MICROBIAL MODELS:

THE GENETICS OF VIRUSES AND BACTERIA

Viruses and bacteria are the simplest biological systems serve as microbial models.

Microbiologists provided most of the evidence that genes are made of DNA, and they worked out most of the major steps in DNA replication, transcription, and translation.

Bacteria are prokaryotic organisms, with cells much smaller and more simply organized. Fig 18.1.

Viruses are smaller and simpler still, being little more than genes in a protein coat.

Viral genetics

Researchers discovered viruses by studying a plant disease
Tobacco mosaic disease stunts the growth and mottles plant leaves.
Several scientists determined that the disease could be transmitted from plant to plant by spraying the sap from an infected plant to a healthy one, even after the sap had been filtered through a filter that should have removed bacteria. It was also determined that it could reproduce only within the host, could not be cultivated on nutrient media, and was not inactivated by alcohol, generally lethal to bacteria.

In 1935, Stanley crystallized the pathogen, the tobacco mosaic virus (TMV). Fig 18.9b.

A virus is a genome enclosed in a protective coat

Viruses are not cells. They are infectious particles consisting of nucleic acid encased in a protein coat, and, in some cases, a membranous envelope. Fig. 18.2.

Viral genomes may consist of double-stranded DNA, single-stranded DNA, double-stranded RNA, or single-stranded RNA, depending on the specific type of virus. The smallest viruses have only four genes, while the largest have several hundred.

The capsid is a protein shell enclosing the viral genome. Capsids are built of a large number of protein subunits called capsomeres, but with limited diversity.

Some viruses have viral envelopes, membranes cloaking their capsids.
These envelopes are derived from the membrane of the host cell.
They also have some viral proteins and glycoproteins.
The most complex capsids are found in viruses that infect bacteria, called bacteriophages or phages. T-even phages may infect E. coli.

Viruses can reproduce only within a host cell. Fig 18.3.

Viruses are obligate intracellular parasites. Viruses lack the enzymes for metabolism or ribosomes for protein synthesis.

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

Viruses identify host cells by a "lock-and-key" fit between proteins on the outside of virus and specific receptor molecules on the host's surface.

Some viruses (like the rabies virus) have a broad enough host range to infect several species, while others infect only a single species.

Most viruses of eukaryotes attack specific tissues.

Human cold viruses - upper respiratory tract.
The AIDS virus - white blood cells.

A viral infection begins when the genome of the virus enters the host cell.
Once inside, the viral genome commandeers its host, reprogramming the cell to copy viral nucleic acid and manufacture proteins from the viral genome.
The nucleic acid molecules and capsomeres then self-assemble into viral particles and exit the cell.

Phages reproduce using lytic or lysogenic cycles

Some double-stranded DNA viruses can reproduce by two alternative mechanisms: the lytic cycle and the lysogenic cycle.

In the lytic cycle (Fig. 18.4), the phage reproductive cycle culminates in the death of the host, when the bacterium lyses and releases the phages produced to infect others.

 

Virulent phages reproduce only by a lytic cycle.

Bacterial defenses:

1. Natural selection favors bacterial mutants with receptors sites that are no longer recognized by a particular type of phage.
2. Bacteria produce restriction nucleases that recognize and cut up foreign DNA, including certain phage DNA.
3. Modifications to the bacteria's own DNA prevent its destruction by restriction nucleases.
In the lysogenic cycle, the phage genome replicates without destroying the host cell.

Temperate phages, like phage lambda, use both lytic and lysogenic cycles. Fig 18.5. Movie!

During a lytic cycle, the viral genes immediately turn the host cell into a virus-producing factory, and the cell soon lyses and releases its viral products.

During the lysogenic cycle, 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 its genes codes for a protein that represses most other prophage genes.

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

Occasionally, the viral genome exits the bacterial chromosome and initiates a lytic cycle.

This switch from lysogenic to lytic may be initiated by an environmental trigger.

Animal viruses very diverse

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. Viruses equipped with an outer envelope use the envelope to enter the host cell. Fig 18.6.

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.

The viral genome duplicates and directs the host's protein synthesis machinery to synthesize capsomeres with free ribosomes and glycoproteins with bound ribosomes.

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. These enveloped viruses do not necessarily kill the host cell.
Some viruses have envelopes that are not derived from plasma membrane. The envelope of the herpesvirus is derived from the nuclear envelope of the host.

These double-stranded DNA viruses reproduce within the cell nucleus using viral and cellular enzymes to replicate and transcribe their DNA.
Herpesvirus DNA may become integrated into the cell's genome as a provirus, which remains latent within the nucleus until triggered by physical or emotional stress to leave the genome and initiate active viral production.

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 mRNA and for a complementary RNA. This complementary strand is the template for the synthesis of additional copies of genome RNA.

All viruses that require RNA -> RNA synthesis to make mRNA use a viral enzyme that is packaged with the genome inside the capsid.

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

These carry an enzyme, reverse transcriptase, which 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. Fig. 18.7.
The viral particle includes an envelope with glycoproteins for binding to specific types of white blood cells, a capsid containing two identical RNA strands as its genome and two copies of reverse transcriptase.

Some viruses damage or kill cells by triggering the release of hydrolytic enzymes from lysosomes.

Some viruses cause the infected cell to produce toxins that lead to disease symptoms.

Others have molecular components, such as envelope proteins, that are toxic.

In some cases, viral damage is easily repaired (respiratory epithelium after a cold), but in others, infection causes permanent damage (nerve cells after polio).

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

Vaccines.

The first vaccine, using cowpox, was developed in the late 1700s by Edward Jenner to prevent smallpox. Many others have since been developed.

Antibiotics don't work against viruses.
Antivirals - recently developed drugs to combat some viruses, mostly by interfering with viral nucleic acid synthesis.
AZT interferes with reverse transcriptase of HIV.
Acyclovir inhibits herpesvirus DNA synthesis.

Emergent viruses. Fig 18.8.

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

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

The emergence of these new viral diseases is due to three processes:

mutation
RNA viruses have high mutation rates because replication of their nucleic acid lacks proofreading.
Influenza strains

spread of existing viruses from one species to another
It is estimated that about three-quarters of new human diseases have originated in other animals.
For example, hantavirus, which killed dozens of people in 1993, normally infects rodents, especially deer mice.

Dissemination of a viral disease from a small, isolated population.
AIDS, present only in small populations in Africa, went unnamed and unnoticed for decades before spreading around the world.
Affordable international travel, blood transfusion technology, sexual promiscuity, and the abuse of intravenous drugs, allowed a previously rare disease to become a global scourge.

Tumor viruses include retrovirus, papilloma virus, adenovirus, and herpesvirus types.

The hepatitis B virus is associated with liver cancer.
The Epstein-Barr virus, which causes infectious mononucleosis, has been linked to several types of cancer in parts of Africa, notably Burkitt's lymphoma.
Papilloma viruses are associated with cervical cancers.
The HTLV-1 retrovirus causes a type of adult leukemia.

All tumor viruses transform cells into cancer cells after integration of viral nucleic acid into host DNA.
Viruses may carry oncogenes that trigger cancerous characteristics in cells.
These oncogenes are often versions of proto-oncogenes that generally code for growth factors or proteins involved in growth factor function.

In other cases, a tumor virus transforms a cell by turning on or increasing the expression of proto-oncogenes.
It is likely that most tumor viruses cause cancer only in combination with other mutagenic events.

Plant viruses Fig 18.9.

Most are RNA viruses with rod-shaped capsids produced by a spiral of capsomeres.

Plant viral diseases are spread by two major routes.

In horizontal transmission, a plant is infected with the virus by an external source.

In vertical transmission, a plant inherits a viral infection from a parent.
This may occurs by asexual propagation or in sexual reproduction via infected seeds.

Viroids and prions

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 can be replicated by the host's cellular enzymes.

Prions are infectious proteins that spread a disease.
They are thought to cause several degenerative brain diseases including scrapie in sheep, "mad cow disease," and Creutzfeldt-Jacob disease in humans.
According to the leading hypothesis, a prion is a misfolded form of a normal brain protein, which then converts normal proteins into the prion version. Fig 18.10.

Viruses living or nonliving?

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, they evolved after the first cells appeared, probably 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.

The evolution of capsid genes may have facilitated the infection of undamaged cells.

Candidates for the original sources of viral genomes include plasmids, small, circular DNA molecules that are separate from chromosomes, and transposons, DNA segments that can move from one location to another within a cell's genome. Both are mobile genetic elements.

The Genetics of Bacteria

Bacteria are very adaptable, both in the evolutionary sense of adaptation via natural selection and the physiological sense of adjustment to changes in the environment by individual bacteria.
The major component of the bacterial genome is one double-stranded, circular DNA molecule.

Tight coiling of the DNA results in a dense region of DNA, called the nucleoid, not bound by a membrane.

Many bacteria have plasmids, much smaller circles of DNA.

Bacterial cells divide by binary fission. Fig 18.11.

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

Genetic recombination

In bacteria, the combining of DNA from two individuals into a single genome.
Recombination occurs through three processes:
Transformation
Transduction
Conjugation

Transformation is the alteration of a bacterial cell's genotype by the uptake of naked, foreign DNA from the surrounding environment.
For example, harmless Streptococcus pneumoniae bacteria can be transformed to pneumonia-causing cells, 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. Fig 18.12.

Many bacterial species have surface proteins that are specialized for the uptake of naked DNA.

Transduction occurs when a phage carries bacterial genes from one host cell to another. Fig 18.13.
In generalized transduction, a small piece of the host cell's degraded DNA is packaged within a capsid, rather than the phage genome.
When this phage attaches to another bacterium, it will inject this foreign DNA into its new host.
This type of transduction transfers bacterial genes at random.

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.
These bacterial genes are injected along with the phage's genome into the next host cell.
Specialized transduction only transfers those genes near the prophage site on the bacterial chromosome.
Both generalized and specialized transduction use phage as a vector to transfer genes between bacteria.
Conjugation transfers genetic material between two bacterial cells that are temporarily joined. Fig 18.15.
One cell ("male") donates DNA and its "mate" ("female") receives the genes.
A sex pilus (Fig 18.14) from the male initially joins the two cells and creates a cytoplasmic bridge between cells.
"Maleness," the ability to form a sex pilus and donate DNA, results from an F factor as a section of the bacterial chromosome or as a plasmid.

Plasmids, including the F plasmid, are small, circular, self-replicating DNA molecules.

Episomes, are a genetic element that can exist as a plasmid or as part of a chromosome. Episomes, like the F plasmid, can undergo reversible incorporation into the cell's chromosome.
Temperate viruses also qualify as episomes.

Plasmid genes are advantageous in stressful conditions.
Recombination exchanges segments of DNA.
This recombinant bacteria has genes from two different cells.

Antibiotic resistance.

The genes conferring resistance are carried by plasmids, specifically the R plasmid (R for resistance).
Some of these genes code for enzymes that specifically destroy certain antibiotics, like tetracycline or ampicillin.
When a bacterial population is exposed to an antibiotic, individuals with the R plasmid will survive and increase in the overall population.
Because R plasmids also have genes that encode for sex pili, they can be transferred from one cell to another by conjugation.

A transposon is a piece of DNA that can move from one location to another in a cell's genome.

Transposon movement occurs as a type of recombination between the transposon and another DNA site, a target site.
In bacteria, the target site may be within the chromosome, from a plasmid to chromosome (or vice versa), or between plasmids.
Transposons can bring multiple copies for antibiotic resistance into a single R plasmid by moving genes to that location from different plasmids.
This explains why some R plasmids convey resistance to many antibiotics.
Some transposons (so called "jumping genes") do jump from one location to another (cut-and-paste translocation).

The simplest bacterial transposon, an insertion sequence, consists only of the DNA necessary for the act of transposition. Fig 18.16.
The insertion sequence consists of the transposase gene, flanked by a pair of inverted repeat sequences.

The transposase enzyme recognizes the inverted repeats as the edges of the transposon and cuts the transposon from its initial site and inserts it into the target site. Fig 18.17.

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.

Composite transposons (complex transposons) include extra genes sandwiched between two insertion sequences. Fig 18.18.
Composite transposons may help bacteria adapt to new environments.
Repeated movements of resistance genes by composite transposition may concentrate several genes for antibiotic resistance onto a single R plasmid.

Transposable genetic elements are important components of eukaryotic genomes as well.
In the 1940s and 1950s Barbara McClintock investigated changes in the color of corn kernels, proposing that the changes in kernel color only made sense if mobile genetic elements moved from other locations in the genome to the genes for kernel color.
When these "controlling elements" were inserted next to the genes for kernel color, they would activate or inactivate those genes.

Individual bacteria may adjust their metabolism to cope with environmental change Fig 18.19

They may vary the number of specific enzyme molecules by regulating gene expression.

They may adjust the activity of enzymes already present.

The operon model for the control of gene expression in bacteria.

An operon consists of three elements:
The genes that it controls.
A promotor region where RNA polymerase first binds.
An operator region between the promotor and the first gene that acts as an "on-off switch."

Repressible enzymes generally function in anabolic pathways, synthesizing end products.
When the end product is present in sufficient quantities, the cell can allocate its resources to other uses.

The trp operon is an example of a repressible operon, one that is inhibited when some tryptophan molecules bind as corepressors to the repressor protein.

Tryptophan absent, repressors inactive, gene transcribed transcribed. Fig 18.20a

Tryptophan present, repressor active, gene not transcribed. Fig 18.20b.

An inducible operon is stimulated when a specific small molecule interacts with a regulatory protein.
Inducible enzymes usually function in catabolic pathways, digesting nutrients to simpler molecules.
By producing the appropriate enzymes only when the nutrient is available, the cell avoids making proteins that have nothing to do.

The lac operon contains genes that code for enzymes that digest lactose.
In the absence of lactose, this operon is off as an active repressor binds to the operator and prevents transcription. Fig 18.21a.
When lactose is present in the cell, the repressor is inactive and the operon is on. Fig 18.21b.

Both repressible and inducible operons demonstrate negative control because active repressors can only have negative effects on transcription.

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, the degree of transcription depends on the concentrations of other substrates. Cellular metabolism first uses glucose.

If glucose levels are low (along with overall energy levels), then cyclic AMP (cAMP) binds to cAMP receptor protein (CRP) which activates transcription. Fig 18.22a

If glucose levels are sufficient and cAMP levels are low (lots of ATP), then the CRP protein has an inactive shape and cannot bind upstream of the lac promotor. Fig 18.22b
The lac operon will be transcribed but at a low level.

For the lac operon, the presence / absence of lactose determines if the operon is on or off.
Overall energy levels in the cell determine the level of transcription, a "volume" control, through CRP.

CRP works on several operons that encode enzymes used in catabolic pathways.
If glucose is present and CRP is inactive, then the synthesis of enzymes that catabolize other compounds is slowed.
If glucose levels are low and CRP is active, then the genes which produce enzymes that catabolize whichever other fuel is present will be transcribed at high levels.