Microbial Genetics

1. Transduction,Bacteriophages, and Gene Transfer Microbial Genetics MI 505 –

2. Bacteriophages • Bacterial viruses • Obligate intracellular parasites • Inject themselves into a host bacterial cell • Take over the host machinery and utilize it for protein synthesis and replication

3. Classification of Bacteriophages • based on two major criteria • phage morphology • nucleic acid properties

4. Major phage families and genera Figure 17.1

5. Reproduction of Double-Stranded DNA Phages: The Lytic Cycle • lytic cycle • phage life cycle that culminates with host cell bursting, releasing virions • virulent phages • phages that lyse their host during the reproductive cycle

6. The One-Step Growth Experiment mix bacterial host and phage  brief incubation (attachment occurs)  dilute greatly (released viruses can’t infect new cells)  over time, collect sample and enumerate viruses

7. latent period – no viruses released from host no virions – either free or within host rise period – viruses released free viruses Figure 17.2

8. Plaque assay • Phage infection and lysis can easily be detected in bacterial cultures grown on agar plates • Typically bacterial cells are cultured in high concentrations on the surface of an agar plate • This produces a “ bacterial lawn” • Phage infection and lysis can be seen as a clear area on the plate. As phage are released they invade neighboring cells and produce a clear area

9. Plaque assay

10. Focus on T4 replication • complex process • highly regulated • some genes expressed early • some genes expressed late • early genes and late genes clustered separately

11. early genes late genes Figure 17.7

12. adsorption and penetration Figure 17.6a

13. Adsorption to the Host Cell and Penetration • receptor sites • specific surface structures on host to which viruses attach • specific for each virus • can be proteins, lipopolysaccharides, techoic acids, etc.

14. T4 empty capsid remains outside of host cell tail tube may form pore in host membrane through which DNA is injected Figure 17.3 penetration mechanism differs from that of other bacteriophages

15. Bacteriophage structure

16. Phage Tour • www.mansfield.ohio-state.edu/.../bgnws020.htm

17. Synthesis of Phage Nucleic Acids and Proteins • sequential process • early mRNA synthesis • synthesis of proteins that enable T4 to take over host cell • phage DNA replication • late mRNA synthesis • encode capsid proteins and other proteins needed for phage assembly

18. some by regular host RNA polymerase others by modified host RNA polymerase some products needed for DNA replication Figure 17.6

19. Synthesis of T4 DNA • contains hydroxymethyl-cytosine (HMC) instead of cytosine • synthesized by two phage encoded enzymes • then HMC glucosylated Figure 17.8

20. HMC glucosylation • protects phage DNA from host restriction endonucleases • enzymes that cleave DNA at specific sequences • restriction • use of restriction endonucleases as a defense mechanism against viral infection

21. Post synthesis events • T4 DNA is terminally redundant • base sequence repeated at both ends • allows for formation of concatamers • long strands of DNA consisting of several units linked together

22. An example of terminal redundancy sticky ends units linked together Figure 17.9

23. during assembly – concatemers are cleaved, generating circularly permuted genomes Figure 17.10

24. synthesized by host RNA polymerase under direction of virus-encoded sigma factor encode capsid proteins and proteins needed for assembly Figure 17.6

25. The Assembly of Phage Particles scaffolding proteins – aid in construction of procapsid Figure 17.11

26. Figure 17.6b2

27. Figure 17.6

28. Release of Phage Particles • T4 • lysis of host brought about by several proteins • e.g., endolysin – attacks peptidoglycan • e.g., holin – produces lesion in cell membrane • other phages • production of enzymes that disrupt cell wall construction

29. focus on two phages X174 filamentous phages Reproduction of Single-Stranded DNA Phages

30. X174 by usual DNA replication method by rolling-circle mechanism new virions released by lysis of host Figure 17.12

31. M13 • M13 is a filamentous bacteriophage which infects E. coli host. The M13 genome has the following characteristics: • Circular single-stranded DNA • 6400 base pairs long • The genome codes for a total of 10 genes (named using Roman numerals I through X)

32. Bacteriophage PhiX174.

34. Reproduction of RNA Phages • most are plus strand RNA viruses • only one (6) is double-stranded RNA virus • also unusual because is envelope phage

35. ssRNAphages Figure 17.14

36. 6 reproduction • icosahedral virus with segmented genome • capsid contains an RNA polymerase • three distinct double-stranded RNA (dsRNA) segments • each encodes an mRNA • mechanism of synthesis of dsRNA genome is not known

37. Temperate Bacteriophages and Lysogeny • lysogeny • nonlytic relationship between a phage and its host • usually involves integration of phage genome into host DNA • prophage – integrated phage genome • lysogens (lysogenic bacteria) • infected bacterial host • temperate phages • phages able to establish lysogeny

38. Induction • process by which phage reproduction is initiated • results in switch to lytic cycle

39. Lysogenic conversion • change in host phenotype induced by lysogeny • e.g., modification of Salmonella lipopolysaccharide structure • e.g., production of diphtheria toxin by Corynebacterium diphtheriae

40. rate of production of cro and cI gene products determines if lysogeny or lytic cycle occurs Figure 17.17

41. Focus on lambda phage • double-stranded DNA phage • linear genome with cohesive ends • circularizes upon entry into host Figure 17.16

42. Lambda repressor • product of cI gene • blocks transcription of lytic cycle genes, including cro gene Figure 17.18

43. Cro protein • involved in regulating lytic cycle genes • blocks synthesis of lambda repressor Figure 17.20

44. The choice the race lambda repressor wins lysogeny cro wins lysis Figure 17.19

45. If lambda repressor wins… • lambda genome inserted into E. coli genome • integrase • catalyzes integration

46. Figure 17.21

47. Induction • triggered by drop in levels of lambda repressor • caused by exposure to UV light and chemicals that cause DNA damage • excisionase • binds integrase • enables integrase to reverse integration process

48. M13 • Among the simplest helical capsids are those of the well-known bacteriophages of the family Inoviridae, such as M13 and fd - known as Ff phages. These phages are about 900nm long and 9nm in diameter and the particles contain 5 proteins. All are similar and are known collectively as Ff phages - they require the E.coli F pilus for infection

49. M 13 Filamentous Phage

50. M13 • M13 is a filamentous bacteriophage which infects E. coli host. The M13 genome has the following characteristics: • Circular single-stranded DNA • 6400 base pairs long • The genome codes for a total of 10 genes (named using Roman numerals I through X)