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Method Title: Phage Techniques

Contact Person or Lab: Dr. Charles R. Stewart


Contents:

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from: http://www.mansfield.ohio-state.edu/~sabedon/


INTRODUCTION

Bacteriophages, commonly referred to as phages, are viruses that infect bacteria and are important because of their ability to transfer genetic material between cells. The word “phage” comes from the Greek “phagein,” which means “to eat” [1]. Phages are small packets of genetic material that use host cells to replicate and infect other cells, which may or may not be of the same species. Once inside cells, phages can remain as small bits of plasmid DNA or they can incorporate themselves into the host’s genome. Amidst these transfers, phages often pick up fragments of DNA from one genome and carry them into another genome [2].

Phages are important in the field of biotechnology as they have been essential in clarifying many molecular genetic processes. They were key in the discovery that DNA is hereditary material and were also central in the elucidation of transcription and translation processes. Currently, they have proven very useful in the areas of gene therapy and genetic engineering [3].

Current and future research in the field of bacteriophages has shifted towards a focus on phages themselves. Scientists are studying the evolution of phages and their role in natural ecosystems. In addition, researchers are using phages to fight bacterial diseases. Another current area of research aims to remove phage pests in the food industry and understand how phages cause diseases in humans [4].

There is a wealth of information available on phages in literature and on the web. This webpage aims to highlight the basics of phage biology and some relevant applications. For further information on phages, please see the list of references and links at the end of this document.

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PHAGE BIOLOGY

The history of bacteriophages in biology begins with the work of Max Delbrück in the late 1930s. Nobel Laureate Delbrück, originally a physicist, began studying T-even phages as genetic and biochemical experimental systems. His work led others to focus their studies on the structure and assembly of phages. In the late 1960s, Edgar, Kellenberger, Epstein, and collaborators demonstrated that assembly of the T4 phage occurs along specific pathways. Since then, many phages have been studied, including T4, l, P22, T7, T3, Mu, P2, P4, and f29 [5].

Structure

Phages are typically categorized into three structure-based groups: (1) short tails – podoviridae; (2) long contractile tails – myoviridae; (3) long, noncontractile tails – styloviridae. All of these virions have single, linear double-stranded DNA (dsDNA) chromosomes stored in a protein coat shell called the capsid, or head (see picture). The capsid is built by protein molecules along icosahedrally symmetric arrays to form the distinctive shape of phages. The tail extends from one corner of the capsid and interacts with a single host cell. During infection, the distal end of the tail adsorbs to the exterior of the host cell as phage DNA travels through the tail into the cell [5] (see picture).

Other types of phages not discussed here are phages with protein attached to DNA, viruses of archaebacteria, filamentous bacteriophages, lipid-containing bacteriophages, and phages of cyanobacteria.

from: http://www.med.sc.edu:85/mayer/phage.htm

 

Function

The majority of the current understanding of DNA replication comes from the study of bacteriophage replication in E. coli. The methods employed by phages take advantage of two properties of DNA polymerases. Firstly, DNA polymerases operate in the 5’ to 3’ direction. Secondly, they require a free 3’-hydroxyl moiety of DNA or RNA to extend upon. Thus, bacteriophages have evolved to create the 3’-hydroxyl to allow DNA replication. Some of the strategies applied by phages include:

Phages induce gene expression through a variety of mechanisms, though primarily through modulating transcription in bacteria. This is accomplished thorough viral modification of RNA polymerase through non-covalent and covalent alterations during viral development [7].

from: http://www.cellsalive.com

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USES

Phages are very useful in the study of molecular processes in biology of a variety of reasons. One reason phages are so useful in molecular biology is their relatively simple structure and small size. Phages may contain as few as 3 or up to 200 genes [3] (or 19 to166 kbp [5]). They are relatively simple when compared to other tools of genetics. Phages grow rapidly and demonstrate haploidy, thus, progeny of phages are genetically identical. Henceforth, a single phage can provide a great deal of information about an organism [3].


A few of the major uses of phages in molecular biology are listed below. More detailed applications are described in other sections of this webpage.

  1. Cloning vectors
  2. Gene delivery vehicles
  3. Model to studying mechanisms of gene regulation, recombination, morphogenesis, and other genetic processes
  4. Tool to determine phenotypes by incorporation into bacterial genomes [3].

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PHAGE THERAPY

A little known fact is that the first main use of phage therapy was as an antibiotic. Due to the current threat of bioterrorism, there is a significant need for new antibiotics. For example, enterococcus faecium, a bacterium prevalent in hospitals, is resistant to most if not all antibiotics. However, phages are very specific and can be useful to treat animal or plant infections caused by antibiotic-resistant bacterial pathogens [3]. In fact, phages have been used to kill enterococcus faecium in mice [8]. Recently, an enzyme from the bacteriophage g was used to lyse and kill B. anthracis, the bacterium associated with anthrax, with high specificity [9].

Most phages are removed by the innate immune system, but resistant phages selected by experiments are useful in therapy. Phages have been used to treat bacterial infections and some human trials are currently in process. Nonetheless, phage therapy suffers from low public opinion as there have been some failed experiments in the past [3].

 

Please see the following links for examples of phage therapy in action:

Phage Therapy Overview

 

Examples of viruses killing bacteria:

“Set a bug to catch a bug” - New Scientist

“Virus cleans up food poisoning bug” - New Scientist

“Therapy uses viruses as natural antibiotics.” - The Seattle Times

 

Phages save lives in Russia:

“Bacteriophage Therapy: Stalin's Forgotten Cure.” - Science

“Silent Killers: Fantastic Phages?” - CBS News

 

Information on Betty Kutter, a phage therapy expert:

“Evergreen scientist researches 'phage,' an antibiotic alternative” - The Daily Olympian

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PHAGE EXCLUSION SYSTEMS

Phage exclusion systems are mechanisms by which cells kill themselves upon infection by a phage, thereby preventing the spread of phages to other cells in the population. These operate based on proteins of both phages and cells that interact to alter transcription and translation ultimately resulting in cell death. See references for further information [10-12].

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PHAGE DISPLAY

Phage display is used to elucidate protein interactions, molecular evolution, and production of recombinant antibodies. Foreign proteins are expressed on the surface of phages, thereby becoming a means for expression of the protein and replication of the protein code. Furthermore, phage displays allow for the production of variant peptides and screening of many proteins. For additional information, see reference [13].

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PHAGE GENOMICS AND TAXONOMY

Many phages have been sequenced and characterized extensively. A commonly studied phage is the T4 bacteriophage. For more information on its genome, see the following reference [14].

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PHAGES OF LACTIC ACID BACTERIA

These phages are of importance because they infect the bacteria required to produce dairy products and can thus have a significant effect on human health. In fact, dairy phages are the most completely sequenced phage group. For more information, see the following reference [15].

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LINKS FOR MORE INFORMATION

The Phage Phorum

Brief Background of Bacteriophages

General Information about Bacteriophages

Bacteriophage Lecture Notes: Dr. Gene Mayer, MBIM, University of South Carolina

Cells Alive: Oh Goodness, My E. Coli has a Virus!

The Bacteriophage Ecology Group

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REFERENCES

1. An Introduction to Viruses. http://www.dform.com/projects/t4/.

2. Alberts, B., et al., Molecular Biology of the Cell. 4th edition ed. 2002, New York: Garland Science.

3. Stewart, C.R., Phage Techniques, A. Mistry, Editor. 2003: Houston, TX.

4. Campbell, A., The future of bacteriophage biology. Nat Rev Genet, 2003. 4(6): p. 471-7.

5. Casjens, S. and R. Hendrix, Control Mechanisms in dsDNA Bacteriophage Assembly, in The Bacteriophages, R. Calendar, Editor. 1988, Plenum Press: New York. p. 15-91.

6. Keppel, F., O. Fayet, and C. Georgopoulos, Strategies of Bacteriophage DNA Replication, in The Bacteriophages, R. Calendar, Editor. 1988, Plenum Press: New York. p. 145-262.

7. Geiduschek, E.P. and G.A. Kassavetis, Changes in RNA Polymerase, in The Bacteriophages, R. Calendar, Editor. 1988, Plenum Press: New York. p. 93-115.

8. Biswas, B., et al., Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium. Infect Immun, 2002. 70(1): p. 204-10.

9. Schuch, R., D. Nelson, and V.A. Fischetti, A bacteriolytic agent that detects and kills Bacillus anthracis. Nature, 2002. 418(6900): p. 884-9.

10. Georgiou, T., et al., Specific peptide-activated proteolytic cleavage of Escherichia coli elongation factor Tu. Proc Natl Acad Sci U S A, 1998. 95(6): p. 2891-5.

11. Yu, Y.T. and L. Snyder, Translation elongation factor Tu cleaved by a phage-exclusion system. Proc Natl Acad Sci U S A, 1994. 91(2): p. 802-6.

12. Bergsland, K.J., et al., A site in the T4 bacteriophage major head protein gene that can promote the inhibition of all translation in Escherichia coli. J Mol Biol, 1990. 213(3): p. 477-94.

13. Willats, W.G., Phage display: practicalities and prospects. Plant Mol Biol, 2002. 50(6): p. 837-54.

14. Miller, E.S., et al., Bacteriophage T4 genome. Microbiol Mol Biol Rev, 2003. 67(1): p. 86-156, table of contents.

15. Brussow, H., Phages of dairy bacteria. Annu Rev Microbiol, 2001. 55: p. 283-303.

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Author: Amit S. Mistry

November 20, 2003