|MicrobiologyBytes: Virology: Bacteriophages||Updated: August 14, 2008||Search|
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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.
Phages of Lactobacillus are a serious problem for the dairy
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|
|Leviviridae||Levivirus||coliphage MS2||icosahedral||No||1 (+)strand RNA|
|Lipothrixviridae||Lipothrixvirus||Thermoproteus phage 1||rod||Yes||linear d/s DNA|
|Microviridae||Microvirus||coliphage ØX174||icosahedral||No||circular s/s DNA|
|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|
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.
Two classical experiments reveal the processes involved in phage replication:
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).
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:
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:
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.
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:
|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|
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)
Genetic Switch: Phage and Higher Organisms
by Mark Ptashne
This book is a must for molecular biology courses - undergrad and graduate alike.
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