Phages or bacteriophages- overview .pptx

1. Phages

2. There are three even-numbered T-phages (e.g. T2, T4 and T6) and four odd-numbered T- phages (e.g. T1, T3, T5 and T7) that infect E.coli . Out of the seven coliphages of T-series, the virulent T4 phages is most extensively studied . Linear ds DNA genome Lytic phages

3. tadpole shaped and consists of the five important sub-structures such as the head, head-tail connector, tail base plate and fibers (Fig. 18.2). The viral particle is naked icosahedral and tailed Sheath is contractile in some (T2, T4 etc) and non-contractile in some (T7)

4. Unique feature of T4 phage Modified bases are present in genome Contains HMC ( Hydroxymethyl cytosine) in place of C (cytosine) Many of MHC are further glycosylated Such modified bases protect the phage DNA from host restriction enzymes The T4 genome is linear dsDNA and shows what is called terminal redundancy ( , a base sequence is repeated at each end of the molecule) and circular permutations

5. Multiplication  (life cycle) 5 stages adsorption , penetration , synthesis , replication, maturation (assembly) and release

6. Adsorption and Penetration bacteriophages attach to specific receptors present on host cell surface For T-phages, receptor is cell wall lipopolysaccharides or proteins as receptors . A ttachment begins when a tail fiber contacts the appropriate receptor -> . As more tail fibers make contact, the baseplate settles down on the surface-> conformational changes occur in the baseplate and sheath, and the tail sheath reorganizes and becomes shorter and wider-> central tube or core is pushed through the bacterial wall. The baseplate contains the protein gp5, which has lysozyme activity. (aids in penetration of the tube through the peptidoglycan ) Finally, the linear DNA is extruded from the head , through the tail tube, and into the host cell

7. Synthesis and Replication Within 2 minutes after injection of T4 DNA, the E. coli RNA polymerase starts synthesizing T4 mRNA This mRNA is called early mRNA ( made before viral DNAis made .) -> translated into early proteins Role-> a)synthesis of protein factors and enzymes required to take over the host cell and force it to manufacture additional viral constituents. B)Some enzymes degrade host DNA to nucleotides (thereby simultaneously halting host gene expression and providing raw material for viral DNA synthesis.)

8. Within 5 minutes , viral DNAsynthesis commences . DNAreplication is initiated from several origins of replication and it proceeds bidirectionally from each. Viral DNA replication is followed by the synthesis of late mRNAs, which are important in later stages of the infection.

9. DNA replication leads to formation of long DNA molecules called concatemers , which are composed of several genome units linked together in the same Orientation During assembly, concatemers are cleaved such that the genome packaged in the capsid is slightly longer than the T4 gene set. Thus each progeny virus has a genome unit that begins with a different gene. However , if each genome of the progeny viruses was circularized, the sequence of genes in each virion would be the same. Therefore the T4 genome is said to be circularly permuted , and the genetic map of T4 is drawn as circular molecule

10. Assembly of Phage Particles Late mRNA directs the synthesis of three kinds of proteins: (1) phage structural proteins, (2) proteins that help with phage assembly without becoming part of the virion structure , (NSP) and (3) proteins involved in cell lysis and phage release. The baseplate is constructed of 16 gene products , then the tail tube is built on it and the sheath is assembled around the tube . The phage prohead ( procapsid ) is constructed of 10 proteins. The prohead is assembled with the aid of scaffolding proteins that are degraded or removed after construction is completed. A special portal protein is located at the base of the prohead where it connects to the tail . The portal protein participates in DNA packaging, yielding the mature head After completion of the head, it spontaneously combines with the tail assembly. D NA packaging within the T4 prohead is accomplished by a complex of proteins sometimes called the “ packasome .” The packasome consists of the portal protein, also contains terminase complex( which generates double-stranded ends at the ends of the concatemers ) the terminase proteins and phage DNA join with the portal protein -> moves DNA into the prohead , (a process powered by ATP hydrolysis .) The concatemer is cut when the phage head is filled with DNA—a DNA molecule roughly 3% longer than the length of one set of T4 genes.

11. Release of Phage Particles The lysis of E. coli by T4 takes places after about 150 virus particles have accumulated in the host cell Two proteins are involved- T4 lysozyme , holin Lysozyme -attacks peptidoglycan in the host’s cell wall. holin creates holes in the E. coli plasma membrane ,

12. φ X174 , Circular ssDNA genome, icosahedral naked phage of E.coli The life cycle of X174 begins with its attachment to the cell wall of its host. The circular ssDNA genome is injected into the cell , the phage DNA is converted to a double-stranded form called the replicative form (RF) (by the bacterial DNA polymerase) The RF directs the synthesis of more RF copies and genomic plus-strand DNA, both by rolling-circle replication. Synthesis of viral capsid protein takes place, After assembly of virions (packaging of genomic DNA into phage head) Then phage is released by host lysis Synthesis of an enzyme (enzyme E) that blocks peptidoglycan synthesis -> weakens the host cell wall, -> lysis and release the progeny virions .

13. Bacteriophage P1 Temperate phage – E. coli and some enteric bacteria an icosahedral head containing the phage DNA and a 220 nm long tail with tube, contractile sheath, baseplate , and six kinked tail fibers. P1 prophage exists as an autonomous plasmid that is maintained at low copy number

14. P1

15. genome The DNA genome ~ 94 kb linear, ds molecule. terminal redundancy, circular permutation. two origins of replication, oriR ( lysogenic cycle) and oriL ( lytic stage)

16. Genome organisation

17. Attachment and penetration receptor on the host cell - terminal glucose of the LPS core of bacterial OM. P1 virion can inject its DNA into host cell DNA circularizes

18. Lysogeny P1 genome in lysogens exists as an independent circular DNA plasmid- like element carefully coordinates replication and partition of its genome with the genome of the host cell.

19. P1 plasmids and lytically replicating P1 genomes- different ori – sp. Repressor Maintenance of lysogeny - oriL completely repressed. P1 lysogens are extremely stable and are not easily induced

20. PACKAGING BACTERIOPHAGE DNA " Headful Packaging" - leading to the genesis of the circularly permuted, terminally redundant DNA s packaging into head is a multi-length DNA "concatemer" genome size is smaller than a headful, terminally redundant DNA will be packaged.

21. GENERALIZED TRANSDUCTION Transduction is the heritable transfer of bacterial DNA from one cell (the donor) to another (the recipient) by a bacteriophage . Generalized transducing phage particles- contain only bacterial DNA sequences.

22. EVENTS IN THE DONOR CELL packaging mechanism - uses host DNA, instead of concatemeric phage DNA little or no specificity About 30% of the phage particles in a lysate contain host DNA rather than phage DNA. approximately 1 in 1,500 phage particles in a lysate –> host gene