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Encyclopedia > Lambda phage
Enterobacteria phage λ

Virus classification
Group: Group I (dsDNA)
Order: Caudovirales
Family: Siphoviridae
Genus: λ-like viruses
Species: λ Phage

Enterobacteria phage λ (lambda phage) is a temperate bacteriophage that infects Escherichia coli. Once the phage has injected its DNA into its host, the phage DNA may integrate itself into the host cell chromosome. In this state, λ is called a prophage and stays resident within the host's genome, without apparent harm. Thus the prophage is duplicated with every cell division of the host. This is referred to as the "lysogenic pathway". The DNA of the prophage that is expressed in that state codes for proteins that look out for signs of stress in the host cell. Stress can be from starvation, poisons (like antibiotics), or other factors that can damage or destroy the host. At that point, the prophage reactivates, excises itself from the DNA of the host cell and enters its lytic cycle. The reactivated phage takes apart the host's DNA and produces large amounts of its own mRNA, so as to produce prodigious amounts of phage units. When all resources of the host are depleted from building new phages, the host cell is lysed (the cell membrane is broken down), and the new phages are released. Image File history File links No higher resolution available. ... Virus classification involves naming and placing viruses into a taxonomic system. ... A DNA virus is a virus that has DNA as its genetic material and does not use an RNA intermediate during replication. ... Families Myoviridae Podoviridae Siphoviridae The Caudovirales are an order of viruses, comprising the bacteriophages that have tails. ... Genera λ-like viruses T1-like viruses T5-like viruses c2-like viruses L5-like viruses ψM1-like viruses Siphoviridae is a family of double-stranded DNA viruses infecting only bacteria that are characterized by a long non-contractile tail and an isometric capsid (morphotype B1) or a prolate... Look up Λ, λ in Wiktionary, the free dictionary. ... This article or section does not cite its references or sources. ... E. coli redirects here. ... A phage genome inserted as part of the linear structure of the DNA chromosome of a bacterium. ... In biology the genome of an organism is the whole hereditary information of an organism that is encoded in the DNA (or, for some viruses, RNA). ... Lysogeny is the fusion of the nucleic acid of a bacteriophage with that of a host bacterium so that the potential exists for the newly integrated genetic material to be transmitted to daughter cells at each subsequent cell division. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... This article is about extreme malnutrition. ... This article is about the dangerous substance. ... An antibiotic is a drug that kills or slows the growth of bacteria. ... The lytic cycle is one of the two cycles of viral reproduction, the other being the lysogenic cycle. ... The interaction of mRNA in a eukaryote cell. ... This article is about the biological definition of the word Lysis. ... Look up cell membrane in Wiktionary, the free dictionary. ...


The integration of phage λ takes place at a special attachment site in the bacterial genome, called attλ. The sequence of the att site is called attB and consists of the parts B-O-B', whereas the complementary sequence in the circular phage genome is called attP and consists of the parts P-O-P'. The integration itself is a sequential exchange (see genetic recombination) via a Holliday junction and requires both the phage protein Int and the bacterial protein IHF (integration host factor). Both Int and IHF bind to attP and form an intasome, a DNA-protein-complex designed for site-specific recombination of the phage and host DNA. The original BOB' sequence is changed by the integration to B-O-P'-phage DNA-P-O-B'. The phage DNA is now part of the host's genome. Genetic recombination is the process by which a strand of DNA is broken and then joined to the end of a different DNA molecule. ... A Holliday junction is a mobile junction between four strands of DNA. The structure is named after Robin Holliday, who proposed it in 1964 to account for a particular type of exchange of genetic information in yeast known as homologous recombination. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... // In site-specific-recombination, DNA strand exchange takes place between segments possessing only a limited degree of sequence homology (Kolb 2002; Coates et al. ...


Lambda phage was discovered by Esther Lederberg in 1951.[1] It has been a rich source for useful tools in molecular biology, including its application as a vector for the cloning of recombinant DNA, the use of its site specific recombinase, int, for the shuffling of cloned DNAs by the 'Gateway' method, and the application of its Red operon, including the proteins Red alpha (also called 'exo'), beta and gamma in the DNA engineering method called recombineering. Esther Miriam Lederberg (nee Zimmer, December 18, 1922 - November 11, 2006) was an American microbiologist and immunologist and pioneer of bacterial genetics. ... Molecular biology is the study of biology at a molecular level. ... Recombinant DNA (rDNA) is an artificial DNA sequence resulting from the combination of different DNA sequences. ... Recombineering (recombinogenic engineering) is a genetic and molecular biology technique based on homologous recombination systems in E. coli to modify DNA. The term was first coined in 2001 by Ellis et al (Proc. ...

Contents

Repressor

The repressor found in the phage lambda is a notable example of the level of control possible over gene expression by a very simple system. It forms a 'binary switch' with two genes under mutually exclusive expression. A repressor is a DNA-binding protein that regulates the expression of one or more genes by decreasing the rate of transcription. ...


In the following paragraphs, we will write genes in italics and their associated proteins in Roman. For instance, cI refers to the gene, while cI is the resulting protein encoded by that gene. The lambda repressor gene system consists of (from left to right on the chromosome):

  • cI gene
  • OR3
  • OR2
  • OR1
  • cro gene

The lambda repressor is a dimer also known as the cI protein. It regulates the transcription of the cI protein and the Cro protein. cI protein is a repressor protein found in lambda phage that shuts off PL and PR. Categories: Stub ...


The life cycle of lambda phages is controlled by cI and Cro proteins. The lambda phage will remain in the lysogenic state if cI proteins predominate, but will be transformed into the lytic cycle if cro proteins predominate. Lysogeny is the fusion of the nucleic acid of a bacteriophage with that of a host bacterium so that the potential exists for the newly integrated genetic material to be transmitted to daughter cells at each subsequent cell division. ... The lytic cycle is one of the two cycles of viral reproduction, the other being the lysogenic cycle. ...


The cI dimer may bind to any of three operators, OR1, OR2, and OR3, in the order OR1 > OR2 > OR3. Binding of a cI dimer to OR1 enhances binding of a second cI dimer to OR2, an effect called cooperativity. Thus, OR1 and OR2 are almost always simultaneously occupied by cI. However, this does not increase the affinity between cI and OR3, which will be occupied only when the cI concentration is high. Cooperativity is a phenomenon in biology displayed by enzymes or receptors that have multiple binding sites. ...

  • In the absence of cI proteins, the cro gene may be transcribed.
  • In the presence of cI proteins, only the cI gene may be transcribed.
  • At high concentration of cI, transcriptions of both genes are repressed.

Lifecycle

Schematic representation of the genome of the bacteriophage lambda.
  1. Bacteriophage Lambda binds to the target E. coli cell, the tail tip binding to a maltose receptor.
  2. The linear phage genome is injected into the cell, and immediately circularises.
  3. Transcription starts, from the L, R and R' promoters producing the 'immediate early' transcripts.
  4. Initially these produce N, Cro and a short inactive protein.
  5. Cro binds to OR3 preventing access to the RM promoter preventing transcription and production of cI. N binds to the two Nut sites, one in the N gene, and one in the cro gene.
  6. The N bound in the L and R open reading frames extends the reading frames. The early translation products of these transcripts (the 'late early' transcripts) are more N and Cro along with cII and cIII.
  7. cIII binds to cII partially preventing protease vulnerability. On initial infection, the stability of cII determines the lifestyle of the phage. In cells with abundant nutrients, protease activity is high, which breaks down cII. This leads to the lytic lifestyle. In cells with limited nutrients, protease activity is low, making cII stable. This leads to the lysogenic lifestyle. Once the lysogenic pathway is initiated, whether the prophage is excised from the DNA and a lytic cycle begins depends on the amount of stress on the cell: see "Induction" below.

Image File history File links Download high resolution version (725x1024, 170 KB) Summary By Richard Wheeler (Zephyris) 2005; Schematic representation of the genome of the bacteriophage lambda. ... Image File history File links Download high resolution version (725x1024, 170 KB) Summary By Richard Wheeler (Zephyris) 2005; Schematic representation of the genome of the bacteriophage lambda. ... Maltose, or malt sugar, is a disaccharide formed from two units of glucose joined with an α(1→4) linkage. ... A promoter is a regulatory region of DNA located upstream (towards the 5 region) of a gene, providing a control point for regulated gene transcription. ...

Lytic Lifestyle

  1. The 'late early' transcripts continue being written, including xis, int, Q and genes for replication of the lambda genome.
  2. The lambda genome is replicated in preparation for daughter phage production.
  3. Q binds to Qut sites.
  4. Transcription from the R' promoter can now extend to produce mRNA for the lysis and the structural proteins.
  5. Structural proteins and phage genomes self assemble into new phage particles.
  6. Lytic proteins build sufficiently far in concentration to cause cell lysis, and the mature phage particles escape.

[xis and int regulation of insertion and excision]

  1. xis and int are found on the same piece of mRNA so approximately equal concentrations of xis and int proteins are produced. This results (initially) in the excision of any inserted genomes from the host genome.
  2. The mRNA from the L promoter forms a stable secondary structure with a bobby pin loop in the sib section of the mRNA. This targets the 3' end of the mRNA for RNAaseIII degradation, so a lower effective concentration of xis mRNA than int mRNA is found, so higher concentrations of xis than int.
  3. Higher concentrations of xis than int result in no insertion or excision of phage genomes, the evolutionarily favoured action - leaving any pre-insterted phage genomes inserted (so reducing competition) and preventing the insertion of the phage genome into the genome of a doomed host.

Lysenogenic (or Lysogenic) Lifestyle

  1. The 'late early' transcripts continue being written, including xis, int, Q and genes for replication of the lambda genome. Although the stablized CII also acts to promote transcription from the pRE, pI and pAQ promoters.
  2. The pAQ promoter produces antisense mRNA to the Q gene message of the pR promoter transcript thereby switching off Q production. The pRE promoter produces antisense mRNA to the cro section of the pR promoter transcript, turning down cro production, along with activating sense mRNA for cI, turning on CI repressor production. The pI promoter produces mRNA for Int, resulting in high concentrations of Int protein.
  3. No Q results in no extension of the pR' promoter's reading frame, so no lytic or structural proteins are made. Elevated levels of Int (to much higher than that of xis) result in the insertion of the lambda genome into the hosts genome (see diagram). Production of CI leads to the binding of CI to the OR1 OR2 sites in the pR promoter, turning off pR, cro and other early gene production. cI also binds to the pL promoter, turning off transcription there too.
  4. Lack of cro leaves the OR3 site unbound, so transcription from the pRM promoter may occur, maintaining levels of cI.
  5. Lack of transcription from the pL and pR promoters leads to no further production of cII and cIII.
  6. As cII and cIII concentrations decrease, transcription from the pAQ, pRE and pI stop being promoted since they are no longer needed.
  7. Only the pRM and pR' promoters are left active, the producing CI protein and the latter a short inactive transcript. The genome is inserted in the host and remains there in a dormant state.

Induction

  1. The host cell, containing a dormant phage genome, experiences DNA damage due to a high stress environment, and starts to undergo the SOS response.
  2. RecA (a cellular protein) detects DNA damage and becomes activated. It is now RecA*, a highly specific co-protease.
  3. Normally RecA* binds LexA (a transcription repressor), activating LexA auto-protease activity,which destroys LexA repressor allowing production of DNA repair proteins. In lysogenic cells this response is hijacked, and RecA* stimulates cI autocleavage.
  4. Cleaved cI can no longer dimerise, and loses its affinity for DNA binding.
  5. The pR and pL promoters are no longer repressed and switch on, and the cell returns to the lytic sequence of expression events (note that cII is not stable in cells undergoing the SOS response). There is however one notable difference.

In genetics the SOS response is a DNA repair system involving RecA protein. ... A micrograph of ongoing gene transcription of ribosomal RNA illustrating the growing primary transcripts. ... DNA damage resulting in multiple broken chromosomes DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. ...

Prophage Induction of Phage λ

The gene regulatory circuitry of phage λ is among the best-understood circuits at the mechanistic level. This circuitry involves several interesting regulatory behaviors. An infected cell undergoes a decision between two alternative pathways, the lytic and lysogenic pathways. If the latter is followed, the lysogenic state is established and maintained. While this state is highly stable, it can switch to the lytic pathway in the process of prophage induction, which occurs when the host SOS response is triggered by DNA damage.[2] For other uses, see Gene (disambiguation). ... A phage genome inserted as part of the linear structure of the DNA chromosome of a bacterium. ...


[Control of phage genome excision in induction]

Schematic representation of the insertion of the bacteriophage lambda. Note how sib is displaced by the recombination event from the N extended pL promoter open reading frame.
Schematic representation of the insertion of the bacteriophage lambda. Note how sib is displaced by the recombination event from the N extended pL promoter open reading frame.
  1. The phage genome is still inserted in the host genome and needs excision for DNA replication to occur. The sib section beyond the normal pL promoter transcript is, however, no longer included in this reading frame (see diagram).
  2. No sib domain on the pL promoter mRNA results in no hairpin loop on the 3' end, and the transcript is no longer targeted for RNAaseIII degradation.
  3. The new intact transcript has one copy of both xis and int, so approximately equal concentrations of xis and int proteins are produced.
  4. Equal concentrations of xis and int result in the excision of the inserted genome from the host genome for replication and later phage production.

Image File history File links Download high resolution version (2409x1024, 182 KB) Summary By Richard Wheeler (Zephyris) 2005; Schematic representation of the insertion of the bacteriophage lambda. ... Image File history File links Download high resolution version (2409x1024, 182 KB) Summary By Richard Wheeler (Zephyris) 2005; Schematic representation of the insertion of the bacteriophage lambda. ...

Protein Function Overview

cro; (Control of Repressor's Opperator) Transcription inhibitor, binds OR3, OR2 and OR1 (affinity OR3 > OR2 > OR1, ie. preferentially binds OR3). At low concentrations blocks the pRM promoter (preventing cI production). At high concentrations downregulates its own production through OR2 and OR1 binding.


cI; (Clear 1) Transcription inhibitor, binds OR1, OR2 and OR3 (affinity OR1 > OR2 > OR3, ie. prefferentially binds OR1). At low concentrations blocks the pR promoter (preventing cro production). At high concentrations downregulates its own production through OR2 and OR3 binding. Repressor also inhibits transcription from the pL promoter. Susceptible to cleavage by RecA* in cells undergoing the SOS response.


cII; (Clear 2) Transcription activator. Activates transcription from the pAQ, pRE and pI promoters. Low stability due to succeptability to cellular HflB (FtsH) proteases (especially in healthy cells and cells undergoing the SOS response.


cIII;(Clear 3) HflB (FtsH) binding protein, protects cII from degradation by proteases.


N; (aNtiterminator) RNA binding protein and RNA polymerase cofactor, binds RNA (at Nut sites) and transfers onto the nascent RNApol that just transcribed the nut site. This RNApol modification prevents its recognition of termination sites, so normal RNA polymerase termination signals are ignored and RNA synthesis continues into distal phage genes. This article does not cite any references or sources. ... A cofactor is the following: In mathematics a cofactor is the minor of an element of a square matrix. ...


Q; DNA binding protein and RNApol cofactor, binds DNA (at Qut sites) and transfers onto the initiating RNApol. This RNApol modification alters its recognition of termination sequences, so normal ones are ignored; special Q termination sequences some 20,000 bp away are effective.


xis; (eXcISion) excisionase and Int protein regulator, manages excision and insertion of phage genome into the host's genome.


int; (INTegration) Int protein, manages insertion of phage genome into the host's genome. In Conditions of low int concentration there is no effect. If xis is low in concentration and int high then this leads to the insertion of the phage genome. If xis and int have high (and approximately equal) concentrations this leads to the excision of phage genomes from the host's genome.


A, B, C, D, E, F, Z, U, V, G, T, H, M, L, K, I, J [Shown on diagram as head and tail, A-F code for phage head genes, Z-J code for phage tail genes. The order shown here is as found on the genome, reading in a clockwise direction]; structural proteins, self assemble with the phage genome into daughter phage particles.


S, R [Shown on diagram as lysis. The order shown here is as found on the genome, reading in a clockwise direction]; cause the host cell to undergo lysis at high enough concentrations.


OP [Shown on diagram as O replication P]; DNA replication functions, promotes the specific replication of only the phage genome.


sib [not a protein, but a vital conserved DNA sequence]; Forms a stable hairpin loop structure in transcribed mRNA beyond int. Attracts degradation of mRNA by RNAaseIII.


attP [not a protein, but a conserved DNA sequence]; point of action of Int and Xis in integration and excision of the phage genome into the host's genome. Corresponding attB found in the host's genome at the point of insertion.


References

  1. ^ Lederberg, 1951, Microbial Genetics Bulletin.
  2. ^ Mc Grath S and van Sinderen D (editors). (2007). Bacteriophage: Genetics and Molecular Biology, 1st ed., Caister Academic Press. ISBN 978-1-904455-14-1 . 

Further Reading

  • Ptashne and Gann, A Genetic Switch, 2nd edition

  Results from FactBites:
 
Lambda phage - Wikipedia, the free encyclopedia (2008 words)
Enterobacteria phage λ (lambda phage) is a temperate bacteriophage that infects Escherichia coli.
Schematic representation of the genome of the bacteriophage lambda.
Schematic representation of the insertion of the bacteriophage lambda.
  More results at FactBites »

 
 

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