Earn upto Rs. 9,000 pm checking Emails. Join now!

Enter your email address:

Delivered by FeedBurner

Wednesday, November 21, 2007

Bacteriophages M.sc Biotech

MicrobiologyBytes: Virology: Bacteriophages
Updated: September 11, 2007
Search
Bacteriophages
The Importance of Bacteriophages:
Historical:
Frederick Twort (1915) and Felix d'Herelle (1917) were the first to recognize viruses which infect bacteria, which d'Herelle called bacteriophages (eaters of bacteria). In the 1930s and subsequent decades, pioneering virologists such as Luria, Delbruck and many others utilized these viruses as model systems to investigate many aspects of virology, including virus structure, genetics, replication, etc. These relatively simple agents have since been very important in the development of our understanding of all types of viruses, including those of man which are much more difficult to propagate and study. They are still a paradigm for many areas of biology, especially gene expression (See Bacteriophage λ).
Environmental:
Bacteriophages, like bacteria, are very common in all natural environments and are directly related to the numbers of bacteria present. They are thus very common in soil and have shaped the evolution of bacteria.
Industrial/Economic:
Phages of Lactobacillus are a serious problem for the dairy industry.Medical - phage typing (e.g. Staphylococcus); antibacterials - Flemming.Recombinant DNA vectors - cloning, expression, enzymes (T4 DNA ligase)
Pathogenesis, e.g:
Bille E. et al. 2005 A chromosomally integrated bacteriophage in invasive meningococci. J Exp Med. 201: 1905-1913.
Waldor MK, Friedman DI. Phage regulatory circuits and virulence gene expression. Curr Opin Microbiol. Jun 2005.
Groisman EA, Casadesus J. The origin and evolution of human pathogens. Mol Microbiol. 2005 56: 1-7.

Diversity
There are at least 12 distinct groups of bacteriophages, which are very diverse structurally and genetically; the best known ones are the common phages of E.coli:
Family or Group:
Genera:
Type Member:
Particle Morphology:
Envelope:
Genome:
Corticoviridae
Corticovirus
PM2
isometric
No
supercoiled d/s DNA
Cystoviridae
Cystovirus
Ø6
isometric
Yes
3 segments d/s RNA
Inoviridae
Inovirus
coliphage fd
rod
No
circular s/s DNA
Plectrovirus
Acholeplasma phage
Leviviridae
Levivirus
coliphage MS2
icosahedral
No
1 (+)strand RNA
Allolevirus
coliphage Qbeta
Lipothrixviridae
Lipothrixvirus
Thermoproteus phage 1
rod
Yes
linear d/s DNA
Microviridae
Microvirus
coliphage ØX174
icosahedral
No
circular s/s DNA
Spirovirus
Spiroplasma phages

Mac-1 phage
Myoviridae

coliphage T4
tailed phage
No
linear d/s DNA
Plasmaviridae
Plasmavirus
Acholeplasma phage
pleiomorphic
Yes
Circular d/s DNA
Podoviridae

coliphage T7
tailed phage
No
linear d/s DNA
Siphoviridae
lambda phage group
coliphage lambda
tailed phage
No
linear d/s DNA
Sulpholobus shibatae virus

SSV-1
lemon-shaped
No
circular d/s DNA
Tectiviridae
Tectivirus
phage PRD1
icosahedral
No
linear d/s dna
Replication:
Essentially similar to other viruses. After attachment (or "adsorption") to a specific receptor in the bacterial cell wall, the phage genome enters the cell. The capsid proteins are usually stripped off and remain outside the cell. T-even phage tails have a contractile sheath and function "like a syringe". The "head and tail" morphology of some is unique to phages and not found among other groups of viruses and is related to their mode of cell penetration:
Other "round" phages (icosahedral or lipid-coated) penetrate the bacterial cell wall by adhering to flagellae or pili and being "drawn in" to the cell.
After entry into the cell, many phage genomes are degraded and destroyed. Since phage are so prevalent in the environment, bacteria have specific mechanisms to protect themselves against infection with phage - "restriction/ modification" systems which depend on the recognition and destruction of foreign (unmodified - methylated or glucosylated) DNA.
Surviving phage genomes sequester the cellular apparatus for gene expression (transcription and translation) to various extents.
Virulent vs. Temperate Phages
Virulent phages do not integrate their genetic material into the host cell chromosome and usually kill the host cells (lytic infection) (e.g. T-phages of E.coli).
Temperate phages may integrate into the host DNA, causing LYSOGENY.

Two classical experiments reveal the processes involved in phage replication:
The Single Burst Experiment
(Ellis E.L, Delbruck M. 'The growth of bacteriophage' J.Gen.Physiol. 22: 365-384, 1939): (You can perform this experiment online!)

The Hershey-Chase Experiment (1952):
Lysogeny:
The indefinite persistence of phage genomes within bacterial cells in the absence of a productive infection but with the potential to produce progeny phage under certain circumstances was first recognised in the 1920's.On infection with a temperate phage, vegetative replication (lysis) occurs in most cells, but a few become persistently infected due to integration of the phage genome. These lysogenized cells are immune (not resistant!) to superinfection by the same phage due to repression of transcription caused by the resident prophage (See Bacteriophage λ) .
Phage Genetics
On rare occasions, lysogeny permits the transfer of bacterial genes between cells by TRANSDUCTION. There are two forms:
Generalized Transduction: bacterial rather than phage DNA is packaged into a phage head. When another cell is infected, the bacterial DNA is injected and in a proportion of cases, may be incorporated into the chromosome by homologous recombination, replacing the existing genes. Frequency 105 - 108 per cell. More than one gene may be cotransduced - limit = packaging size = ~50kbp = ~1% of bacterial chromosome.
Abortive transduction occurs when the new DNA does not integrate into the chromosome - may affect the phenotype transiently but is not replicated and is eventually lost.
Specialized Transduction: Results from inaccurate excision of an integrated prophage; some phage DNA is lost and some bacterial genes are picked up and carried to the next host - therefore phage are usually defective (non-infectious) and require replication-competent helper phage to replicate, depending on which phage genes are lost.
Transposition and Insertional Mutagenesis: A few temperate phage, such as Mu, act as transposons and move from site to site in the bacterial chromosome. New insertions may result of the inactivation of bacterial genes (promoters or coding sequences).The above mechanisms have been very important in bacterial genetics, but are now largely superseded by molecular biology, in which phage remain important as cloning vectors (M13, lambda, cosmids).

Bacteriophage λ
Control of Prokaryote Gene Expression
Genetic control in prokaryotes is exercised both at the level of transcription and at subsequent (post-transcriptional) stages of gene expression. Control of gene expression in eukaryotic cells is, in general, more complex than in prokaryotic cells, and involves a 'multilayered' approach in which diverse control mechanisms exert their effects at multiple levels.
The initiation of transcription is regulated primarily in a negative fashion by the synthesis of trans-acting repressor proteins, which bind to operator sequences upstream of protein coding sequences. Collections of metabollically-related genes are grouped together and co-ordinately controlled as 'operons' in this way. Transcription of these operons typically produces a polycistronic mRNA which encodes several different proteins.
During subsequent stages of expression, transcription is also regulated by a number of mechanisms which act, in Mark Ptashne's famous phrase, as 'genetic switches', turning on or off the transcription of different genes. Such mechanisms include:
Antitermination: controlled by trans-acting factors which promote the synthesis of longer transcripts encoding additional genetic information.
Modification of RNA polymerase: Bacterial sigma factors are apoproteins which affect the specificity of the RNA polymerase holoenzyme for different promoters. Several bacteriophages, e.g. phage SP01 of Bacillus subtilis, encode proteins which function as alternative sigma factors, sequestering RNA polymerase and altering the rate at which phage genes are transcribed. Phage T4 of E.coli encodes an enzyme which carries out a covalent modification (ADP-ribosylation) of the host cell RNA polymerase. This is believed to eliminate the requirement of the polymerase holoenzyme for sigma factor and to achieve a similar effect to the production of modified sigma factors by other bacteriophages.
At a post-transcriptional level, gene expression in bacteria is also regulated by control of translation. The best known virus examples of this phenomenon come from study of bacteriophages of the family Leviviridae, such as R17, MS2 and Q-beta. In these phages, the secondary structure of the single-stranded RNA phage genome regulates both the quantities of different phage proteins which are translated, but in addition, also operates temporal control of a switch in the ratios between the different proteins produced in infected cells.
Control of Expression in Bacteriophage λ
The genome of phage λ has probably been studied in more detail than any other and illustrates several of the mechanisms described above, including the action of repressor proteins in regulating lysogeny versus lytic replication, and antitermination of transcription by phage-encoded trans-acting factors. Such has been the impact of these discoveries that no discussion of the control of virus gene expression could be considered complete without detailed examination of this phage.
Lambda was discovered at the Pasteur Institute by Andre Lwoff (who later helped to initiate the idea of operons in bacteria) when he observed that some strains of E.coli , when irradiated with ultraviolet light, stopped growing and subsequently lysed, releasing a crop of bacteriophage particles. Together with Francois Jacob and Jaques Monod, he subsequently showed that all the cells of these bacterial strains carried the bacteriophage in a dormant form, known as a prophage, and that the phage could be made to alternate between the lysogenic (non-productive) and lytic (productive) growth cycles. After many years of study, our understanding of lambda has been refined into a picture which represents one of the best understood and most elegant genetic control systems yet to be investigated.
A simplified genetic map of lambda:
For regulation of the growth cycle of the phage, the structural genes encoding the head and tail components of the virus capsid can be ignored. The pertinent components involved in genetic control are:
PL is the promoter responsible for transcription of the left-hand side of the lambda genome, including N and cIII.
OL is a short non-coding region of the phage genome (approximately 50bp) which lies between the cI and N genes next to PL.
PR is the promoter responsible for transcription of the right-hand side of the lambda genome, including cro, cII and the genes encoding the structural proteins.
OR is a short non-coding region of the phage genome (approximately 50bp) which lies between the cI and cro genes next to PR.
cI is transcribed from its own promoter and encodes a repressor protein of 236 amino acids which binds to OR, preventing transcription of cro but allowing transcription of cI, and to OL, preventing transcription of N and the other genes in the left-hand end of the genome.
cII and cIII encode activator proteins which bind to the genome, enhancing the transcription of the cI gene.
cro encodes a 66 amino acid protein which binds to OR, blocking binding of the repressor to this site.
N encodes an antiterminator protein which acts as an alternative rho factor for host cell RNA polymerase, modifying its activity and permitting extensive transcription from PL and PR.
Q is an antiterminator similar to N, but only permits extended transcription from PR.
In a newly infected cell, N and cro are transcribed from PL and PR respectively:
The N protein allows RNA polymerase to transcribe a number of phage genes, including those responsible for DNA recombination and integration of the prophage, as well as cII and cIII. The N protein acts as a positive transcription regulator.
In the absence of the N protein, the RNA polymerase holoenzyme stops at certain sequences located at the end of the N and Q genes, known as nut and qut sites, respectively. However, RNA polymerase-N protein complexes are able to overcome this restriction and permit full transcription from PL and PR. The RNA polymerase-Q protein complex results in extended transcription from PR only.
As levels of the cII and cIII proteins in the cell build up, transcription of the cI repressor gene from its own promoter is turned on. It is at this point that the critical event occurs which determines the outcome of the infection:
The cII protein is constantly degraded by proteases present in the cell. If levels of cII remain below a critical level, transcription from PR and PL continues and the phage undergoes a productive replication cycle which culminates in lysis of the cell and the release of phage particles. This is the sequence of events which occurs in the vast majority of cells which become infected.
In a few rare instances, the concentration of cII protein builds up, transcription of cI is enhanced and intracellular levels of the cI repressor protein rise. The repressor binds to OR and OL, which prevents transcription of all phage genes (and in particular cro - see below) except itself. The level of cI protein is maintained automatically by a negative feedback mechanism, since at high concentrations the repressor also binds to the left-hand end of OR and prevents transcription of cI:
This autoregulation of cI synthesis keeps the cell in a stable state of lysogeny. If this is the case, how do such cells ever leave this state and enter a productive, lytic replication cycle? Physiological stress and particularly ultraviolet irradiation of cells results in the induction of a host cell protein, RecA. This protein, whose normal function is to induce the expression of cellular genes which permit the cell to adapt to and survive in altered environmental conditions, cleaves the cI repressor protein.In itself, this would not be sufficient to prevent the cell re-entering the lysogenic state. However, when repressor protein is not bound to OR, cro is transcribed from PR. This protein also binds to OR, but unlike cI, which preferentially binds to the right-hand end of OR, the cro protein binds preferentially to the left-hand end of OR, preventing the transcription of cI and enhancing its own transcription in a positive feedback loop. Thus, the phage is locked into a lytic cycle and cannot return to the lysogenic state.

Structure and Replication of Bacteriophage M13
Among the simplest helical capsids are those of the well-known bacteriophages of the family Inoviridae, such as M13 and fd - known as Ff phages. These phages are about 900nm long and 9nm in diameter and the particles contain 5 proteins. All are similar and are known collectively as Ff phages - they require the E.coli F pilus for infection:
M13 particle:
Structure:
The major coat protein is the product of phage gene 8 (g8p) and there are 2,700 - 3,000 copies of this protein per particle, together with approximately 5 copies each of four minor capsid proteins, g3p, g6p, g7p and g9p which are located at the ends of the filamentous particle.The primary structure of the major coat protein g8p explains many of the properties of the particle. Mature molecules of g8p consist of approximately 50 amino acid residues (a signal sequence of 23 amino acids is cleaved from the precursor protein during its translocation into the outer membrane of the host bacterium), and is almost entirely alpha-helical in structure so that the molecule forms a short rod. There are three distinct domains within this rod:
A negatively-charged region at the amino terminal end which contains acidic amino acid residues and which forms the outer, hydrophilic surface of the virus particle
A basic, positively-charged region at the carboxyl terminal end which lines the inside of the protein cylinder adjacent to the negatively-charged DNA genome
A hydrophobic region which is responsible for interactions between the g8p subunits which allow the formation of and stabilize the phage particle.
Ff phage particles are held together by the hydrophobic interactions between the coat protein subunits and this is demonstrated by the fact that the particles fall apart in the presence of chloroform, even though they do not contain any lipid component. The g8p subunits in successive turns of the helix interlock with the subunits in the turn below, are tilted at an angle of approximately 20° to the long axis of the particle and have been described as overlapping one another 'like the scales of a fish'. The value of µ (protein subunits per complete helix turn) is 4.5 and p (axial rise per subunit) = 1.5nm.
Since the phage DNA is packaged inside the core of the helical particle, the length of the particle is dependent on the length of the genome. In all Ff phage preparations the following forms occur:
Polyphage: containing more than one genome length of DNA
Miniphage: deleted forms containing 0.2-0.5 phage genome lengths of DNA
Maxiphage: genetically defective forms but containing more than one phage genome length of DNA
This plastic property of these filamentous particles has been exploited by molecular biologists to develop the M13 genome as a cloning vector - insertion of foreign DNA into the non-essential inter-genic region results in recombinant phage particles which are longer than the wild-type filaments. Unlike most viruses, there is no sharp cut-off genome-length at which the genome can no longer be packaged into the particle.However, as M13 genome size increases, the efficiency of replication declines such that while recombinant phage genomes 1-10% longer than the wild-type do not appear to be significantly disadvantaged, those 10-50% longer than the wild-type replicate significantly more slowly and above 50% increase over the normal genome length it becomes progressively more difficult to isolate recombinant phage. This property has been exploited in M13 cloning vectors.
M13 genome:
Gene:
Product Size:
Function:
1
35-40kd
NS membrane protein; few copies/cell; interacts with host fip gene product (thioredoxin); required for assembly
2
46kd
Site- & strand-specific endonuclease/topoisomerase; ~103 copies/cell; required for replication of RF
X (10)
12kd (111aa)
N-terminal fragment of gene 2; ~500 molecules/cell; required for replication of RF
3
42kd
5 copies at one end of particle; required for correct morphogenesis of unit-length particles; N-terminal domain binds to F pilus of host cell (receptor)
4
49kd
NS membrane protein; few copies/cell; required for assembly
5
10kd (87aa)
Major structural protein during replication; ~105 copies/cell; controls expression of g2p; binds to DNA; replaced by g8p during assembly; controls switch from RF replication to progeny (+)stand synthesis
6
12kd (112aa)
~5 copies at same end of particle to g3p; involved in attachment & morphogenesis
7/9
3.5kd
~5 copies at opposite end of particle to g3p/g6p; involved in assembly
8
5kd (50aa)
Major coat protein: ~2700-3000 copies/virion
Replication:
Attachment/Penetration:
Ff phages are 'male-specific', i.e. they require the F pilus on the surface of E. coli for infection. The first event in infection is an interaction between g3p, located at one end of the filament together with g6p, and the end of the F pilus.This interaction causes a conformational changes in g8p: Initially, its structure changes from 100% alpha-helix to 85% alpha-helix and this causes the filament to shorten. The end of the particle attached to the F pilus flares open, exposing the phage DNA.Subsequently, a second conformational change in the g8p subunits reduces its alpha-helical content from 85% to 50%, causing the phage particle to form a hollow spheroid about 40nm in diameter and expelling the phage DNA, thus initiating the infection of the host cell.g8p is stripped off and ends up in the inner cell membrane, where it may possibly be stored and reused to produce new particles.
Genome Replication:
Infecting (+)strand DNA is converted in d/s 'RF' form by host cell enzymes, which together with g2p build up a pool of RF DNA in the cell. Virus proteins are synthesized from this pool of DNA.Later in the infection, the concentration of g5p in the cell builds up. This protein binds to newly formed (+)strands, causing a switch from RF to (+)strand replication (coated with g5p).
Assembly/Release:
Assembly occurs at the inner membrane of the cell. DNA is extruded into the periplasmic space and the g5p coat is replaced by g8p. g5p is recycled to form further particles (efficient!).
These are temperate (i.e. non-lytic) phages - establish permanent infections without lysogeny. ~300 particles/cell/generation are produced; titres in infected cultures up to 5x1012/ml (~150mg/ml) - good recombinant vectors.
Detailed notes for these documents can be found in Chapters 2 & 5 of Principles of Molecular Virology.


Standard Version: The 4th edition contains new material on virus structure, virus evolution, zoonoses, bushmeat, SARS and bioterrorism, CD-ROM with FLASH animations, virtual interactive tutorials and experiments, self-assessment questions, useful online resources, along with the glossary, classification of subcellular infectious agents and history of virology. (Amazon.co.uk)

Instructors Version: The 4th edition contains new material on virus structure, virus evolution, zoonoses, bushmeat, SARS and bioterrorism, CD-ROM with all the Standard Version content plus all the figures from the book in electronic form and a PowerPoint slide set with complete lecture notes to aid in course preparation. (Amazon.co.uk)


A Genetic Switch: Phage and Higher Organismsby Mark PtashneThis book is a must for molecular biology courses - undergrad and graduate alike.Top of Form 1
Bottom of Form 1

No comments:

 
Thanks

Total Pageviews