Bacteriophage T4

T4 is a bacteriophage that infects Escherichia coli bacteria. The T4 phage is a member of the T-even phages, a group including enterobacteriophagesT2 and T6.

It is one of the largest phages, encoding roughly 200 genes and was the first prokaryotic organism providing evidence of gene splicing through presence of introns in the genome. T4 is capable of undergoing only a lytic lifecycle and not the lysogenic lifecycle.

History of T4.

T4 has been extensively studied and has a rich history in the advancement of genetics. Some of the first essential ideas of genetics came from studies using T4 including: the basis of genetic code, even ribosomes, mRNA, and the codon. It is surprising that science has learned so much using a virus which is extremely complex. Nonetheless, we owe much of our current knowledge to T4.

Genome and Structure

The elongated isocahedral head of T4 phage contains linear double-stranded DNA genome about 169 kbp long and encodes 289 proteins including two accesory proteins, HOC (highly antigenic outer capsid proteins) and SOC (small outer capsid proteins). Binding of SOC stablizes the capsid and resist osmotic shocks.

The T4 genome is terminally redundant and is first replicated as a unit, then several genomic units are recombined end-to-end to form a concatemer. When packaged, the concatemer is cut at unspecific positions of the same length, leading to several genomes that represent circular permutations of the original. The genome is AT-rich and contains modified bases in the form of 5 hydroxy-methyl-cytosine, rather than cytosin, which protect the phage DNA from many host restriction systems and from phage-encoded nucleases that degrade cytosine-containing host DNA during infection. The T4 genome bears eukaryote-like intron sequences.

The T4 tail fibres, pins and base plates are involved in binding to the lipo-polysaccharide receptor of the E.Coli. The T4’s tail is hollow so that it can pass its nucleic acid into the cell it is infecting after attachment. The tail attaches to a host cell with the help of tail fibres.

The life cycle of T4

1. T4 adsorption and Injection

Phage binds to the lipo-polysaccharide and tryptophan receptor on the bacterium. T4 looks for a susceptible bacterium with its tail fibers. The tail fibers recognise the membrane first. The phage once bound to the cell, the base plate goes under conformational change which causes the sheath to contract piercing the cell membrane allowing the viral core to enter the cell, and release the dsDNA viral genome.  The outer sheath contracts driving the internal tail tube in to the cell. The Gene product, gp5 (known as tail lysozome) facilitates digestion of the peptidoglycon layer of the bacterium to reach the inner membrane.

The trans-membrane electrochemical potential is required for transfer of T4 DNA to cytoplasm. The empty capsid remains extracellular.

2. Replication and expression of genes.

Once T4 DNA is in the cytoplasm, it specifies a highly organised and coordinated program of gene expression with the help of 3 promoters; Pe (early genes), Pm (middle gens) and PL (late genes). Some of the first genes which are transcribed, called immediate-early, encode enzymes which break down host DNA. Host DNA is broken down in order to use host nucleotides to produce more viral DNA. It also encode the proteins needed for DNA synthesis and to build the capsid and tail structures.

The host enzyme, RNA polymerase, transcribe and translate the expression of early genes. Early genes rely on the transcription apparatus of the host, being transcribed from normal sigma70 promoters.

Delayed early and middle genes encode the 20 proteins which are involved in viral replication. In fact, T4 encodes nearly all of its own replicative machinery. There are two distinct replicative processes of T4 which are outlined below:

  1. Stage 1 – Replication occurs in a bidirectional manner with multiple origins of replication within the genome. The first several rounds of replication are initiated by RNA primers synthesized by the host RNA polymerase. These RNA primers can travel to their complimentary region in dsDNA and displace the other strand to produce a structure called an R-loop. The attached RNA can now act as a primer for the leading strand of DNA replication. The lagging strand is then synthesized using the replicative helicase, gp41. DNA replication is finished by gp30 which is the T4 encoded ligase as well as the host DNA ligase. Several minutes after infection, host RNA primers cannot be used because the promoting recognition specificity is altered on the host RNA polymerase and recombinant-dependent replication (RDR) is favored. The mechanism of RDR is outlined in stage 2.
  2. Stage 2 – At this point the virus uses its own replicative machinery to transcribe its late genes with a process known as the gp45 sliding clamp model. The primers for leading strand synthesis are recombination intermediates instead of RNAs made by RNA polymerase. 

Early gene products alter the host RNA polymerase in two ways for expression of middle genes. Modifies RNA polymerase will not stop translation and over shoot to reach Pm (middle gene promoter). The enzyme is there by able to read through a transcription terminator by anti-termination mechanism to express genes downstream of early genes and is modofied to recognise middle promoters.

Transcription of the late gene is coupled to replication. Promoters for these genes again differ from sigma70 promoters and require an alternative sigma factor, encoded by T4 regulatory gene, gp55, to activate transcription. Binding of gene, gp55 to host RNA polumerase allows the enzyme to recognise specifically T4 late gene promoters. Other T4 gene products, gp44, gp45 and gp62 are additionally required for late gene transcription.

3. Assembly and Release.

Once late genes are expressed, the viral base plate is first assembled, this then attaches to the tail and tail fiber proteins. These three different protein pathways combine to form a mature T4 phage capsid. DNA is packaged into the mature capsid protein by packaging and cutting the concatemers using a terminase complex found at the end of the concatemerized DNA strand. The terminase complex binds to the capsid head and moves DNA into the empty capsid head. The capsid also encases necessary enzymes for future infections such as virally encoded DNA polymerase.

The viral encoded enzyme holin (gpt) creates the holes in the inner membrane of the bacterial host-cell to allow lysozymes to exit and degrade the peptidoglycan cell wall. Cell lysis subsequently follows releasing a shower of bacteriophage progeny into the extracellular space.

Lambda Phage


Lambda phage 
is a temperate bacteriophage that infects Escherichia coli.. It have alternative replication pathways: Lysogenic or Lytic pathways.

History of Lambda Phage

In 1950, Esther Lederberg, an American microbiologist, was performing experiments on E. coli mixtures. She happened to observe streaks of mixtures of two types of E. coli strains that seemed to have been nibbled on and had viral plaque. One E. coli strain had been treated with ultraviolet light, so the damage was more apparent. It was later determined that this was caused by bacterial viruses, which replicated and spread resulting in cell destruction. The discovery led to the employment of Lambda phage as a model organism in microbial genetics as well as in molecular genetics.

Structure and Genome.

Lambda phage is a virus particle consisting of a Icosahedral head, measuring around 50-60 nanometers in diameter, containing double-stranded linear DNA as its genetic material, and a flexible tail that is around 150 nanometers long and may and may contain tail fibers.Lambda phage.jpg

Lambda contains a linear double stranded DNA genome of 48.5 KB.

Lambda phage exists in two form. Inside the phage it is linear double stranded DNA; inside host double stranded circular DNA molecule.

 

The life cycle of lambda

1. Lambda adsorption

Phage identify a host bacterium by binding or adsorption to a specific structure on the surface of the cell. Many different cell surface structures can be used as binding sites.

Lambda binds to an outer membrane protein called LamB via a protein that resides at the tip of the lambda tail called the J protein. LamB normally functions in the binding and uptake of the sugars maltose and Maltodextrin.

 

2. Lambda DNA Injection

Initially, lambda binds to LamB and the binding is reversible. This step requires only the lambda tail and the LamB protein.

Next, the bound phage undergoes a change and the binding to lag LamB becomes irreversible. This nature of the change is unknown but it requires that a phage head be attached to the phage tail.

Next, the lambda DNA is ejected from the phage and taken up by the bacterium. The DNA in the phage head is very tightly packed. If the condensed state of the phage DNA is stabilized, ejection if the DNA does not occur.

In addition to LamB, lambda also uses an inner membrane protein called Pstm to gain entry to the cytoplasm.

 

3. Protecting the lambda genome in the cytoplasm of the bacteria.

Lambda contains a linear double-stranded DNA molecule in its capsid. In the bacterium cytoplasm, dsDNA molecules are subjected to degradation by exonucleases that need a free end to digest the DNA.

The first event that happens to newly injected lambda DNA is that the DNA circularizes to prevent it from being degraded.

How circulation of DNA occurs?
Lambda has a specific site on its DNA, termed Cos sites (cohesive sites), which is used to circularize the DNA. The cos site is a 22bp sequence that is cut asymmetrically when the lambda DNA is packed. These cos sites are complementary to each other. A host enzyme, DNA ligase, seals the nicks at either end of the cos site generating a covalently closed, circular lambda genome. Basically two cos sites: cosL and cosR.

Restriction Alleviation.

This is a uniqueproperty of this virus.Lambda phage is very small.It mimics the methylation system and its own DNA is methylated. Because of this restriction enzyme will not cleave that DNA and are protected.

Restriction enzyme actually recognises a specific DNA sequence and cleaves the DNA on the both strands. The cut or digested DNA is sensitive to nucleases that degrade DNA. The modification part of the system is a protein that specifically modifies the DNA sequence recognise by the restriction enzyme and prevent the DNA from being digested.

This restriction or modification system allows a bacterium to tell DNA from its own species from foreign DNA.

 

4. What happens to the lambda genome after it is stabilized.

In case of lambda all the genes involved in specific functions are clamped together. SubattPxisint is the gene resp possible for recombination. The genome contain six major promoters. Function of Promoter is to promote transcription by binding to RNA polymerase.

PL – promoter leftward
PR – Promoter rightward
PRE – Promoter for repressor establishment.
PRM – Promoter for repressor maintenance.
PI – Promoter for integration
PR1 – Secondary rightward Promoter

CI – Repressor
Cro – Anti repressor
Q, N – Anti terminator proteins
Nut R – Nutrilisation site (rightward)
Nut L – Nutrilisation site (leftward)
tRI – Terminator right ward
tL – Terminator left ward
PL/OL – Promoter and operator (rightward)
PR/OR – Promoter and operator (leftward)
O, P – DNA synthesis
Paq – Promoter anti q
q nut – Q nutrilisation site

S helps in transcription of R to peptidoglycon, altimately weakening cell membrane for lysis.

After the genome is circularised and supercoiled (The host encoded enzyme, DNA gyrase, supwrcoils the lambda molecule), transcription begins from PL and PR. A series of genes known as early genes are transcribed and translated. These early genes are namely N and Cro. These genes products are the initial proteins needed for further phage development.

E coli RNA polymerase interacts with PL to give rise to short mRNA transcript that is translated into the N protein. Transcription from PR leads to Cro protein.

The N protein is able to extend transcription when RNA polymerase encounters a sequence in the DNA that tells it to stop. For that reason, N is called an anti termination protein. N allows RNA polymerase to transcript through tL and tR1 termination signals resulting in the synthesis of longer mRNA transcripts.

The longer transcript from PR encode the O, P and Cll proteins and a small amount of another anti terminator, the Q protein.

From PL, Clll the recombination proteins and a small amount of Xis and Int are made. Cll and Clll are reffered delayed early proteins. Cll helps in expression of promoters. Clll protects Cll from host proteases.

When N is bind to nut R1 / nut L1 the termination of R1 and L1 occurs. N protein anti terminates by binding to RNA polymerase after a specific base pair sequence, has been transcribed into mRNA. This sequence is called nut for Neutralization.

At this point, all of the players needed to make the lytic-lysogenic decision have been made. Cll and Clll are needed for lysogenic growth. The O and P proteins are used for replicating the lambda DNA.

Operater OR is further divided into OR3, OR2 and OR1. There are will be competition between Cro and Cl in competing in respect to binding. Cro likes to bind at OR3, if it bind it prevent binding of Cl. When N is bind to nutR1 and nutL1 the termination of R1 and L1 is not occurred and it continues dissociation. If no dissociation O,P,Q,S and R is produced. Function of O and P is to synthesise DNA, so phage DNA is synthesized.

DNA synthesis occurs on lytic cycle. Q protein is going to bind Qnut site, termination will not occur so expression of S and R occurs. S and R are responsible for lysis of the cell.

 

5. Lambda and the lytic-lysogenic decision:

At the most basic level, the decision depends on the amount of two phage encoded proteins called Cl and Cro, and their binding to their promoter control regions.

When Cl is bound, the expression of the lytic gene is repressed and the phage follows the lysogenic pathway. For this reason Cl is also known as Cl repressor or lambda repressor. The expression and binding of Cro will lead to lytic development. Cro is made from PR and Cl made from either PRE or PRM. Both Cro and Cl binds to the same DNA sequences called promoters.

Lambda contains two operators that bind Cro and Cl.
OR – overlaps PRM and PR promoters.
OL – overlaps with PL.

OR is the major player in the lytic-lysogenic decision, while OL is not a part of the decision. OR is composed of 17 base pair sequences called OR1, OR2 and OR3. Cl repressor binds to OR1, ten times better than it binds to OR2 and OR3. At increasing concentrations of Cl, it will bind to OR2 and eventually OR3.

When Cl repressor binds to OR, it stimulates the PRM Promoter and the production of Cl repressor and inhibits the PR promoter and the production of Cro, leading to lysogeny.

Cro binds to OR3 first, then OR2 and finally at high concentration to OR1. When Cro is bound to OR, it inhibits the PRM Promoter and the production of Cl, leading to lytic growth.

How does the phage switch between thses development pathways?

The major protein involved in the switch ia another phage -encoded protein called Cll. Cll activates the PRE and PI promoters. This leads to production of repressor, Cl and the integrase protein, which is also needed for lysogeny. Cll is unstable and rapidly degraded by the host-encoded HfIA protease. Inactivate Cll leads to lytic growth. Cll can be protected by the phage- encoded protein Clll. Active Cll leads to lysogrni. Cycle or growth.

 

The lambda lysogenic pathway

If active Cll prevails, it activates PRE and PI promoters leading to production of repressor CI and the integrase protein. Eventually Cl activates PRM ensuring that a continuous supply of CI is made.

Lambda recombines into the chromosomes using a specific site on the phage called attP and a specific site on bacterial chromosome called attB. When the lambda DNA is in the chromosome, it is bound by attL and attR, which are hybrid attP/attB sites.

Once the lambda DNA is recombined into the chromosome, it is replicated and stably inherited by daughter cells as part of the bacterial chromosome. It stays in quiescent.

What prevent the expression of the late genes coding for lytic function?

The expression of late genes is prevented by the action of the lambda repressor, Cl. Lambda repressor binding to the operator sequences OR and OL blocks transcription from PL and PR. Since PR is blocked, the lambda Q protein is not made and transcription of the late genes does not occur.

 

The lambda lytic cycle.

If enough of the Q proteins accumulates in the cell, RNA Polymerase will continue its transcription from the third promoter, PR1 located infront of the Q gene. This extends transcription in to the late genes located downstream of Q. The late genes encode the proteins needed to complete the lytic infection including the head, tail, and lysis proteins.

The Q proteins which is made from PR, when N is present is a second anti-termination protein. It acts on the qut site and allows transcription through tR1. Q is necessary for synthesis of the head and tail genes.

After the infecting lambda DNA has been converted to a double-stranded circular molecule, it replicates from a specific origin using both the phage encoded O and P proteins and bacterial encoded proteins. This is called theta replication. Later in lytic development, lambda switches to a second mode of replication called rolling circle replication. Rolling circle replication produces long DNA molecules containing multiple phage genomes called concatomers. These concatomers are packed int to phage heads and mature phages are produced. 

The lambda R and S proteins are required for lambda to release progeny phages into the environment. The R protein is an endolysin that degrades the peptidoglycan cell wall and allow phage to escape from the cell. The S proteins forms a hole in the inner membrane to allow the endolysin to gain excess to cellwall. After the hole is made, approx 100 intact lambda phage particles are released. The entire lytic cycle lasts~ 35 minutes.

 

 

Induction of lambda by the SOS System.

When a lambda lysogen is treated with UV ~ 35 minutes later te cells lyse and release phages. UV damages the DNA and triggers a cellular response called SOS response to deal with this damage. The single stranded DNA from the damaged DNA activates RecA. Activated recA interacts with Cl, leading to cleavage of Cl and induction of the lambda lysogen. Activated RecA also interact with LexA and leads to LecA. Inactivation which leads to expression of number of genes, whose products repair the DNA damage in the cell. Cleavage of Cl, leads to expression of the phage lytic genes and phage production. The rational for this response is that lambda does not want to risk staying in the cell that has DNA damaged and may not survive. 

 

Superinfection:

If a cell is a lambda lysogen, another lambda phage that infects is not able undergo lytic development and produce phage. The incoming phage can inject its DNA, however the DNA is immediately shutdown and no transcription or translation of the lambda initiates. The Lysogens are immune to infection by another lambda phage particle, which is called superinfection. Superinfection is blocked because the lysogen is continuosly producing Cl repressor.

The lysogen actually produces more repressor than it needs to shut down one phage. This extra repressor binds to the superinfecting phage DNA at OL and OR and prevents transcription from PL and PR,

 

Applications of Lambda Phage 

The lambda phage has different applications, most of which are related to DNA cloning. This is because lambda phage can be used as a vector for generating recombinant DNA, which are combined DNA sequences that result from using laboratory techniques like molecular cloning to assemble genetic material from several sources. The site-specific recombinase of lambda phage can be used for shuffling cloned DNAs via the gateway cloning system, a molecular biology technique that ensures the effective transfer of DNA fragments between plasmids.

The lambda phage’s ability to mediate genetic recombincation is due to its red operon, which is a functioning unit of genomic DNA that has a cluster of genes controlled by a promoter or a single regulatory signal. This red operon can be expressed to yield the proteins red alpha (or exo), beta, and gamma, which can be used in recombination-mediated genetic engineering, a method commonly employed in bacterial genetics, generation of target vectors, and DNA modification.

Undoubtedly, the lambda phage is a powerful genetic tool that can be used in many important studies.

Bacteriophage P1 – Structure and Life Cycle.

P1 is a temperate bacteriophage (phage) that infects Escherichia coli and a some other bacteria. When undergoing a lysogenic cycle the phage genome exists as a autonomous plasmid, that is maintained at low copy number, in the bacterium. It is not integrated in to host genome.

Structure and Genome:

P1 has an icosahedral “head” containing the phage DNA attached to a 220nm long tube, contractile tail or shealth, and base plate with six tail fibers.

In a same virion population, 80% of virions have a head diameter of 85nm, while the other 20% have a diameter of 65nm.

Genome :

The genome of P1 Phage is linear double stranded DNA Which is moderately large, about 94 Kbp. The genome is longer, about 120Kbs, than the actual length when in viral particle as it is created by cutting an appropriately sized fragment from a concatemeric DNA chain having multiple copies of the genome. Due to this the ends of the DNA molecule are identical and are referred to as being “terminally redundant”. It has large, about 15 kbp, terminal redundancy. 

Population of phages exhibit Circular Permutation. Means that a given linear molecule can start at any location on the circular genome.  It is another consequence of DNA being cut out of a concatemer.

Though, in the viral particle it is in the form of a linear double stranded DNA molecule, once inserted into the host it circularizes because of the its large terminal redundancy mentioned before and replicates as a plasmid.

The genome contains two origins of replication, oriR which replicates it during the lysogenic cycle and oriL which replicates it during the lytic stage. The genome of P1 has 112 protein coding and 5 untranslated genes. It even encodes 3 of its own tRNAs which are expressed in the lytic stage.

 

The Life Cycle of P1 Phage

Temperate phage, such as P1, have the ability to exist within the bacterial cell they infect in two different ways. In lysogeny, P1 can exist within a bacterial cell as a circular DNA in that it exists by replicating as if it were a plasmid and does not cause cell death. Alternatively, in its lytic phase, P1 can promote cell lysis during growth resulting in host cell death.

During lysogeny new phage particles are not produced. In contrast, during lytic growth many new phage particles are assembled and released from the cell. By alternating between these two modes of infection, P1 can survive during extreme nutritional conditions that may be imposed upon the bacterial host in which it exists.

1. Absorption, Injection and protection of the genome.

P1 absorbs to the receptors on the host cells – terminal glucose on the lipopolysaccharide present on the outer surface of the outer membrane of the host cell. P1 can contract its tail and inject its DNA into a wide range of species but cannot replicate in these species. 

Once inside the cell, P1 DNA circularizes by homologous recombination because these DNA when packed it is packed by Head full Mechanism. This can be done either by host or phage recombination system. In head full mechanism it requires a certain amount or length of genes in the sequence to get packed. So when phage DNA is packed, Some extra genes about 107% to 112% of the phage genome is incorporated in to the capsid. This ensures that the minimum Kb of gene required to get packed is available by ensuring between 7% to 12% homology at the end; a property called Terminal Redundancy. The terminal redundancy is used to circularize the genome.

 Sequence repeated in the both terminal of the genome sequence is called Terminal Redundancy.

 

 2. P1 DNA Replication and Phage Assembly. (Lytic Cycle)

The P1 plasmid has a separate origin of replication (oriL) that is activated during the lytic cycle. Like Lambda, early P1 replication takes place by the theta mode of replication. Later in infection, P1 switch to rolling circle replication. Rolling circle replication produces concatemers for packaging into phage heads. 

Polymeric Structure of complete genome (Multiple repeats of a nucleotide sequence) end to end is known as concatemer.

At approx 45 minutes after the infection, the cells are filled with concatemers of phage DNA, assembled phage heads, and assembled phage tails. Now complete phage must take place. A protein made from phage genome recognizes a site on the concatomers of phage DNA called the pac site. The protein cuts the DNA, making a double-stranded end. This end is inserted into a phage head. The DNA continues to be pushed inside the head until the head is full, a process called head-full packaging.

Once the first phage head is full, another empty phage starts packaging. Experiments shows five head-full of DNA can be packaged sequentially from a single pac site at 100% efficiency. An additional five head-fulls of DNA can be packed although the efficiency gradually decreases to only 5%. While each phage head contains the same genes, the gene order changes. This is known as circular permutation of the genome. 

Circularly permuted means that the order of the genes on each DNA molecule is different but the every DNA molecule contains the same genes.

P1 genome are both circularly permuted and terminally redundant. 

Terminal redundancy means that the same sequences are present on the both end of one DNA molecule.

A- Complete genome, B- Concatemer, C- Shows how concatemer is cut and packed in the head. Each shows circularly permutation and terminally redundancy. 

 

After the head is full of DNA, a double stranded cut is made and a tail is attached. This part of phage development is very much an assembly line. Once the complete virions are assembled, the host cell is lysed, releasing the viral particles.

3.The Location of the P1 Prophage in a lysogen (Lysogenic Cycle)

Prophages can be physically located in one or two places in a lysogen. In case of Lambda, the phage genome is recombined into the bacterial chromosome. P1 contains an origin for DNA replication and once the phage genome is converted to circular, double-stranded DNA, and is maintained in the cytoplasm as a stably inherited extra-chromosomal piece of DNA or plasmid. 

In P1, no integration of DNA, no int/xis System for recombination, So coordinated DNA replication along with the host DNA occurs. Coordinated DNA replication occurs, in order to maintain its population in all daughter cells. If not, when multiplied by binary fission only one daughter cell will posses the phage DNA and another daughter cell does not. 

4. P1 Transducing Particles

One unusual aspect of P1 development is the formation of transducing particles or phage particles that contain chromosomal DNA instead of phage DNA. 

E-Coli chromosomes contains many pseudopac sites or sites that can be used ti initiate packaging of host chromosomal DNA into maturing phage. Theses pseudopac sites are used much less frequently than the phage pac sites but they are used; The resulting phage carry random pieces of the chromosome in place of phage genomes.

The ability to package any piece of chromosomal DNA instead of phage DNA makes P1 a generalized transducing gene. 

More about Transduction click Here

 

 

One step multiplication curve for bacteriophages.

The single-step growth experiment of Ellis and Delbruck demonstrates the cyclic replication of the phage. These authors devised a method to demonstrate only a single step of the many steps of phage replication. Essentially they drastically diluted the mixture after attachment of phage to bacteria, so when the infected cells lysed, no new host cells could be found for a second round of infection. A number of modifications have been introduced since the original experiment was reported. For instance, instead of diluting the initial bacterium:phage mixture, antibodies specific for the phage attachment apparatus may be added to the mixture to ‘neutralize’ and thus render all of the unadsorbed phage unable to adsorb to any bacterium. image

How do you perform this experiment:

Bacteriophages are infected with a very large number of phage particles: The large number of phage ensures all bacteriem are rapidly infected. The high level of infection is called multiplicity of infection (MOI) and can be achieved with a phage to host ratio of 5 to 10 plaque forming units (PFU) per cell. Adsorption of virions to cells is allowed to proceed for suitable time : To replicate, a virus should induce its host to synthesize components that are necessary for the assembly of new virus particles. The virus accomplishes this process by first attaching to the host (adsorption) and then injecting its nucleic acid into the cell (injection or penetration). The viral DNA can stay free in the cell and be replicated as such, or it can be incorporated into the host chromosome and be replicated simultaneously with it. Viral proteins are next synthesized with the host’s machinery under the direction of viral DNA and the new virus particles are assembled mechanically. These particles can find their way out of the cell or lyse the cell and be released into the medium, ready to infect new cells.

The phage/cell mixture is then diluted synchronizing the infection or adding antivirus antiserum. This stop the absorption  of virions to cells that are infected and also prevent infection of new cells other than those that has been infected. Antivirus antiserum contains antibodies directed against the virus. It binds and occupy all the attachment sites of the viral particles so that no new bacteria cells can attach a virus. As the infection of bacteriophages is synchronised, the interaction of virus with the cell population can be seen as a single interaction between phage and the cell. In order to visualize  the infection over time, samples are removed at specified intervals and plated to quantitate the phage present in the culture.

At the start of the experiment, the plaque count is relatively constant over a time period because each infected bacterium will yield only one plaque. A rise in plaque forming units (pfu) to a plateau level occurs as bacteria are lysed and the newly synthesized phage are released into the medium. These phage particles fail to meet susceptible bacteria (due to the dilution of the adsorption mixture) and thus remain free in the culture fluid. The average number of phage released per bacterium is called the burst size and this value may be calculated from the data. The burst size varies in accordance with the specific virus, and may range from 10 to 100 for the DNA transducing phages to approximately 20,000 pfu for the RNA viruses. Plaque assay for bacteriophages are performed by mixing the phage into a layer of bacteria which are spread out as an overlay on the surface of an agar plate. As the plate is incubated, the bacteria grow and they become visible as a turbid layer on the plate. When a phage infects bacteria cells, a zone of Lysis or growth inhibition can occur. This produces clear zone in the bacterial lawn known as plaque. Each plaque originates from a single phage particle. If the number of phage particles was monitored during growth, a growth curve could be drawn which would be similar to that of the bacterial growth curve except in the last stage.

The phage growth curve starts with a latent or eclipse period (similar to the bacterial lag phase). During this phase, the infection, adsorption, injection and syntheses of new viral DNA and protein coat occur. The next phase is called the maturation or release stage (similar to the log phase in bacteria) when new phage particles are assembled and released. The cycle can then start over with the infection of new cells. In this manner, the shape of the curve would look step-wise and that is why the process is called “one-step phage growth curve”. image

 

Stages of one step multiplication curve:

Eclipse: or initial period can be defined as the time period taken for the appearance of first intracellular phages. No phage particles can be detected during this period as the phages are being uncoated and  phage DNA is being injected foe replication.

Synthetic period: During synthetic period intracellular particles are being produced. As in the Eclipse period, there are no phage particles released during the synthetic period.

Latent Period: The first two periods are combined in the third period known as latent period. The latent period is described as the time period prior to the release of infection particles or appearance of extra cellular phages.

In the latent period, attachment, entry, replication, transcription, translation and assembly of progeny phages occur.   

Rise Period: In this period lysis occurs and extracellular phages appear and they increase in number of concentration of bacteriophages rises.

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  • Burst Size: Average yield of infectious virus per cell is called burst size.
  • Burst Size = Final titre of virus / Initial viral titre.
  • There is much variation in bursts size between different kind of cells.
  • In one study with phage, a burst size of 170 was obtained when growing bacteria cells and value of 20 was obtained with resting bacteria cells. This is because rapid growing cells means, its cell machinery are active and are metabolically active than the resting bacteria cell. So yield of infectious virus increases with growing cells than resting bacteria cell.
  • Extra step, lysis from without (LO): LO described as an early lysis of bacteria induced by high-multiplicity virion adsorption and that occurs without phage production and leads to killing of bacteria. LO can be induced by adding chloroform, which break open the host cell and the intracellular phages are released. It is an artificial lysis. 

Lysogenic Cycle

image

Step 1: Adsorption
Attachment of adsorption of tail fibres of the phage on to a specific receptor site on the bacterial cell wall.

Step 2: Injection
Injection of viral genome into the host through the hollow tubes of the tail.

Step 3: Integration of viral genome to the host genome.
After entry of viral genome, it gets integrated into the bacterial genome of the host. The viral genome integrated into the bacterial genome is termed Prophage.

Step 4: Viral genome synthesis
Viral genome replicates along with the bacterial genome replication and pass on to the daughter cells.

Step 5: Induction of Lytic cycle
Occasionally, integrated viral genome detaches and released into the bacterial cytoplasm.  This dissociation is called induction and Lytic cycle is followed releasing mature lysogenic phages. Induction can be induced using UV rays or heat treatment.

Lytic Cycle

image

Step 1: Absorption
Attachment of absorption of tail fibres of the phages on to a specific receptor site on the bacterial cell wall.

Step 2: Injection
Injection of viral genome into the host through the hollow tubes of the tail.

Step 3: Protein synthesis
Inside the host, the viral genome directs the synthesis of viral proteins using the machinery of the host. Viral genome generally encodes for some enzymes and coat proteins.

Step 4: Viral genome synthesis
Viral genome replicates inside the host making several copies of it self.

Step 5 & 6: Packaging and release
The viral genome gets packed inside the protein coat.

These intact mature infectious particles are called virions. The crowding of virions inside the host ultimately cause cell lysis and liberation of mature viral particles – about 200 mature phages are liberated.

Mode of Multiplication of Bacteriophages

Two modes of multiplication cycle in Bacteriophages namely lytic cycle and lysogenic cycle.

image

Lytic cycle :
Lytic cycle or lytic phages called as Virulent phages. Multiples inside the host bacterium and new viral particles comes out by lysing  or by rupturing the host bacterial cell wall.
Example: T phages, T2,  T4, T6 etc…

Lysogenic cycle :
Lysogenic cycle or lysogenic phages called as temperate phages. It does not undergo multiplication or induce lysis, here the viral DNA get integrated into the bacterial DNA without causing lysis.
Example: Lambda phages.

The life cycle of a bacteriophage

Phage must carry out specific set of reactions in order to make more of themselves. 

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First, the phage must be able to recognise a bacterium that can multiply in by binding to the bacterial cell surface.
Next, the phage must inject it’s genome and the genome must be protected from the bacterial nucleases in the cytoplasm.

The phage genome must be replicated transcribed and translated so that a large number of genomes, capsid proteins, and tail proteins, if present, are produced at the same time. Complete phage particles are then assembled and the phage must get back out of bacterium.

Classification of Bacteriophages

Broadly pages can be classified as either virulent or temperate.

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The process of phage infecting a bacterium and producing progeny is referred to as lytic Infection. A virulent phage subverts the cellular apparatus of its bacteria host for multiplification, and release of progeny virions. Eg. T4.

Temperate phages have alternative replication cycles: lytic infection or a lysogenic pathways.

Lysogenic pathway is a pathway in which phage remains latent in the host. Phages are capable of maintaining their chromosome in a stable, silent state within the bacteria. When the bacterium contains a silent phage chromosome, it is referred to as a Lysogen. The incorporated phage genome is referred to as a Prophage.

Bacteriophages – Overview

Bacteriophages are also known as bacterial Viruses or phages .They are the viruses that Infect bacteria. phages are first discovered independently by Frederick w.Twort in great britain C 1915) and Felix d’ Herelle In France (1917). Like all viruses, phages are Viruses that consist of a core of genetic material surrounded by a protein coat Or capsid.

The phage genomemay be DNA or RNA, single or doublestranded, circular or linear, generally present as a single copy. Morphologically varies from simple, Icosahedral and filamentous phage to more complex tailed phages with 9n Icosahedral head.

They are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery.