Microbiology Exam 4/Final Exam Study Guide
Microbiology Exam 4/Final Exam Study Guide MICR 3050
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This 79 page Study Guide was uploaded by Toni Franken on Tuesday April 26, 2016. The Study Guide belongs to MICR 3050 at Clemson University taught by Dr. Whitehead in Spring 2016. Since its upload, it has received 89 views. For similar materials see General Microbiology in Microbiology at Clemson University.
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MICR 3050 Exam 4/Final Study Guide Dr. Whitehead, Clemson University Units 1 – 4 Modeled around Dr. Whitehead’s Recommended Topics for study, color coded and organized by chapter. (Unit 4 is at the very beginning, followed by units 1 – 3) UNIT 4 Chapter 6: Viruses General Information about Viruses: o Cannot reproduce on their own – not considered living for that reason. There are viruses that can infect all cell types. There are those that infect eukaryotic organisms (animals, plants, fungi), some that infect archaea (little known about them – mainly due to our previous idea that bacteria and archaea were very similar), and those that infect bacteria (bacteriophages). o Only active in host cells. There is only one known exception to this. They are obligate intracellular parasites. They MUST have a host cell in order to continue function. o Classify according, mainly, by DNA or RNA as their genetic information. Single or double stranded, linear, circular, or segmented. Viruses DO NOT have ribosomes. Cannot be classified in the same way as other organisms. o Virion is the complete virus particle: This includes the following: Nucleic acid Protein coat surrounding the nucleic acid Sometimes (not always) external layers in addition to that. The bare minimum is the nucleic acid and protein coat. o There is a great deal of diversity in viruses, ranging from genetic material and shape. Rabies virus has a bullet shape (rhabdovirus family). Also have a variety of other shapes and sizes. Some viruses (not most) are big enough to see with light microscopes. Also a wide range of external structure, changing how the immune system responds to them. o The absolute simplest form a virus possible that is still classified as such is called a nonenveloped virus. Just the protein coat (capsid) and the nucleic acid. Nucelocapsid – the nucleic information and the protein coat together. o Some viruses have additional components. An enveloped virus is a little more complex. There is a membrane outside of the nucleocapsid – can be spiked – have spikes extending from the surface of the virus. One of the more common types of virus capsids are helical capsids – long, narrow, hollow tubelike structure. The tobacco mosaic virus has this type of coat. They are made up of a variety of protein subunits of helical capsids – these are called protomers. The size of the protomers themselves play into the width of the capsid. The length of the capsid is believed to be determined by the length of the viral genome. Viruses tend to be efficient, and the capsid is thought to just be used to protect the viral genome. Icosahedral capsid – made up of triangular shapes – this is considered to be very efficient. Have special subunits that are called capsomers, which are proteins – pentamers and hexamers are the two main types of capsomers. Pentamers are capsomers made of five protomers. Hexamers are capsomers made of six protomers. In a helical capsid, we have protomers alone that assemble to make the capsid. In an icosahedral capsid, we have protomers that assemble to make capsomers, that then assemble o Complex Capsids – Vaccinia (smallpox family) – doesn’t have a classic shape for viruses. Does not fall into helical or icosahedral. Has an oval shape. o Viral envelopes – usually an outer, flexible, membranous layer made of lipids and carbohydrates. Many of these factors are hostderived so viruses can make themselves look like the host to protect from immune system. Envelope proteins may project from the envelope surface – called spikes or peplomers (proteins coded by the viral genome). They have functions that are specific to the virus itself. o Spikes can help with attachment to host cells, can have enzymatic activity (exact activity is virus dependent), spikes can be involved in viral genetic replication (replication of nucleic acids), and can also be used for the identification of viruses (by humans in the lab). This is commonly used in the influenza virus. Names such as H1N1 stand for spike proteins on the surface of the influenza virus. H stands for hemagglutinin. Hemagglutinin is involved with attachment to the host cell or surface. N stands for nuraminidase. Neuraminidase has to do with viral release from the cell. The 1s stand for the type of protein. Describe bacteriophages. o Bacteriophages (T4, or phages) have an icosahedral portion on top , then a helical portion lower. The bacteriophages keep the genetic information in the capsid (icosahedral) head – the tail part then acts as an injector, so the virus just injects its genetic information directly into the bacterial cell. Distinguish between virulent (lytic) and temperate (lysogenic) bacteriophages and their life cycles using T4 phage and lambda phage as examples, respectively. o Step 1: The virus has to attach to a host cell – very first step. If a virus can’t attach, it can’t invade. There are certain individuals that appear to be immune to HIV infection due to the fact that they don’t have certain receptors on their cells that are required for the virus to attach. Some viruses require more than just attachment. o Step 2: Entry – the entire viral agent may enter, or just the genetic info can enter. Don’t typically see an enveloped virus entering the cell. Depending on what entered, the next step may or may not happen. o Step 3: Uncoating of genome – if only genetic info entered the cell, this does not happen. However, if the entire virus entered the host cell, the protein coat must be removed so the viral DNA or RNA is free to be worked on by the host enzymes. One way or another you’ll end up with transcription and translation of the viral genome. o Step 4: Synthesis – synthesis of the viral genome. o Step 5: Assembly – new viruses must be assembled from the replicated genome pieces. o Step 6: Release – some viruses are released due to the lysis of the host cell. For enveloped viruses, when they go to leave, they do “budding” where there is fusion of the host cell membrane around the virus. The virus takes up some of the host cell membrane to become part of their envelope. May be released a few at a time. o There are two different types/classes of bacteriophages: multiple viruses within each of these. Designated as virulent or temperate. Virulent Bacteriophages are incredibly destructive to the host cell. They enter, replicate, and then lyse the host cell so that they may be released. That is their only option. The most common example of this is the T4 bacteriophage. First, have attachment. From there, the genetic info is injected into the host cell. The only thing that gets in the host cell is the viral nucleic acids. Regulation is very important – has to do when certain viral genes are transcribed. Tehre are often two different points where transcription would occur. Early mRNA transcription and late mRNA. If the bacteria cares nothing about keeping the host cell alive, it basically destroys the bacterial DNA, and only uses it to replicate the phage DNA. Get transcription and translation, synthesis, and assembly. Get formation of virions, and release through lysis. This can happen in about 22 minutes – the doubling time of E. coli. Temperate Phages: Temperate phages have two different lifecycle options. Lytic pathway (act just like a virulent bacteriophage). They have attachment, entry, replication, synthesis, assembly, and lysis. Lysogenic pathway, where for a period of time, the viral DNA (or RNA) will be incorporated into the host cell chromosome, and will just hang out through bacterial replication. Every time the bacterial chromosome is replicated, the viral genome is also. The lysogenic pathway is a way for the virus to remain inside the host cell without destroying it. Typically what happens is that at some point in time, these cells that have the lysogenic virus in them will undergo induction. Usually a stress will trigger the viral DNA to exit the chromosome and enter into a lytic pathway. Explain lysogeny and how the lytic cycle may be induced in lysogens. o Lysogeny: Nonlytic relationship between a phage and its host – no lysis of the host cell. o Prophage: The integrated bacteriophage genome – uses an integrase enzyme to be incorporated into the chromosome of the host cell. Replicates whenever the host genome replicates. o Lysogen: The infected bacterial host – appears normal. A prophage may change the phenotype of its host. This change is called lysogenic conversion – presence of the prophage causes changes in the physical characteristics of the lysogen. The phage may switch from lysogenic to lytic cycle upon induction. Lysogenic conversion – change in a host cell due to presence of prophage. May change the LPS of gram negatives, or may change the proteins on the surface (possibly so no other viruses can infect?). Other pathogens have also picked up diseasecausing elements through lysogenic conversion o Induction is the process of a phage switching from lysogenic to lytic pathways. Induction is usually triggered by stresses on the host cell or in the environment. These stresses may be exposure to UV light, chemical mutagens that cause DNA damage, or nutritional deprivation. Once these are detected, the viral genome switches to the lytic pathway. Excisionase: An enzyme that binds the integrase enzyme, which allows for reverse integration. This is where the viral genome is cut out of the bacterial genome where it was added. This is Excision, or removal, from the bacterial chromosome. The cell can then undergo the normal lytic pathway. Describe the effects of animal viruses on their hosts. o Cytopathic effects (CPEs): Often include a change in cell's morphology such as fusion with adjacent cells to form polykaryocytes as well as the synthesis of nuclear and cytoplasmic inclusion bodies. Inclusion bodies – microscopically these are visible sites of viral assembly or cellular damage. They are often used as a diagnostic tool. Examples include: Virions in the nucleus (Adenovirus) Virions in the cytoplasm (Rhabdovirus negri bodies of rabies virus) Host cells may also exhibit proliferation of membranes, shrinking of the nucleus, formation of vacuoles in the cytoplasm, or may even undergo programmed cell death (celled Apoptosis). Viruses may halt or alter host cell DNA synthesis, transcription, and/or protein synthesis. o Cytocidal Effects: Associated with changes in cell morphology, physiology and are important for the complete viral replication and transformation. These almost always lead to the death of the host cell. Cytopathic effects can be cytocidal, such as inducing apoptosis, or preventing the synthesis of substances needed by the host cell. Many times, cytocidal effects are those that interrupt important metabolic pathways, thus preventing the cell from functioning normally. There are two types of cytocidal infections: Productive infections, where additional infectious viruses are produced. One example of a productive cytocidal infection is the herpes virus. Abortive infections do not produce infectious viruses, and only kill the host cell. o Animal Viruses can undergo 4 different types of infection: Acute, latent, chronic, or cancerous. See the next section for more information. Distinguish between acute, latent, and chronic viral infections (know examples of each): o Acute Viral Infection: There is rapid multiplication of the virus that very quickly leads to cell death, and the release of more viruses. These diseases progress very rapidly, and often resolve themselves quickly either through death or recovery. Influenza and the common cold (rhinovirus) are good examples of this. An acute viral infection may cause an inapparent infection, or one that is not easily detected due to low or absent symptoms. This happens due to the action of the immune system. o Latent viral infection: The virus stops reproducing and remains dormant for some time. During latency, symptoms, antivirusantibodies, and viruses are not detectable. They do not cause disease regularly, but may be activated by a stressor. Some examples of a latent viral infection are shingles (the chickenpox virus stays dormant in the host system) and cold sores (herpes simplex virus). o Chronic viral infection: The virus almost always detectable, but the clinical symptoms may be mild or absent for long periods of time, making it very hard to identify. Hosts often are infected with the virus for the rest of their lives, and it commonly causes fatality. HIV is an example of a chronic, aka persistent, viral infection. o Cancer: More can be found two sections down, but some viruses can cause changes in their host cell that cause unchecked replication and growth. The cell itself is transformed to be abnormal. HPV is commonly known to cause cancer in humans. Understand the replication process of HIV (a retrovirus). o Retrovirus action is simply the idea that the virus has RNA as its genetic material, and must reverse transcribe to create DNA from its genome before injecting it into the host cell. The Human Immunodeficiency Virus does just that. o 1. The virus must enter the host cell. o 2. The virus must uncoat its RNA for access to reverse transcription. o 3. Reverse Transcription takes place, creating double stranded DNA from the viral single stranded RNA. o 4. The DNA goes to the nucleus, where it is incorporated into the host DNA. This forms a provirus, where the viral DNA is incorporated into the host cell DNA. o 5. Transcription takes place, creating viral RNA. o 6. Encapsulation of the viral RNA takes place to create a nucleocapsid. o 7. The virus undergoes budding, where the nucleocapsid takes some of the host’s cytoplasmic membrane. This makes the HIV virus much harder to identify and treat. o 8. The virus particle is released into the body, where it can travel to infect more host cells. o NOTE: The HIV virus is known to be incredibly adaptive. This makes it difficult to treat. Explain the role of viruses in causing cancer (oncoviruses). o Oncoviruses typically cause tumor formation through two different mechanisms: The tumor virus can introduce and express a "transforming" gene either through the integration of DNA or RNA into the host genome. In other words, the virus is carrying the gene that causes the tumor formation. The tumor virus can alter expression on preexisting genes of the host. In other words, the virus action causes the upregulation of normal genes, called oncogenes, to protooncogenes. These caused unchecked cell growth and replication, and can also introduce greater error in genetic material. o Some examples of Oncoviruses are the HPV virus, Hepatitis B and C, and the Epstein Barr Virus. How might one cultivate viruses? What effects might viruses have on host cells? o Viruses are incredibly difficult to cultivate in a lab setting. Bacteriophages may be cultivated in broth or agar that is inoculated with bacterial cells, such as E. coli. In a broth culture, you can actually visualize phages in action by the clearing of the broth, or the loss of rabidity. This is due to the lysis of cells by the phages. On agar cultures, you can observe plaques. Plaques are cleared zones on a lawn of bacteria that result from the total lysis of bacterial cells by viruses in that spot. Animal Viruses are much more difficult to cultivate in a lab setting due to ethical issues that come along with it. Mice are commonly used as hosts for viral agents. Rabbit testicles have been used in the past to cultivate especially fastidious viruses. The testes would be injected with the virus, allowed to replicate, and then the rabbits would be castrated, and you would have a live viral culture for a time. o Cytopathic effects (CPEs): Often include a change in cell's morphology such as fusion with adjacent cells to form polykaryocytes as well as the synthesis of nuclear and cytoplasmic inclusion bodies. Inclusion bodies – microscopically these are visible sites of viral assembly or cellular damage. They are often used as a diagnostic tool. Examples include: Virions in the nucleus (Adenovirus) Virions in the cytoplasm (Rhabdovirus negri bodies of rabies virus) Host cells may also exhibit proliferation of membranes, shrinking of the nucleus, formation of vacuoles in the cytoplasm, or may even undergo programmed cell death (celled Apoptosis). Viruses may halt or alter host cell DNA synthesis, transcription, and/or protein synthesis. o Cytocidal Effects: Associated with changes in cell morphology, physiology and are important for the complete viral replication and transformation. These almost always lead to the death of the host cell. Cytopathic effects can be cytocidal, such as inducing apoptosis, or preventing the synthesis of substances needed by the host cell. Many times, cytocidal effects are those that interrupt important metabolic pathways, thus preventing the cell from functioning normally. There are two types of cytocidal infections: Productive infections, where additional infectious viruses are produced. One example of a productive cytocidal infection is the herpes virus. Abortive infections do not produce infectious viruses, and only kill the host cell. Explain the ways in which one might obtain a count of the viruses in a solution. o Direct Counts: You could use an electron microscope to do direct counts of viral particles. However, not every facility has access to electron microscopes, and this can be very time and money consuming. o Indirect Counts: Plaque Assay: This is what we did in lab, where you inoculate a soft agar bacterial culture with a dilution of bacteriophages. You then pour the agar into a plate, and incubate the plate. You count the number of plaques to figure out the number of Plaque forming units. A countable plate of plaques is between 25 and 250 plaques. Each individual plaque is assumed to have been created by a single virus that has replicated. You would then take the number and multiply it by the dilution factor to figure out the number of viruses in the original sample. Hemagglutination Assay (animal viruses): This is done using animal blood, and determines the highest dilution of virus that causes red blood cells to agglutinate, or clump together. Endpoint Method: This method basically uses live organisms (bacteria or animals), and involves observing the population to see where the dose of virus is large enough to destroy 50% of the host cells, or kill 50% of the animal population in question. This is called LD50 Dilutions of the virus are made until it reaches that point. Chapters 16 and 17: Mechanisms of Genetic Variation Miscellaneous Information: o Nucleoid: The location of the chromosome and associated proteins within a bacterial cell. In most cases this is not membrane bound. There are a few exceptions to this. This is just where this material is concentrated and condensed. It is usually an irregularly shaped region, and usually contains one, closed circular, double stranded DNA molecule. If you were to take the DNA of E. coli and stretch it out, it would be about 500x longer than the bacterial cell itself. Very highly condensed. The circular chromosome carries the majority of genetic information. You can also have plasmids – extrachromosomal DNA (DNA that is located outside the chromosome o Plasmids are usually small, closed circular DNA molecules that replicate and exist independently of the chromosome. Often do not replicate at the same time as the chromosome. They contain few genes, and are nonessential. Some bacteria have many plasmids, others only have 1, and some have no plasmids at all. There are usually less than 30 genes or so on a plasmid. Typically the information is nonessential, meaning they can survive without it. Plasmids typically give some kind of advantage. Very commonly see antibiotic resistance genes on plasmids. Just because a bacterium has a plasmid, that doesn’t mean it keeps it. If a cell is “cured” it has lost the plasmid. This might happen because the plasmid is not replicated when the chromosome is, so it doesn’t get passed to the daughter cell. If the plasmid replicates slowly, this easily happens through generation turnover. There are some exceptions – linear plasmids exist. Know the basic structure of DNA. o DNA: Composed of 4 building nucleotides or bases, where A is complementary to T, and C is complementary to G. DNA is the genetic material of life – discovered and shown by Griffith in 1928. Avery, MacLeod and McCarthy in 1944. Hershey and Chase. DNA structure was discovered by Watson and Crick in the 1950’s. Strands run 3’ to 5’, and the complementary strand runs 5’ to 3’ against it. There is a strong sugarphosphate backbone that keeps the DNA together, and forms the double helix shape. Understand the terms associated with bacterial genetics. o Gene: A portion of DNA that encodes either for a polypeptide, rRNA, or tRNA. Codes for something else. o Genotype: What the genetic information actually says – the gene or genes an organism actually has. o Phenotype: The physical characteristics an organism is actually showing. Complicated relationship with genotype. o Wildtype Strain: Just whatever strain can be isolated from nature – the one we found “out in the wild.” This doesn’t tell you anything about how normal that strain is, it’s just the one found in the environment. o Mutation: A stable change in the genotype of an organism. An inherited change from generation to generation in the DNA sequence. The genotype is definitely changed, but the phenotype may or may not be changed. Just because the DNA sequence changes, that doesn’t mean the physical characteristics change. Explain how mutations arise. o Spontaneously: This type of mutation happens in almost all living organism – any time DNA is replicated, it is possible for a mistake to be made, and if it sticks around, is technically a mutation. In some cases, spontaneous mutations have led to advantageous things for the organism (such as antibiotic resistance). o Induced: Exposure to a mutagen (chemical or physical agent that can change the DNA sequence) that causes a change in the DNA sequence that is heritable. Can purposely expose a bacteria to a mutagen, but we don’t know where mutation will occur. Sitedirected mutagenesis: Nothing random about this mutation method. Go in and change a specific nucleotide in the genetic sequence. Can change it according to the location you are targeting, and changing the specific base. This is done only in a laboratory setting. Mutations can be caused by agents that directly damage DNA (mutagens): have base analogs, DNA modifying agents, and intercalating agents. Base analogs: Agents that are very similar to nucleotides – these will get incorporated into the DNA during DNA replication if bacteria are grown in the presence of base analogs. The problem with this is that base analogs don’t bind to other nucleotides like other nucleotides do. 5 Bromouracil will be incorporated into the DNA as if it is a Thymine. However, it instead binds with Guanine. Over time, this becomes a stable mutation. DNA Modifying Agents: Correct nucleotides are put into DNA during replication. These modifying agents go in and change the nucleotides, chemically altering them. Includes Hydroxylamine, which can alter C nucleotides so they no longer bind with Gs. Can make it bind to Thymine instead of Guanine. Intercalating Agents: These agents wiggle their way into the DNA and cause the entire structure of the DNA to be thrown off. Because of this disrupted shape of the DNA, we can have other issues. Acridine orange is an example. Compare and contrast point and frameshift mutations and their effects. o Point Mutations: Alteration of a single pair of nucleotides – one nucleotide or pair being changed. Have three types – nonsense, missense, silent. Transition: Swap one purine for another (As and Gs) or Pyrimidines for pyrimidines (T and C and U). Transversion: Tend to be rarer, occurs when a purine is swapped for pyrimidine, or vice versa. Silent mutation: The DNA sequence changed, but the amino acid that the triplet coded was the same, so we can’t see the effects of the mutation. Nonesense Mutation: A change in a base pair causes for a new Stop codon to be created. Translation will stop entirely. The closer to the beginning of the gene, the more influential a nonsense mutation is. If the nonsense mutation happens close to the end of the gene, the protein may be hardly affected, and may still be functional. Missesne Mutation: Where the change in the DNA sequence leads to the production of a different amino acid. This creates an entirely different polypeptide. The effect of a missense mutation is variable – depends which amino acid is replaced, and where. Could have a nonfunctional protein, or could still have functionality. o Frameshift: Insertion or Deletion: Tend to be more problematic – if you throw off the triplets of a DNA sequence, you completely change the protein sequence from that point on. This is called a frameshift mutation. More devastating mutation – have the insertion or deletion of a single nucleotide. However, the genetic code is read in triplets of nucleotides, so a single shift in the genetic code throws off the entire sequence after it. If you have an addition, you have a +1 reading frame. If you have a deletion, you have a 1 reading frame. o Larger Mutations: Mutations involving more than just a single nucleotide base. Insertions: Insertion of an entire segment of genetic material. Deletions: Removal of an entire segment off genetic material Inversions: A piece of genetic material is taken out, flipped around, and inserted back in. Problems with regulation. Duplications: Where a sequence of genetic material is replicated and placed next to the original piece. Translocations: The movement of a strand of genetic material from one location on the chromosome to another, or to a completely different chromosome. Distinguish between the various point mutations. o See in the section above. (nonsense, missense, silent, Transition, Transversion). Understand the expression of mutations (forward vs. reverse). o Wildtype: Most prevalent form of the gene, typically. o Forward Mutations: What we classically think of when we think of mutations – went form a wildtype to a mutant. Something changed to make an organism different than the normal individuals of its population. o Reverse Mutations: Where we had a mutant, now we’ve reverted back to the wildtype. There was a mutation in an organism, but somehow its offspring did not inherit that mutation. Revertants: Organisms that are known to have had their mutation reversed. This can happen in multiple locations. Samesite Revertants: Had a mutation that occurred, which was then simply reversed. Secondsite Revertants (suppressor mutations): There is a mutation somewhere else in the genome (maybe somewhere else in the gene or chromosome), that undoes the effects of the first mutation. Basically, they suppress the effects of the first mutation. An example might be, if a mutation caused inhibition of a gene, a second mutation happens that destroys the inhibition material, allowing the gene to be expressed. Describe the phenotypic effects of mutations in bacteria. o Morphological: Change in colonial or cellular morphology – you can actually see the change in the shape or structure of the colony or cell itself. o Lethal: Changes that kill the organism. Usually happens because a mutation has destroyed or inhibited an essential gene and its function. o Conditional Mutation: Mutations that really only matter under certain conditions. Classically we look at changes in heatshock proteins. Won’t see the effects until we try to grow the organism at higher temperatures. At normal temperatures, the mutation will go unnoticed. o Biochemical effects: Something about the biochemistry of the cell has been changed. A pathway for production of material is interrupted, for example. Auxotrophic Mutants: Generally looking at organisms that have mutated to be unable to make its own amino acids or other macromolecule. For instance, a lysine auxotroph would need lysine in the medium to survive because it can’t make its own. Prototroph: The strain from which the auxotroph arose. In the above lysine example, the prototroph strain can make its own lysine. o Resistance: Can have antibiotic, competition, or chemical resistance mutations. Often are classified as biochemical changes. Distinguish between prototrophs and auxotrophs. o See above. Describe how to detect and isolate mutants (including the replica plating technique). o Mutant Detection: Observation of changes in phenotype in the mutants (screening). Usually just used when you’re trying to find the mutant. Can be looking at colony morphology, o Selectable Mutations: Confer some type of advantage to the organisms that possess them (for example: drug resistance). We can then attempt to isolate organisms based on them having a particular characteristic. Find conditions where only one form of the organism will grow. Replica Plating: Can be used to detect auxotrophic mutants. The idea is that you have a plate of cells that are grown on a complete medium. You know that everything a cell could need is in this medium, and you have colonies on the plate. Perhaps you expose them to a mutagen, so you suspect you have some auxotrophs on the plate. So, you’d take two more plates, one of which is complete, and one of which is a medium that is deficient in what you expect the auxotrophs to be unable to create. You use a stamp (typically made of velvet), and transfer the original colonies to these new plates. You incubate the plates and look for colonies that grow on the complete medium, but not the deficient plate. If you see this, you can take them from the complete plate to study them as auxotrophs. Know the difference between screening and selection. o Screening: When screening for a phenotype, you are allowing for all of the organisms to grow, and only observing if your mutation is displayed. For instance, on a plate that changes colony color if lactose is fermented, you would look for cells in the colony that either do, or don’t ferment lactose. One is the wildtype, the other is a mutant. o Selection: This is used to actually isolate ONLY the mutants. This was what we did in lab with the plasmid/transformation exercise. You give a bacterial culture conditions that allow ONLY mutants to grow. For instance, only mutants that contain resistance to an antibiotic would grow on a plate containing that antibiotic. The wildtype bacteria would not grow. Describe the possible fates of donor DNA during horizontal gene transfer. o As a refresher, here is some information about horizontal gene transfer: Vertical gene transfer: transfer of genetics from one generation to the next. Microbes can go through horizontal gene transfer (not possible in most (almost all) eukaryotes). Transfer of genetic information between two organisms living at the same time. Not from generation to generation. There are 3 basic pathways for horizontal gene transfer in bacteria. Transformation: The ability of some bacteria to pick up extracellular DNA from the environment. Usually happens due to lysis of other cells, putting the DNA into the environment. Some cells can take this DNA up, and if they are able to do so, they are called competent. Transduction: Kind of an accidental process – transfer of genetic material from one bacteria to the next due to a phage infection. Have a bacteriophage that comes and infects the first cell. When it goes over and infects the next cell, some of the genetic information from the first cell gets passed to the second cell. There are multiple types of this. Conjugation: Talked about a little bit so far. Usually involves the formation of a sex pilus. The plasmid in one cell (donor) has genes that encode to create a sex pilus. The donor basically has a tube that can form between it and a recipient, and can pass genetic information to the recipient. What can be picked up/passed from one cell to another could be plasmids. Or, it could just be a piece of DNA that came from a chromosome. What happens to that DNA depends on the type of DNA it is, and the factors of the host cell. There are generally four possibilities. o When a recipient cell receives DNA from somewhere else – 2 possibilities that give you stable recombinants, 2 possibilities that do not get you stable recombinants: DNA that the cell picked up is similar to DNA that is already in the cell genome. This can result in recombination, where external DNA will get incorporated into the host cell chromosome. This results in a Stable Recombinant. Once that new DNA is incorporated, every time the cell replicates, this piece of DNA will also replicate. The cell picked up a plasmid – if the plasmid you picked up is capable of replicating on its own, and it replicates at a fairly decent speed, it probably will not get incorporated into the host cell chromosome, but every time the cell replicates, the plasmid might replicate as well. This would also be considered a stable recombinant. The cell picked up a plasmid, but that plasmid is not capable of replicating on its own. The host cell probably doesn’t have the factors that force the plasmid to replicate. There are some plasmids that have to have cell factors that make it replicate. This usually results in the plasmid being lost from the population, and you end up with no stable recombinants. Cell enzymes are used to destroy foreign DNA – probably a defense mechanism against phage infection. Will not get any stable recombinants. This process is called Restriction. The enzymes are called Restriction enzymes. We have been able to use them in the laboratory setting. Describe homologous recombination in bacteria. o Genetic Recombination – this occurs AFTER horizontal gene transfer has already occurred. This is where one or more nucleic acid molecules are rearranged or combined to produce a new nucleotide sequence. This results in a changed genotype, the actual genetic sequence has changed. There are multiple types. Homologous recombination: Most common type that occurs. This is due to sequence similarity between the part that is foreign, and the cell DNA. Also referred to as crossing over, but it is NOT the same as what happens during Meiosis. The idea that we have long stretches of sequence similarity between some foreign piece of DNA, and the bacterial chromosome. The name homologous tells you that you have to have sequence homology between the two pieces. Have to have similarity, not a random event. In order for this to happen, we have to first have “nicking” happen. There are two pieces of double stranded DNA, the chromosome and the foreign piece. We have to cut part of those strands so that recombination can occur. This involves an endonuclease – an enzyme that can cut inside a nucleic acid sequence – which comes in and cuts one of the strands of DNA, not both of them. Single Stranded DNA Binding Protein comes in to stabilize the loose end. Binds to the free DNA to help prevent degradation of it. From there, RecA protein comes in and helps the single strand “invade” the portion of the host cell chromosome where we are trying to get recombination to occur. This process is called strand invasion. Have foreign DNA come in and bind partially to the host cell chromosome. This process continues, and you end up with the “crossover event.” You have portions of the foreign DNA, and portions of the host cell chromosome that are interacting with each other. This crossing is sometimes called a Holiday Junction. This is the site where recombination will occur. You have another enzyme, known as resolvase that comes in to help resolve the crossover area (holiday junction). The result of this is usually that part of the foreign DNA becomes incorporated into the recipient DNA, and part of the chromosome DNA gets incorporated to the foreign piece of DNA. Sitespecific recombination: Will not talk about this so much. Doesn’t depend on sequence similarity. This is just due to the fact that there are areas of the genome that are more prone to recombination. Compare and contrast insertion sequences and transposons. o Transposition: Not dependent of long regions of sequence similarity. This is based on certain genetic elements – dependent on insertion sequences or transposons. This is done by transposable elements. Transposable elements are pieces of DNA that can participate in transposition. It gets a little more specific than that, there are specific types. They can be moved around in the chromosome, usually to specific sites. They all have a gene that are responsible for the transposase production. All are bound by inverted terminal repeats, and flanked by direct repeats. These repeats have a lot to do with their ability to move, and where they get incorporated. [GAGTCAT(transposon)TACGAGT] = basic structure, with both inverted repeats and flanking direct repeats. There are two types of transposable elements: Insertion Sequences: The simplest type. They are small relative to other transposable elements and only code for proteins implicated in the transposition activity. This protein is often transposase, which allows the insertion sequence to move. Transposons: Often abbreviated as Tn. These sequences carry all of the same stuff as an insertion sequence, but also carry other genes with them. They often carry antibiotic resistance in bacteria, for instance. Understand the mechanisms of transposition (simple and replicative). o Simple: Known for having other genes than just the flanking repeats and the inverted repeats. Transposons may carry antibiotic resistance genes. They can move within a bacterial genetic sequence, but also between organisms. Act through a cut and paste mechanism. Gets cut from one part of the genome, and inserted into another part of the genome. o Replicative: Same three basic elements as the simple mechanism, but now we also have a resolvase gene, which is needed due to the mechanism that these transposons move around. Act through a copy and paste mechanism. This is similar to homologous recombination – happens where a portion of the transposon is copied, and then inserted into a different area of the genome. Have the transposon in both the original spot, and the new spot. Need resolvase to close the crossover region. Describe bacterial plasmids, their significance in a bacterial host, and their role in horizontal gene transfer. o Bacterial Plasmids: Separate circular piece of genetic material (DNA) within the bacterial cell. Plasmids may or may not be incorporated into the cell genome. Episomes is the term for a plasmid that gets incorporated into the host cell chromosome. Must have an ori: This is the origin of replication, where a plasmid will begin replication. They have a distinct copy number: The number of times you will find a plasmid within a cell. Some plasmids are single copy plasmids, which means you’ll find only one in the cell. There are also multicopy plasmids that can be present in much larger number. They carry genes that may confer a selective advantage: A number of pathogens causing virulence factors. These factors help the pathogen cause infection, but aren’t needed for basic survival. o R plasmids, also called R factors: These are called R plasmids/factors because they carry a tremendous number of genes for resistance to antimicrobials. Some are conjugative (passable through conjugation), which means some are also F plasmids. They are usually not episomes, they usually stay as selfreplicating plasmids. o Fertility (F) Plasmids: also called F factors – conjugative plasmids (can be passed through conjugation). Have a tra region, which typically contains the genes for a sex pilus. Can transfer copies of themselves to other bacteria during conjugation. If a plasmid has a number of resistance genes, but also has the tra region, it is also an F plasmid. These plasmids can be episomes. Once they get transferred through conjugation, they may incorporate into the cell genome. If there is a plasmid that has a tra region, but no resistance genes, it is just an F plasmid. Be able to read a plasmid map. o Need to be able to recognize the basics, such as the ori as the origin of replication, and the Tn that is a transposon, and IS that are insertion sequences. All of the rest of the other genes are typically resistance genes. If there is a plasmid that has a tra region, but no resistance genes, it is just an F plasmid Describe the process of bacterial conjugation. o Conjugation: The idea that you have contact (direct) dependent DNA transfer. A donor cell gives DNA to a recipient cell. The donor cell is typically an F+ cell. The donor has an F+ plasmid that creates a sex pilus, and then transfers the F+ plasmid. The donor starts as F+ and ends as F+. The recipient starts as F and ends as F+. The cells usually start by touching each other. Once contact happens, the formation of the sex pilus occurs. The pilus retracts after it forms, and brings that back in touch with each other so DNA transfer can happen more readily. A single strand from the plasmid gets nicked, and is passed to the recipient. The plasmid DNA is replicated at the same time in both cells, and you get double stranded copies of the plasmid in both cells. Understand the process of transformation (natural and artificial) and the concept of cell competence. o Competent cells are cells that are able to pick up DNA from the environment. Natural competence is that cells can just do this in their natural environment, and it may be triggered by stress. They, on their own, can pick up DNA from their environment. We have ways of creating competent cells in the lab. We can force cells to pick up external DNA. This competence is named after the way you force cells to make them competent. Chemical: Chemically competent cells. Electricity: Electric Competent cells. This plays an important role in horizontal gene transfer – usually results from the lysis of one cell, releasing DNA in the environment. o Laboratory competency: Might also be called artificial transformation – you’re forcing the cell to take up the external DNA. Many of the techniques boil down to that you temporarily form holes in the cell envelope. The cells take up anything else in the medium. You damage the cell envelope enough that anything can be taken in for a period of time. You’re just stacking the odds in your favor. Whenever this is done, there is a huge amount of cell death that occurs. If you get too many holes, or the cell can’t repair fast enough, the cell will probably die. The success rate is very low. One method of artificial transformation is a heat shock method – take them through a series of hot and cold steps, and causes slight degredation of the cell envelope. Electroporation – pretreatment of cells occurs, but also put into a device that allows giving a shock to the cells to open the pores of their envelope. Know the significance of the following abbreviations: RecA, IS, Tn, F+, F−, tra, ori. o RecA: Abbreviation for the protein that assists in homologous recombination. o IS: Intron sequence – abbreviation on a plasmid map that indicates an intron. o Tn: Transposon sequence – abbreviation on a plasmid map that indicates a transposon, which typically carries resistance genes with it. o F+: Indicates a plasmid that is capable of initiation of conjugation. Fertility plasmid is present. o F: Indicates a cell that is not capable of initiation of conjugation. Fertility plasmid is not present. o Tra: Abbreviation on a plasmid map that indicates o Ori: Abbreviation on a plasmid map that indicates an origin of replication. All plasmids should have this. Compare and contrast generalized and specialized transduction. o Transduction – transfer of bacterial genes by viruses – ultimately fatal for the phage. Virulent bacteriophages (reproduce using a lytic life cycle) and temperate bacteriophages (reproduce using a lysogenic or lytic life cycle) can be involved in this. A mistake usually occurs during the lysogenic life cycle of the temperate phages that allows for transduction. There are two types. Generalized transduction: Phages that are capable of this can carry any and all bacterial genes and transfer them to another. No specificity. Specialized Transduction: Phages are only able to pick up genes from specific locations in the bacterial genome. This is carried out only by temperate phages that have established lysogeny. Only specific portion of bacterial genome is transferred. When the virus goes to switch to the lytic life cycle, they cut themselves out of their location in the bacterial genome. This means that ONLY the genes from around that site can get excised with the viral genome. You have potential for transduction, and creates a defective phage that cannot replicate. If everything goes per normal, this will not happen, and the viral genome will just cut itself out of the bacterial genome, and go on to replicate and lyse the cell. If a mistake occurs, the viroids can be assembled, but they contain the bacterial genes, and leave behind viral g
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