BIS 1A Handout 18 - Bacterial and Viral Genetics

Microbial Model Systems

Viruses and bacteria are the simplest biological systems-microbial models in which scientists find life's fundamental molecular mechanisms in their most basic, accessible forms.

Molecular biology was born in the laboratories of microbiologists studying viruses and bacteria.

In addition, viruses and bacteria have unique genetic features with implications for understanding the diseases that they cause.

Bacteria are prokaryotic organisms, with cells that are much smaller and more simply organized than those of eukaryotes, such as plants and animals.

Viruses are smaller and simpler still, lacking the structure and metabolic machinery of cells.

Most viruses are little more than aggregates of nucleic acids and protein.

A virus has a genome but can reproduce only within a host cell

A virus is a genome enclosed in a protective coat.

In 1935, Wendell Stanley isolated the first virus, the tobacco mosaic virus (TMV).

Stanley's discovery that some viruses could be crystallized was puzzling because not even the simplest cells can aggregate into regular crystals.

Viruses are infectious particles consisting of nucleic acid encased in a protein coat and, in some cases, a membranous envelope.

The tiniest viruses are only 20 nm in diameter-smaller than a ribosome.

The genome of viruses may consist of double-stranded DNA, single-stranded DNA, double-stranded RNA, or single-stranded RNA, depending on the kind of virus.

The viral genome is usually organized as a single linear or circular molecule of nucleic acid.

The smallest viruses have only four genes, while the largest have several hundred.

The capsid is the protein shell enclosing the viral genome.

Capsids are built of a large number of protein subunits called capsomeres.

The capsid of the tobacco mosaic virus has more than 1,000 identical capsomeres.

A membranous envelope surrounds the capsids of flu (influenza) viruses.

These viral envelopes are derived from the membrane of the host cell.

They also have some host cell viral proteins and glycoproteins, as well as molecules of viral origin.

Some viruses carry a few viral enzyme within their capsids.

The most complex capsids are found in viruses that infect bacteria, called bacteriophages or phages.

Viruses can reproduce only within a host cell.

Viruses are obligate intracellular parasites.

Viruses lack the enzymes for metabolism and the ribosomes for protein synthesis.

Each type of virus can infect only a limited range of host cells, called its host range.

This host specificity depends on the evolution of recognition systems by the virus.

Viruses identify host cells by proteins on the outside of the virus, which bind specific receptor molecules on the host's surface (which evolved for functions that benefit the host).

West Nile virus can infect mosquitoes, birds, horses, and humans.

Measles virus can infect only humans.

Human cold viruses infect only the cells lining the upper respiratory tract.

The AIDS virus binds only to certain white blood cells.

Once inside the host cell, the viral genome commands the cell to copy viral nucleic acid and manufacture proteins from the viral genome.

The host provides nucleotides, ribosomes, tRNAs, amino acids, ATP, and other components for making the viral components dictated by viral genes.

RNA viruses use special virus-encoded polymerases that can use RNA as a template.

The nucleic acid molecules and capsomeres then self-assemble into viral particles and exit the cell.

Tobacco mosaic virus RNA and capsomeres can be assembled to form complete viruses if the components are mixed together under the right conditions.

The simplest type of viral reproductive cycle ends with the exit of many viruses from the infected host cell, a process that usually damages or destroys the host cell.

Phages reproduce using lytic or lysogenic cycles.

Research on phages led to the discovery that some double-stranded DNA viruses can reproduce by two alternative mechanisms: the lytic cycle and the lysogenic cycle.

In the lytic cycle, the phage reproductive cycle culminates in the death of the host.

In the last stage, the bacterium lyses (breaks open) and releases the phages produced within the cell to infect others.

Each of these phages can infect a healthy cell.

In the lysogenic cycle, the phage genome replicates without destroying the host cell.

Temperate phages, like phage lambda, use both lytic and lysogenic cycles.

The viral DNA molecule is incorporated by genetic recombination into a specific site on the host cell's chromosome.

In this prophage stage, one of the viral genes codes for a protein that represses most other prophage genes.

Every time the host divides, it copies the phage DNA and passes the copies to daughter cells.

The term lysogenic implies that prophages are capable of a lytic cycle.

That happens when the viral genome exits the bacterial chromosome and initiates a lytic cycle.

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

Many variations on the basic scheme of viral infection and reproduction are represented among animal viruses.

One key variable is the type of nucleic acid that serves as a virus's genetic material.

Another variable is the presence or absence of a membranous envelope derived from the host cell membrane.

Most animal viruses with RNA genomes have an envelope, as do some with DNA genomes.

Viruses equipped with an outer envelope use the envelope to enter the host cell.

Glycoproteins on the envelope bind to specific receptors on the host's membrane.

The envelope fuses with the host's membrane, transporting the capsid and viral genome inside.

After the capsid and viral genome self-assemble, they bud from the host cell covered with an envelope derived from the host's plasma membrane, including viral glycoproteins.

The envelope of the herpesvirus is derived from the nuclear envelope of the host.

The viruses that use RNA as the genetic material are quite diverse, especially those that infect animals.

In some with single-stranded RNA (class IV), the genome acts as mRNA and is translated directly.

In others (class V), the RNA genome serves as a template for complementary RNA strands, which function both as mRNA and as templates for the synthesis of additional copies of genome RNA.

Retroviruses (class VI) have the most complicated life cycles.

These carry an enzyme called reverse transcriptase that transcribes DNA from an RNA template.

The newly made DNA is inserted as a provirus into a chromosome in the animal cell.

The host's RNA polymerase transcribes the viral DNA into more RNA molecules.

These can function both as mRNA for the synthesis of viral proteins and as genomes for new virus particles released from the cell.

Human immunodeficiency virus (HIV), the virus that causes AIDS (acquired immunodeficiency syndrome) is a retrovirus.

Viruses may have evolved from other mobile genetic elements.

An isolated virus is biologically inert, and yet it has a genetic program written in the universal language of life.

Because viruses depend on cells for their own propagation, it is reasonable to assume that they evolved after the first cells appeared.

Most molecular biologists favor the hypothesis that viruses originated from fragments of cellular nucleic acids that could move from one cell to another.

A viral genome usually has more in common with the genome of its host than with those of viruses infecting other hosts.

Perhaps the earliest viruses were naked bits of nucleic acids that passed between cells via injured cell surfaces.

Candidates for the original sources of viral genomes include plasmids and transposable elements.

Plasmids are small, circular DNA molecules that are separate from chromosomes.

Plasmids, found in bacteria and in yeast, and are occasionally transferred between cells.

Transposable elements are DNA segments that can move from one location to another within a cell's genome.

Viruses, viroids, and prions are formidable pathogens in animals and plants

Many of the temporary symptoms associated with a viral infection result from the body's own efforts at defending itself against infection.

The immune system is a complex and critical part of the body's natural defense mechanism against viral and other infections.

Modern medicine has developed vaccines, harmless variants or derivatives of pathogenic microbes that stimulate the immune system to mount defenses against the actual pathogen.

Antibiotics, which can kill bacteria by inhibiting enzymes or processes specific to bacteria, are powerless against viruses, which have few or no enzymes of their own.

Most antiviral drugs resemble nucleosides and interfere with viral nucleic acid synthesis.

New viral diseases are emerging.

In recent years, several emerging viruses have risen to prominence.

HIV, the AIDS virus, seemed to appear suddenly in the early 1980s.

Each year new strains of influenza virus cause millions to miss work or class, and deaths are not uncommon.

The deadly Ebola virus has caused hemorrhagic fevers in central Africa periodically since 1976.

A more recent viral disease is severe acute respiratory syndrome (SARS).

These emerging viruses are generally not new. Rather, they are existing viruses that mutate, spread to new host species, or expand their host territory.

Changes in host behavior and environmental changes can increase the viral traffic responsible for emerging disease.

Destruction of forests to expand cropland may bring humans into contact with other animals that may host viruses that can infect humans.

Plant viruses are serious agricultural pests.

More than 2,000 types of viral diseases of plants are known.

Once a virus starts reproducing inside a plant cell, viral particles can spread throughout the plant by passing through plasmodesmata.

Proteins encoded by viral genes can alter the diameter of plasmodesmata to allow passage of viral proteins or genomes.

Agricultural scientists have focused their efforts largely on reducing the incidence and transmission of viral disease and in breeding resistant plant varieties.

Viroids and prions are the simplest infectious agents.

Viroids, smaller and simpler than even viruses, consist of tiny molecules of naked circular RNA that infect plants.

Their several hundred nucleotides do not encode for proteins but cause errors in the regulatory systems that control plant growth.

Prions are infectious proteins that spread disease.

They appear to cause several degenerative brain diseases including scrapie in sheep, "mad cow disease," and Creutzfeldt-Jakob disease in humans.

Prions are likely transmitted in food.

According to the leading hypothesis, a prion is a misfolded form of a normal brain protein.

When the prion gets into a cell with the normal form of the protein, the prion can convert the normal protein into the prion version, creating a chain reaction that increases their numbers.

Rapid reproduction, mutation, and genetic recombination contribute to the genetic diversity of bacteria

The best-studied bacterium is Escherichia coli, a species that lives in the human colon

The major component of the bacterial genome is one double-stranded, circular DNA molecule that is associated with a small amount of protein.

For E. coli, the chromosomal DNA consists of about 4.6 million nucleotide pairs with about 4,400 genes.

This is 100 times more DNA than in a typical virus and 1,000 times less than in a typical eukaryote cell.

In addition, many bacteria have plasmids, much smaller circles of DNA.

Each plasmid has only a small number of genes, from just a few to several dozen.

Bacteria proliferate very rapidly in a favorable natural or laboratory environment.

Under optimal laboratory conditions, E. coli can divide every 20 minutes, producing a colony of 107 to 108 bacteria in as little as 12 hours.

Through binary fission, most of the bacteria in a colony are genetically identical to the parent cell.

However, the spontaneous mutation rate of E. coli is 1 × 10"7 mutations per gene per cell division.

This produces about 2,000 bacteria per day in the human colon that have a new mutation.

New mutations, though individually rare, can have a significant impact on genetic diversity when reproductive rates are very high because of short generation spans.

In contrast, organisms with slower reproduction rates (like humans) create genetic variation primarily by sexual recombination of existing alleles.

Genetic recombination produces new bacterial strains.

In addition to mutation, genetic recombination generates diversity within bacterial populations.

Here, recombination is defined as the combining of DNA from two individuals into a single genome.

Bacterial recombination occurs through three processes: transformation, transduction, and conjugation.

Transformation is the alteration of a bacterial cell's genotype by the uptake of foreign DNA from the surrounding environment.

This occurs when a live nonpathogenic cell takes up a piece of DNA that happens to include the allele for pathogenicity from dead, broken-open pathogenic cells.

The foreign allele replaces the native allele in the bacterial chromosome by genetic recombination.

In biotechnology, this technique has been used to introduce foreign DNA into E. coli.

Transduction occurs when a phage carries bacterial genes from one host cell to another as a result of aberrations in the phage reproductive cycle.

In generalized transduction, bacterial genes are randomly transferred from one bacterial cell to another.

Some of this DNA can subsequently replace the homologous region of the second cell.

Specialized transduction occurs via a temperate phage.

When the prophage viral genome is excised from the chromosome, it sometimes takes with it a small region of adjacent bacterial DNA.

Specialized transduction only transfers those genes near the prophage site on the bacterial chromosome.

Sometimes known as bacterial "sex," conjugation transfers genetic material between two bacterial cells that are temporarily joined.

The transfer is one-way. One cell ("male") donates DNA and its "mate" ("female") receives the genes.

A sex pilus from the male initially joins the two cells and creates a cytoplasmic mating bridge between cells.

"Maleness," the ability to form a sex pilus and donate DNA, results from an F (for fertility) factor as a section of the bacterial chromosome or as a plasmid.

Plasmids usually have only a few genes, which are not required for normal survival and reproduction of the bacterium.

However, plasmid genes may be advantageous in stressful conditions.

The F plasmid facilitates genetic recombination when environmental conditions no longer favor existing strains.

Recombination exchanges segments of DNA.

The resulting recombinant bacterium has genes from two different cells.

The DNA of a single cell can also undergo recombination due to movement of transposable genetic elements or transposable elements within the cell's genome.

Unlike plasmids or prophages, transposable elements never exist independently but are always part of chromosomal or plasmid DNA.

During transposition, the transposable element moves from one location to another in a cell's genome.

Transposable elements may move by a "copy and paste" mechanism, in which the transposable element replicates at its original site, and the copy inserts elsewhere.

Most transposable elements can move to many alternative locations in the DNA, potentially moving genes to a site where genes of that sort have never before existed.

The simplest transposable elements, called insertion sequences, exist only in bacteria.

An insertion sequence contains a single gene that codes for transposase, an enzyme that catalyzes movement of the insertion sequence from one site to another within the genome.

Insertion sequences cause mutations when they happen to land within the coding sequence of a gene or within a DNA region that regulates gene expression.

Insertion sequences account for 1.5% of the E. coli genome, but a mutation in a particular gene by transposition is rare, occurring about once in every 10 million generations.

This is about the same rate as spontaneous mutations from external factors.

Transposable elements longer and more complex than insertion sequences are called transposons.

In addition to the DNA required for transposition, transposons include extra genes such as genes for antibiotic resistance.

While insertion sequences may not benefit bacteria in any specific way, transposons may help bacteria adapt to new environments.

For example, a single R plasmid may carry several genes for resistance to different antibiotics.

This is explained by transposons, which can add a gene for antibiotic resistance to a plasmid already carrying genes for resistance to other antibiotics.

Transposable elements are also important components of eukaryotic genomes.

Individual bacteria respond to environmental change by regulating their gene expression

An individual bacterium can cope with environmental fluctuations by exerting metabolic control.

First, cells can vary the number of specific enzyme molecules they make by regulating gene expression.

Second, cells can adjust the activity of enzymes already present (for example, by feedback inhibition).

The tryptophan biosynthesis pathway demonstrates both levels of control.

If tryptophan levels are high, some of the tryptophan molecules can inhibit the first enzyme in the pathway.

If the abundance of tryptophan continues, the cell can stop synthesizing additional enzymes in this pathway by blocking transcription of the genes for these enzymes.

The basic mechanism for this control of gene expression in bacteria, the operon model, was discovered in 1961 by François Jacob and Jacques Monod.

E. coli synthesizes tryptophan from a precursor molecule in a series of steps, with each reaction catalyzed by a specific enzyme.

The five genes coding for these enzymes are clustered together on the bacterial chromosome, served by a single promoter.

The mRNA is punctuated with start and stop codons that signal where the coding sequence for each polypeptide begins and ends.

A key advantage of grouping genes of related functions into one transcription unit is that a single "on-off switch" can control a cluster of functionally related genes.

When an E. coli cell must make tryptophan for itself, all the enzymes are synthesized at one time.

The switch is a segment of DNA called an operator.

The operator, located between the promoter and the enzyme-coding genes, controls the access of RNA polymerase to the genes.

The operator, the promoter, and the genes they control constitute an operon.

By itself, an operon is on and RNA polymerase can bind to the promoter and transcribe the genes.

However, if a repressor protein, a product of a regulatory gene, binds to the operator, it can prevent transcription of the operon's genes.

Each repressor protein recognizes and binds only to the operator of a certain operon.

Binding by the repressor to the operator is reversible.

Repressors contain allosteric sites that change shape depending on the binding of other molecules.

In the case of the trp, or tryptophan, operon, when concentrations of tryptophan in the cell are high, some tryptophan molecules bind as a corepressor to the repressor protein.

This activates the repressor and turns the operon off.

At low levels of tryptophan, most of the repressors are inactive, and the operon is transcribed.

The trp operon is an example of a repressible operon, one that is inhibited when a specific small molecule binds allosterically to a regulatory protein.

In contrast, an inducible operon is stimulated when a specific small molecule interacts with a regulatory protein.

In inducible operons, the regulatory protein is active, and the operon is off.

Allosteric binding by an inducer molecule makes the regulatory protein inactive, and the operon is turned on.

The lac operon contains a series of genes that code for enzymes that play a major role in the hydrolysis and metabolism of lactose (milk sugar).

In the absence of lactose, this operon is off, as an active repressor binds to the operator and prevents transcription.

When lactose is present in the cell, allolactose, an isomer of lactose, acts as the inducer.

This inactivates the repressor, and the lac operon can be transcribed.

Both repressible and inducible operons demonstrate negative control because active repressors switch off the active form of the repressor protein.

Positive gene control occurs when an activator molecule interacts directly with the genome to switch transcription on.

Even if the lac operon is turned on by the presence of allolactose, the degree of transcription depends on the concentrations of other substrates.

If glucose levels are low, then cyclic AMP (cAMP) accumulates.

The regulatory protein catabolite activator protein (CAP) is an activator of transcription.

When cAMP is abundant, it binds to CAP, and the regulatory protein assumes its active shape and can bind to a specific site at the upstream end of the lac promoter.

The attachment of CAP to the promoter directly stimulates gene expression.

Thus, this mechanism qualifies as positive regulation.

CAP works on several operons that encode enzymes used in catabolic pathways.

If glucose is present and CAP is inactive, then the synthesis of enzymes that catabolize other compounds is slowed.

If glucose levels are low and CAP is active, then the genes that produce enzymes that catabolize whichever other fuel is present will be transcribed at high levels.