Phage Biology Phage are viruses that grow on bacterial cells [bacteriophage = bacteria eater].

1. Phage Biology Phage are viruses that grow on bacterial cells [bacteriophage = bacteria eater].

2. Icosahedral tail-less Icosahedral tailed Filamentous There are 3 basic types of phage structure. Head diameter ~ 50nm ~ 300nm in length

3. Icosahedral tailed phage (T4) attacking an E. coli cell

4. Icosahedral tail-less phage f2 attached along side of F pilus structure on E. coli host cell. Filamentous M13 phage attached to tip of F pilus.

5. Phage can be detected (and counted) by a plaque assay. 1. Mix phage suspension and bacterial cells. 2. Add molten cooled soft agar. 3. Pour on nutrient agar plate. Phage particle Top agar Base agar Plaque Lawn

6. Plaques of phage T4 on a lawn of E. coli host cells.

7. Phage can replicate in 2 ways: (1) by multiplying in a host cell (lytic growth) (2) by integrating their genome into the bacterial chromosome (lysogenic growth).

8. Phage adsorbs to specific receptor on host cell surface and injects DNA. Lytic growth Lysogenic growth Induction Phage DNA integrates into bacterial chromosome. Phage multiplies and bursts host cell.

9. A phage genome inserted into a bacterial chromosome is called a prophage. A bacterium carrying a prophage is called a lysogen. A repressor switches off phage genes. A lysogen is immune to superinfection by the the same type of phage. Prophage induction results in phage multiplication and host cell lysis.

10. Virulent phage are only capable of lytic growth. Temperate phage can undergo lytic or lysogenic growth. 90% of all known phage are temperate.

11. Transduction transfer of bacterial DNA from one cell to another by a phage particle. leu+ thr - leu - thr+

12. Phage conversion Some prophages carry genes that “convert” the host bacterium (confer new properties). Many bacterial protein toxin genes are carried by converting phages Bacterial species Toxin Corynebacterium diphtheriae Diphtheria toxin Vibrio cholerae Cholera toxin Streptococcus pyogenes Erythrogenic toxin Escherichia coli O157 Vero cytotoxin Clostridium botulinum Botulin (neurotoxin)

13. Pseudomembrane in throat of diphtheria patient. The gene for diphtheria toxin is located within a prophage in the Corynebacterium diphtheriae genome.

14. Host restriction and modification Restriction enzymes - endonucleases that cut specific sequences in DNA -restrict the growth of bacteriophage by degrading phage DNA. Modification enzymes protect host DNA by methylating sensitive sites

15. Type I restriction enzymes bind to specific sites, migrate along DNA for 1000 to 5000 bases then cut randomly. Type II restriction enzymes cut specific sites e. g. Eco RI ------GAATTC------ ------CTTAAG------ ---G AATTC--- ---CTTAA G---

16. Eco RI methylase modifies these sites. * ------GAATTC------ ------CTTAAG------ * Methylated adenine base

17. Type III restriction enzymes bind to specific sites but cut DNA 24 to 26 bases downstream. A few phage genomes escape destruction. The phage DNA is then modified and progeny are resistant to the restriction enzyme.