1. Syeda Naushin Tabassum _14126003
Bacteriophage
Sharika Ferdous _
Syeda Naushin Tabassum _

2. Background and History

3. Contributions in History
Introduction
Bacteriophages are a different class of organisms who are commonly found in places where bacteria is in abundance as bacteriophages are viruses who infect bacteria. Among this particular group of organisms, T-even are most widely studied.
Contributions in History
Research on T-even phages lead to three major concepts that are the basis of biological sciences. They are:
the concept that only nucleic acid of the virus entered the cell proving that DNA is the genetic material
the concept of restriction and modification which led to the discovery of restriction enzymes and their application in Genetic Engineering
last but not the least, Viruses causes killing of the host cells

4. T4 Bacteriophage

5. T4 Structure
Bacteriophage T4 falls under the t-even phages and is structurally complex. It consists of :
an icosahedral head (200nm long, nm wide)
a collar
a complex tail consisting of a helical tube
a contractile sheath surrounding tail
a complex endplate/base-plate
tail fibers
tail pins

6. T4 Genome double-stranded, linear DNAgenome
can be divided into 3 parts encoding early, middle and late proteins
does not produce its own RNA polymerase
encodes for proteins that modify the specificity of the host RNA polymerase
encodes for anti-sigma factors
ends of the DNA molecule are terminally redundant
DNA is packaged into capsid using the headful mechanism

7. Circular Permutation
“During T4 DNA replication, the newly synthesized phage DNA undergoes recombination, forming long concatamers (linear molecules of several genomes attached to one another).
The concatamers are processed into pieces representing one "headful" of DNA. This length is one genome plus about 5000 bp present at both ends.
Individual pieces that are packaged into phage heads have different terminal repeats, but every piece has a complete genome plus the duplicated sequence at the ends. This arrangement is said to represent a "circular permutation" of the T4 genome.” (*)
* The text has been taken from

8. Fig: Circular Permutation and concatamer formation of T4 DNA

9. T4 Gene Expression
The DNA of T4 contains an unusual base, 5-hydroxymethylcytosine.
The enzymes required to synthesize this is formed after phage infection. T4 also encodes large amounts of some enzymes having similar functions to those of host DNA replication enzymes. Overall the T4 genes can be divided into 3 groups:
early genes– early proteins
middle genes– middle proteins
late genes– late proteins
T4 does not produce its own RNA polymerase so T4 mRNA synthesis is controlled by host RNA polymerase that is sequentially modified by T4 proteins which results in the regulation of their gene expression.

10. Fig: Time course of events in T4 infection and gene expression.
T4 gene expression
Fig: Time course of events in T4 infection and gene expression.

11. Gene Products Early proteins Middle proteins Late proteins
Nuclease (breaks down host DNA)
Proteins inhibiting host restriction system
RNA dependent RNA polymerase
Proteins stopping functions of host RNA polymerase
DNA polymerase
Helicase
Primase
Endonuclease
DNA ligase
Capsid protein
Envelope protein
Head, tail ,collar, base plate, tail fiber proteins
Lysozyme
Proteins necessary for self assembly
Enzymes involved in maturation
Endolysin for host cell lysis

12. T7 Bacteriophage

13. Introduction: Structure:
After the discovery of the T-even phages, or more specifically T4 Bacteriophage, among the T-odd phages, T7 Bacteriophage was more widely studied then the other members of the group. This group of bacteriophages are small DNA viruses that infect Escherichia coli (E.coli). Sometimes, strains of Shigella and Pasteurella can also be potential host for T7 Bacteriophage.
Structure:
The Structure of T7 Bacteriophage is not as big and complicated as T4 Bacteriophage, however, the virion has an icosahedral head and a very small tail. The Genome is a linear double-stranded DNA molecule of 39,936 base pairs. About 92% of the DNA of T7 encoded proteins.
The genome composed of 40bp with 160bp of terminal repeats at both ends.

14. Genome

15. T7 Gene Expression Genome Gene functions
T7 RNAP expression, host interference
Class I
Class II
Class III
host DNA digestion, T7 DNA replication
T7 particle formation, DNA maturation and host lysis

16. Gene Products Class Expression time Significance I
4 to 8 minutes after infection
Made by using cell RNA polymerase to form immediate early proteins II
6 to 15 minutes after infection
Made by using T7 RNA polymerase for phage DNA replication
III
15 minutes to lysis
Made by using T7 RNA polymerase to form late proteins

17. Lifecycle of Bacteriophage

18. Fig: Life cycle of Bacteriophages from attachment to replication to release

19. T- Phage life cycle
Essentially there are 5 steps that constitute the life cycle of a bacteriophage. These events set forth when collision occurs between a virion and its host. They include:
Adsorption and Penetration
Gene Expression
synthesis of proteins and nucleic acids– Replication
Assembly
Release

20. Adsorption and Penetration
first step in infection
mediated by tail fibers
attaches to specific host receptor– Ompc, LPS, pili
reversible attachment
Penetration:
irreversible attachment
contraction of sheath and tail tube
injection of genetic material with enzymatic degradation

21. Replication
Within minutes, bacterial ribosome start translating viral mRNA into protein. Some phages encodes for their own RNA polymerase to make mRNA that are translated into proteins. Some phages like T7 produce their own RNA polymerase.
The host’s normal synthesis of proteins and nucleic acids is disrupted, and is forced to manufacture viral products instead. These products go on to become part of new virions.
The synthesis and events that take place are as follows:
replicate own DNA
transcribe its own DNA
replicate and recombine its own DNA to form a concatemaric structure
headful packaging

22. Assembly
The assembly of phage particles is A complex self-assembly process.
Late mRNA ( produced after DNA replication) directs the synthesis of three kinds of proteins:
(1) phage structural proteins,
(2) proteins that help with phage assembly and
(3) proteins involved in cell lysis and phage release.

23. Release
Many phages lyse their host cells at the end of the intracellular phase. The lysis of E. coli by T takes place (after ~ 22 minutes for T even and 30 minutes for T odd) at 37°C, releasing ~ 300 T phage particles per infected bacterial cell.
Several T proteins are involved in this process:
Endolysin that attacks the cell wall peptidoglycan.
Holin produces a plasma membrane lesion that stops respiration and allows the endolysin to attack the peptidoglycan ,forms holes in the membrane and lysis.

24. M13 Bacteriophage

25. M13 Structure
contain a nucleo-capsid which is thin and long
contains circular single-stranded DNA genome which is coated by single layer of about 2700 subunits of major coat proteins (gp8) giving it a filamentous appearance
one end of the filament has M13 minor proteins bound– gp9, gp7
on the other end are bound M13 minor proteins– gp3 and gp6
the strong native structure is maintained by hydrophobic interaction of individual coat proteins.

26. Genome Architecture

27. Genome Architecture Gene Protein Function II gp II DNA Replication X
gp X V
gp V
Binding ssDNA
VIII
gp VIII
Major capsid protein
III
gp III
Minor capsid protein VI
gp VI
VII
gp VII IX
gp IX I
gp I
Assembly IV
gp IV XI
gp XI

28. Lifecycle of M13 Bacteriophage

29. Lifecycle of M13
The life cycle of an M13 phage starts with adsorption of the phage particles at the tip of the baterial F pilus, mediated by gp3 viral adsorption protein. The sequence of events are as follows:
phage is subsequently transported to cell surface of host by pilus retraction
viral gp3 play a role in facilitating the entry of phage genome into cell by pore formation
host encoded outer membrane proteins, tolq, tolr and tola can mediate uncoating and penetration
the major and minor proteins are stripped off and deposited in the inner membrane
phage genome is injected into cell cytoplasm
the genome is replicated and newly synthesized virions are assembled and release.

30. Fig: Lifecycle of bacteriophage M13

31. Replication and Synthesis
Single stranded genome on entry is converted to double-stranded, supercoiled covalently closed replicative form (RF) by host enzyme
RF acts as template strand for rolling cycle replication
Rolling cycle controlled by gp2 and gp10 to give phage encoded 11 proteins
At later stage, gp2, gp5 and gp10 regulate level of RF DNA and ssdna
Therefore, gp5 is determining protein for M13 DNA replication by rolling cycle
At high gp5 concentrations, it binds to new viral strands separating them from replication machinery and pulling them for assembly

32. Assembly and Release

33. ASSEMBLY AND RELEASE
The filamentous bacteriophages is released by extrusion from host cells rather lysis of host cell. Infected cell continue to grow and divide while extruding the progeny viruses in medium
Assembly occurs in between the inner and outer membrane of host
Three morphogenic protein gp1,gp4 and gp11 and host protein thirodexin are involved in assembly process
Gp1 and gp11 are associated with inner membrane and form a part of extrusion channel through the inner membrane
Gp4 are associated with outer membrane and form a part of extrusion channel through the outer membrane
All the newly synthesized phage proteins are inserted in inner membrane associated with gp1

34. Gp5-ssDNA complex interact with gp1 which in turn open the extrusion channel
Phage assembly initiated by binding of gp7 and gp9 mediated by gp1
Then each gp5 protein of gp5-ssDNA is replaced by major coat protein gp8
The gp8 molecules fit to gather with other gp8 molecules surrounding the phage DNA
The final termination step in assembly occurs by adding gp3 and gp6 to close the phage filament which is released into the medium

35. Release of Phages

36. Applications of M13

37. Ideal Cloning Vector
The phage M13 can be extensively used as a cloning vector because it has certain features that are in favor it. They include:
plasmid like replicative form– can be handled in laboratory like a plasmid
does not have any strict limit for insertion– can tolerate insertion of large fragments of foreign DNA
genome is single-stranded DNA– ideal template for sequencing
does not lyse host cell– pure viral DNA can be isolated

38. Thank You

39. Any Questions?