Editing Lambda phage (section)

== Life cycle ==
=== Infection ===
[[File:MANXYZ permease Step 4.jpg|thumb|450px|right|upright|Lambda phage J protein interaction with the LamB porin]]

Lambda phage is a non-contractile tailed phage, meaning during an infection event it cannot 'force' its DNA through a bacterial cell membrane. It must instead use an existing pathway to invade the host cell, having evolved the tip of its tail to interact with a specific pore to allow entry of its DNA to the hosts.
# Bacteriophage Lambda binds to an ''E. coli'' cell by means of its J protein in the tail tip. The J protein interacts with the maltose outer membrane [[porin (protein)|porin]] (the product of the ''lamB'' gene) of ''E. coli'',<ref>{{cite journal | vauthors = Werts C, Michel V, Hofnung M, Charbit A | title = Adsorption of bacteriophage lambda on the LamB protein of Escherichia coli K-12: point mutations in gene J of lambda responsible for extended host range | journal = Journal of Bacteriology | volume = 176 | issue = 4 | pages = 941–947 | date = February 1994 | pmid = 8106335 | pmc = 205142 | doi = 10.1128/jb.176.4.941-947.1994 }}</ref> a porin molecule, which is part of the [[maltose]] operon.
# The linear phage genome is injected through the outer membrane.
# The DNA passes through the mannose permease complex in the inner membrane<ref>{{cite journal | vauthors = Erni B, Zanolari B, Kocher HP | title = The mannose permease of Escherichia coli consists of three different proteins. Amino acid sequence and function in sugar transport, sugar phosphorylation, and penetration of phage lambda DNA | journal = The Journal of Biological Chemistry | volume = 262 | issue = 11 | pages = 5238–5247 | date = April 1987 | pmid = 2951378 | doi = 10.1016/S0021-9258(18)61180-9 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Liu X, Zeng J, Huang K, Wang J | title = Structure of the mannose transporter of the bacterial phosphotransferase system | journal = Cell Research | volume = 29 | issue = 8 | pages = 680–682 | date = August 2019 | pmid = 31209249 | pmc = 6796895 | doi = 10.1038/s41422-019-0194-z }}</ref>  (encoded by the manXYZ genes) and immediately circularises using the ''cos'' sites, 12-base G-C-rich cohesive "sticky ends". The single-strand viral DNA ends are ligated by host [[DNA ligase]].  It is not generally appreciated that the 12 bp lambda cohesive ends were the subject of the first direct nucleotide sequencing of a biological DNA.<ref name="src3"/>

[[File:MANXYZ permease Step 10.jpg|thumb|350px|right|Lambda phage DNA injection into the cell membrane using Mannose PTS permease (a sugar transporting system) as a mechanism of entry into the cytoplasm]]

# Host [[DNA gyrase]] puts negative [[supercoil]]s in the circular chromosome, causing A-T-rich regions to unwind and drive transcription.
# Transcription starts from the constitutive ''P<sub>L</sub>'', ''P<sub>R</sub>'' and ''P<sub>R'</sub>'' [[promoter (biology)|promoters]] producing the 'immediate early' transcripts. At first, these express the ''N'' and ''cro'' genes, producing N, Cro and a short inactive protein.

[[File:N protien.svg|thumb|200px|Early activation events involving N protein]]

# Cro binds to ''OR3'', preventing access to the ''P<sub>RM</sub>'' promoter, preventing expression of the ''cI'' gene. N binds to the two ''Nut'' (N utilisation) sites, one in the ''N'' gene in the ''P<sub>L</sub>'' reading frame, and one in the ''cro'' gene in the ''P<sub>R</sub>'' reading frame.
# The N protein is an [[antiterminator]], and functions by engaging the transcribing [[RNA polymerase]] at specific sites of the nascently transcribed mRNA. When [[RNA polymerase]] transcribes these regions, it recruits 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.<ref>{{cite journal | vauthors = Kobiler O, Rokney A, Oppenheim AB | title = Phage lambda CIII: a protease inhibitor regulating the lysis-lysogeny decision | journal = PLOS ONE | volume = 2 | issue = 4 | pages = e363 | date = April 2007 | pmid = 17426811 | pmc = 1838920 | doi = 10.1371/journal.pone.0000363 | doi-access = free | bibcode = 2007PLoSO...2..363K }}</ref>
On initial infection, the stability of [[CII protein|cII]] determines the lifestyle of the phage; stable cII will lead to the lysogenic pathway, whereas if [[CII protein|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).<ref>{{cite book |last1=Henkin |first1=Tina M. |last2=Peters |first2=Joseph E. |title=Snyder and Champness molecular genetics of bacteria |date=2020 |publisher=John Wiley & Sons, Inc |location=Hoboken, NJ |isbn=9781555819750 |pages=293–294 |edition=Fifth |chapter=Bacteriophages and Transduction}}</ref>

==== N antitermination ====
{{multiple image
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|footer =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<ref name="Korlach 2008">{{cite journal | vauthors = Santangelo TJ, Artsimovitch I | title = Termination and antitermination: RNA polymerase runs a stop sign | journal = Nature Reviews. Microbiology | volume = 9 | issue = 5 | pages = 319–329 | date = May 2011 | pmid = 21478900 | pmc = 3125153 | doi = 10.1038/nrmicro2560 }}</ref>

| image1    =Nantitermination1.jpg

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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 RNA sequences are located close to the 5' end of the pL and pR transcripts. When transcribed, each sequence forms a hairpin loop structure that the N protein can bind to.
# N protein binds to boxB in each transcript, and contacts the transcribing RNA polymerase via RNA looping. The N-RNAP complex is stabilized by subsequent binding of several host Nus (N utilisation substance) proteins (which include transcription termination/antitermination factors and, bizarrely, a ribosome subunit).
# The entire complex (including the bound ''Nut'' site on the mRNA) continues transcription, and can skip through termination sequences.

===Lytic life cycle===
{{Main|Lytic cycle}}
[[File:LambdaPlaques.jpg|thumb|Lysis plaques of lambda phage on ''[[Escherichia coli|E. coli]]'' bacteria]]
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.{{citation needed|date=October 2022}}
# 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|"Repressor" section]]), repressing synthesis from the ''P<sub>RM</sub>'' 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 ''P<sub>R'</sub>'' 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 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====
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 ''P<sub>R’</sub>''.<ref name="src3"/>

Pr is the promoter for rightward transcription, and the cro gene is a regulator gene. The cro gene will encode for the Cro protein that will then repress Prm promoter.  Once Pr transcription is underway the Q gene will then be transcribed at the far end of the operon for rightward transcription. The Q gene is a regulator gene found on this operon, which will control the expression of later genes for rightward transcription. Once the gene's regulatory proteins allow for expression, the Q protein will then act as an anti-terminator. This will then allow for the rest of the operon to be read through until it reaches the transcription terminator. Thus allowing expression of later genes in the operon, and leading to the expression of the lytic cycle.<ref>{{cite journal | vauthors = Thomason LC, Schiltz CJ, Court C, Hosford CJ, Adams MC, Chappie JS, Court DL | title = Bacteriophage λ RexA and RexB functions assist the transition from lysogeny to lytic growth | journal = Molecular Microbiology | volume = 116 | issue = 4 | pages = 1044–1063 | date = October 2021 | pmid = 34379857 | pmc = 8541928 | doi = 10.1111/mmi.14792 }}</ref>

Pr promoter has been found to activate the origin in the use of rightward transcription, but the whole picture of this is still somewhat misunderstood. Given there are some caveats to this, for instance this process is different for other phages such as N15 phage, which may encode for DNA polymerase. Another example is the P22 phage may replace the p gene, which encodes for an essential replication protein for something that is capable of encoding for a DnaB helices.<ref name="src3"/>

====Lytic replication====
# For the first few replication cycles, the lambda genome undergoes [[Theta structure|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 DNA polymerase.
# Soon, the phage switches to a [[rolling circle replication]] similar to that used by phage M13. The DNA is nicked and the 3’ end serves as a primer. Note that this does not release single copies of the phage genome but rather one long molecule with many copies of the genome: a [[concatemer]].
# These concatemers are cleaved at their ''cos'' sites as they are packaged. Packaging cannot occur from circular phage DNA, only from concatomeric DNA.

====Q antitermination====
{{multiple image
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|footer =  The Q protein modifies the RNA polymerase at the promoter region and is  recruited to RNA polymerase. The Q protein turns into a RNA polymerase subunit after it is recruitment to RNAP and modifies the enzyme into a processive state.  Note that NusA can stimulate the activity of the Q protein.<ref name="Korlach 2008">{{cite journal | vauthors = Santangelo TJ, Artsimovitch I | title = Termination and antitermination: RNA polymerase runs a stop sign | journal = Nature Reviews. Microbiology | volume = 9 | issue = 5 | pages = 319–329 | date = May 2011 | pmid = 21478900 | pmc = 3125153 | doi = 10.1038/nrmicro2560 }}</ref>

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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 mRNA sequence.<ref name="Deighan, P. and Hochschild, A.">{{cite journal | vauthors = Deighan P, Hochschild A | title = The bacteriophage lambdaQ anti-terminator protein regulates late gene expression as a stable component of the transcription elongation complex | journal = Molecular Microbiology | volume = 63 | issue = 3 | pages = 911–920 | date = February 2007 | pmid = 17302807 | doi = 10.1111/j.1365-2958.2006.05563.x | doi-access = free }}</ref>
# The ''Qut'' site is very close to the ''P<sub>R’</sub>'' promoter, close enough that the σ factor has not been released from the RNA polymerase holoenzyme. Part of the ''Qut'' site resembles the -10 [[Pribnow box]], causing the holoenzyme to pause.
# Q protein then binds and displaces part of the σ factor and transcription re-initiates.
# The head and tail genes are transcribed and the corresponding proteins self-assemble.

====Leftward transcription====
[[File:Phage Lambda int xis Retroregulation.jpg|thumb|right|300px|Diagram showing the retro-regulation process that yields a higher concentration of xis compared to int. The mRNA transcript is digested by bacterial RNase starting from the cleaved hairpin loop at sib.]]
Leftward transcription expresses the ''gam,'' ''xis'', ''bar'' and ''int'' genes.<ref name="src3"/> Gam 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.{{Citation needed|date=November 2023}}

===== Leftward transcription process =====

# Lambda phage inserts chromosome into the cytoplasm of the host bacterial cell.
# The phage chromosome is inserted to the host bacterial chromosome through DNA ligase.
# Transcription of the phage chromosome proceeds leftward when the host RNA polymerase attaches to promotor site ''p''L resulting in the translation of gene ''N.''
## Gene N acts a regulatory gene that results in RNA polymerase being unable to recognize translation-termination sites.<ref name="pmid6241940">{{cite journal | vauthors = Brammar WJ, Hadfield C | title = A programme for the construction of a lambda phage | journal = Journal of Embryology and Experimental Morphology | volume = 83 | issue = Suppl | pages = 75–88 | date = November 1984 | pmid = 6241940 | doi = | url = }}</ref>

===== Leftward Transcription mutations =====
Leftward transcription is believed to result in a deletion mutation of the ''rap'' gene resulting in a lack of growth of lambda phage. This is due to RNA polymerase attaching to pL promoter site instead of the pR promotor site. Leftward transcription results in ''bar''I and ''bar''II transcription on the left operon. Bar  positive phenotype is present when the ''rap'' gene is absent. The lack of growth of lambda phage is believed to occur due to a temperature sensitivity resulting in inhibition of growth.<ref>{{cite journal | vauthors = Guzmán P, Guarneros G | title = Phage genetic sites involved in lambda growth inhibition by the Escherichia coli rap mutant | journal = Genetics | volume = 121 | issue = 3 | pages = 401–409 | date = March 1989 | pmid = 2523838 | pmc = 1203628 | doi = 10.1093/genetics/121.3.401 }}</ref>

===== xis and int regulation of insertion and excision =====
# ''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.
# The mRNA from the ''P<sub>L</sub>'' promoter forms a stable secondary structure with a [[stem-loop]] in the ''sib'' section of the mRNA. This targets the 3' (''sib'') end of the mRNA for RNAaseIII degradation, which results in a lower effective concentration of ''int'' mRNA than ''xis'' mRNA (as the ''int'' cistron is nearer to the ''sib'' sequence than the ''xis'' cistron is to the ''sib'' sequence), so a higher concentrations of ''xis'' than ''int'' is observed.
# Higher concentrations of ''xis'' than ''int'' result in no insertion or excision of phage genomes, the evolutionarily favoured action - leaving any pre-inserted phage genomes inserted (so reducing competition) and preventing the insertion of the phage genome into the genome of a doomed host.

===Lysogenic (or lysenogenic) life cycle===
{{Main|Lysogenic cycle}}
The lysogenic lifecycle begins once the cI 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 ''P<sub>RE</sub>'', ''P<sub>I</sub>'' and ''P<sub>antiq</sub>'' promoters.
# The ''P<sub>antiq</sub>'' promoter produces antisense mRNA to the ''Q'' gene message of the ''P<sub>R</sub>'' promoter transcript, thereby switching off Q production. The ''P<sub>RE</sub>'' promoter produces antisense mRNA to the cro section of the ''P<sub>R</sub>'' promoter transcript, turning down cro production, and has a transcript of the ''cI'' gene. This is expressed, turning on cI repressor production. The ''P<sub>I</sub>'' promoter expresses the ''int'' gene, resulting in high concentrations of Int protein. This int protein integrates the phage DNA into the host chromosome (see "Prophage Integration").
# No Q results in no extension of the ''P<sub>R'</sub>'' promoter's reading frame, so no lytic or structural proteins are made. Elevated levels of int (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'' and ''OR2'' sites in the ''P<sub>R</sub>'' promoter, turning off  ''cro'' and other early gene expression. cI also binds to the ''P<sub>L</sub>'' promoter, turning off transcription there too.
# Lack of cro leaves the ''OR3'' site unbound, so transcription from the ''P<sub>RM</sub>'' promoter may occur, maintaining levels of cI.
# Lack of transcription from the ''P<sub>L</sub>'' and ''P<sub>R</sub>'' promoters leads to no further production of cII and cIII.
# As cII and cIII concentrations decrease, transcription from the ''P<sub>antiq</sub>'', ''P<sub>RE</sub>'' and ''P<sub>I</sub>'' stop being promoted since they are no longer needed.
# Only the ''P<sub>RM</sub>'' and ''P<sub>R'</sub>'' promoters are left active, the former producing cI protein and the latter a short inactive transcript. The genome remains inserted into the host genome in a dormant state.

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]], [[poison]]s (like [[antibiotics]]), or other factors that can damage or destroy the host. In response to stress, the activated prophage is excised from the DNA of the host cell by one of the newly expressed gene products and enters its lytic pathway.

====Prophage integration====
The integration of phage λ takes place at a special attachment site in the bacterial and phage genomes, called ''att<sup>λ</sup>''. 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 [[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 B-O-B' 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.<ref>{{cite journal | vauthors = Groth AC, Calos MP | title = Phage integrases: biology and applications | journal = Journal of Molecular Biology | volume = 335 | issue = 3 | pages = 667–678 | date = January 2004 | pmid = 14687564 | doi = 10.1016/j.jmb.2003.09.082 }}</ref>

====Maintenance of lysogeny====
[[File:Phage Lambda Integration Excision.jpg|thumb|right|upright=1.75|A simplified representation of the integration/excision paradigm and the major genes involved.]]
* Lysogeny is maintained solely by cI. cI represses transcription from ''P<sub>L</sub>'' and ''P<sub>R</sub>'' while upregulating and controlling its own expression from ''P<sub>RM</sub>''. It is therefore the only protein expressed by lysogenic phage.
[[File:Polymerase cl protien.svg|thumb|Lysogen repressors and polymerase bound to OR1 and recruits OR2, which will activate PRM and shutdown PR.]]
* This is coordinated by the ''P<sub>L</sub>'' and ''P<sub>R</sub>'' operators. Both operators have three binding sites for cI: ''OL1'', ''OL2'', and ''OL3'' for ''P<sub>L</sub>'', and ''OR1'', ''OR2'' and ''OR3'' for ''P<sub>R</sub>''.
* cI binds most favorably to ''OR1''; binding here inhibits transcription from ''P<sub>R</sub>''. 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 ''P<sub>RM</sub>'', as the N terminal domain of cI on ''OR2'' tightens the binding of RNA polymerase to ''P<sub>RM</sub>'' and hence cI stimulates its own transcription. When it is present at a much higher concentration, it also binds to ''OR3'', inhibiting transcription from ''P<sub>RM</sub>'', thus regulating its own levels in a [[negative feedback]] loop.
* cI binding to the ''P<sub>L</sub>'' 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 DNA between them to tetramerise.
* The presence of cI causes immunity to superinfection by other lambda phages, as it will inhibit their ''P<sub>L</sub>'' and ''P<sub>R</sub>'' promoters.

====Induction====
[[File:Phage Lambda SwitchStates.jpg|thumb|200px|upright=1.5|Transcriptional state of the P<sub>RM</sub> and P<sub>R</sub> 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 [[SOS response]] of the host is otherwise stimulated leads to induction.
# The host cell, containing a dormant phage genome, experiences DNA damage due to a high stress environment, and starts to undergo the [[SOS response]].
# RecA (a cellular protein) detects DNA damage and becomes activated. It is now RecA*, a highly specific co-protease.
# Normally RecA* binds LexA (a [[transcription (genetics)|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. This is because cI mimics the structure of LexA at the autocleavage site.
# Cleaved cI can no longer dimerise, and loses its affinity for DNA binding.
# The ''P<sub>R</sub>'' and ''P<sub>L</sub>'' 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.
[[File:Lambda phage LexA inihibition.svg|thumb|right|200px|The function of LexA in the SOS response.  LexA expression leads to inhibition of various genes including LexA.]]

====Control of phage genome excision in induction====
# The phage genome is still inserted in the host genome and needs excision for DNA replication to occur. The ''sib'' section beyond the normal ''P<sub>L</sub>'' promoter transcript is, however, no longer included in this reading frame (see diagram).
# No ''sib'' domain on the ''P<sub>L</sub>'' promoter mRNA results in no hairpin loop on the 3' end, and the transcript is no longer targeted for RNAaseIII degradation.
# The new intact transcript has one  copy of both ''xis'' and ''int'', so approximately equal concentrations of xis and int proteins are produced.
# Equal concentrations of xis and int result in the excision of the inserted genome from the host genome for replication and later phage production.