Bacteriophage lambda life cycle's strategy
- 1. Bacteriophages Devlina Sengupta Department of Microbiology Kanchrapara College
- 2. Diversity • Bacteriophages are the most abundant living organisms on the planet, with an estimated number of 1031 total viral particles (virions). They outnumber bacteria in most ecological niches where they have been studied (Weinbauer, 2004). • Bacteriophages have different roles in the biosphere. • Bacteriophages are classified according to their morphology and type on nucleic acid. There is a great variety of combinations and consequently a wide range of bacteriophage types. However, the following six families are by far the most common among bacteriophages (Krupovic et al., 2016): Myoviridae: This kind of phage is characterized by having a cubic capsid (which can be elongated or icosahedral) and a long contractile tail. Their capsid diameter can vary from 65 nm up to 100 nm. As for the genetic material, the members of this group present linear double stranded (ds) DNA. A typical member would be the bacteriophage T4. Siphoviridae: They present an icosahedral capsid and a long non-contractile tail. They usually have a capsid with a diameter between 50 nm and 70 nm. Like the Myoviridae, they have linear dsDNA. Examples of this group are phage T5 and phage lambda. Podoviridae: In this case, the characteristic morphology of the group consists of an icosahedral capsid and a short non-contractile tail. Podoviridae usually have a width of 60 nm up to 65 nm. Just like the previous groups, they present linear dsDNA. A representative member of this group is the bacteriophage T7. Microviridae: They are bacteriophages that only have an icosahedral capsid and present no tail. The diameter of each virion is around 25-30 nm. In this case, their nucleic acid is circular single stranded (ss) DNA. Within the Microviridae we can find the bacteriophage X174. ᶲ Inoviridae: This group presents a filamentous or rod-shaped morphology which is very different from the rest of groups, as the diameter is around 7 nm but they are up to 2000 nm long. They also present circular ssDNA. A typical member of this group is the phage M13.
- 4. Classification of viruses • Bacteriophages with DNA Genome: A. Genome is double-stranded DNA, generally linear: 1. Family Myoviridae: Icosahedral, isometric or elongated head with a helical rigid tail containing a contractile sheath, tail plate, tail fibres and spikes. Example: E. coli phage (coliphage) T2, T4, T6. 2. Family Styloviridae: Icosahedral, isometric head with a long flexible tail without a contractile sheath. Tail fibres may or may not be present. Example: E. coli phage T1, T5. 3. Family Pedoviridae: Icosahedral, isometric head with a sheath-less tail which is shorter than the head. Tail fibres may or may not be present. Example: E. coli phage T3, T7. 4. Family Corticoviridae: Icosahedral, isometric head without a tail. Capsid contains lipid in addition to protein. The genome is closed circular ds-DNA. Example: Pseudomonas phage MP2. 5. Family Tectiviridae: Icosahedral, isometric head without tail. Virion contains double capsid. Example: phage PRD 1. 6. Family Plasmaviridae: Virion plemorphic, Enveloped. Example Phage MV-L2. B. Genome single-stranded DNA: 1. Family Microviridae: Icosahedral, isometric head without tail or envelope. Example: E. coli phage φX174 having a circular ss-DNA genome. 2. Family Inoviridae: Helical symmetry; virions long rod shaped or filamentous; non-enveloped; genome circular ss-DNA. Example: E. coli phage fd and bacteriophage MV-L1. • Bacteriophages with RNA Genome: A. Genome double-stranded RNA: 1. Family Cystoviridae: Capsid icosahedral, enveloped; ds-RNA is segmented; virions more or less spherical, about 90-100 nm in diameter. Example: Phage φ6.
- 6. One step multiplication curve • It can be seen also that the kinetics of appearance of intracellular phage particles are linear, not exponential. This is consistent with particles being produced by assembly from component parts, rather than by binary fission. • One-step growth analysis soon became adapted for studying the replication of animal viruses. The experiment begins with removal of the medium from the cell monolayer and addition of virus in a small volume to promote rapid adsorption. • After 1 h, unabsorbed inoculum containing virus particles is removed, the cells are washed, and ∼ fresh medium is added. • At different times after infection, samples of the cell culture supernatant are collected and the virus titer is determined. • The kinetics of intracellular virus production can be monitored by removing the medium containing extracellular particles, scraping the cells into fresh medium, and lysing them. • The results of a one-step growth experiment establish a number of important features about viral replication. • In the example the first 11 h after infection constitutes the eclipse period, during which the viral nucleic acid is uncoated from its protective shell and no infectious virus can be detected inside cells.
- 8. Lytic & lysogenic phages • The first indication that two alternative outcomes of infection by the same virus are possible was with bacteriophage λ. This was followed by the discovery of other bacteriophage which can also adopt two different types of infectious process. • Bacteriophage that are able to adopt either a lytic or a lysogenic replication cycle are called temperate phage. Several studies have shown that the phage DNA is inserted in the bacterial host genome during lysogeny. In this state the virus DNA is replicated with the host chromosome and is called a prophage. • The bacteria carrying the phage DNA are called lysogens. Phage λ remains a key example of latency and our understanding of the molecular events which occur during both lytic and lysogenic replication have formed the basis for our understanding of gene expression and latency. • When phage λ enters a bacterium it must initiate either a lytic or a lysogenic cycle. In case of lysogeny, following attachment, the phage λ linear genomic DNA is introduced by an injection mechanism into the E. coli and is immediately converted into a covalently closed dsDNA circle by host enzymes. • Circularization is possible because the phage λ dsDNA genome contains 12 bases of single-stranded DNA at either end of the linear molecule which are complementary called Cos site. • During lytic replication the circular phage DNA replicates independently of the host DNA and does not integrate into the genome of the host.
- 9. Mechanism of lytic cycle • The phage λ genome contains two principal promoters which are recognized by the host cell DNA-dependent RNA polymerase which immediately begins transcription of mRNA. • One of the promoters PL directs transcription in a leftwards direction and generates an mRNA that terminates at the end of the gene encoding the N protein. • The other promoter PR directs transcription in a rightwards direction to encode the protein Cro. However, termination of transcription at the end of the cro gene is not absolute and some mRNAs extend through the cII, O, and P genes. • The proteins are then translated from the polycistronic mRNA. The N protein causes the RNA polymerase to transcribe through the regions of DNA at the ends of the N, cro, and P genes, where it had previously stopped, to generate polycistronic mRNAs encoding several proteins. • The mRNA from PL extends through the N, cIII, xis, and int genes, and mRNA from PR extends through the cro, cII, O, P, and Q genes. • N protein thus acts as a transcriptional anti-terminator and allows expression of additional genes. These events occur before phage λ DNA synthesis and are referred to as immediate early (N and cro) and early (cIII, xis, and int from PL, and Q gene from PR) gene expression. • The Cro and Q proteins are important for the next phase of gene expression. This occurs after phage λ DNA synthesis and is therefore, by definition, a late event in the replication cycle. • Immediately on infection, and at the same time as PL and PR are utilized, host cell RNA polymerase recognizes a third promoter region in phage λ DNA, PR ′, located immediately after the Q gene. However, transcription is terminated just downstream to synthesize a very short mRNA. This short mRNA does not encode a protein. The Q protein is an anti-terminator which causes the RNA polymerase molecules initiating transcription at PR ′ to ignore the termination signal and to continue mRNA synthesis. • The resulting mRNA is extremely long and extends through the genes encoding the structural proteins which make up the phage head and tail. • At the same time as the Q protein is exerting its activity then Cro protein is also at work. The Cro protein binds to the phage λ DNA at the operator elements (OL and OR, respectively) of the promoters PL and PR. By doing this Cro inhibits transcription from these promoters, stopping production of the early mRNAs. Sufficient Q protein is present to ensure that PR ′, which is not affected by Cro protein. • The result is that the only mRNA found at late times encodes the structural proteins which can package the newly synthesized phage λ DNA into progeny virions utilizing the cos site.
- 10. Mechanism of lysogeny • The initial events in the process of lysogeny are identical to those seen in a lytic infection. The circularized phage DNA is transcribed from the two major promoters, PL and PR, and also from PR . • Transcription from PL and PR makes the mRNAs encoding the N and Cro proteins, with small amounts of the cII, O, and P proteins. Subsequently, the anti-terminator action of the N protein results in the synthesis of cIII, Xis, Int, and Q proteins and larger quantities of cII, O, and P. • The cII protein acts as a gene activator, directing the host RNA polymerase to begin transcription at two promoters which would otherwise be inactive. These are PRE (promoter for repressor expression) and Pint (promoter for integrase expression). • The mRNA initiated at Pint directs the synthesis of larger quantities of the Int protein which is responsible for integrating the phage λ DNA into the host chromosome. • PRE directs transcription of a single gene, cI. The cI protein, usually referred to as the cI, or lambda repressor, is critical in lysogeny and phage with mutations in the cI gene can only replicate lytically. The cI protein binds to OL and OR, inhibiting transcription from PL and PR and preventing production of the early proteins. By inhibiting synthesis of the early proteins, the cI protein prevents the subsequent appearance of the late, structural, proteins and, consequently, of infectious particles. • In order to ensure that its own synthesis is not prevented by an absence of cII protein, the cI protein also directs RNA polymerase to an additional promoter, PRM (promoter for repressor maintenance), which initiates transcription of the cI gene alone. • The action of the cII protein in activating expression from PRE has an additional consequence. The mRNA transcribed from PRE contains some sequences that are antisense to those of the mRNA from PR encoding the Cro protein. Hybridization of these will prevent translation of the cro gene mRNA and reduce the level of Cro protein in the bacterium.
- 11. The genes involved in the lytic cycle are indicated. The positions of the phage promoters and transcriptional terminators involved in the lytic cycle are shown together with the mRNAs produced at immediate early, early, and late times. (a) The cII protein activates the PRE and Pint promoters to produce mRNAs for the cI and Int proteins, respectively. mRNA from PRE contains sequences antisense to the cro gene. (b) The cI protein binds OL and OR, inactivating PL and PR, respectively, while activating PRM, ensuring its own continued synthesis throughout lysogeny. (c) The steady state of cI gene expression during lysogeny
- 12. Regulation between lysis & lysogeny • Several aspects of what determines the choice between lysis and lysogeny remain unclear, but a key factor is the balance between the various repressors and activators produced early in the phage’s interaction with the bacterium, and amongst these, the role of the cII protein is critical. • If cII is very active cI protein will be produced in large amounts. This will efficiently inhibit synthesis of all genes except its own, and lysogeny will result. • On the other hand, if the cII protein is poorly expressed or has low activity, very low levels of cI protein will be present. The Cro protein will inhibit the activity of PL and PR and the synthesis of the cII protein, amongst others, will be significantly reduced. • Without sufficient cII protein PRE will not be strongly activated and synthesis of the cI protein will, in turn, be further reduced. In this case the lytic pathway will follow. • The cII protein is susceptible to proteolytic degradation by host enzymes. The levels of host proteases are affected by many factors, especially growth conditions. Healthy bacteria grown in rich medium contain high levels of proteases and when infected are more likely to support lytic replication; vice versa for nutritionally deprived bacteria. • It should be clear that it is to the advantage of the phage to undertake a lytic infection in healthy bacteria which will contain high levels of ATP and are equipped to synthesize the virus proteins. When bacteria are nutritionally deficient it is to the advantage of the phage to establish itself as a lysogen and wait until conditions improve. • The cIII protein also plays a role in the choice between lysis and lysogeny. The function of cIII is to protect cII from proteolytic degradation. This protection is not complete since cII can be destroyed even in the presence of cIII. However, if cIII is absent or not functional cII is almost always degraded and the phage can only undergo a
- 13. Regulation of transcription in lambda phage • The integration of phage λ DNA into the host chromosome is carried out by the Int protein. The integrated DNA is referred to as a prophage and genetic mapping studies showed that there is only one site of integration, adjacent to the galactose (gal) operon in the E. coli genome at a position referred to as the lambda attachment (att λ) locus. • The position of the recombination site in the phage DNA lies downstream of the int gene and the region is referred to as the att P site. The bacterial and phage attachment sites, att λ and att P, contain identical tracts of 15 base-pairs indicating that homologous recombination occurs during the integration event. • Lysis & excision of integrated DNA can be induced to take place by application of ultraviolet irradiation of a lysogenic culture of E. coli. Several other stimuli, such as treatment with potent mutagens, also induce the phage. This is termed induction of the lysogen. • During this response E.coli responds by SOS response caused by a critical protein called Rec A protein. The function of the Rec A protein is to mediate recombination between DNA molecules. However, when the bacterium is subjected to stress, such as irradiation with ultraviolet light, the Rec A protein alters its activity to become a specific protease that cleaves a repressor called Lex A. The Lex A protein represses expression from a range of genes and its cleavage removes this repression giving expression of the genes. • Rec A protein also cleaves the phage λ cI repressor protein, and when this occurs the promoters PL and PR become functional, transcribing the N, cro, and other genes involved in lysis. • While the integration event requires only the Int protein, for excision to occur the xis and int genes must both be transcribed as both proteins are required to act together. Campbell’s model for the insertion of phage λ DNA into the bacterial genome by reciprocal recombination between phage and host DNA.
- 14. Rare error prone excision of λ DNA