In this article we will discuss about the infection cycles of lytic, single-stranded and temperate bacteriophages.
Contents
Infection Cycle of Lytic Bacteriophages:
The lytic cycle of these bacteriophages can be divided into several stages, like attachment and adsorption, penetration, intercellular changes resulting in the synthesis of viral components, assembly of progeny virions and release from the infected cell. These steps are described taking the E. coli phage T4 as a model system.
Phage T4 of E. coli B has an icosahedral, bi-pyramidal hexagonal head attached to a straight tail comprising of a central, rigid, hollow core surrounded by a contractile sheath. The sheath consists of protein subunits joined to each other to form a helical structure. The distal end of the core is attached to a hexagonal base plate containing six short spikes and six long tail fibres, each with a kink in the middle.
(i) Attachment and Adsorption:
The host bacterium, E. coli B, has specific attachment sites on the cell wall for phage T4, though for other phages of E. coli, like T2, T5 and T6 the receptors are located on the outer membrane. It has been observed that selective removal of the outer-membrane lipopolysaccharides makes the bacteria resistant to these phages, whereas E. coli protoplasts, devoid of both outer membrane and cell wall, are unable to adsorb any bacteriophage. Some coliphages, like phage fd can attach to sex pili instead of the main cell body.
Meeting of a phage and its host bacterium occurs through accidental collision between the two. No chemical attractant is known to be involved. Attachment begins with the adsorption of the tip of one of the tail fibres to the receptor site on the cell surface. It is followed by adsorption of the other tail fibres making the attachment more firm. However, attachment with tail fibres is not sufficient. A more intimate contact is established by attachment with the base plate helped by the spikes. This makes the attachment of the phage to the host cell irreversible.
(ii) Penetration:
Bacteriophage T4 or other large phages do not physically enter into the host cells, as the animal or plant viruses do. Phages inject their nucleic acid into the cytoplasm of the host cell. In case of T-even phages (T2, T4, T6 etc.), the ds-linear DNA present in the phage head is injected through the hollow core of the tail into the bacterial cell.
The phage is held tightly on the cell surface, while the sheath surrounding the hollow core contracts forcing the rigid core to penetrate the outer membrane and the cell wall. A viral lysozyme released during penetration attacks the murein wall, thereby helping the process.
The viral DNA along with some associated proteins pass into the host cell. The main feature of phage infection is that all other parts of the virus remain outside the infected cell except the phage DNA and the associated proteins present in the phage head.
The penetration is diagrammatically shown in Fig. 6.16:
That in phage infection, the protein capsid including the head and the tail with all its parts remain outside the infected cell, and only the nucleic acid enters into the cell was elegantly proved by a classical experiment conducted by Hershey and Chase (1953). The essential feature of the experiment is briefly described here and in Fig. 6.17.
The coliphage T2 was used in the Hershey and Chase experiment which infects E. coli B. In two sets of T2 phage, one was labelled with radioactive 32P by growing the host bacteria in 32P-labelled phosphate containing medium and then the phage was allowed to infect the labelled bacteria, so that the phage DNA became labelled with 32P.
In another set, the phage was labelled with 35S by growing E. coli B in 35S-labelled sulfate containing medium and, subsequently, allowing the phage to grow in the labelled bacteria. As sulfur is absent in DNA, but present in protein, and phosphorus is absent in protein, but present in nucleic acids, the two sets of phages contained 32P-labelled nucleic acid core in one set and 35S-labelled capsid in the other set.
The two sets of labelled phages were allowed to infect the host bacteria separately. The unabsorbed phages were removed by centrifugation, the bacterial mass was suspended and the adsorbed phages were dislodged by violent shaking in a blender. Then the distribution of radioactivity in the bacteria and the suspending medium containing the empty capsids was measured.
The results showed that in the set in which the bacteria were infected with 35S-labelled phage, the major portion of radioactivity (-97%) was present in the suspending medium and only about 3% in the bacterial cells. The small amount of radioactivity in the cells could be accounted for by the DNA- associated proteins which were injected along with DNA.
On the other hand, when the bacteria were infected by 32-labelled phage, the radioactivity was detected almost exclusively in the bacterial cells and practically no activity in the suspending medium. The experiment thus convincingly proved that in bacteriophage infection, only DNA enters into the host cells, leaving the coat outside.
Hershey and Chase experiment also proved another very important matter. It showed that for replication of phage, only DNA is necessary and the capsid proteins do not play any role in reproduction of progeny phages. In other words, the genetic information necessary for building up of new phage particles is located in DNA. Thus, the experiment also adduced evidence to prove that DNA is the genetic material.
(iii) Synthesis of Phage Components:
Synthesis of phage components in the infected host bacteria occurs by a large number of sequential steps controlled by the viral nucleic acid. These steps initiated by the entry of the phage nucleic acid and ending in the appearance of extracellular progeny phages constitute the phage multiplication cycle. The multiplication cycle has been best studied in the E. coli phage T4.
The presence of an unusual base, viz. hydroxymethyl cytosine (HMC) in the DNA of T-even bacteriophages, makes it possible to follow the behaviour of the phage DNA in the infected cells. The DNA of these phages contains HMC in place of cytosine. HMC of T4 phage DNA is often glucosylated by addition of glucose to the hydroxyl group (Fig. 6.18).
The multiplication cycle of the lytic phages in host bacteria can be divided into several phases. These phases were first clearly defined by the classical experiments of Delbriick (1930) and is known as one-step growth experiment. The objective of these experiments was to study the kinetics of lytic phage development following infection of host bacteria.
The ability of lytic phages to form clear, more or less circular zones, called plaques on confluent growth of the host bacterium in a Petri dish was utilized. A plaque is taken to originate from a single ‘plaque forming unit’ (PFU) present in the original phage suspension plated. Number of PFU/ml of the suspension is called its plaque-titre.
For these experiments, a dilute suspension of phage T2 was mixed with a broth culture of E. coli B and the phages were allowed to infect bacteria. After some time (about two minutes) the un-adsorbed phages were neutralized by adding specific antiserum, making them ineffective in infecting bacteria. The mixture was then highly diluted with fresh broth and incubated at 37°C. Samples were withdrawn at intervals and the plaque-titre of the sample was determined.
A plot of PFU/ml against time in minutes produced a sigmoid curve which could be divided into several phases (Fig. 6.19). During the first 10 minutes or so, no phage particles could be recovered even by disrupting the bacteria by treating with chloroform. This period was called the eclipse phase.
The eclipse phase ends with the appearance of the first intracellular phage virion initiating the next phase, the latent phase. During this phase, mature phage virions accumulate within the infected cell, but they are not released extracellularly.
The latent phase terminates with the appearance of phages in the medium due to lysis of the infected bacteria. This is known as the rise period. During this period the PFU/ml increases sharply until a maximum value is reached indicating that all the infected bacteria had undergone lysis releasing the maximum number of phage virions in the extracellular medium. Since, in these experiments, a bacterial population was treated, all the bacteria were not infected at the same time and were not lysed at the same time.
This explains the gradual increase in extracellular phage-titre during the rise period. The number of phages released from a single infected bacterium is known as the burst size. In case of T2 phage infecting E. coli B, the burst size is approximately 100.
It is to be noted that a plaque is formed because the phage particles released by lysis of a single bacterium infects the neighbouring host cells leading to their lysis and the process continues like a chain reaction until a visible clear zone is produced which is known as a plaque. Thus, each plaque represents an infected bacterium, and not a single phage.
The entry of phage DNA into the host cell causes a profound change in the host synthetic processes. Synthesis of bacterial DNA stops almost immediately and is soon replaced by viral synthesis. The viral DNA takes over control and, as a result, the synthesis of DNA, RNA and proteins of the host stops and the biochemical machinery is diverted for synthesis of viral products.
Synthesis of viral products begins with transcription of viral m-RNAs from the parental phage DNA catalysed by the host RNA-polymerase. These early m-RNAs are translated by the host enzymes and ribosomes to produce viral proteins which take part in degradation of the host DNA to the stage of nucleotides which arc subsequently used for synthesis of viral DNA.
Some of the early proteins are used for modification of the bacterial RNA polymerase so that the enzyme can recognize the promoters of phage DNA. An important early protein is the viral DNA polymerase for replication of the parental phage DNA. T4 DNA contains an unusual base, hydroxymethyl cytosine (HMC) in glucosylated form.
Although the other nucleotides necessary for synthesis of phage DNA are derived from the degraded host DNA, HMC has to be synthesized anew. Synthesis of this nucleotide base is catalysed by two enzymes encoded in the phage DNA. Glucosylation of HMC protects it from the host restriction endonucleases. Thus, as observed in case of animal viruses, the synthetic processes in infected cells can be temporally distinguished into an early phase and a late phase.
The two phases can be demarcated by the commencement of phage DNA synthesis with the help of phage-specific DNA polymerase synthesized during the early phase. The formation of late proteins requires DNA replication.
The late messengers transcribed from the progeny DNA molecules code for the coat proteins, like that of the head, the tail, the base plate, tail fibres and also the enzyme, phage lysozyme required for lysis of the bacterial cell wall during release of the progeny viruses from the infected cell.
All these synthetic processes take place during the first 10 to 12 minutes of infection which constitute the eclipse phase. During this period no intracellular phage particle is detected. Appearance of intracellular phages begins when sufficient number of phage DNA molecules and the component protein molecules accumulate in the infected cell.
(iv) Assembly:
Assembly of mature phage virions in the host cell is a complex process involving many steps controlled by viral genes. Numerous proteins participate in the morphogenesis of a complex phage capsid as that of T4 or other T phages. All these proteins are mainly late proteins, except three which are internal proteins.
T4 phage DNA is formed as a long linear intermediate containing several T4 genomes covalently linked end to end to produce a concatemer. At the end of DNA replication, these intermediates i.e. concatemers are cleaved into pieces that correspond to the size of mature DNA contained in a T4 head.
The cleaved pieces have repetitive ends in T4 phage as shown in Fig. 6.20:
The T4-DNA is about 500 μm long. This long molecule must be condensed and efficiently packed into the phage head which measures only 65 nm in width and 100 nm in length. During maturation phase, the components of the progeny phage virions are assembled to form the capsid and the nucleic acid is introduced into the phage head.
A rigorous genetic control is observed in the assembly of mature phages. The capsid is assembled through three independent lines in step-wise manner. These lines produce the phage head in one, the tail in another and the tail fibres in the third.
The phage head is built up from several proteins of which P23 is the main constituent. Other proteins are then sequentially added to give the final form of the phage head. In another assembly line, the base plate is first formed and the central hollow core of the tail is built on it followed by the assembly of the contractile sheath around the core. The tail fibres,-each of which consists of a proximal part and a distal part, joined to each other with a kink, are separately assembled and are attached to the base plate in an independent assembly line.
Finally, the assembly of mature phage virion requires the long DNA molecule to be folded and packed into the empty phage head. The exact mechanism of DNA packaging is not known. Probably, a head protein binds to the DNA concatemer and trans-locates the DNA into the head.
The protein probably has also cleavage activity and it determines the length of the DNA which fills the empty head cavity. Special internal proteins appear to be involved in cross-linking of the long DNA molecule into closely packed layers.
The assembly lines of T4-phage is diagrammatically shown in Fig. 6.21:
(v) Release of Virions From Infected Cells:
The T-even bacteriophages as well as many others are released by lysis of their host cells. At least three viral genes appear to be involved in causing lysis in case of E. coli cells infected by phage T4. One gene product is lysozyme which attacks the murein layer of the cell. Another gene promotes lysis by modifying the cytoplasmic membrane and stopping cellular metabolism. The action of these gene products facilitates the passage of lysozyme to reach the cell wall. Some bacteriophages, like the filamentous ones, come out of the infected cells without causing lysis.
The multiplication cycle of T4 phage can be diagrammatically represented in a simplified way as shown in Fig. 6.22:
Single-Stranded Bacteriophages:
(i) Single-Stranded DNA Phages:
The single-stranded DNA phages can have either icosahedral symmetry or helical symmetry. Both types have single stranded circular DNA as genetic material. The best known among the icosahedral type is bacteriophage φX174 which is a lytic phage infecting E. coli.
On the other hand, among the more well-known circular ss-DNA helical phages are fd, fi, M13 etc. also infecting E. coli. They do not causp lysis of the host cell. Also, they differ from other phages in their mode of entry. The intact virions enter into the cell.
The single-stranded DNA phages differ from other phages in that they depend heavily on the host and, therefore, they do not generally inhibit the biosynthetic processes of the hosts. This is particularly evident in the linear non-lytic phages like fd or fi, where the phages are formed continuously in the growing host cells and are released by extrusion without lysis.
(a) Bacteriophage φX174:
The best known ss-DNA phage, φX174 has an icosahedral virion without a tail belonging to the Family Microviridae. The genome has a molecular weight of 1.6 x 106 Daltons containing 4,500 nucleotides encoding nine genes. The DNA contains uracil in place of thymine. It has a (+) DNA strand.
The circular ss-DNA is injected into the host cell and is attached at a site on the host membrane from which it is never transferred to the progeny phage virions. The attached phage DNA is immediately converted to a ds-DNA by synthesizing a complementary (-) strand with the help of a host DNA-polymerase. This double-stranded circular DNA is the replicative form (RF).
Next, the RF is transcribed into (+) m-RNAs which are translated into viral proteins. The parental RF remains attached at its site and is then replicated repeatedly to produce daughter RFs. The daughter RFs then replicate to produce single-stranded (+) DNA strands which are incorporated in the progeny phage virions. The linear (+) DNA strands are circularized before packaging.
The progeny phage virions are assembled and released from the bacterial host (E. coli) by lysis. However, 0X174 does not produce lysozyme. The lytic cycle is completed within 16 to 18 minutes. Several hundred progeny phage virions are released from each infected cell.
The multiplication cycle of 0X174 is shown in Fig. 6.23:
(b) Single-stranded filamentous phages:
Single-stranded DNA containing filamentous phages having a helical symmetry belong to the Family Inoviridae. Examples of these phages are phages fd and M 13, both grow in E. coli strains. The phage fd is about 6 nm in diameter and 900 to 1,900 nm long.
It has a single-stranded circular DNA genome. It differs from other bacteriophages in that the whole virion enters into the cell. Also, on maturity the virions are not released by lysis of the host cell. The host cell continues to live and continuously produce the phage. The progeny phage virions are extruded from the host cell.
Like that of φX174, the ss-DNA of these phages is converted to a double-stranded replicative form which replicates to produce more RF. The multiplication of phage DNA occurs in actively growing host cells. The coat proteins are synthesized and are inserted into the host cell membrane. They are organized around the viral ss-DNA as they are extruded from the infected cell producing intact phage virions. The ss-DNA coliphage f1 also behaves in a similar manner.
(ii) RNA Bacteriophages:
There are some bacteriophages which possess RNA instead of DNA as genetic material. Most of them have a single-stranded RNA genome. The only ds-RNA bacteriophage is the φ6 which infects Pseudomonas phaseolicola. It has an enveloped icosahedral virion and belongs to the Family Cystoviridae.
There are several ss-RNA bacteriophages, like f2, R17, MS2 and Qβ. They are small, icosahedral viruses without a tail. The genome size is about 1.1 x 106 Daltons, encoding only three or four proteins. The genes located on a single-stranded (+) RNA code for a maturation protein, a capsid protomer protein and an RNA dependent RNA polymerase (replicase). Some of the ss-RNA phages infecting E. coli, like f2, MS2 and Qβ attach to the F-pili (sex pili) of F’-cells and inject the ss-RNA.
The viral RNA replicates in the infected host cell via a replicative form (RF) consisting of a ds-RNA. The parental (+) strand immediately after entry acts as a messenger RNA for production of the replicase protein. The replicase then copies the (+) strand into a (-) strand and the ds-RNA (RF) is produced. The RF then produces many copies of (+) RNA, most of which are utilized as messenger RNA to synthesise viral proteins including the capsid protomers. The rest of the (+) RNA strands are packaged into new progeny viruses. The new phage virions are ultimately released by lysis of the host cells, lysis being caused by a phage coded enzyme.
The multiplication cycle of ss-RNA phages is shown in Fig. VI-24:
Infection Cycle of Temperate Bacteriophages and Lysogeny:
The bacteriophages described so far included the virulent types, most of which infect the hosts leading to a lytic Cycle resulting in lysis. Many phages — perhaps the majority of them — show a different form of parasitism. These bacteriophages, known as temperate, do not cause immediate lysis of the infected host, but enter into a sort of Symbiotic relationship, called lysogeny.
The injected phage DNA persist in the host cell as a prophage for generations and under suitable conditions the viral DNA in the lysogenic cells is induced to multiply like virulent phages leading to lysis and are liberated to attack new host cells. The temperate phages when they infect appropriate bacterial strains may show two alternative responses.
One of the options is to carry out a lytic cycle like a virulent phage, and the other is to enter into a lysogenic state. When the lysogenic host cells are disrupted artificially, no infectious phages are recovered which shows that the phage must be present in a non-infectious state.
A characteristic feature of the temperate phages is that they produce turbid plaques on a lawn of the lysogenic bacterial host. In contrast, the lytic virulent phages produce clear plaques. A turbid plaque is produced because only some cells of the host are lysed at any given time producing progeny phages, while the majority of the population enters into lysogenic state.
Another important feature of lysogenic bacteria is that they are immune to infection by the same or related bacteriophages, though not immune to attack by other unrelated phages. Such immunity has been ascribed to the changes in the lipopolysaccharide surface layer of the infected bacteria eliminating the receptor sites of the host cells for attachment of phages e.g. in the epsilon phage of Salmonella.
Sometimes, lysogenic hosts acquire new properties which are absent in uninfected bacteria. This is known as lysogenic conversion. For example, toxin production of Corynebacterium diphtheriae is due to the temperate β-phage.
Bacteria cured of this phage are non-toxigenic, because the gene encoding the exotoxin protein is carried by the P-phage. Similar toxigenic phages have been found in Clostridium botulinum, Vibrio cholerae and streptococci causing scarlet fever.
The most widely known temperate phage is the lambda (λ) phage infecting E. coli K12. It is a double-stranded DNA virus with an icosahedral head, about 55 nm in diameter and a long tail (-180 nm) without a contractile sheath. The tail is provided with a thin tail fibre at its distal end.
The double- stranded DNA of the virion is linear with 12 nucleotide long single-stranded cohesive ends at the 5′(P) ends. The cohesive ends are complementary and with the help of these single-stranded segments, the linear molecule can form a circular ds-DNA molecule with two staggered nicks which are sealed in vivo by DNA ligase. The linear virion DNA produces a covalently closed circular DNA soon after entering the host cell.
These structural features are shown in Fig. 6.25:
After circularization, the A-DNA may function in two alternative ways. In one, the phage DNA can initiate a vegetative or lytic cycle involving replication of the circular DNA, transcription and translation and ending with assembly of progeny phages and release. In the other alternative way, the phage DNA after circularization is integrated into the bacterial chromosome at a specific site to become a prophage and the host bacterium becomes lysogenic.
In this state, the phage DNA becomes a part of the bacterial DNA and it replicates along with the latter. The genes controlling vegetative multiplication leading to the lytic cycle of the phage remain inhibited by a repressor, called A-repressor. The vegetative multiplication of A-phage and its lysogenic cycle are described separately.
A simplified representation of the alternative pathway is shown in Fig. 6.26:
(i) The Vegetative or Lytic Cycle:
After attachment to E. coli K12 cell, A-phage injects its linear DNA into the host cell. Attachment is helped by the single tail fibre. After entry, the linear ds-DNA is converted to a circular molecule with the help of the single-stranded cohesive ends.
The phage DNA then is attached to a membrane site of the host cell and starts replication. The replication starts near the origin and proceeds in both directions symmetrically and terminates when the two replicating forks meet each other going through a typical Ө (theta) configuration.
The daughter DNA molecules derived from the parental DNA then replicate in the cytoplasm by rolling-circle model producing long linear concatemers. From these concatemers containing several A-genomes, appropriate lengths with single- stranded cohesive ends are cut by specific enzymes and packaged into phage heads. The transcription of phage DNA produces messengers coding the capsid and other phage proteins.
Transcription of the phage DNA is initiated by a host polymerase which binds to two promoters on the A-DNA transcribing two different strands in opposite directions. The early genes transcribed are those coding regulatory proteins controlling the lytic cycle. More than 40 genes have been mapped on the A-DNA and most of them are clustered according to their functions, such as synthesis of head proteins, tail proteins, synthesis of viral DNA, cell lysis, lysogeny etc.
A simplified representation of the A-DNA is shown in Fig. 6.27. When sufficient numbers of virion components have been synthesized, the progeny phages are assembled and released by cell lysis. The liberated A-virions then infect new host cells and can either carry out another lytic cycle or can enter into a lysogenic cycle.
Generally, only a small proportion of the infected cells are subjected to lysis and the major proportion enters into lysogenic relationship. In the latter group, the circularized A-DNA is integrated into the host chromosome.
(ii) The Lysogenic Cycle:
The λ-phage after attachment to the host cell injects its DNA into the cytoplasm and the DNA is circularized in the usual way. When the phage enters into a lysogenic relationship, the genes controlling phage multiplication and lysis of the host cell are inhibited by the λ-repressor produced by a phage gene, cl. The lambda-repressor is an acidic protein having a molecular weight of 26,000 Daltons and containing 236 amino acid residues. It specifically binds to two different operators of the λ-genome, thereby blocking their transcription.
These two operators, called OL and OR, are involved in initiation of transcription of phage genes controlling the multiplication of phages. The repression of OL and OR by the λ-repressor stops synthesis of two important proteins, ell and cIII which are essential for initiation of phage multiplication. This ensures the establishment of the lysogenic state.
Lysogenization of λ-phage is accomplished by integration of the circular phage DNA into the bacterial DNA. Integration is catalysed by an enzyme, integrase coded by the int gene of the λ-genome. This enzyme can recognize a specific site in the phage DNA as well as a site in the bacterial DNA and it catalyses a site-specific recombination resulting in integration. In the process, the circular λ-DNA is inserted as a linear DNA into the host DNA at a specific site between the galactose and biotin operons. The inserted phage DNA then becomes the prophage (Fig. 6.28).
As prophage, the X-DNA replicates as a part of the host DNA and is perpetuated indefinitely. However, each lysogenic bacterium also inherits the capacity to produce phage particles, though this capacity is not expressed frequently. A lysogenic bacterium produces phage particles through the process of induction. Such induction may occur spontaneously at a low frequency in about one out of 102 to 105 cells, or may be due to external agencies, like UV, X-rays, γ-rays and certain chemical mutagens.
On induction, the prophage is released from the bacterial chromosome as vegetative λ-DNA in circular form. The excision of prophage occurs by reversal of the integration process and catalysed by a phage-coded enzyme, called excisionase. The corresponding gene is called xis gene. The freed λ-DNA then carries out the lytic cycle, as in the case of vegetative multiplication.
The key factor in lytic induction is the reduction in the level of λ-repressor and the increased synthesis of another protein, called the croprotein which initiates the process of synthesis of viral components. The drop in the concentration of λ-repressor is caused by degradation of the repressor protein by a proteolytic enzyme produced by the rec A gene of E. coli.
The gene is apparently activated by the exposure to the inducing agent e.g. UV to synthesise a rec A protein having a proteolytic activity. The enzyme cleaves λ-repressor to an inactive form which can no longer bind to the promoter inhibiting transcription of the lytic genes.
As a result the cro gene is activated to produce cro-protein which acts as a repressor inhibiting the synthesis of A-repressor. Thus, the two proteins, viz. λ-repressor and cro-protein, are mutually antagonistic in their action. They compete with each other in maintaining the lysogenic state and the vegetative state of the λ-DNA. This aspect has been treated in more details below.
Most of the temperate phages infect their respective hosts and their DNA is integrated into the bacterial chromosome at fixed locations. For example, A-DNA is inserted into E. coli K 12 chromosome between the galactose and biotin operons (Fig. 6.28).
Such integration is effected through a single reciprocal crossing-over event involving both the circular λ-DNA and the circular E. coli chromosome. Some phages like P2 of E. coli, can insert its DNA into several sites of the host chromosome. Phage μ (mu), on the other hand, has no specific site of insertion in the E. coli chromosome and its DNA can be inserted at any site of the host chromosome causing inactivation of that site (mutation). The name of the phage μ (mu) has been derived from its ability to cause random mutations of the infected host cells.
In some temperate phages, like E. coli phage PI, the viral DNA is not integrated into the host chromosome and remains separate as a plasmid in the infected host cell.
Lysogeny has an important genetical implication, because the temperate phages can act as agents for gene transfer in the bacterial hosts they infect by a process known as specialized transduction. When a temperate phage as prophage is excised from its host chromosome, it can include by mistake a part of the host DNA. After lysis the phages can infect host cells and transfer the portion of bacterial DNA to them.
(iii) Choice between Lysogeny and Vegetative Multiplication:
When A-phage DNA enters into a host cell, transcription of the phage DNA starts immediately.
The gene order in the relevant portion of the A-DNA and their products are diagrammatically shown in Fig. 6.29:
Transcription starts from PR and PL. The N-protein is produced and it allows transcription of cIII leftward and cro-gene, cll and other early genes, O, P, Q etc. Both cro and cII proteins accumulate in the infected cell at the early stage. These two proteins have a critical role in the choice between lysogeny and lytic cycle.
The cII protein activates cI gene to produce the λ-repressor which prevents transcription of all genes required for lytic growth. The cro-protein, on the other hand, inhibits λ-repressor synthesis by blocking the promoter (PRM) from where the repressor is made during lysogenic cycle (PRM = Promoter for repressor maintenance).
Whether a λ-phage infection will lead to lysogeny or to a lytic cycle depends on which of the two proteins, λ-repressor or cro-protein wins the competition. In case of lytic development, the cro-protein predominates and it occupies the operators, OR and OL preventing synthesis of repressor m-RNA from promoter PRM, but allowing rightward transcription of the early and late genes.
Alternatively, when the infection leads to the development of lysogeny, the cl gene product i.e. the λ-repressor predominates. It binds to OR and OL controlling the promoters PR and PRM. cll protein is synthesized and it stimulates synthesis of m-RNA for the enzyme integrase which catalyses incorporation of λ-DNA in the E. coli chromosome. Moreover, λ-repressor can also stimulate its own synthesis by binding to 0R causing more m-RNA to be transcribed from PRM. Thus, the two alternative pathways are chiefly controlled by the two proteins, A-repressor and the cro-protein.
The sequence of events after entry of A-DNA into the host cell can be described by a flow-sheet shown in Fig. 6.30:
