proteins, E. coli can be artificially induced to take up foreign DNA by incubating the bacteria in a culture medium that has a high concentration of calcium ions.
• This technique of artificially inducing transformations is used by the biotechnology industry to introduce foreign genes into bacterial genomes, so that bacterial cells can produce proteins characteristic of other species (e.g. insulin and human growth hormonw).

B. Transduction

Transduction = Gene transfer from one bacterium to antoher by a bacteriophage.

Generalized transduction = Transduction that occurs when random pieces of host cell DBA are packaged within a phage capsid during the lytic cycle of a phage.

• This process can transfer almost any host gene and little or no phage genes.
• When the phage particle infects a new host cell, the donor cell DNA can recombine with the recipient cell DNA.

Specialized transduction = Transduction that occurs when a prophage excises from the bacterial chromosome and carries with it some host genes adjacent to the excision site. Also known as restricted transduction .

• Carried out only by temperature phages.
• Differs from general transduction in that:
• Specific host genes and most phage genes are packed into the same virion.
• Transducted bacterial genes are restricted to specific genes adjacent to the prophage insertion site. In general transduction, host genes are randomly selected and almost any host gene can be transferred.

C. Conjugation and Plasmids

Conjugation = The direct transfer of genes between two cells that are temporarily joined.

· Discoveres by Joshua Lederberg and Edward Tatum.

· Conjugation in E. coli is one of the best studied examples:

·

A DNA-donating E. coli cell extends

External appendages called sex pili.

Sex pili attach DNA-receiving cell.

A cytoplasmic bridge forms through which

DNA transfer occurs.

The ability to form sex pili and to transfer DNA is conferred by gens in a plasmid called the F plasmid.

1. General Characteristics of Plasmids

Plasmids = A small double-stranded ring of DNA that carries extrachromosomal genes in some bacteria.

• Plasmids have only a few genes, and they are not requires for survival and reproduction.
• Plasmid genes can be beneficial in stressful environments. Examples include the F plasmid, which confers ability to conjugate; and the R plasmid, which confers antibiotic resistance.

These small circular DNA molecules replicate independently:

• Some plasmids under more relaxed control can replicate in synchrony with the bacterial chromosome, so only a few are present in the cell.
• Some plasmids under more relaxed control can replicate on their own schedule, so the number of plasmids, in the cell at any one time can vary from only a few to as many as 100.

Some plasmids are episomes that can reversibly incorporate in the cell’s chromosome.

Episomes = Genetic elements that can replicate wither independently as free molecules in the cytoplasm or as integrated parts of the main bacterial chromosome.

• Examples include some plamids, as well as, temperate viruses such as lambda phage.
• Temperate phage genomes replicate separately in the cytoplasm during a lytic cycle, and as an integral part of the host’s chromosme during a lysogenic cycle.

While plasmids and viruses can both be episomes, they differ in that:

• Plasmids, unlike viruses, lack an extracellular stage.
• Plasmids are generally beneficial to the cell, while viruses are parasites that usually harm their hosts.

2. The F Plasmid and Conjugation

The F plasmid (F for fertility) has about 25 genes, most of which are involved in the production of sex pili.

· Bacterial cells that contain the F factor and can donate DNA (“male”) are called F⁺ cells.

· The F factor replicates in synchrony with chromosomal DNA, so the F⁺ factor is inheritable; that is, division of an F⁺ cell results in two F⁺ daughter cells.

· Cells without the F factor are designated F^- (“female”).

During conjugation between and F⁺ and an F^- bacterium:

· The F factor replicates by rolling circle replication. The 5’ end of the copy peels off the circular plasmid and is transferred in linear form.

· The F⁺ cell transfers a copy of its F factor to the F^- partner, and the F^- cell becomes F⁺

· The donor cell remains F⁺, with its original DNA intact.

The F factor is an episome and occasionally inserts into the bacterial chromosme.

· Integrated F factor genes are still expressed.

· Cells with integrated F factors are called Hfr cells (high frequency of recombination).

Conjugation can occur between and Hfr and an F^- bacterium.

· As the integrated F factor of the Hfr cell transfers to the F^- cell, it pulls the bacterial chromosme behind its leading end.

· The F factor always opens up at the same point for a particular Hfr strain. As rolling circle replication proceeds , the sequence of chromosomal genes behind the leading 5’ end is always the same.

· The conjugation bridge usually breaks before the entire chromosome and tail end of the F factor can be transferred. As a result:

o Only some bacterial genes are donated.

o The recipient F^- cell does not become a F⁺ cell, because only part of the F factor is transferred.

o The recipient cell becomes a partial diplois.

o Recombination occurs between the Hfr chromosomal fragment and the F^- cell. Homologous strand exchange results in a recombinant F^- cell.

o Asexual reproduction of the recombinant F^- cell produces a bacteril colony that is genetically different from both original parental cells.

3. Interrupting Conjugation to Map Bacterial Chromosomes.

Scientists have mapped gene sequence around the E. coli chromosome by using several features of conjugation.

· A specific strain of Hfr bacteria always transfers genes in the same sequence.

· The sequence of gene transfer results from where the F factor is inserted and how it is oriented in the bacterial chromosome.

· The duration of conjugation determines how many chromosomal genes will be transferred.

By artificially interrupting conjugation at different time intervals, geneticists can rpughly determone gene location on the bacterial chromosome. The experimental steps are outlined below.

1. Liquid cultures of an Hfr strain and an F^- strain with different alleles are mixed together.

2. At successive time intervals, a sample is taken and agitated in a blender to disrupt conjugating pairs.

3. Bacteria are then cultured and genetic analysis of the recombinants indicates which genes were transferred during the time period allowed for conjugation.

4. Gene sequence and relative distances between genes are deduced from the above information.

4. R Plasmids and Antibiotic Resistance

One class of nonepisomal plasmids, the R plasmids (for resistance), carry genes that confer resistance to certain antibiotics.

· Some carry up to genes for resistance to antibiotics.

· During conjugation, some mobilize their own transfer to nonresistant cells.

· Increased antibiotic use has selected for antibiotic resistant bacterial strains carrying the R plasmid.

· Additionally, R plasmids can transfer resistance genes to bacteria of different species including pathogenic strains. As a consequence, resistant strain of pathigens are becoming more common.

D, Transposons

Pieces of DNA called transposable genetic elements, can actually move from one location to another in a cell’s genome.

Transposons = DNA sequences that can move from one chromosomal site to another.

• Occur as natural agents of genetic change in both prokaryotic and eukaryotic organisms.
• Were first proposed in the 1940’s by Barbara McClintock, who deduced their existence in maize. Decades later, the importance of her discovery was recognized’ in 1983, at the age of 81, she received the Nobel Prize for her work.

There are two patterns of transposition: a) conservative transposition and b) replicative transposition.

Conservative transposition = Movement of preexisting genes from one genomic location to another; the transposon’s genes are not replicated before the move, so the number of gene copies is conserved.

Replicative transposition = Movement of gene copies from their original site of replication to another location in the genome, so the transposon’s genes are inserted at some new site without being lost from the original site.

Transposition is fundamentally different from all other mechanisms of genetic recombination, because transposons may scatter certain genes throughout the genome with no apparent single, specific target.

• All other mechanisms of genetic recombination depend upon homologous strand exchange: meiotic crossing over eukaryotes; and transformation, transformation, trnasduction, and conjugation in prokaryotes.
• Insertion of episomic plasmids into chromosomes is also site specific, even though it does not require an extensive stretch of DNA homologous to the plasmid.

1. Insertion Sequences

The simplest transposons are insertion sequences.

Insertion sequences (IS) = The simplest transposons, which contain only the genes necessary for the process of transposition. Insertion sequence DNA includes two essential types of nucleotide sequences:

1. Nucleotide sequence coding for transposase.
2. Inverted repeats.

Transposase = Enzyme that catalyzes insertion of transposons into new chromosomal sites.

• The transposase gene in an insertion sequence is flanked by inverted repeats.

Inverted repeats (IR) = Short noncoding nucleotide sequences of DNA that are repeated in reverse order on opposite ends of a transposon. For example:

Note that each base sequence (IR) is repeated in reverse, on the DNA strand opposite the inverted repeat at the other end. Inverted repeats:

• Contain only 20 to 40 nucleotide pairs.
• Are recognition sites for transposase.

Transposase catalyzes the recombination by:

• Binding to the inverted repeats and holding them close together.
• Cutting and resealing DNA required for insertion of the transposon at a new site.

Insertion of transposons also requires other enzymes, such as DNA polymerase. For example,

• At the target site, transposase makes staggered cuts in the two DNA strands, leavingshort segments of unpaired DNA at each end of the cut.
• Transposase inserts the transposon into the open target site.
• DNA polymerase helps from direct repeats, which flan transposons in their target site. Gaps in the two DNA strands fill in when nucleotides base pair with the exposed single-stranded regions.

Direct repeats = Two or more identical DNA sequences in the same molecule.

· The transposition process creates direct repeats that flank transposons in their targert site.

Transposed insertion sequences are likely to somehow alter the cell’s phenotype; they may:

· Cause mutations by interrupting coding sequences for proteins.

· Increase or decrease a protein’s production by inserting within regulatory regions that control transcription rates.

Transposition of insertion sequences probably plays a significant role in bacterial evolutuon as a source of genetic variation.

· Though insertion sequences only rarely cause mutatiuons (about one ine every 10⁶ generations), the mutation rate from transpositions is about the same as the mutation rate from extrinsic causes, such as radiation and chemical mutagens.

2. Complex Transposons and R Plasmids

Complex transposons = Transposons which include additional genetic material besides that required for transposition; consist of one or more genes flanked by insertion sequences.

· The additional DNA may have any nucleotide sequence.

· Can insert into almost any stretch of DNA since their insertion is not dependent upon DNA sequence homology.

· Generate genetic diversity in bacteria by moving genes form one chromosome, or even one species, to another. This diversity may help bacteria adapt to new environmental conditions.

· An example is a transposon that carries a bacterial gene for antibiotic resistance.

Examples of genetic elements that contain one or more complex transpososns include:

· F factor.

· R plasmids.

· DNA version of the retrovirus genome.

XI. The control of gene expression enables individual bacteria to adjust their metabolism to environmental change.

Genes switch on and off as conditions in the intracellular environment change. Bacterial cells have two main ways of controlling metabolism:

1. Regulation of enzyme activity. The catalytic activity of many enzymes increases or decreases in response to chemical cues.

· For example, the end product of an anbolic pathway may turn off its own production by inhibiting activity of an enzyme at the beginning of the pathway (feedback inhibition).

· Useful for immediate short-term response.

2. Regulation of gene expression. Enzyme concentration may rise and fall in response to cellular metabolic changes that switch genes on or off.

· For example, accumalation of product may trigger a mechanism that inhibits transcrption of mRNA production by genes that code for an enzyme at the beginning of the pathway (gene repression).

· Slower to take effect than feedback inhibition, but is more economical for the cell. It prevents unneeded protein synthesis for enzymes, as well as, unneeded pathway product.

An example illustrating regulation of a metabolic pathway is the tryptophan pathway in E. coli. Mechanismis for gene regulaiton were first discovered for E. coli, and current understanding of such regulatory mechanisms at the molecular level is still limited to bacterial systems.

A. Operons: The Basic Concept

Regulated genes can be switced on or off depending on the cell’s metabolic needs. From their research on thecontrol of lactose metabolism in E. coli, Francois Jacob and Jacques Monod proposed a mechanism for the control of gene expression, the operon concept.

Structural gene = Gene that codes for a polypeptide.

Operon = A regulated cluster of adjacent structural genes with related functions.

· Common in bacteria and phages.

· Has a single promoter region, so an RNA polymerase will transcribe all structural genes on an all-or-none bais.

· Transcription produces a single polycistronic mRNA with coding sequences for all enzymes in a metabolic pathway (e.g. tryptophan pathway in E. coli).

Polycistronic mRNA = A large mRNA molecule that is a transcrpt of several genes.

· Is translated into separate polypepetides.

· Contains stop and start codons for the translation of each polypeptide.

Grouping structural genes into operons is advantageous because:

· Expression of these genes can be coordinated. When a cell needs the product of a metaboloc pathway. all the necessary enzymes are synthesized at one time.

· The entire operon can be controlled by a single operator.

Operator = A DNA segment between a operon’s promoter and structural genes, which controls access of RNA polymerase to structural genes.

· Sometimes overlaps the transcription starting point for the operon's first structural gene.

· Acts as an on/off switch for movement of RNA polymerase and transcription of the operon's structural genes.

What determines whether an operator is in the "on" or "off' mode? By itself, the operator

is on; it is switched off by a protein repressor. '

Repressor = Specific protein that binds to an operator and blocks transcription of the operon.

• Blocks attachment of RNA polymerase to the promoter.

• Is similar to an enzyme in that it:

ð Has an active site with a specific conformation, which discriminates among operators.

ð Repressor proteins are specific only for operators of certain operons.

ð Binds reversibly to DNA.

=> May have an allosteric site in addition to its DNA-binding site. • Repressors are encoded by regulatory genes.

Regulatol@@ genes = Genes that code for repressor or regulators of other genes.

ð Are often located some distance away from the operons they control.

o Are involved in switching on or off the transcription of structural genes by the -following process:

Transcription of the regulatory gene

products

mRNA

translated into

Regulatory protein

binds to

Operator

represses or activates

Transcription of operon's structural genes

Regulatory genes are continually transcribed, so their activity depends upon how efficient their promoters are in binding RNA polymerase.

· They produce repressor molecules continuously, but slowly.

· Operons are still expressed even though repressor molecules are always present, because repressors are not always capable of blocking transcription; they alternate between inactive and active conformations.

A repressor's activity depends upon the presence of key metabolites in the cell.

· Regulation of the trp operon in E. coli is an example of how a matabolite cues a repressor.

· Repressible enzymes cataltze the anabolic pathway that produces tryptophan, an amino acid.

· Tryptophan accumaltion represses synthesis of the enzymes that catalyze its production.

How does tryptophan activate the repressor protein?

· The repressor protein, which normally has a low affinity for the operator, has DNA binding site plus an allosteric site specific for tryptophan.

· When tryptophan binds to the repressor's allosteric site, it activates the repressor causing it to change its conformation.

· The activated repressor binds to the operator, which switches the trp operon off.

· Tryptophan functions in this regulatory system as a corepressor.

Corepressor = A molecule, usually a metabolite, that binds to a repressor protein, causing the repressor to change into is active conformation.

· Only the repressor-corepressor complex can attach to the operator and turn off the operon.

· When tryptophan concentrations drop, it is less likely to be bound to repressor protein. The trp operon, once free from repression, begins transcription.

· As concentrations of tryptophan rise, it turns off its own production by activating the repressor.

· Enzymes of the tryptophan pathway are said to be repressible.

B. Repressible Versus Inducible Enzymes: Two Types of Negative Gene Regulation

Repressible ena\zymes = Enzymes which have their synthesis inhibited by a metabolite (e.g. tryptophan).

Inducible enzymes = Enzymes which have their synthesis stimutalted or induced by specific metabolites.

Some operons can be switched on or induced by specific metabolites (e.g. lac operon in E. coli).

· E. coli can metabolize the disaccharide lactose. Once lactose is transported into the cell b-galactosidase cleaves lactose into glucose and galactose.

Lactose b-galactosidase glucose + galactose

(disaccharide) (monosaccharides)

· When E. coli is in a lactose-free medium, it only contains a few b-galactosidase molecules.

· When lactose is added to the medium, E. coli increases the nu,ber of mRNA molecules coding for b-galactosidase. These mRNA molecules are quickly translated into thousands of b-galactosidase molecules.

· Lactose metabolism in E. coli is programmed by the lac operon which has three structural genes:

1. lac Z - Codes for b-galactosidase which hydrolyzes lactose.

2. lac Y – Codes for permease, a membrane protein that ransports lactose inot the cell.

3. lac A - Codes for transacetylase, an enzyme that has no known role in lactose metabolism.

· The lac operon has a single promoter and operator. The lac repressor is innately active, so it attaches to the operon without a corepressor.

· Allolactose, an isomer of lactose, acts as an inducer to turn on the lac operon:

Allolactose

Binds to repressor

Inactivated repressor loses affinity for lac operon.

Operon is transcribed.

Enzymes for lactose metabolism are produced.

Differences between repressible and inducible operons reflect differences in the pathways they control.

Repressible Enzymes	Inducible Enzymes
Their genes are switched on until a specific metabolite activates the repressor.	Their genes are switched off until a specific metabolite inactivates the repressor.
Generally function in anabolic pathways.	Function in catabolic pathways.
Pathway end products switches off its own production by repressing enzyme synthesis.	Enzyme synthesis is switched on by the nutrient the pathway uses.

Repressible and inducible operons share similar features of gene regulation. In both cases:

· Specific repressor proteins control gene expression.

· Repressors can assume an active conformation that blocks transcription and an inactive conformation that allows transcription.

· Which form the repressor assumes depends upon cues from a metabolite.

Both systems are thus examples of negative control.

· Binding of active repressor to an operator always turns off structural gene expression.

· The lac operon is a system with negative control, because allolactose does not interact directly with the genome. The derepression allolactose cause us indirect, by freeing the lac operon from the repressos’s negative effect.

Positive control of a regulatory system occurs only if an activator molecule interacts directly with the genome to turn in transcription.

C. An Example of Positive Gene Regulation

The lac operon I sunder dual regulation which includes negative control by repressor protein and positive control by catabolite activator protein.

CAP (catabolite activator protein) = A protein that binds within an operon’s promoter region and enhances the promoter’s affinity for RNA polymerase.

· It is necessary for the normal expression of the lac operon. (Even if allolactose is present to inactivate the repressor, transcription proceeds slowly because the promoter has such a low affinity for RNA polymerase.)

· It is a positive regulator because it directly interacts with the genome to stimulate gene expression.

· It can bind to the promoter only if glucose is absent from the cell.

E. coli preferentially uses glucose over lactose as a substrate for glycolysis. So, normal expression of the lac operon requires:

· Presence of lactose.

· Absence of glucose.

How is CAP affected by the absence or presence of glucose?

· When glucose is missing, the cell accumulates cyclic AMP 9cAMP), a nucleotide derived from AYP. CAMP activates CAP so that it can bind to the lac promoter.

· When glucose concetration rises, glucose catabolism decreases the intracellular concentration of cAMP. Thus, cAMP releases CAP.

Glucose concentration

cAMP concentration rises cAMP becomes scarce

cAMP binds to CAP CAP loses its cAMP

cAMP-CAP complex binds CAP disemgages

to lac promoter from the lac promoter.

Efficient transcription Slowed transcription of

of lac operon. Lac operon.

In this dual regulation of the lac operon:

· Negative control by the repressor determines whether or not the oerpn will transcribe the structural genes.

· Posistive control by CAP determined the rate of transcription.

E. coli economizes on RNA and protein synthesis with the help of these negative and positive controls.

· CAP is an activator of several different operons that program catabolic pathways.

· Glucose’s presence deactivates CAP. This, in turn, slows synthesis of those enzymes a cell needs to use catabolites other than glucose.

· E. coli preferentially uses glucose as its primary carbon and energy source, and the enzymes for glucose catabolism are coded for by unregulated genes that are continously transcribed (constitutive).

· Consequently, when glucose is present, CAP does not work and the cell’s systems for using secondary energy sources are inactive.

When glucose is absent, the cell metabolizes alternate energy sources.

· The cAMP level rises, CAP is activated and transcription begins of operons that program the use of alternate energy sources (e.g. lactose).

· Which operon is actually transcribed depends upon which nutrients are available to the cell. For example, if lactose is present, the lac operon will be switched on as allolactose inactivates the repressor.