M13 phage
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M13 is a filamentous bacteriophage composed of circular single stranded DNA (ssDNA 6407 nucleotides long) encapsulated in approximately 2700 copies of the major coat protein P8, and capped with 5 copies of two different minor coat proteins (P9, P6, P3) on the ends. The minor coat protein P3 attaches to the receptor at the tip of the F-pilus of the host E.coli. Infection with filamentous phages is not lethal, instead the infection cause turbid plaques in E.coli. It is a non-lytic virus which has been studied for its uses in nanostructures and nanotechnology.
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[edit] Introduction
How many genetically-encoded creatures exist in every milliliter of sea water? 100? 1000? More? It turns out that bacteria are by far the best represented life form, numbering up to a million cells/ml. If each cell is assumed to harbor the DNA content of pedestrian E. coli MG1655, then that means 10^12th base pairs of DNA/ml. This thriving gene pool is even more remarkable in light of the fact that each ml of sea water contains approximately 10^10th viruses that infect bacteria, aka bacteriophage or “phage” for short. These destroy half the world's bacterial population every 48 hours [1]. Given the huge number of bacteriophage that exist, it's probably not surprising that most of the earth’s bacteriophage are completely uncharacterized, though massive genome sequencing efforts are underway.
A few bacteriophage are exquisitely well characterized. Indeed, the study of phage laid much of the groundwork for our current understanding of genetics and molecular principles in biology. These principles carry over to the biology of more complex cells (Jacques Monod famously said “What is true for Escherichia coli is true for the elephant” [Francois Jacob (1988)]). M13 is a member of the filamentous phage family. It has a long (~900 nm), narrow (~20 nm) protein coat that encases a small (~6.4 kb) single stranded DNA genome. The genome encodes 11 proteins, five of which are exposed on the phage’s protein coat and six of which are involved in phage maturation inside its E. coli host.
[edit] Phage Particles
The phage coat is primarily assembled from a 50 amino acid protein called pVIII (or p8), which is sensibly enough encoded by gene VIII (or g8) in the phage genome. For a wild type M13 particle, it takes about ~2700 copies of p8 to make the ~900 nm long coat. The coat's dimensions are flexible though and the number of p8 copies adjusts to accommodate the size of the single stranded genome it packages. For example, when the phage genome was mutated to reduce its number of DNA bases (from 6.4 kb to 221 bp) [2] , then the p8 coat “shrink wraps" around the reduced genome, decreasing the number of p8 copies to less than 100. Electron micrographs of the resulting “microphage” and its wild type parent are shown below. And what about the upper limit to the length of the phage particle? Anecdotally, viable phage seems to top out at approximately twice the natural DNA content. However, deletion of a phage protein (p3) prevents full escape from the host E. coli, and phage that are 10-20X the normal length with several copies of the phage genome can be seen shedding from the E. coli host.
There are four other proteins on the phage surface, two of which have been extensively studied. At one end of the filament are five copies of the surface exposed pIX (p9) and a more buried companion protein, pVII (p7). If p8 forms the shaft of the phage, p9 and p7 form the “blunt” end that’s seen in the micrographs. These proteins are some of the smallest known (only 33 and 32 amino acids), though some additional residues can be added to the N-terminal portion of each which are then presented on the “outside” of the phage coat (much more on this technique later). At the other end of the phage particle are five copies of the surface exposed pIII (p3) and its less exposed accessory protein, pVI (p6). These form the rounded tip of the phage and are the first proteins to interact with the E. coli host during infection. p3 is also the last point of contact with the host as new phage bud from the bacterial surface.
[edit] Phage Life-Cycle
The general stages to a viral life cycle are: infection, replication of the viral genome, assembly of new viral particles and then release of the progeny particles from the host. Filamentous phage use a bacterial structure known as the F pilus to infect E. coli, with the M13 p3 tip contacting the TolA protein on the bacterial pilus. The phage genome is then transferred to the cytoplasm of the bacterial cell where resident proteins convert the single stranded DNA genome to a double stranded replicative form (“RF”). This DNA then serves as a template for expression of the phage genes.
Two phage gene products play critical roles in the next stage of the phage life cycle, namely amplification of the genome. pII (aka p2) nicks the double stranded form of the genome to initiate replication of the + strand. Without p2, no replication of the phage genome can occur. Host enzymes copy the replicated + strand, resulting in more copies of double stranded phage DNA. pV (aka p5) competes with double stranded DNA formation by sequestering copies of the + stranded DNA into a protein/DNA complex destined for packaging into new phage particles. Interestingly there is one additional phage-encoded protein, pX (p10), that is important for regulating the number of double stranded genomes in the bacterial host. Without p10 no + strands can accumulate. What's particularly interesting about p10 is that it's identical to the C-terminal portion of p2 since the gene for p10 is within the gene for p2 and the protein arises from transcription initiation within gene 2. This makes the manipulation of p10 inextricably linked to manipulation of p2 (an engineering headache) but it also makes for a compact and efficient phage in nature.
Phage maturation requires the phage-encoded proteins pIV (p4), pI (p1) and its translational restart product pXI (p11). Multiple copies (on the order of 12 or 14) of p4 assemble in the outer membrane into a stable, i.e. detergent resistant, barrel-shaped structure. Similarly a handful of the p1 and p11 proteins (5 or 6 copies of each) assemble in the bacterial inner membrane, and genetic evidence suggests C-terminal portions of p1 and p11 interact with the N-terminal portion of p4 in the periplasm. Together the p1, p11, p4 complex forms channels through which mature phage are secreted from the bacterial host.
To initiate phage secretion, two of the minor phage coat proteins, p9 and p7, are thought to interact with the p5-single stranded DNA complex at a region of the DNA called the packaging sequence (aka PS). The p5 proteins covering the single stranded DNA are then replaced by p8 proteins that are embedded in the bacterial membrane and the growing phage filament is threaded through the p1, p11, p4 channel. This replacement of p5 by p8 explains the microphage data presented earlier...making very clear how the size of the phage particle is determined by the number of bases the phage packages. Once the phage DNA has been fully coated with p8, the secretion terminates by adding the p3/p6 cap, and the new phage detaches from the bacterial surface. How long does all this take? Amazingly, new M13 phage particles are secreted within 10 minutes from a newly infected host and can arise at a rate of 1000/cell within the first hour of infection. Also amazing is how the bacterial host can continue to grow and divide, allowing this process to continue indefinitely.
[edit] Replication in E. coli
Below are steps involved with replication of M13 in E. coli.
- Viral (+) strand DNA enters cytoplasm
- Complementary (-) strand is synthesized by bacterial enzymes
- Gyrase, a type II topoisomerase, acts on double-stranded DNA and catalyzes formation of negative supercoils in double-stranded DNA
- Final product is parental replicative form (RF) DNA
- A phage protein, pII, nicks the (+) strand in the RF
- 3'-hydroxyl acts as a primer in the creation of new viral strand
- pII circulizes displaced viral (+) strand DNA
- Pool of progeny double-stranded RF molecules produced
- Negative strand of RF is template of transcription
- mRNAs are translated into the phage proteins
Phage proteins in the cytoplasm are pII, pX, and pV, and they are part of the replication process of DNA. The other phage proteins are synthesized and inserted into the cytoplasmic or outer membranes.
- pV dimers bind newly synthesized single-stranded DNA and prevent conversion to RF DNA
- RF DNA synthesis continues and amount of pV reaches critical concentration
- DNA replication switches to synthesis of single-stranded (+) viral DNA
- pV-DNA structures from about 800 nm long and 8 nm in diamter
- pV-DNA complex is substrate in phage assembly reaction
Assembly of phage is associated with the membrane of bacteria and requires five capsid proteins, three assembly proteins, ATP, a proton motive force, and at least one bacterial protein, thioredoxin.
[edit] Display of Peptides and Proteins
George Smith showed that fragments of EcoRI endonuclease could fuse to amino-terminal portion of pIII.
[edit] References
- 20.109(S07): Start-up Genome Engineering [3]
- Phage Display: A Laboratory Manual