Life Cycles of Lambda: Lytic and Lysogenic | Microbiology

Lambda consists of two types of life cycles, the lytic and lysogenic.

Lytic Cycle:

The events of lytic cycle, starting with adsorption, at 37°C occurs as below:

Time (t) = 0 minutes: Phage adsorbed and DNA is injected

= 3 minutes: First early mRNA is synthesized

= 5 minutes: Two classes of early mRNA are synthesized

= 6 minutes: DNA replication begins

= 9 minutes: Synthesis of late mRNA begins

= 10 minutes: Synthesis of structural protein occurs

= 22 minutes: First phage particle is completed

= 45 minutes: Lysis of bacterial cell envelope and release of progeny phage.

Life cycle of phage λ is 45 minutes long, as compared to 22-25 minutes long life cycle of T4 phage. In general the life cycle of most phages at 37°C varies between 22 and 60 minutes. After injection, the linear phage DNA is circularized.

The cos site results in a circular form of DNA which starts the two events:

(i) Transcription and translation of DNA, and

(ii) Replication of DNA.

As a result of replication numerous DNA molecules are formed in the form of concatemers. Structural proteins for head and tail are synthesized through translation of mRNA. During maturation assembly of phage particles occurs. The lytic cycle ends with lysis of host cell envelope and releases lambda particle.

The events of lytic cycle are discussed in detail as below:

(i) Circularization of Phage DNA:

After injection the linear phage DNA is delivered into bacterial cell. The cohesive ends form hydrogen bonds. The E.coli DNA seals the breaks and the DNA is converted into closed circle within three minutes after infection.

(ii) Transcription:

After circularization of phage DNA, transcription begins by the modified RNA polymerase of host. The modification enables the polymerase to ignore certain termination sites. Fig. 18.15 shows a detailed version of genetic map of phage.

There are three regulatory genes era, N and Q, three promoters, left promoter (PL), right promoter (PR1 and PR2). The DNA replication genes O and P, and five termination sites (tL1, tR1, tR2, tR3 and tR4). The L and R series are transcribed leftward and rightward, respectively from the complementary DNA strands.

On the basis of transcription the genes are grouped into three classes, immediate early genes (N and cro), delayed early genes (located left to N e.g. cIII, gam, red, xis and int, and right to cro e.g. cll, O, P and Q) and late genes.

Consequently three classes of mRNAs are transcribed e.g. immediate early, delayed early and late mRNAs. Early mRNAs are transcribed from both left and right strands, whereas late mRNAs are transcribed from left to right, and the left transcribes from right to left.

First of all O and P genes are transcribed whose products are necessary for DNA synthesis. This is followed by transcription of gene coding for structural proteins, packaging system and finally lytic proteins.

Before transcription of O and P genes, two immediate early mRNA transcripts are formed that codes for regulatory proteins responsible for turning ‘on’ and ‘off to the leftward transcription and rightward transcription when ever required.

The phage has two promoters, PL and PR which initiates the synthesis of RNA molecules L1 and L2. Initially transcription terminates at the sites tL1 and tR1. L1 encodes gene product N (gpN) which is the delayed early gene product and a major regulatory factor that controls certain regions of DNA when it is transcribed.

After synthesis, gpN binds to nutL and nutR sites present at left and right side of the promoters. When RNA polymerase moves along with the DNA, it picks up the gpN. The gpN enables the polymerase to ignore the termination sites (tL1 and tR1) resulting in longer transcripts L1 and L2. Therefore, the gpN acts as anti-terminator and neutralizes the effect of tL1 and tR2. Hence, the gpN controls the expression of most vital function.

After inhibition of termination, transcription occurs in leftward direction and extends upto b2 region, and the rightward transcription extends upto Q gene. The rightward transcription permits the synthesis of O and P gene products. The leftward transcript consists of a red region that codes for two proteins required for genetic recombination.

The O, P and red proteins have catalytic property; therefore, they are not made continuously. When sufficient amount of gpcro (gene product of cro) encoded in R1 is available, it binds to leftward operator (OL), and the repressor activity of cro turns off the synthesis of all leftward mRNA.

The tR3 is the termination site of mRNA even after modification by gpN. Therefore, the rightward transcription terminates at tR3. However, during the time of early transcription sufficient amount of mRNA is produced by rightward transcription. Thus the O and P proteins become sufficient for DNA replication.

After binding of gpcro to OL the concentration of gpcro increases, and also binds to OR and turns off the rightward mRNA synthesis. Therefore, wasteful synthesis of O and P proteins does not occur, and aberrant and deleterious DNA synthesis are also checked.

The gene product of cll (gpcll) is encoded in the same transcript containing O and P during the early transcription. The sufficient amount of gpcll acts as late promoter and delays late mRNA synthesis. After turning of rightward transcription by gpcro, the gpcll is not synthesized.

This relieves the inhibition of late mRNA synthesis. At this time the gpQ (a positive regulator) is required that begins the late mRNA synthesis and neutralizes the third right terminator (tR3) and allow to proceed transcription through vegetative genes to J gene and into the b2 region of DNA molecule.

The gpQ turns on late mRNA synthesis which translates structural and assembly proteins, maturation system and the lysis enzymes. The gpQ is also called anti-terminator which binds the qut sequence and taken up by RNA polymerase. Therefore, the RNA polymerase ignores tR4. Taking advantage of it R4 is extended and forms R5 transcript of late mRNA which synthesizes the head, tail and lysis proteins.

(iii) Replication:

The phage λ replicates autonomously during lytic as well as lysogenic cycles by using only exogenous precursors. The host chromosome is not degraded by the phage, unlike T4 phage.

The replication is accomplished in two stages, early replication and late replication stages:

(a) Early replication (theta mode of replication):

During early replication the circular DNA molecule is associated with host’s cell membrane, and replicates to produce circular copies of DNA molecule. On the genome an origin for replication (ori site) is situated within O gene.

The gpO and gpP nick the circular DNA at ori site. Replication is bidirectional and proceeds in opposite directions from the ori site (Fig. 18.16A). In another temperate phage P2, replication is unidirec­tional. The replication fork moves around the circle and forms the Greek letter theta (0); therefore, it is called theta mode of replication.

The two branch points are called replication forks at which the non-replicated original duplex joins the two daughter chromosomes. At the end of replication two identical copies of circular DNA molecule are formed. Thus the theta mode increases the number of templates for transcription and further replication. For the first time Cairns (1963) reported the theta mode of replication in E. coli.

(b) Late replication (rolling circle mode of replication):

After the synthesis of circular copies of DNA the progenies dissociate from the cell membrane and switch over from theta to rolling circle model of replication. By the time heads and tails have been synthesized and the sequence- specific cutting system called the terminase (Ter) system (Ter proteins are the components of an empty head) becomes active resulting in predominance of rolling circles.

A nick is made at a point on outer strand of duplex (Fig. 18.16 B). Circle rolls and a new strand is synthesized at 3′ end. The 5′ end single stranded DNA is displaced. Finally, the displaced strand contains a long ssDNA molecule of one parental strand and other newly synthesized strand. The rolling circle has the two types of cos sites, one in the circle and the second in the linear branch. In the linear branch there are several cos sites. Such branch is called concatemeric branch.

During replication the cos site of rolling circle does not open because in the open strand replication cannot occur. If two cos sites are present in circular DNA, one is cut by Ter system resulting in free end in concatemer and removal of phage DNA. Thus, Ter-cutting requires two cos sites or one cos site and a free cohesive end on a single DNA molecule. The Ter system was first identified-by genetic analysis of tandem di-lysogens i.e. cells having two adjacent prophage.

The cut dsDNA of phage thus liberated from the concatemeric branch contains 12 nucleotide long ssDNA that acts as cohesive ends. The unit length genome synthesized by this mechanism contains exactly the phage genome. The gpgam is required to inhibit recBC endonuclease V of the host. Otherwise the concatemers would be broken down.

The two process cutting at cos sites and packaging of phage genome are coupled.

(iv) Assembly:

Hohn and Katsura (1977) have described about the structure and assembly of phage. During the process of maturation, the phage particles (head and tail) are independently assembled. The genes encoding for DNA maturation and phage head proteins are: nul, A, W, B, C, nu3, D, E and F. The genAes that code for phage tail are: Z, U, V, G, H, M, K, L, I and J. There are bacterial genes (groES and groEL) that also help in assembly of phage particles.

Different steps in DNA packaging in phage λ head assembly have been described by Kaiser et al. (1975). The process of assembly begins with aggregation of several copies of four head proteins which built up a scaffolded pro-head (Fig.18.17). The pro-head is only a sphere supported with an internal supporting system. Therefore, it looks like a wheel. Many phages form this type of scaffolded pro-head.

In the second stage, gene product of groES and groEL genes i.e. GroEs and GroEL proteins interact and form GroES-GroEL complex. This complex binds to scaffold prohead. The scaffolding is removed by bacterial protease. In some phages, scaffolding falls away and is reused. Gene products of nul and A (gpnul and gpA) that contain Ter system interact a short base-sequence near one cos site with a point on the head.

Later on, it becomes the region for head-tail attachment. The phage DNA folds into the head. A change in conformation in E protein occurs after a small amount of DNA enters into the head. The gpE and the changed E protein causes the formation of icosahedral head. The gpF1 plays a role in expansion of spherical particles to icosahedron. A small amount of gpD enters into head which is filled with λ DNA by the unknown mechanism.

When the next cos site reaches the head during the process of filling, it is cut by Ter system generating the sticky ends. The unpacked DNA is released from the filled head. Insertion of phage λ DNA occurs till the cos site comes. The fully packed particle is called black particle.

During this process tail is assembled by several tail proteins, and terminated by a head-tail connector protein. The complete tail is bound to the head through the short piece of ssDNA and head protein present in the neck.

The free ssDNA of released DNA binds to the neck of the second pro-head, and so on. In this way the complete phage particles are formed. Murialdo (1991) has reviewed the bacteriophage λ DNA maturation and packaging.

(v) Lysis:

Inside the bacterial cell about 100 particles are assembled within an hour. The two genes S and R of λ take part in bacterial lysis. The gpS stops metabolism of bacterial cell, and gpR lyses the cell wall. Finally progenies are released from the destroyed cell.

Lysogenic Cycle:

The alternative cycle of phage X where progenies are not produced is called lysogenic pathway. The phage genome is integrated into bacterial chromosomes. The host cell survives for indefinite time. The host cells that contain integrated phage DNA, i.e. prophage, is called the lysogen.

The prophage multiplies for several generations. The prophage is excised from the bacterial chromo­somes due to stimulation by UV irradiation or mitomycin C. After excision of DNA, the phage leads lytic cycle and the host cell is killed.

The lysogenic bacteria bear the two key features, immunity to super-infection by other phage λ and induction under certain environmental conditions to enter into lytic cycle. The immunity to super-infection and establishment of lysogeny in lysogens is conferred in the presence of λ repressor coded by cl gene. In a lysogen, repressor is always synthesized to bind operators OL and OP resulting in blocking of RNA polymerase activity.

Thus the repressor prevents the transcription of all prophage genes except its own. As a result of blocking of PL, transcription of N gene does not occur. Similarly blocking of PR prevents the early transcriptional genes i.e. O, P and O genes.

The gpcll activates the specific promotor site (PRE) for transcription of cl gene and synthesis of the repressor (gpcl). Therefore, the maintenance of lysogenic state requires that the synthesis of repressor must be continued.

Establishment of lysogenic state occurs as described below:

(i) Integration (Insertion):

The process of firmly joining of phage DNA with bacterial chromosomes is called integration or insertion (Fig. 18.18). The significant features of insertion mechanism have been given by Allen Campbell in 1962 who obtained a λ prophage map by three factor crosses with two I genes and a gal (galactose utilizing) gene marker of bacterial chromosomes.

The two important features of the Campbell model are:

(i) The formation of a circular DNA from the linear DNA, and

(ii) At specific loci in a phage and bacterial DNA, a single reciprocal recombination results in the insertion of phage DNA into the bacterial chromosome.

(a) The Attachment sites:

The specific loci are called the attachment sites (Fig.18.18). The attachment site of phage is designed as attP which consists of two halves, P.P. Similarly, the attachment site of bacterial chromosomes designated as attB, and its two halves as B.B.

The dots (.) between the two att sites are the points where crossing over occurs. This point has a common base pairs and designated as O. Thus the complete att sites are designated as POP’ and BOB’. The phage att site is located between the int and J genes, and bacterial att site is situated between gal (galactose) and bio (biotin) genes.

(b) Mechanism of Integration:

The essence of mechanism which is called Campbell model is the circularization of DNA followed by physical breakage and rejoining of phage and bacterial DNA between the POP’ and BOB’. The Ter endonuclease makes staggered nicks in A, DNA. The gpcll stimulates transcription of the int gene at the same time as that of cl gene.

The int gene codes for synthesis of an integrase enzyme which becomes in plentiful before I repressor turns off transcription. The attPOP’ and attBOB’ match each other. The integrase with the help of a special bacterial protein catalyses the physical exchange of viral and bacterial DNA strand.

The circular DNA is integrated into the E.coli chromosomes as a linear DNA between gal and bio genes, and is called prophage (Fig 18.18). The phage DNA joins with bacterial chromosomes by covalent bonds. The process by which the X DNA is inserted into the bacterial chromosome is called site-specific recombination. The point recognises the att sites and brings about the reciprocal crossing over between the POP’ and BOB’ sites.

(ii) Replication:

The bacterium containing a complete set of phage genes is called lysogen and the life cycle as lysogenic cycle. The process of formation of a lysogen by a temperate phage is called lysogenization. Now the prophage replicates normally under the control of the bacterium by normal bacterial replication mechanism. The replication prophage contributes to viral growth and produces phage particles.

However, integration is not an absolute requirement for lysogeny. In E.coli phage, P1 is similar to X which circularizes after infection and starts synthesizing repressor. Therefore, it remains as an independent circular DNA molecule in the lysogen and replicates as the chromosome. After the bacterial cell divides, the daughter cell contains one or two copies of phage genome.

(iii) Excision:

When the host cell is unable to survive, the λ prophage leaves the E.coli genome and begins the production of new phages. This process is known as induction which is triggered by a drop in X repressor level. Whenever, the repressor will decline, the lytic cycle will commence. In addition, induction occurs in response to environmental factors e.g. UV light or chemical mutagens that damage host DNA.

This damage causes the synthesis of recA protein, which acts as protease and cleaves repressor chain between the two domains. RecA protein binds to X repressor and stimulates it to proteolytically cleave itself. An early gene (xis gene) codes for synthesis of excision are that binds to the integrase (int gene product) and enables it to excise the prophage. Thus, the excision is the reversal process of integration (Fig 18.18).

After excision phage is converted to its circular form and enters the lytic cycle. During the course of excision, as a result of mistake, the bacterial gal or bio gene remains included in phage genome. This mistake occurs at a frequency of one in a million.