The synthesis of viral RNA and viral proteins often displays a biphasic or multiphasic pattern. First thing transcribed is early RNA species (starts after ~1-2 mins, peaks at 5-7 mins), then early proteins (happens slightly after early RNA), then synthesis of phage DNA (starts at ~6-7 mins, peaks at 35 mins right before lysis occurs), then late RNA (starts slightly after phage DNA, also peaks right before lysis), then late proteins (starts slightly after late RNA, also peaks right before lysis). Then it all stops because lysis of bacteria takes place and contents are emptied.
*Early mRNA is synthesized from 1-12 mins after infection. Late mRNA is synthesized from 8-35mins after infection*
Early genes - code for proteins required for viral DNA replication (e.g., a specific DNA polymerase or polymerase component)
Late genes - code for the structural components (capsid, tail fibers) of the virus, and the lysis proteins
How does the virus decide whether to replicate itself and kill its host cell or to shut off the lytic cycle in order to form a stable relationship with the cell? E. coli bacteriophage λ is the classical model for understanding the decision process (and also the ancestor of the phage that causes hemolytic-uremic syndrome).
A. Lytic growth of λ
- λ is a double-stranded DNA virus containing about 50 genes. Following infection of the cell, the ends of the linear virus genome come together, forming a closed circle. Transcription of the viral genes occurs in three phases, all of which require E. coli host RNA polymerase. Key sites are the left and right promoters, and late promoters, depicted as PL, PR, and Plate, respectively.
EARLY gene expression - initiates at PL and PR and ends at terminators found downstream of the promoters. Early gene expression leads to the production of the N protein.
MIDDLE gene expression - initiates from PL and PR but requires the N protein, which acts as a transcriptional "antiterminator," allowing RNA polymerase to bypass the early terminators because N protein binds to the terminator site and blocks termination, allowing for the production of longer transcripts. In the presence of N protein, leftward transcription proceeds through the int gene; rightward transcription proceeds through the Q gene. In addition to the Q protein, middle genes encode phage DNA replication proteins (O, P), integration and excision proteins (Int, Xis), and the regulatory protein CII.
LATE gene expression - initiates from Plate promoter just upstream of late genes and requires the Q protein (encoded in middle RNA) to act as an antiterminator of transcription from Plate. Late genes encode phage structural components and the host lysis enzymes.
***The only gene not transcribed in the lytic cycle is the cI gene, which is the repressor of lytic growth. If cI protein is activated, it represses lytic growth, need two promoters that were previously not active to become functional, PRM and PRE.
When phage λ infects a cell that is growing slowly (for instance, a cell growing on a poor carbon source), the phage opts to repress itself rather than try to make any progeny phage and lyse the host cell. Under poor growth conditions, the phage has evolved mechanisms to shut itself off before committing to replication and destruction of the host cell-the result of initiating late gene expression. To choose the lysogenic response, the phage needs to do two things quickly: synthesize a high concentration of CI repressor and synthesize the Int protein, a DNA recombination protein, in order to integrate the phage genomic DNA into the bacterial host chromosome. The response to the nutritional status of the host cell is mediated through the phage regulatory protein, CII, and a host protease that uses CII as a substrate.
i) Initial synthesis of the λ repressor, CI, depends on the presence of the CII protein, a transcriptional activator that binds to PRE, the "promoter for repressor establishment."
During an infection that will lead to lysogeny, the early and middle stages of phage gene expression occur in exactly the same manner as for a lytic infection. If environmental conditions promote lysogeny, the CII protein builds up and binds at PRE, acting as positive regulator, leading to high levels of CI repressor expression. The CI repressor binds immediately to PL and PR, shutting off all early and middle gene expression of the phage, thereby preventing lytic infection.
ii) Maintenance of repression is due to CI binding at PRM. Once repression is established (PR and PL are shut off) there is no more synthesis of CII.Thus, the concentration of CII protein will diminish as the host cells grow and divide, and there will no longer enough CII to stimulate expression at PRE. In order to maintain repression of the lytic cycle, phage λ has a second mechanism for synthesizing the repressor, another promoter, PRM. This promoter requires the λ CI repressor to act as a positive regulator for its own synthesis. PRM can only be used when there is already repressor protein in the cell. The location of PRM is interesting as it is within the PR site, but drives transcription
in the opposite direction of PR.
iii) Whether a phage initiates lysogeny depends on the nutritional status of the cell. The CII protein is exquisitely sensitive to a certain host protease whose levels fluctuate in
response to the host's nutritional status. When cells are growing in rich medium, there is high expression of a protease and the CII protein is quickly degraded. In the absence of CII, there is no activation of the PRE promoter and the phage can enter into the lytic cycle. Conversely,
when the cells are growing in a poor medium, the is very little protease sparing CII, allowing it to activate transcription at PRE and express the λ repressor, CI. As a result, lysogeny ensues and the phage genome integrates into the bacterial chromosome.
POOR MEDIUM -> LOW PROTEASE AND Q VIA ACTIVATED CII-> Phage undergoes LYSOGENY
RICH MEDIUM -> HIGH PROTEASE AND Q VIA INACTIVATED CII-> Phage undergoes LYTIC GROWTH
Surprisingly, once the phage DNA has become repressed, the growth state of the cell no longer affects repressor synthesis (because CII is no longer present and CI, which is not a substrate of the protease can activate its own synthesis). The return to the lytic cycle can only occur if the repressor protein is inactivated or destroyed. The mechanism of phage induction depends on the cell's system for responding to DNA damage. Damage to DNA activates the cellular protein RecA, which, in turn, binds to certain kinds of bacterial repressors and causes them to autoproteolyze. This is termed the "SOS" response. SOS is bacterial DNA repair genes. Sits on operons repressed by the LexA protein unless there is DNA damage. The repressors of many temperate phages are inactivated by autoproteolysis upon interaction with activated RecA* protein (this is not a general feature of bacterial repressors).
Once CI, the λ repressor, is destroyed, phage gene expression begins in the usual way via transcription initiating at PL and PR. Among the middle genes are int and xis, which encode the viral integrase and a specific excision protein (Xis). These two proteins work together to carry out a recombination reaction that removes the phage DNA from the chromosome as a circle that is identical to the original phage DNA molecule that had previously integrated. After induction:
1. Phage genes are expressed.
2. Phage development proceeds and can lead to excision from the chromosome and lysis of the cell.
Enterohemorrhagic E. coli strain O157:H7 and its relatives (E. coli O104:H4, Shigella) have been responsible for several outbreaks of severe food poisoning, currently causing over 70,000 cases a year of bloody colitis in the United States alone. The damage to the intestines is the result of the production of a toxin called Shiga-like toxin. After DNA damage, activated RecA protein, phage repressor undergoes autoproteolysis, the phage is induced. Expression of the toxin genes, stx A and stx B, is greatly enhanced by the Q protein, expressed upon induction of the prophage. The stx toxin genes are inserted within the
late gene region of the prophage and can be transcribed from the phage Late promoter. It's unknown if the expression of the stx genes in these bacteria is due to a higher-than-normal rate of spontaneous induction of the prophage or whether the stx genes have a cryptic promoter (insensitive to the phage repressor) that is constitutively expressing low levels of toxin. What is known is that the severity of the illness in many of the patients dramatically increased upon administration of certain antibiotics that target and damage bacterial DNA, as this causes the induction of prophages and results in a massive rise in transcription and expression of the stx toxin genes. Because an increase in Shiga-like toxin can lead to HUS, kidney failure, and subsequent death, it is contraindicated to prescribe antibiotics for enterohemorrhagic E. coli infections.
10th EditionCain, Campbell, Minorsky, Reece, Urry, Wasserman 11th EditionLisa A. Urry, Michael L. Cain, Peter V Minorsky, Steven A. Wasserman 5th EditionCharlotte W. Pratt, Donald Voet, Judith G. Voet 1st EditionJanet L. Hopson, Postlethwait