ENTEROBACTERIA PHAGE λ (LAMBDA PHAGE , COLIPHAGE λ) is a bacterial
virus, or bacteriophage , that infects the bacterial species
The phage particle consists of a head (also known as a capsid ), a
tail, and tail fibers (see image of virus below). The head contains
the phage's double-strand linear
* 1 Anatomy
* 2 Life cycle
* 2.1 Infection
* 2.1.1 N antitermination
* 2.2 Lytic life cycle
* 2.2.1 Rightward transcription
* 188.8.131.52 Lytic replication * 184.108.40.206 Q antitermination
* 2.2.2 Leftward transcription
* 220.127.116.11 xis and int regulation of insertion and excision
* 2.3 Lysogenic (or lysenogenic) life cycle
* 2.3.3 Induction
* 18.104.22.168 Control of phage genome excision in induction
* 3 Multiplicity reactivation and prophage reactivation
Bacteriophage lambda virion (schematic). Protein names and their copy numbers in the virion particle are shown. The presence of the L and M proteins in the virion is still unclear.
The virus particle consists of a head and a tail that can have tail
fibers. The whole particle consists of 12–14 different proteins with
more than 1000 protein molecules total and one
The GENOME contains 48,490 base pairs of double-stranded, linear DNA,
with 12-base single-strand segments at both 5' ends. These two
single-stranded segments are the "sticky ends" of what is called the
cos site. The cos site circularizes the
Early activation events involving N protein
* CRO binds to OR3, preventing access to the PRM promoter, preventing expression of the cI gene. N binds to the two Nut (N utilisation) sites, one in the N gene in the PL reading frame, and one in the cro gene in the PR reading frame. * The N protein is an antiterminator , and functions to extend the reading frames to which it is bound. When RNA polymerase transcribes these regions, it recruits the N and forms a complex with several host NUS proteins. This complex skips through most termination sequences. The extended transcripts (the 'late early' transcripts) include the N and CRO genes along with CII and CIII genes, and XIS, INT, O, P and Q genes discussed later. * The CIII protein acts to protect the CII protein from proteolysis by FTSH (a membrane-bound essential E. coli protease) by acting as a competitive inhibitor. This inhibition can induce a bacteriostatic state, which favours lysogeny. cIII also directly stabilises the cII protein.
On initial infection, the stability of cII determines the lifestyle of the phage; stable cII will lead to the lysogenic pathway, whereas if cII is degraded the phage will go into the lytic pathway. Low temperature, starvation of the cells and high multiplicity of infection (MOI) are known to favor lysogeny (see later discussion).
N Antitermination requires the assembly of a large ribonucleoprotein complex to effectively prolong the anti-termination process, without the full complex the RNA polymerase is able to bypass only a single terminator
This occurs without the N PROTEIN interacting with the DNA; the protein instead binds to the freshly transcribed mRNA. Nut sites contain 3 conserved "boxes," of which only BOXB is essential.
* The boxB
LYTIC LIFE CYCLE
Main article: Lytic cycle
This is the lifecycle that the phage follows following most infections, where the cII protein does not reach a high enough concentration due to degradation, so does not activate its promoters.
* The 'late early' transcripts continue being written, including XIS, INT, Q and genes for replication of the lambda genome (OP). Cro dominates the repressor site (see "Repressor" section ), repressing synthesis from the PRM promoter (which is a promoter of the lysogenic cycle). * The O and P PROTEINS initiate replication of the phage chromosome (see " Lytic Replication"). * Q, another antiterminator , binds to QUT sites. * Transcription from the PR' promoter can now extend to produce mRNA for the lysis and the head and tail proteins. * Structural proteins and phage genomes self-assemble into new phage particles. * Products of the lysis genes S,R, RZ and RZ1 cause cell lysis. S is a holin , a small membrane protein that, at a time determined by the sequence of the protein, suddenly makes holes in the membrane. R is an endolysin , an enzyme that escapes through the S holes and cleaves the cell wall. Rz and Rz1 are membrane proteins that form a complex that somehow destroys the outer membrane, after the endolysin has degraded the cell wall. For wild-type lambda, lysis occurs at about 50 minutes after the start of infection and releases around 100 virions.
Rightward transcription expresses the O, P and Q genes. O and P are responsible for initiating replication, and Q is another antiterminator that allows the expression of head, tail, and lysis genes from PR’.
* For the first few replication cycles, the lambda genome undergoes
θ replication (circle-to-circle).
* This is initiated at the ORI site located in the O gene. O protein
binds the ori site, and P protein binds the DnaB subunit of the host
replication machinery as well as binding O. This effectively
commandeers the host
The Q protein modifies the
RNA polymerase at the
promoter region and is recruited to
Q is similar to N in its effect: Q binds to
RNA polymerase in Qut
sites and the resulting complex can ignore terminators, however the
mechanism is very different; the Q protein first associates with a DNA
sequence rather than an m
* The Qut site is very close to the PR’ promoter, close enough
that the σ factor has not been released from the
Leftward transcription expresses the gam, red, xis, and int genes. Gam and red proteins are involved in recombination. Gam is also important in that it inhibits the host RecBCD nuclease from degrading the 3’ ends in rolling circle replication. Int and xis are integration and excision proteins vital to lysogeny.
xis And Int Regulation Of Insertion And Excision
Diagram showing the retro-regulation process that yields a
higher concentration of xis compared to int. The m
* 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
* The m
LYSOGENIC (OR LYSENOGENIC) LIFE CYCLE
Main article: Lysogenic cycle
The lysogenic lifecycle begins once the CII protein reaches a high enough concentration to activate its promoters, after a small number of infections.
* The 'late early' transcripts continue being written, including
XIS, INT, Q and genes for replication of the lambda genome.
* The stabilized cII acts to promote transcription from the PRE, PI
and PANTIQ promoters.
* The Pantiq promoter produces antisense m
The prophage is duplicated with every subsequent cell division of the
host. The phage genes expressed in this dormant state code for
proteins that repress expression of other phage genes (such as the
structural and lysis genes) in order to prevent entry into the lytic
cycle. These repressive proteins are broken down when the host cell is
under stress, resulting in the expression of the repressed phage
genes. Stress can be from starvation , poisons (like antibiotics ), or
other factors that can damage or destroy the host. In response to
stress, the activated prophage is excised from the
The INTEGRATION of phage λ takes place at a special ATTachment site
in the bacterial and phage genomes, called attλ. The sequence of the
bacterial ATT site is called attB, between the gal and bio operons,
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
Maintenance Of Lysogeny
A simplified representation of the integration/excision paradigm and the major genes involved.
* Lysogeny is maintained solely by CI. cI represses transcription from PL and PR while upregulating and controlling its own expression from PRM. It is therefore the only protein expressed by lysogenic phage.
Lysogen repressors and polymerase bound to OR1 and recruits OR2, which will activate PRM and shutdown PR.
* This is coordinated by the PL and PR operators. Both operators
have three binding sites for cI: OL1, OL2, and OL3 for PL, and OR1,
OR2 and OR3 for PR.
* cI binds most favorably to OR1; binding here inhibits
transcription from PR. As cI easily dimerises, the binding of cI to
OR1 greatly increases the affinity of the binding of cI to OR2, and
this happens almost immediately after OR1 binding. This activates
transcription in the other direction from PRM, as the N terminal
domain of cI on OR2 tightens the binding of
RNA polymerase to PRM and
hence cI stimulates its own transcription. When it is present at a
much higher concentration, it also binds to OR3, inhibiting
transcription from PRM, thus regulating its own levels in a negative
* cI binding to the PL operator is very similar, except that it has
no direct effect on cI transcription. As an additional repression of
its own expression, however, cI dimers bound to OR3 and OL3 bend the
Transcriptional state of the PRM and PR promoter regions during a lysogenic state vs induced, early lytic state.
The classic induction of a lysogen involved irradiating the infected
cells with UV light. Any situation where a lysogen undergoes DNA
damage or the
* The host cell, containing a dormant phage genome, experiences DNA
damage due to a high stress environment, and starts to undergo the SOS
RecA (a cellular protein) detects
The function of LexA in the SOS response. LexA expression leads to inhibition of various genes including LexA.
* The phage genome is still inserted in the host genome and needs
MULTIPLICITY REACTIVATION AND PROPHAGE REACTIVATION
Multiplicity reactivation (MR) is the process by which multiple viral genomes, each containing inactivating genome damage, interact within an infected cell to form a viable viral genome. MR was originally discovered with phage T4, but was subsequently found in phage λ (as well as in numerous other bacterial and mammalian viruses ). MR of phage λ inactivated by UV light depends on the recombination function of either the host or of the infecting phage. Absence of both recombination systems leads to a loss of MR.
Survival of UV-irradiated phage λ is increased when the E. coli host
is lysogenic for an homologous prophage, a phenomenon termed prophage
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, as discovered by Barbara J. Meyer .
The lambda repressor gene system consists of (from left to right on the chromosome):
* cI gene * OR3 * OR2 * OR1 * cro gene
Visual representation of repressor tetramer/octamer binding to phage lambda L and R operator sites (stable lysogenic state)
The lambda repressor is a self assembling dimer also known as the cI
protein . It binds
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.
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.
At high concentrations of cI, the dimers will also bind to operators OL1 and OL2 (which are over 2 kb downstream from the R operators). When cI dimers are bound to OL1, OL2, OR1, and OR2 a loop is induced in the DNA, allowing these dimers to bind together to form an octamer. This is a phenomenon called long-range cooperativity. Upon formation of the octamer, cI dimers may cooperatively bind to OL3 and OR3, repressing transcription of cI. This autonegative regulation ensures a stable minimum concentration of the repressor molecule and, should SOS signals arise, allows for more efficient prophage induction.
* 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.
Some base pairs with serve a dual function with promoter and operator for either cl and cro proteins. Protein cl turned ON, with repressor bound to OR2 polymerase binding is increased and turn OFF OR1. Lysogen repression all 3 sites bound is a low occurrence due to OR3 weak binding affinity. OR1 repression increases binding affinity to OR2 due to repressor-repressor interaction. Increased concentrations of repressor increase binding.
PROTEIN FUNCTION OVERVIEW
PROTEIN FUNCTION IN LIFE CYCLE PROMOTER REGION DESCRIPTION
CIII Regulatory protein CIII. Lysogeny, cII Stability PL (Clear 3) HflB (FtsH) binding protein, protects cII from degradation by proteases.
CII Lysogeny, Transcription activator PR (Clear 2) Activates transcription from the PAQ, PRE and PI promoters, transcribing cI and int. Low stability due to susceptibility to cellular HflB (FtsH) proteases (especially in healthy cells and cells undergoing the SOS response). High levels of cII will push the phage toward integration and lysogeny while low levels of cII will result in lysis.
Repressor, Maintenance of Lysogeny
(Clear 1) Transcription inhibitor, binds OR1, OR2 and OR3 (affinity
OR1 > OR2 = OR3, i.e. preferentially binds OR1). At low concentrations
blocks the PR promoter (preventing cro production). At high
concentrations downregulates its own production through OR3 binding.
CRO Lysis, Control of Repressor's Operator PR Transcription inhibitor, binds OR3, OR2 and OR1 (affinity OR3 > OR2 = OR1, i.e. 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. No cooperative binding (c.f. below for cI binding)
S Lysis PR' Holin , a membrane protein that perforates the membrane during lysis.
RZ AND RZ1 Lysis PR' Forms a membrane protein complex that destroys the outer cell membrane following the cell wall degradation by endolysin. Spanin, Rz1(outer membrane subunit) and Rz(inner membrane subunit).
D Lysis PR' Head decoration protein.
E Lysis PR' Major head protein.
C Lysis PR' Minor capsid protein.
A Lysis PR' Large terminase protein.
J Lysis PR' Host specificity protein J.
M V U G L T Z Lysis PR' Minor tail protein M.
K Lysis PR' Probable endopeptidase.
H Lysis PR' Tail tape measure protein H.
I Lysis PR' Tail assembly protein I.
FI Lysis PR' DNA-packing protein FI.
FII Lysis PR' Tail attachment protein.
TFA Lysis PR' Tail fiber assembly protein.
Antitermination for Transcription of Late Early Genes
Antiterminator , RNA-binding protein and
RNA polymerase cofactor,
Antitermination for Transcription of Late
LYTIC OR LYSOGENIC?
Diagram of temperate phage life cycle, showing both lytic and lysogenic cycles.
An important distinction here is that between the two decisions;
lysogeny and lysis on infection, and continuing lysogeny or lysis from
a prophage. The latter is determined solely by the activation of RecA
In cells with sufficient 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. cIII appears to stabilize cII, both directly and by acting as a competitive inhibitor to the relevant proteases. This means that a cell "in trouble", i.e. lacking in nutrients and in a more dormant state, is more likely to lysogenise. This would be selected for because the phage can now lie dormant in the bacterium until it falls on better times, and so the phage can create more copies of itself with the additional resources available and with the more likely proximity of further infectable cells.
A full biophysical model for lambda's lysis-lysogeny decision remains to be developed. Computer modeling and simulation suggest that random processes during infection drive the selection of lysis or lysogeny within individual cells. However, recent experiments suggest that physical differences among cells, that exist prior to infection, predetermine whether a cell will lyse or become a lysogen.
LAMBDA AS A GENETIC TOOL
* ^ Esther M. Zimmer Lederberg: Published Works
* ^ Esther Lederberg, "Lysogenicity in Eescherichia coli strain
* James Watson, Tania Baker, Stephen Bell, Alexander Gann, Michael
Levine, Richard Losick " Molecular Biology of the Gene (International
Edition)" - 6th Edition