Bacteriophage by Prof. Kunal Upadhyay Rajkot India
Kunal H. Upadhyay Lecturer in MicrobiologyGyanyagna College of Science & Management Dt. 07/02/2009 Lecture for HNGU, Patan
Infectious agents Need electron microscope No nucleus, organelles, cytoplasm – acellular Not harmed by antibiotics Invade susceptible host cell & take over Obligate intracellular parasites Uses host cell for replication Host specificity
Nucleic acid core DNA or RNA – single or double stranded; linear, circular, segmented Capsid Protein outer coat Covers N.A. & protects; determines shape of virus; Envelope (no envelope – naked) Bilayer membrane outside capsid Acquired when BUDDING from host(nuclear or plasma membrane) May have SPIKES (glycoproteins) - aid in attachment Keeps virus hidden from host defenses
Largest virus = smallest bacterium Most viruses – specific shape Helical – ribbon-like protein in spiral around nuclear core Icosahedral – most common polyhedral shape; 20 triangular faces Bacteriophages – Viruses that infect bacteria More complex shape – heads, tails, tail fibers – aids in attachment
Host Range – spectrum of hosts a virus can infect; all living cells Viruses are limited to one host type, one cell type, or tissue type EX. Poliovirus – can grow in monkey kidney cells; infects ONLY HUMANS Rabies – attacks CNS of many warm blooded animals Viral specificity - particular kind of cell virus infects
1. Attachment – determined by receptor sites on host cell & attachment structures on virus’ capsid or envelope 2. Appropriate host enzymes & proteins in host cell available to virus 3. Can replicated virus be released from host cell to spread infection
Type & structure of N.A. Method of replication Host range Chemical & physical characteristics Family – highest taxonomic level English name , not binomial name Virus families based on: N.A. type Capsid shape Envelope Size
One step growth experiment reveals events during a single infectious cycle Adsorption Latent period (includes eclipse) Rise Burst size: the number of particles released per infected cell
Adsorption DNA injection Synthesis of early mRNA Degradation of host DNA Synthesis of phage DNA Synthesis of late mRNA Assembly Host cell lysis and release of ~300 virions Involves holin and endolysin
3 steps procedure: Sheath Contraction Unplugging of Core Tube Injection of DNA
Sheath is made up of 144 subunits of gp 19. They are arranged in 24 layers & each contains 6 subunits. Sheath contraction is ATP dependent process & sheath will be contracted to 12 layers with 12 subunits in each. So length will be half while width will be doubled.
Gp e is attached on tip of tail fibers It has lysozyme activity It helps in drilling in membrane
Circular DNA will get converted to linear during injection Diameter of linear DNA is 18nm while diameter of core tube is 20 nm Then DNA will pass through channel created by gp e & inserted in Host. After entering in host it will undergo circularization
Adsorption DNA injection Energy independent These steps require energy
Early Late Gene Gene s sImmediate Delayed Quasi Late True Late Early Early
Immediate Early genes : 0 to 3.75mins Delayed Early genes : 3.75 to 5mins Quasi Late genes : 5 to 11mins True Late genes : 11 to release Early genes are expressed in counterclockwise direction Late genes are expressed in clockwise direction
Adsorption DNA injection Synthesis of early mRNA Transcribed using host RNA polymerase, new proteins include modifiers of transcriptional specificity, especially, a new sigma factor (s), that recognizes only phage promoters Other early genes encode proteins required to take over host cell and synthesize viral nucleic acids
Adsorption DNA injection Synthesis of early mRNA Degradation of host DNA Phage DNA is protected from degradation by incorporation of hydroxymethyl cytosine (HMC), and glucosylation. Phage DNA is protected from degradation by viral nucleases and host restriction enzymes.
169 kb double stranded, linear molecule (genome 166 kb pairs) Encodes 144 genes Terminally redundant - 3 kb pairs (2% at left end repeated at the right end) Circularly permuted (circular DNA cut at different points yields linear DNA, different permutations) Replication forms concatemers Tandemly linked genome copies
Requires activity of rII & S genes. rII gene triggers the lysis of host at 22nd minute of infection S gene is responsible for delay in lysis until maturation process gets completed. gp e is also involved in lysis as it is lysozyme in nature
2. Entry-nucleic acid is inserted into host cell 3. Replication-viral components are made 4. Assembly-new viruses are assembled1. Attachment: virusconnects to host cell 5. Release-host cell membranes are destroyed by viral enzymes. New viruses are released and free to destroy other cells.
No genes specific for host cell disruption ФX174 has overlapping genes (coding region of one overlaps another) Replicative form (RF) is double stranded circle fd is filamentous phage that does not kill host RF is double stranded circle
Small (<4 kb) genome, 3-4 genes Positive sense ssRNA genome encodes replicase to copy RNA (RNA-dependent RNA polymerase) Regulation of gene expression by RNA secondary structure; alterations permit ribosome binding
Sigma factor cascade (T4) Gene order/slow injection (T7) Access to translational start sites (RNA phages) Antisense RNA (l, P1) Antiterminators (l) Frameshift allows expression of 2nd protein (FX174)
Temperate phages can convert host to lysogen Establishment likely with high level of infection of bacterial culture, starvation Stable association of phage DNA with bacterial cell (prophage) Normal growth and division of lysogen Environmental cues may induce lytic cycle
A) Attachment-virus connects to host cellB) Injection-viral nucleic acid is inserted into host cell and is incorporated into the host cell’s DNA as a Prophage. It can remain dormant for days, months, or even years.C) Host cells replicate both the host cells DNA and the Prophage.D) The “new” host cells continue to survive.
A) Attachment- B) Injection-viral C) Host cellsvirus connects to nucleic acid is inserted replicates both the D) The “new” host cell into host cell and is host cells DNA and host cells the Prophage. continue to incorporated into the host cell’s DNA as a survive. Prophage. It can remain dormant for days, months, or even years.
Double stranded DNA genome of 49 kb Linear DNA has cohesive ends, circularizes Early gene expression from leftward and rightward promoters determines dominance of cI or cro If cI dominates, lysogeny If cro wins, lytic cycle