Bacteriophages

Lysogen

Bacterium carrying the prophage is a ______________.

Ex. 1. "I got it. This E coli is susceptible to phage 11682, etc" and now, that's it--this person who got the infection last week also had this specific E. coli serotype 2. See who walked in 2 weeks ago, carrying this type of staph aureus and you are able to quarantine them, etc. *Good for epidemiology because you can find out who is responsible for this outbreak

Can you give an example of how we are able to ID serotypes of bacteria?

1. Viruses in general are composed of 2 molecules. Nucleic acid and protein. *Lytic viruses you should remember can be RNA or DNA, unlike temperate phages which can only be DNA

Composition of viruses

Infection by phage produces many progeny and breaks open (lyses) the host bacterium

Define: Lysis

-LYSOGENIC CONVERSION is the term applied to the new properties that a bacterium acquires as a result of expression of the integrated prophage genes *E.x. Non pathogenic bacteria becoming pathogenic

Define: Lysogenic conversion

After infection, the phage DNA integrates into the host genome and resides there passively - No progeny - No lysis of the host - But subsequently, lyse (lysogeny)

Define: Lysogeny

1. A method used for detecting single strains of bacteria. It is used to trace the source of outbreaks of infections. 2. The viruses that infect bacteria are called bacteriophages ("phages" for short) and some of these can only infect a single strain of bacteria. 3. These phages are used to identify different strains of bacteria within a single species.

Define: Phage typing

A coliphage is a type of bacteriophage that infects Escherichia coli.

Define: coliphage

Cell lysis releases the phages (lysozyme used to weaken bacterial cell wall during lysis)

Explain the fifth step (Release) in replication of a lytic bacteriophage

-Tail fibers of bacteriophage find some receptor in cell wall of bacteria that they are able to attach -LPS, PTG, or flagella/pili may serve as receptors to attach phage to cell. Varies quite a bit--and has to absorb receptors

Explain the first step (Adsorption) in replication of a lytic bacteriophage?

Maturation - the assembly of viral parts into complete virus particles. *Phage DNA and proteins are synthesized into new phages

Explain the forth step (maturation) in replication of a lytic bacteriophage?

-The penetration stage refers to penetration of bacteriophage nucleic acid into the bacterial cell. * 1 big difference between bacteriophages and viruses that infect us (animal cells) is that with bacteriophage, only nucleic acid gets into the cell...everything else stays outside (protein components).

Explain the second step (penetration) in replication of a lytic bacteriophage?

Biosynthesis - production of: i.) early proteins - enzymes for new phage genomes and ii.) late proteins - capsomeres, lysozyme

Explain the third step (biosynthesis) in replication of a lytic bacteriophage?

1. virus gets nucleotides from host DNA to make its own DNA in virus progeny. 2. Cleaves bacterial chromosome DNA and uses nucleotides to assemble to make viral DNA, not bacterial DNA

First piece of viral DNA gets into bacterial cell--where does it get nucleotides to make its own DNA in virus progeny?

-Lysogenic cycle = temperate phage. -DNA from the lysogenic bacteriophage penetrates into the bacterial cell, circularizes, and then integrates into the host chromosome. *Prophage = integrated DNA of bacteriophage. . -The bacterial cell can reproduce over and over again as the cell doubles, more phage made as it doubles! -Then phage wants to disintegrate and go through lytic cycle--ticking time bomb. Will want to break free eventually. That's lysogenic.

Give a brief overview of what happens when a bacteriophage goes through the lysogenic stage?

1. Get that tube, and dilute it--1000/ml. 100/ml. 2. Make serial dilutions. 3. Take sample from each diluted tube and place on a petri plate 4. There is bacterial lawn on the petri plate, and drop suspension from tube onto this bacterial lawn 5. Count viruses by counting holes (plaque) in that lawn of bacteria. ex. There's 15 or so holes/plaques here--15 plaques = suspension had 15 viruses in it.

Given test tube with broth in it, there may be 10million or 10billion phages--how do you count them?

Exotoxins in bacteria as a result of being infected with prophage. So prophage has genes for its own reproduction, but also genes for toxin production and other things.

How do exotoxins became introduced into a bacterial cell?

Lysogens are IMMUNE to further infection by similar phage because the phage functions are REPRESSED

Lysogens are ______1______ to further infection by similar phage because the phage functions are ________2___________

TRUE *Let's make up a number: 100 different genes in the prophage, maybe 50 involved in replication cycle (early/late enzymes) but also a chunk of genes that encode for toxins, that make us very sick and are responsible for infections.

T/F: In addition to the lysogenic bacteriophages genes for its own reproduction, it often caries genes for toxin production

FALSE *ALL viruses have to be one or the other, either DNA [OR] RNA

T/F: It is possible to have a virus that has both RNA and DNA

TRUE

T/F: Let's say this bacteria is diptheria bacterium. This bacterium isn't pathogenic--it cannot cause diptheria. But if it's infected with prophage that carries gene for diptheria toxin, this bacteria can now produce diptheria.

TRUE

T/F: Lytic phages can either be RNA or DNA.

TRUE

T/F: Phage injects DNA, goes into the cell and it circularizes first before entering the lytic cycle or the lysogenic cycle.

TRUE

T/F: Sometimes in the lysogenic cycle when the prophage disintegrates from chromosome, by mistake it may take along the bacterial DNA that is adjacent to it with it. Whereas in the lytic cycle, almost any gene of the 1000's in bacterial cell may be picked up and brought to another bacterial cell

TRUE

T/F: Sometimes you can have hundreds of T2 phages on 1 cell--but usually only 1 phage will inject its nucleic acid into bacterial cells

TRUE

T/F: T-even group bacteriophages are virulent viruses that carry out a lytic cycle of infection in prokaryotes.

TRUE *Temperate phages integrate their DNA into the host DNA of the bacterium... cannot be RNA, RNA will not integrate into host genome

T/F: temperate phages ALWAYS have DNA not RNA

T2 phage infects E coli through tail fibers--something on the tip of these tail fibers that recognizes something on surface of E coli to which it can bind and attach

T2 phage infects _________?

galactose gene ***Integrate phage DNA into host chromosome now called a prophage (gets replicated when the chromosome replicates)

The lambda phage (lysogenic bacteriophage) that attacks E coli always inserts at the same site near a locus for the _____________ gene.

Prophage

The phage genome integrated into the host bacterial genome is a _________________

The replication of bacteriophages is a FIVE-step process

The replication of lytic bacteriophages is a _______-step process

Viruses that infect bacteria

What are bacteriophages?

1. Diphtheria 2. Botulinum 3. Cholera 4. Erythrogenic toxins

What are examples of toxins that are encoded by the genes of the integrated bacteriophage (prophage)?

-infect bacterial cells -replicate to large numbers (progeny) -cause the cell to lyse (burst) - lytic cycle

What are the characteristics of a bacteriophage that is a LYTIC PHAGE?

-infect bacterial cells -DNA integrates into the host genome - resides there -Do not cause the cell to lyse (at least not right away) - lysogenic cycle

What are the characteristics of a bacteriophages that is a temperate phage?

1. Adsorption - Attachment by tail fibers 2. Penetration 3. Biosynthesis 4. Maturation 5. Release

What are the five stages in the replication of a lytic bacteriophages?

T2: 1. head 2. neck 3. collar 4. sheath 5. base plate 6. tail fibers

What are the structural components of a T2 bacteriophage?

1. Lytic phages - e.g. - T2 2. Temperate phages - e.g. - lambda

What are the two classifications of bacteriophages?

1. Phosphorous is an element that is in nucleic acid but never in proteins or amino acids 2. Sulfur is in amino acids an never in nucleic acids 3. So, they used isotope P32 to label nucleic acids and S35 to label amino acids 4. They attached the radio-labeled phage to E.coli and let them grow/inject their products into the bacteria 5.What they established is that only P32 was able to get into the bacterial cell but never S35 meaning that the nucleic acid component was the only thing injected from the phage into the bacterial cell

What experiment did Hershey and Chase do to determine if nucleic acid and/or proteins from the bacteriophage entered the host bacterial cell?

Integration of foreign DNA into resident DNA = recombination

What is genetic recombination when referring to the lysogenic bacteria and host bacterial cell?

-Made up of a capsid: protein coat -Inside the head= DNA/RNA of the phage -Individual proteins that make up capsid are called capsomeres

What is the 'head' of a bacteriophage made up of?

1. A culture of the strain is grown in the agar and dried. 2. A grid is drawn on the base of the petri dish to mark out different regions. 3. Inoculation of each square of the grid is done by a different phage. 4. The phage drops are allowed to dry and are incubated: 5. The susceptible phage regions will show a circular clearing where the bacteria have been lysed, and this is used in differentiation

What is the process of phage typing?

T2 capsid: -Icosahedral symmetry -- 20 triangles - form an icosahedron -Sphere

What is the shape of the T2 bacteriophage head?

The sheath of T2 will act like a muscle/syringe and push DNA from the head into the cytoplasm of the bacterial cell *happens within nanoseconds

What structure of the bacteriophage T2 is responsible for contracting like a muscle/syringe to get its DNA into E coli?

1. The prophage will consist of only lambda DNA (infectious phage primarily produced) 2. The prophage will consist of lambda DNA and the galactose gene from the adjacent strand of DNA in the hosts chromosome (transducing, non infectious lambda phage rarely produced)

When the lambda gene is being disintegrated from the bacterial chromosome it has two fates, what are they?

In lab we can shine UV light on bacteria to induce disintegration of prophage so it can replicate and go through lytic cycle

When we work with lysogenic bacteriophages in lab, how could we speed up the process of disintegration of the prophage?

1. Capsomeres are formed inside bacterial cells that are infected--these capsomeres are somehow self-assembled and surround a nucleic acid. 2. Maybe 1 type of proteins or different capsomeres formed depending upon the phage.

Where are the capsomeres that come together to make the capsid (protein coat around 'head' of bacteriophage) produced?

1. Bacteria are fairly simple systems, and viruses that infect bacteria--learn this process of replication cycle--it's easier to study in a bacterial cell 2. Another important reason: think about what they do--they infect bacteria, and eventually will cause bacteria to lyse--may be a great way to treat bacteria that cause infections rather than using antibiotics 3. Bacteriophages are also very specific... E coli only infects E Coli. If we know what type of bacteria we are infected with, maybe we can use a bacteriophage to destroy these.

Why do we study bacteriophages?

-PHAGE TYPING. -This procedure involves determining the sensitivity (+ or -) of a strain to a variety of standard bacteriophages.

Within a species of staphylococci, individual strains can be identified by a procedure called ____________. What does this procedure involve?

1. Different serotypes = different properties 2.Some are harmless, pathogenic, or very deadly. 3. Try to ID bacteria, especially pathogenic ones by phage typing. 4. By ID'ing which phage will infect which serotype, we will see if it's one serotype or another and see if they're pathogenic or not.

Within each species of bacteria, there's different serotypes. How can you ID each of them if you couldn't actually visualize them? What properties?

INDUCTION of the lysogen leads to EXCISION of the prophage, REPLICATION of the phage DNA, production of more phage and LYSIS of the host bacterium.

______1_______ of the lysogen leads to _________2________ of the prophage, _______3_______ of the phage DNA, production of more phage and ________4_______ of the host bacterium.