Bacteriophage
What are the two ways a bacteriophage can replicate?
Two ways to replicate: Way that you already know is caleld lytic cycle. Phage gets in, replicates itself up to 100/200 phages. Lyses open bacteria and then gets out . Viral genes fully expressed. Virulent phage (lytic phage) MS2 is lytic it has a lytic enyme to break open E.coli Lysogenic cycle Phage DNA inserted into the host (like HIV) chromosome sometimes at a specific site- prophage. Site specific recombination system is something that can cut DNA and then insert and ligate it. It is a like a ligase and restriction enzyme rolled up into one thing. Looks for certain sequence patterns. Once DNA is inserted into the host, it becomes part of host E. Coli/bacterial chromosome, and replicates when bacteria replicates. So if bacteria divides 2,4,8 you get 2,4,8 prophages. Once phage is inside chromosome - its caleld a prophage. Is very similar to how HIV works, it even uses a similar enzyme called integrase for part of that job. Most of phage functions are switched off. Temperate phage - Once it is integrated it just sits there - Good trick for bacteriophage. If it can't be bothered killing all the bacteria, it can just take a holidays and insert itself into chromosome and sit there for a few months.
What is the restriction modification system that bacteria use against phages?
Restriciton modification system: Every bacteria has an enzyme that chops up DNA Each bacteria has its own restriction enzyme. That restriction enzyme doesn't cut its own dna because it methalates its own DNA which stops restrictio nenzume fro mworking. But when phage comes in its restriction enzyme will cut every 4000 base pairs and will chop up that phage DNA into quite a few different bits. The resitriction modeification system came from phages, the bacteria protects its own DNA by methylating but chops up any invading DNA using that enzyme.
How does recombination occur with phage and bacteria?
Site specific recombination system. This is the attachment phage, Attachment P and attachment B for bacteria. This is usually a conserved sequence in the E. coli that the phage recognises. These two join up, so we get cutting (restriction bit) and then lygation and production of the prophase. Its restriction enzyme and integrae.
History of bacteriphages
1915-1917: discovered by Twort and d'Herelle independently 1920: combat pathogenic bacteria, bacterial strain typing. Not only to kill infections but also used to type strains 1940-1970: foundation of molecular biology. Restriction enzymes, cloning, plasmids all sorts of things, mapping of E. Coli chromosomes. Many nobel przies won by people working with these phages Phages found in over 140 bacterial genera, pretty much every bacteria has a few and iscovring more all the time Over 5100 phages examined by EM Bacteriophage is derived from Greek - Bacteria eater
Who discovered bacteriophages? how did they discover them?
1915-1917: discovered by Twort and d'Herelle independentlyHe discovered bacteriophages is 1918: This guy travelled all over the world and tried to kill locuses with bacteria The nobel prize of immunilogy told him that he was crazy and there was no such thing as a bacteriophage, and he wasn't even a properly trained scientistl Acientist acedemia establishemnet poo pood him for a while But he proved them wrong Was the first person to think about using bacteriophages to treat bacterial infections
What is used to classify a phage
4900 tailed phages, 5100 phages in total, so only 190 other (non tailed viruses) Pahge for salmonella will only affect salmonella, phage for E. Coli will only infect E. Coli. That's how they are used for typing - one phage per species. The classification is worked out by: What bacteria does it infect What genome does it have Its morphology Ability to lysogenize A temperate virus is also a lysogenic virus. Got over 110 families of virus now to infect animals. A lot less here, but still a lot of viruses out there When he did sequencing of bacterial genes they had to clone genes in M13 vector so that you can have nice single strand which increased base pair reading from 200 to 400.
What are some of the advantages of bacteriophages and antibiotics?
Advantages of bacteriophages: Very specific - affects the targetted bacterim only Change of developing secondary infections reduced No side effects Phage able to reproduce as long as host bacteria is available - therefore repeated administration is greatly reduced. Development of resistance may lead to attenuation of virulence Selection of new phages can overcome resistance and is easy Production is simple and relativey inexpensive Advantages of antibiotics: Can be used without knowing the ID of the bacteria
What is the structure of a phage?
DNA sits inside sheath. It sits on top of the bacteria, it will inject a bacteria with its DNA through this sheath and the bacteria phages held on to the bacteria using these tail fibres and tail pins. Tail fibres attach to specific receptors. This digs into the membrane of the bacteria (outer membrane) the DNA is injected out of there, through the sheath and into the bacteria. The capsid never enters the bacteria, its on the outside, only the DNA goes in. and then it starts to take over the bacteria cell. Can attach to LPS, pili (MS2 gets into bacteria through pili) and lipoproteins. Many structures they can use to get into cell.
What are some of the disadvantages of bacteriophages and antibiotics?
Disadvantages of bacteriophages: The disease causing bacteria must be identified before therapy Disadvantages of Antibiotics: Non-specific action that targets pathogen and normal flora- this can affect microbial balance leading to secondary infections Multiple side effects: yeast infection, allergies, intestinal disorders Repeated administration is needed Antibiotic resistant bacteria remains pathogenic Development of new antibiotics takes years Production is expensive
Describe and explain injection of nucleic acid in lytic cycle
Hershey and Chase Experiments - 1952 Phage DNA carries genetic information into the cells. All of this protein structural stuff never gets into the cell. Only the Dna does. This famous hershey chase experiment done in 1952, basically proved that DNA are not protein, it's the genetic material of life. Labeled protein with S35 and DNA with P32, and only DNA labelled P32 was passed down to subsequent generations. S35 couldn't see after first infection. Phage has pins and legs to help it attach the bacteria, and then get injection, tail contraction penetration pushes the injection into the bacteria and then DNA goes down. DNA then needs to do something.
Describe and explain Adsorption
Highly specific: Each phage tends to like its own species of bacteria Interaction between tail and cell surface receptors (can be lipoproteins or LPS) Host range mutation may occur in phages - If a phage has a mutation, it can on rare occasion can switch the species barrier. It might mutate, say before it couldn't get into one bacteria, but because it has mutated it will have a slightly different tail pin and then it can get into a different species of bacteria. Host range can increase through mutation under certain circumstances. E Coli vs phages When it bursts open will get about 200 phages coming out. Bacteria aren't very big only about a micron. Our cells are about 20-40 microns, so how many viruses coing out of our cells? Way ore then 200, sometimes 25-50 thousand coming out of a human cell.
What are some mechanisms bacteria use as resistance against phages?
If you are using bacteria to make yoghurt or cheese, worst thing you can get in factory is a phge Once bacteria gets in, it kills all good bacteria This has plagued bacteria for years and year so started engineering phage resistance into bacteria. Blocking of phage DNA injection Abortive infection systems - interfering with phage DNA replication, DNA transcription, phage development, and morphogenesis - stopping phage assembling into virion The evolutionary arms race between bacteria and their phages has been well documented by dairy microbiologists. Geneticaly engineered phage resistance: Per-phage encoded resistance Iby the bacteria) say phage needs pili to get into bacteria, get rid of pili. Antisense RNA (Crispa/cas): Bacteria can colelct a bit of phage DNA to use against phages. This lead to development of Crispa Cas system for DNA editing. Bacteria colelcts sequence data from a phage and can use it to attack the phage.
What is a plague? how does it form?
If you plate out bacteria, there will be a lawn of bacteria (Which is a whole lot of bacteria) If you infect them with a bacteriophage, what you will see are these clear areas called plaques. We do this stuff wth animal viruses as well except infecting cells rather than bacteria. And this is where phages have infected cells with bacteria, and have created a small zone of clearing known as a plaque. You can use these plaques to count how many pahges. One plaque = one phage. Plaque formaiton is very important. It's the only way to see bacteriophage except for if you have an electron microscope.
explain and describe steps of transcription and translation process. how does bacteriophage transcript and translate genetic information?
Immediately the early genes (like in herpes viruses) the pacteriphage doesn't just make all its gene products all in one go. It has three ways, early midle and late. Say it made the late genes first before it has replicated its DNA, it means its made a lysis protein that has killed bacteria before it has even replicated. So can't have all genes made in one go. Have to do sequential gene expression. Human viruses work similarly to this way. Immediate early and delayed early genes - shut off cellular macromolecule synthesis of bacteria, tell bacteria to stop making their own proteins. The get replicaiton of phage DNA Middle Genes - transcribed throughout infection DNA replication and recombination - Then get integrase and those enzymes needed in lysogenic phase, that will get phage DNA into chromosome of bacteria Late genes - capsid protein and enzymes for lysing the cells. Each capsid has at least 180 proteins (maybe 240 if a big phage) that's a lot of proteins, so need to make a lot of capsids at the end and package DNA up into the capsid, then lysing enzyme and out the DNA goes. All viruses are parasites- what they really paristise is the ribosomal machinery of the cell. Viruses can't make own protein, so need cel lto make proteins. They hijack ribosomes- they tell the cell to stop making own proteins to make the viral proteins and most abundant are strctural ones, T4 is main component of the head. The capsid protein is main protein of capsid head
what are the steps of the lytic cycle?
Infection of most phages, every phage pretty much has a lytic cycle but some phages have lytic and lysogenic. Lytic cycle is easy with 5 stages: 1. Adsorption - Attaches itself to surface of bacteria 2. Injection of genomic nucleic acid, down sheath of bacteria. 3. transcription and translation 4. replication of DNA 5. Maturation and Release: The phage structure comes together, packages together the DNA and then releases (usually by lysis) of the phage into the environment to infect more bacteria.
What is phiX174
It's very small phage no tail, icocahedral. It's genome is very small - only 5000 base pairs. Phi X174 one of the most simplest viruses. Has enough nucleic acid to code 3-5 gene products while the more complex phages may code over 100 gene produces. The phage phiX174 was the first organism to have its entire nucleotide sequence determined, a feat that was accomplished by Frederick Sanger and colleagues in 1977.
What is M13?
M13: Circular ss DNA filamentous. No lysogenic cycle. Worm like phage
What are some applications of bacteriophages?
Model system of molecular biology (many nobel prizes have been won using the phage Cloning and expression, Phage display systems, Phage typing (typing salmonella - classic example), Phage therapy: phages as natural, self-replicating antibiotics. People ditched working with phages when anti-biotics came out. Conventional bacteriophage therapy: In early days bacteriophage therapy was used by making bacteriophage preperation and are effective agaisnt P. aeruginosa, E. Coli S. aureus, Streptococcus and Proteus. The first reviwed report of the therapeutic efficacy of PahgoBioDerm (cocktail of lytic bacterophages) was bublished. 107 patients with ulcers - failed to resposne to conventiona ltherapy (comin in diabetics with skin ulcer from bacteria) With PhagoBioDerm - Ulcers healed completely in 67 (70%). Was basically a bandage with a little bit of medication with phages in it. Nasty legion on this guys back, put phage bioderm over the top and thenits cleared out after couple of weeks. This is a staphoryious skin infection. It lives all over our skin but some people get infected by it. It's still used today to treat skin lesions. Antimicrobial wound dressing biodegradable polymeric film (artificial skin) containing bacteriophages.
phage morphology and genomes
Most phages are double stranded and look like this. Over 95% of all phages have head tail configuration. This is icosahedral head and has a sheath here which is a helical structure. These are main families shown here Most of these phages have a 40, 50, 100 thousand base pairs of DNA which means they can make about 100-200 proteins. That's a lot more than human viruses. That's a lot more proteins than most viruses that would infect us would make apart from herpes which makes about 150-200. Can get every congifugration - double stranded Rna, Dna, circular. Lots of configurations of genomes similarly to human viruses.
What is myoviridae (What is T4 and T7?)?
Myoviridae P2, T2 and T4 are most famous family of phages. Can get single stranded phage DNA which are very small. linear ds DNA, icosahedral, lysogenic cycle. These are the head-tail phages they can have a lysogenic cycle
Use Phage P1 as an example to explain transduction
P1 is a phage famous for transduction - used to map E. Coli genome. Transduction is the transfer of bacterial DNA from one bacteria to another, usually same species, because phages are species specfici but phage is the vector that does the job of using a bit of DNA from one E. Coli into another E. coli. About 1/10,000 of the P1 phage particules carry chromosomal DNA - on the end of the phage DNA from E. Coli. From the E. coli. They have own DNA (94, 000 base pairs) plus a little bit maybe 10,000 or 5,000 from the E. Coli it has just infected. It can do this because it has terminal redundancy on either end of its genome. It's linear in the virion but in the cell it circularises for replication Say you insert DNA from E. Coli into the genome, the phage will still function because it has the genes for function on this end, so it has 15kb of DNA which is duplicated which means it can capture E. Coli DNA in the other bit. Termed terminal redundancy. Importance of this is that DNA can be inserted into it without infecting the phage. The phage can still functin and carry out its lifecycle. The foreign DNA can be moved around from bacteria to bacteria. Some cells will gain a gene from the other E. Coli. So recipient cells have encorporated donor DNA and thorugh recombination will push it into their chromosome and have gained a piece of DNA from original E. Coli. The phage was thing that did the job of mocing it from one E. Coli to another. The phage components - this phage here is infecting the recepient bacteria by inserting through recombination a little bit of DNA from a bug into transuced bacteria to gain function from new gene.
What is Phi 6?
Phi 6 - this is a bacteriphage but it is thought that some of the human viruses actually came originally, it's the ancestor of some human viruses. A few of these around, but not that common.
What is lysogeny?
The indefinite persistence of the phage DNA in the host cells, without phage production Temeperate phages: can produce the phenomenon of lysogeny which means it higes in DNA of E.Coli Prophages: the integrated non-infectious form of phages (this is the result of lysogeny) replicates when bug replicates and is a very clever way to hide.
Use lambda as an example to explain gene suppression in lysogenic cycle
The life cycle of lmbda phages is controlled by cl and Cro proteins. The lambda phage will remain in the lysogenic state if cl proteins predominate - when nutrients are low C1is stable and leads to lysogenic cycle (these are bad conditions) Cl repressor turns off phage transcriptor Integrase catalyzes integration of lambda DNA into bacterial chromosome via short sites of homology (site-specific recombination) - prophage The phage will transform to the lytic cycle if cro proteins predominate. Lets say food suddenly comes up, the phage now wants to come out. And knows if it comes out now ther will be lots of bacteria around. When nutrients are high, more proteases are in the cell and leads to breakdown of repressor cl-leading to lytic cycle. Allowing for more Cro, which leads to lytic cycle.
Explain how genes of phage are suppressed in lysogenic stage
When phage enters chromosome, it only makes a few proteins. Maybe 2 or 3, not many. Very limited transcription. This is a control pannel. Main mechanism is repression of phage genome usually in bad times. If bacteria is struggling for food for example, it will go into lysogenic stage and hide away. Its struggling and phage doesn't want to struggle. Makes proteins that will stop any transcription of left and right area of the genome. Most phage genes except for repressive genes are shut off. Repressed phage genome - repression is specific. If you have prophage in your genome can't have infection of another phage. Makes few proteins in lysogenic stage, usually just to repress genome.
What is lambda?
linear ss DNA, icosahedral, lysogenic cycle Used in many molecular biology experiments. Used to make gene libraries in the 90s of bacteria and other things. Hepititis C was discovered from a gene library using bacteriophage lamda in 1987ish
What is MS2?
ssRNA icosahedral, released by lysis. Single stranded Rna genome - liviviridae MS2 Used as a control in molecular biology assays or final diagnostic assays. We can count it, plate it out, can use it as a control. It is used to test things like hepofilters in airlines. The air-con system in a plane goes through a hepofilter that keeps all viruses out. To check that filter is working using MS2. it's a small virus (only 25 nm) almost smallest virus you can get, icosahedral, 180 proteins make up the shell of MS2. that MS2 phage goes into clefciella or E. Coli through the pilus. It uses pilus to enter the bacteria. This phage only has 4 genes, a very small genome. This protein helps package RNA into the icocahedron of the phage. Capsid protein has 180 of those to form together to form the capsid. The RNA dependent RNA polymerase which replicates the RNA genome with help of a couple of E. Coli proteins. Lysis protein - after the infection the phage needs to get out of the bacteria so it can infect more, so it has a lysis protein that will break open bacteria to release the phage. This phage gets into the E. Coli by attaching and using the pili. Can see MS2 guys holding onto pili (in the photo) this is how the phage uses the F-pili to get inside. They have some sort of attraction or bonding that gets the phage onto the pili and it will get phage into that cell.