chapter 18 the genetics of viruses and bacteria. tobacco mosaic virus in the late 19th century,...
TRANSCRIPT
Chapter 18
The Genetics of Viruses and Bacteria
Tobacco Mosaic VirusIn the late 19th century, scientists
were working with a disease that infected the tobacco plant.
At first it was believed to be a bacterium.
Touching two plants together caused disease in both plants.
However, scientists couldn’t isolate and culture the infectious agent it so they concluded it must be too small to isolate, and maybe it was producing a toxin poisonous to the plants.
Tobacco Mosaic Virus
Eventually, scientists were able to crystallize the substance and it was determined to be a virus using electron microscopy.
Viruses
They are extremely small--20nm in diameter.
Some are DNA viruses, others are RNA viruses.
The DNA or RNA can be single stranded, double stranded, linear or circular.
Small viruses have 4 genes, large ones several hundred.
Viruses Viruses come in many different shapes and sizes. Regardless of the size, protein coat that
encapsulates their genome is called a capsid. Capsides are built from protein subunits called
capsomeres.
Viruses
Viruses only reproduce inside of cells.They lack all cellular machinery to
carry out any function such as reproduction or metabolism.
They only thing they can do is get their genes inside of cells and take them over.
Viruses have a host range. Some are very specific, others are non-specific.
VirusesMost viruses have proteins on the
outside that interact with specific receptor molecules on the surface of cells.
This “lock-and-key” system is what limits the specificity of a virus to certain cell types.
Once the virus attaches to the cell, it injects its DNA and takes over the control of the cell. It now uses the cell’s machinery to build
new virus particles.
Viruses
It sounds strange, but once the viral nucleic acids and capsomeres are formed, many viruses spontaneously assemble and hundreds of thousands of viral particles leave the cell, usually destroying it. This viral destruction of cells is often the
cause of the viral related symptoms we feel when we are infected.
Virus Reproduction-An Overview
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Virus Reproduction
Phages are the best understood of all viruses.
Viruses reproduce using one of two mechanisms: 1. The lytic cycle. 2. The lysogenic cycle.
1. The Lytic Cycle
When a phage reproduces and ultimately kills the host cell, it is said to use the lytic cycle.
The lytic cycle is the last stage of infection, lysing the cell and releasing phages.
Phages that reproduce using only the lytic cycle are said to be virulent phages.
1. The Lytic Cycle
There are 5 main steps to the lytic cycle of a common phage--T4. 1. Attachment. 2. Injection of genetic material and
control of the cell. 3. Synthesis of viral genomes and
proteins. 4. Assembly of viruses. 5. Release of phages.
The entire cycle lasts about 20-30 min.
1. The Lytic Cycle
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A Game of Cat and Mouse
The main reason why viruses haven’t wiped out all bacteria and other cells is because surface receptors on cells often mutate preventing attachment of viruses.
On the other hand, viruses often mutate enabling them to adapt and attach to the surfaces of cells displaying these new receptors.
A Game of Cat and Mouse
Another defense many cells have is the ability to detect foreign genetic material and cut it up with degradative enzymes (restriction endonucleases).
Some phage mutants produce nucleic acid that is not recognizable by restriction enzymes and can thus take over a cell’s function.
2. The Lysogenic CycleThe lysogenic cycle is yet another
reason why viruses haven’t taken over all cells.
In this cycle, the phage genome is replicated without destroying the cell.
Some phages are called temperate phages because they use both the lysogenic and lytic modes of reproduction.
phage is an example.
Phage Lifecycle
The phage lifecycle is as follows: 1. Attachment. 2. Injection of DNA into cells. 3. Phage DNA “circulizes.”
Environmental factors determine the lytic or lysogenic cycles.
Lytic cycle: 4. Phage DNA and proteins are made. 5. Cell lyses releasing phages.
Phage Lifecycle The phage lifecycle is as follows:
1. Attachment. 2. Injection of DNA into cells. 3. Phage DNA “circulizes.”
Environmental factors determine the lytic or lysogenic cycles.
Lysogenic cycle: 4. Phage DNA integrates with bacterial DNA. 5. Bacteria reproduce normally transmitting all
DNA to both cells, crossing over occurs. 6. Many cell divisions produce many cells with
phage DNA and altered phenotypes.
2. The Lysogenic Cycle
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Lytic or LysogenicThe cycle by which a temperate phage
reproduces is largely due to environmental signals.
A temperate phage can use the lysogenic cycle to incorporate its genome into a bacterium’s and many copies of the phage DNA can be made as the bacterium divides. When something in the environment triggers a switchover to the lytic cycle, the phage then destroys the cell(s).
The Ways in Which Viruses Infect Cells
One of the key variables is whether the genetic material is DNA or RNA, and whether or not it is single or double stranded.
Many bacteriophages are not RNA viruses and lack membranous envelopes.
Most animal viruses with RNA genomes have envelopes.
Animal Viruses with RNA Genomes and Viral EnvelopesAnimal viruses with viral envelopes
use them to enter host cells. The envelopes contain viral glycoproteins that attach to the surface of host cells.
When the virus enters the cell, takes it over and begins synthesizing new virus particles. It uses the cellular enzymes in the ER to synthesize glycoproteins to form a new envelope.
Animal Viruses with RNA Genomes and Viral EnvelopesIn a process similar to exocytosis, the
new viruses wrap themselves in the membrane derived from the host’s plasma membrane and bud off from the cell.
This reproductive cycle doesn’t necessarily kill the host cell like the lytic cycle.
Other Forms of Viral Envelopes
Some viruses reproduce within the nucleus of the cell and don’t use the plasma membrane as an envelope--instead they are derived from the nuclear membrane of the cell.
Many of these viruses have double stranded DNA and use a combination of host and viral enzymes to replicate and transcribe their genomes.
HIV-A Retrovirus
Retroviruses are RNA viruses which reproduce via a DNA intermediate.
The actual flow of information is reversed; it goes RNA to DNA back to RNA.
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Bacteria
Most bacteria have a single, circular, double stranded DNA molecule as their chromosome.
In contrast to eukaryotic chromosomes, bacterial chromosomes are associated with a relatively small amount of protein.
These chromosomes exist in the nucleoid region.
Bacteria
Bacteria are also often associated with small pieces of DNA called plasmids.
Plasmids usually contain DNA fragments that are of benefit to the cell.
Bacteria divide via binary fission after replicating their DNA.
Recall that the DNA replication begins at the origin and moves around until finished.
Bacterial Reproduction
Mutations arise in bacteria at a rate of about 1:10,000,000. This doesn’t seem like much, but considering how prolific they are, this is a high rate.
Example: In the human gut, 20 billion new E. coli cells arise each day. With a 1:10 million mutation rate, this produces about 2000 new mutants that increase the genetic diversity of the population.
Mutations
Mutations are a major source of genetic variation in quickly reproducing organisms.
In slowly reproducing populations, genetic recombination (crossing over) is a source of new variation.
Bacteria also have a means by which recombination occurs and produces new variation.
Evidence for Recombination in E. coli
Researchers used two mutant strains of E. coli.
One could synthesize tryptophan and not arginine.
The other could synthesize arginine and not tryptophan.
Samples from the two strains were mixed together to see if they could grow on minimal medium.
Evidence for Recombination in E. coli
If recombination occurs, some cells from the mixed culture should grow on minimal medium, whereas they should not from the original mutant strain.
Cell colonies grew on minimal medium after being incubated together.
Somehow the cells, when incubated together, exchanged genetic material and gave rise to individuals that could survive on minimal medium.
3 Mechanisms of Genetic Recombination in Bacteria
1. Transformation.2. Transduction.3. Conjugation.
1. Transformation Transformation is the alteration of the
genotype and phenotype of a cell by the uptake of naked DNA from the surroundings.
Recall the Griffith experiment with mice and pneumonia.
This was transformation. The mixture of heat killed smooth and the
living rough produced recombinant cells that became virulent. They were recombinant because they contained genes from two different cells.
2. Transduction
Transduction is another means by which bacterial cells can undergo recombination.
This process employs phages that carry genes from one cell to another.
It often results from irregularities in the phage reproductive cycle. Generalized transduction. Specialized transduction.
2. Transduction The most effective
way in which transduction occurs is when, during the lytic cycle, a piece of host cell DNA gets packaged into a capsid of a newly formed virus accidentally.
2. Transduction When this occurs,
as it sometimes does, the virus will inject only the DNA taken up from the host cell. If this DNA then crosses over and is taken up by the bacterium, recombination is said to occur.
Generalized Transduction
Generalized transduction occurs when bacterial genes are randomly transferred from one bacterial cell to another through a phage intermediate.
It occurs when DNA from a bacterium gets accidentally inserted into a newly formed phage.
Specialized Transduction
In this process, a temperate phage (a phage that can use the lytic or lysogenic mode of reproduction), inserts its genome into a bacterial chromosome becoming a prophage.
Specialized transduction occurs when the prophage picks up a few specific (adjacent) bacterial genes as it exits the chromosome transferring them to a new host cell.
3. Conjugation Conjugation is
basically bacterial sex. It is the direct transfer of genetic information from one cell to the next.
The cells join briefly through the formation of a sex pilus which acts to pull the cells close together.
3. Conjugation
Once the cells are close enough together, a cytoplasmic bridge forms between the two cells and an exchange of genetic information takes place.
The ability to form sex pili depends on F factor which is a small segment of DNA in the bacterial chromosome or on a plasmid.
Plasmids
Plasmids are small pieces of DNA that integrate into bacterial chromosomes and confer some sort of phenotypic change to it.
R plasmids, for instance, often confer antibiotic resistance to various bacteria.
It can also stand alone and replicate independently of the bacterium. When this happens, it is called an episome.
F Plasmids and Conjugation
F plasmids consist of about 25 genes and are responsible for the formation of sex pili.
Cells with F plasmid are F+ which are DNA donors.
Cells without F plasmid are F- and are the DNA recipients.
F plasmids replicate with chromosomal DNA
F Plasmids and Conjugation An F+ plasmid can turn an F- cell into an F+ cell
when the two cells mix genes. One of the two strands of plasmid DNA is
transferred to the F- plasmid. Each of the parental strands now serve as templates in each of the respective cells where DNA replication completes.
F Plasmids and Conjugation
When a donor cell’s F factor gets integrated into its chromosome, chromosomal genes can also get transferred during conjugation.
Cells with F factor built into the chromosome are called Hfr cells (high frequency of recombination), and they are the donors.
F Plasmids and Conjugation Many times when the two cells are joined together during
conjugation, chromosomal DNA is brought into the F- cell.
Random movements of the bacteria often prevent complete transfer of an F+ genome.
The recipient cell now contains small amounts of DNA which often mixes with its DNA. This is the mechanism of recombination.
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Transposable ElementsThe DNA of bacterial cells can also
undergo recombination of its genes due to transposable elements.
Transposable elements are always part of chromosomal or plasmid DNA and never exist alone (unlike phage and plasmid DNA).
Transposition describes the movement of genes and it’s when they move from one spot in the cell’s DNA to another--called a target site.
Transposable Elements
In bacteria, transposable elements can move form one spot within a chromosome to another, from chromosome to plasmid and vice-versa, or from plasmid to plasmid.
When they move, the original and new DNA sites are brought together by DNA folding. Some of them move via cut and paste, while others move via copy and paste.
Transposable Elements
Most transposable elements can move anywhere within a cell’s DNA; some prefer certain areas over others.
The movement of a transposable element is different from the other three mechanisms of genetic mixing. The other require homologous regions of DNA for gene mixing. Transposable elements can insert anywhere.
Transposable Elements
Now that you know what transposable elements essentially are, there are two types: 1. Insertion sequences 2. Transposons
1. Insertion sequencesInsertion sequences are the simplest
of all transposable elements and exist only in bacteria.
They encode for a single enzyme (transposase) that cuts DNA only at specified regions.
They don’t do much of anything. They can cause mutations if they insert themselves in the right spot. Can be considered an intrinsic source of variation.
2. Transposons
Transposons are more complex than insertion sequences.
They carry the gene for transposase and other genes--such as antibiotic resistance.
Transposons often help bacteria adapt to new environments.
Transposons can help explain how R plasmids contain a number of genes that confer resistance to bacteria.
Bacterial Metabolism
Bacteria can fine tune their metabolism to suit their varying metabolic demands.
There are two ways in which the bacterium can control its metabolism: 1. They can adjust the enzymatic activity
within the cell. 2. They can adjust the amount of product
being made by certain enzymes--regulation of gene expression coding for enzymes.
The Operon
An operon is a unit of genetic function common to bacteria and phages that consist of coordinately controlled clusters of genes with similar functions.
An operon consists of an operator, a promoter, and the genes that code for the enzymes they control in a particular pathway.
The Basics of the OperonThe operon is the operator, the
promoter and the genes they control.The operator is the segment of the
DNA that acts as a switch.A repressor is a protein that can bind
to the operon and switch it off. The repressor is usually a product of a
regulatory gene often acting allosterically.Corepressors are small molecules that
cooperate with repressor proteins to switch operons off.
The Basics of the Operon
Inducers are small molecules that inactivate repressors.
Activators are proteins that bind to DNA and stimulate transcription of specific genes.
The Amino Acid Tryptophan: An Example
When the cell lacks tryptophan in its medium, a series of steps can be activated so that the cell can synthesize it.
When tryptophan is synthesized from a precursor molecule, a series of steps are activated and each is catalyzed by a specific enzyme.
The 5 genes that code for the enzymes are clustered together in a single stretch on the bacterial chromosome.
The Amino Acid Tryptophan: An Example
A single promoter serves all 5 genes.This constitutes a transcriptional unit,
and when the mRNA is synthesized, it codes for all five enzymes in the pathway.
The mRNA is punctuated with start and stop codons that signal where the coding sequences begin and end.
Thus, the 5 separate polypeptides can be translated as needed.
The Amino Acid Tryptophan: An Example
The advantage to this is that there is one on-off switch for the series of genes related to tryptophan synthesis.
When the cell needs tryptophan, all enzymes are switched on.
The “switch” is the segment of DNA called the operator.
The operator is positioned within or between the promoter and the enzyme-coding genes.
The operator controls the access of RNA polymerase to the genes.
The Operon The operator, the promoter and the genes
they control make up the operon.
The OperonOperons move back and forth between
2 states: One with a repressor bound, and one
without.Also, most regulatory proteins are
allosterically controlled. That is, there is an active state and an
inactive state. Controlled by the amount of gene product
available.
The trp Operon, a Repressible Operon
The operon is switched off and on by a protein called a repressor.
The operon, in this case, is always on and binding of the repressor blocks the attachment of RNA polymerase to the promoter and thus transcription of the genes.
This blocking action is very specific and has no effect on other genes within the cell.
The trp Operon, a Repressible Operon
In our example using tryptophan, the trp repressor is the product of a regulatory gene (trpR) which is located some distance from the trp operon it is controlling.
It has its own promoter. Regulatory genes are always on and always expressed
within a cell. However, they bind reversibly.
The trp Operon, a Repressible Operon
If the active genes ultimately produce tryptophan, and its available, it will bind to and alter the function of the trp repressor protein.
That is, it binds to the protein, activating it and enabling it to bind to the operon shutting it off.
Tryptophan is a corepressor.
The lac OperonThe lac operon is an example of an
inducible operon.-galactosidase is an enzyme that
breaks down lactose into glucose and galactose.
Most E. coli cells only have a few molecules of -galactosidase available at any one time.
When you add lactose to the medium in which they are growing, the amount of -galactosidase increases sharply.
The lac Operon The gene encoding for -galactosidase is part of
the lac operon. It includes 2 other genes that code for enzymes that
function in lactose metabolism. The whole thing is considered one transcriptional
unit and is under the control of 1 operator and 1 promoter.
The lac Operon The lacI gene is a
regulatory gene located outside of the operon. It encodes for an allosteric repressor protein that can switch off the lac operon by binding to the operator.
The reason it is called an inducible operon is because the lac repressor is active by itself, and binds to the operator and shuts the lac operon off.
The lac Operon To inactivate the repressor, an inducer is needed.
An inducer is a small molecule which binds to and inactivates a repressor.
In the case of the lac operon, allolactose is the inducer.
Allolactose is an isomer of lactose found in small amounts when lactose enters a cell.
The lac Operon When lactose is present, allolactose binds to the
repressor altering its conformation, preventing the ability of the repressor from binding to the operator. When the repressor is unable to bind to the operator, the genes in the lac operon can be transcribed into mRNA which can be translated into lactose-metabolizing enzymes.
Summary
The regulation of the trp and lac operons involves negative control of genes--their operons are switched off by the active form of the repressor gene.
Gene regulation is said to be under positive control only when a regulatory protein interacts directly with the genome to switch transcription on.
Positive Control of the lac Operon
Not only are the lactose utilizing enzymes working and on when lactose is present, but they are also working when glucose is in short supply.
E. coli cells can sense low concentrations of glucose and relay the information to the genome.
It does so with the interaction of an allosteric regulatory protein and a small organic molecule.
Positive Control of the lac Operon
When glucose is scarce, cAMP accumulates in the cell. When accumulation occurs, it interacts with the regulatory protein called catabolite activator protein (CAP).
CAP acts as an activator of transcription.
Positive Control of the lac Operon
When cAMP binds to CAP, CAP assumes an active shape, binds to a specific site on the lac promoter stimulating gene expression.
This is called positive regulation.
Positive Control of the lac Operon
When glucose levels within the cell increase, cAMP falls and CAP detaches from the operon.
When CAP is inactive, transcription of the lac operon proceeds at a low level.
SummaryThe state of the lac repressor
determines whether or not transcription of the lac operon’s genes occurs at all. The state of CAP controls the rate of transcription if the operon is repressor free.
In both cases, when glucose is available, it will be used preferentially by the cell. Lactose enzymes will only be made when lactose is available and glucose is not.
Summary
In our example involving tryptophan, the trp operon, is a repressible operon because its transcription is usually on but can be turned off.
In contrast, the example involving lactose, the lac operon, is an inducible operon. Transcription is normall off, but can be turned on.
Summary When looking at the
figure at the right, keep in mind the following 2 things: Negative control,
binding has a negative effect, it shuts down transcription.
Positive control, binding has a positive effect, it stimulates transcription.
