Biochemistry 3107 - Fall 2002

Bacteriophage

Bacteriophage

Bacteriophage were jointly discovered by Frederick Twort in England and by Felix d'Herelle at the Pasteur Institute in Farance.

Four types of bacteriophage have been widely used in biochemical and molecular biological research. Most of these infect E. coli.

1. T series of DNA bacteriophages

   The T series of bacteriophages had a central role in the development of molecular biology. In 1944, at the instigation of Max Delbruck, the phage group at Cold Spring Harbor agreed to concentrate their research on 7 bacteriophages, all of which were active against E. coli B, which had first been isolated by Demerec and Fano. Up till this point, different scientists worked with different phage - as a result, it was difficult to compare results.

   This decision, while appearing correct at the time, has been criticised since because it led to the neglect of some other important aspects of bacteriophage biology, most notably lysogeny. Nevertheless, the study of the T bacteriophage has contributed a great deal to our understanding of molecular biology and genetic regulation.

    

   • The Hershey-Chase experiment used bacteriophage T2.
     
     
   • Volkin and Astrachan's experiments which led to the discovery of mRNA were based on the study of bacteriophage T2 infection.
     
     
   • Seymour Benzer used bacteriophage T4 in his classic fine structure analysis of the gene.
     
     
   • A bacteriophage T7 promoter has been incorporated into a number of different cloning and expression plasmids such as pTZ.
     
     

   The following table (Table 6.1 from The Emergence of Bacterial Genetics by Thomas D. Brock) summarizes the properties of the seven T-series bacteriophages:

   																														Morphology
   Name										Plaque size																				Head (nm)																				Tail (nm)																				Latent period (min)															Burst size
   T1										medium																				50																				150 x 15																				13															180
   T2										small																				65 x 80																				120 x 20																				21															120
   T3										large																				45																				invisible																				13															300
   T4										small																				65 x 80																				120 x 20																				23.5															300
   T5										small																				100																				tiny																				40															300
   T6										small																				65 x 80																				120 x 20																				25.5															200-300
   T7										large																				45																				invisible																				13															300

   
   

   The genome of all of these phages consists of a single linear molecule of dsDNA. However, circular forms and/or circular permutations exist. All undergo lytic growth exclusively.

   The T-even phages, T2, T4 and T6, are all related serologically and all have large genomes. Seymour Benzer used bacteriophage T4 in his classic fine structure analysis of the gene. It has a large genome 168,895 bp in length. T4 was the first "prokaryotic" organism in which evidence of gene splicing (i.e. introns) was found.

Image shows an electron micrograph of T4 from the ICTV database.


Read the ICTV entry on T4 Read the EMBL database entry on T4

 

The T-odd phages fall into three serological groups: T3 and T7 are related to each other but not to T1 or to T5 which are unrelated. The T7 genome was sequenced in 1983; it is 39,937 bp in length. Bacteriophage T7 has been used for the study of DNA replication because of its linear chromosome and the problems that poses for DNA replication and also because it encodes its own DNA polymerase. A modified form of T7 DNA polymerase has been marketed as the very popular sequencing enzyme Sequenase. T7 promoters also require a special RNA polymerase; as a result, they have been incorporated into a number of cloning/expression vectors.

Read the ICTV entry on T7 Read the EMBL database entry on T7

 

 

 

1. Temperate bacteriophages

   Temperate bacteriophage have "alternative" life-cycles. After infecting a cell, they can undergo a typical lytic growth cycle or they become dormant in a lysogenic growth cycle. The phage is still present in the cell as a prophage and under certain conditions, such as UV irradiation, it becomes active and resumes a lytic growth cycle.

   Bacteriophage lambda, which infects E. coli, is the classic example of a temperate bacteriophage. It has a linear dsDNA genome, which circularizes after infection, of 48,502 bp. Bacteriophage lambda has been one of the work-horses of molecular biology particularly as a model system for understanding gene regulation. It has also played an important role as a cloning vector.

   [27-18]

Image shows an example of lambdoid phage from the ICTV database.


View a diagram of the The Life Cycle Of Bacteriophage Lambda from the Access Excellence Graphics Gallery

Read the ICTV entry on bacteriophage lambda Read the EMBL database entry on bacteriophage lambda

 

Other examples of temperate bacteriophage are P1, P22 (which infects Salmonella), P4 (11624 bp) and Mu.

 

 

1. Small DNA bacteriophages

   The small DNA bacteriophages have ssDNA genomes which replicate as dsDNA intermediates. They encode 10-12 proteins.

   Two groups of these phage can be distinguished:

    

   • Spherical phage

     The spherical phage (PhiX174, G4, S13) are broadly similar to the filamentous phage. The capsid is icosahedral not helical and is not enveloped (these phage lyse the host cell). Their genome consists of a circular ssDNA molecule. A well-known examples is PhiX174, which was the first genome to be sequenced - by Fred Sanger's group in 1976. Its genome of 5386 bp coded for 11 genes, including several examples of overlapping genes. The coding frames for 7 proteins overlap: A* is a truncated form of A; B is coded within A in a different reading frame; K is encoded in a third reading frame at the end of A which extends into and overlaps with that of C; E is coded within D in a different reading frame. These were the first examples of overlapping genes.

     Other relatives of PhiX174 are G4 and S13.

     The image shows the molecular surface of bacteriophage PhiX174, radially depth cued, as solved by X-ray crystallography. This image is from the University of Wisconsin.

     Read the ICTV entry on PhiX174 Read the EMBL database entry on PhiX174

      

      

   • Filamentous phage

     As described above, the filamentous phage (M13, fd, f1) have a filamentous capsid with a circular ssDNA molecule. The genome, 6407 nt, contains 10 genes but none for a lysis protein. Virions are enveloped. The filamentous phage will only infect E. coli cells carrying the F plasmid since the phage must adsorb to the F pilusto gain entry to the cells. Their life-cycle involves a dsDNA intermediate replicative form within the cell which is converted to a ssDNA molecule prior to encapsidation. This conversion is the major reason for the great utility of these phage as a molecular biological laboratory tool: they provide an easy means to prepare ssDNA for DNA sequencing.

     The best known example is bacteriophage M13 which has been adapted for use as a cloning and sequencing vector. The wild-type M13 genome is 6407 bp in length; the modified cloning vector is 7249 bp in length.

     Other relatives of M13 are fd and f1.

     Read the ICTV entry on bacteriophage fd Read the EMBL database entry on bacteriophage fd Read the EMBL database entry on bacteriophage M13

      

      

2. RNA bacteriophages
   

   The ssRNA bacteriophages are the simplest viruses known. Both families (MS2, R17 and f2 form one family; Q beta forms another) have small genomes (3600-4200 nt; MS2 is 3569 nt) that encode 4 proteins. In the MS2 family these are: a capsid coat protein, a replicase, a lysis protein, and an attachment protein that is needed for attachment of the phage to a host cell. In Q beta these are: a capsid coat protein which also has lysis activity, a replicase, a minor virion protein, and an attachment protein. All have linear ssRNA genomes. Because of this, they have served as useful sources of RNA for studies of translation and protein synthesis.

   Examples are Qbeta, MS2 (3569 nt), R17, f2.

   Images show molecular reconstruction of bacteriophage MS2, radially depth cued, as solved by X-ray crystallography. This image is from the University of Wisconsin.

Read the ICTV entry on Q beta Read the ICTV entry on MS2