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

2. 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. 3 Classification of Bacteriophages  based on two major criteria phage morphology nucleic acid properties

4. 4 Figure 17.1 Major phage families and genera

5. 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. 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. 7 free viruses no virions – either free or within host latent period – no viruses released from host rise period – viruses released Figure 17.2

8. 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. 9 Plaque assay

10. 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. 11 Figure 17.7 early genes late genes

12. 12 adsorption and penetration Figure 17.6a

13. 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. 14 Figure 17.3 penetration mechanism differs from that of other bacteriophages empty capsid remains outside of host cell tail tube may form pore in host membrane through which DNA is injected T4

15. 15 Bacteriophage structure

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

17. 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. 18 some by regular host RNA polymerase others by modified host RNA polymerase some products needed for DNA replicati on Figure 17.6

19. 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. 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. 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. 22 Figure 17.9 sticky ends units linked together An example of terminal redundancy

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

24. 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. 25 The Assembly of Phage Particles Figure 17.11 scaffolding proteins – aid in construction of procapsid

26. 26 Figure 17.6b2

27. 27 Figure 17.6

28. 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. 29 Reproduction of Single- Stranded DNA Phages  focus on two phages  X174 filamentous phages

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

31. 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. 32 Bacteriophage PhiX174.

33. 33

34. 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. 35 ssRNA phages Figure 17.14

36. 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. 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. 38 Induction  process by which phage reproduction is initiated  results in switch to lytic cycle

39. 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. 40 Figure 17.17 rate of production of cro and cI gene products determines if lysogeny or lytic cycle occurs

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

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

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

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

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

46. 46 Figure 17.21

47. 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. 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. 49 M 13 Filamentous Phage

50. 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)

51. 51

52. 52  Gene VIII codes for the major structural protein of the bacteriophage particles  Gene III codes for the minor coat protein

53. 53 Infection  The gene VIII protein forms a tubular array of approx. 2,700 identical subunits surrounding the viral genome  Approximately five to eight copies of the gene III protein are located at the ends of the filamentous phage (i.e. genome plus gene VIII assembly)  Allows binding to bacterial "sex" pilus Pilus is a bacterial surface structure of E. coli which harbor the "F factor" extrachromosomal element

54. 54 Infection  Single strand genome (designated '+' strand) attached to pilus enters host cell Major coat protein (gene VIII) stripped off Minor coat protein (gene III) remains attached  Host components convert single strand (+) genome to double stranded circular DNA (called the replicative or "RF" form)

55. 55 Transcription  Transcription begins Series of promoters  Provides a gradient of transcription such that gene nearest the two transcription terminators are transcribed the most Two terminators  One at the end of gene VIII  One at the end of gene IV Transcription of all 10 genes proceeds in same direction

56. 56 Part One  Gene II protein introduces 'nick' in (+) strand  Pol I extends the (+) strand using strand displacement (and the '-' strand as template)  After one trip around the genome the gene II protein nicks again to release a completed (linear) '+' genome  Linear (+) genome is circularized

57. 57 Part Two  During first 15-20 minutes of DNA replication the progeny (+) strands are converted to double stranded (RF) form  These serve as additional templates for further transcription  Gene V protein builds up  This is a single stranded DNA binding protein  Prevents conversion of single (+) strand to the RF form  Now get a buildup of circular single stranded (+) DNA (M13 genome)

58. 58 Summary of Repliation

59. 59 Phage Packaging  Phage packaging  Major coat protein (Gene VIII) present in E. coli membrane  M13 (+) genome, covered in ss binding protein - Gene V protein, move to cell membrane  Gene V protein stripped off and the major coat protein (Gene VIII) covers phage DNA as it is extruded out Packaging process is therefore not linked to any size constraint of the M13 genome Length of the filamentous phage is determined by size of the DNA in the genome Inserts of up 42 Kb have been introduced into M13 genome and packaged (7x genome size)  ~8 copies of the Gene III protein are attached at the end of the extruded genome

60. 60 M13

61. 61 M13 Cloning Vector  M13 was developed into a useful cloning vector by inserting the following elements into the genome:  a gene for the lac repressor (lac I) protein to allow regulation of the lac promoter  the operator-proximal region of the lac Z gene (to allow for a- complementation in a host with operator-proximal deletion of the lac Z gene).  a lac promoter upstream of the lac Z gene  a polylinker (multiple cloning site) region inserted several codons into the lac Z gene  The vectors were named according to the specific polyliner region they contained  The vectors were typically constructed in pairs, with the polylinker regions in opposite orientations

62. 62 M13 Cloning Vector

63. 63 Polylinker Cloning Region

64. 64 Medicine and Phages  www.intralytix.com/sciencemag.htm www.intralytix.com/sciencemag.htm

65. 65 Bacteriophage PhiX174.

66. 66

67. 67 PRD1 phages  Virions consist of a capsid and an internal lipid membrane.  Virus capsid is not enveloped.  Virions are tail-less, but can produce tail-like tubes  Capsid/nucleocapsid is round and exhibits icosahedral symmetry.

68. 68 Adsorption and penetration by other phages PRD1)

69. 69 Structural characteristics  The isometric capsid has a diameter of 63 nm. The capsid shells of virions are composed of two layers.  The outer capsid consists of a smooth, rigid 3 nm thin protein shell and appear to have a hexagonal in outline.  Surface projections are distinct 20 nm long spikes protruding from each apex  Inner capsids consist of a 5-6 nm flexible shell made from a thick lipoprotein vesicle.  The genome forms a tightly packed coil.

70. 70 Genome  The genome is not segmented and contains a single molecule of linear double-stranded DNA.  The complete genome is 147000- 157000 nucleotides long, is fully sequenced and encodes gene 8 for DNA terminal proteins and genes for protein P15 (lytic enzyme).

71. 71 Group A Streptococci( GAS)  Genes activated when macrophages engulf bacterial cells  These phage genes are part of the ability of bacterial cells to avoid destruction

72. 72 M18 strain of GAS  Significant part of genome contains phage genes  Difference in phage genes accounts for differences in pathogenicity

73. 73 Streptococcus canis  Normally a bacterium that harmlessly infects dogs  Treatment with antibiotics for other infections especially fluoroquinolones, causes the activation of phage genes  Induces flesh eating infections and toxic shock

74. 74 Listeria phages  The Gram-positive bacterium Listeria monocytogenes can be found in raw food and causes human disease, The immune- compromised are particularly susceptible, and infection leading to listeric meningitis can be deadly. Listeria monocytogenes  Listeria transducing bacteriophage CU153, shown on the left has a very long tail with two disk-like structures at the distal end (DNA content is about 42Kbp).  Phage P35 shown on the right has a much shorter tail with a single disk-like structure at the distal end.

75. 75 Fluoroquinolones  Cipro that fights Anthrax belongs to this group  Triggers phage genes  Can increase the amount of toxin released  ( Shiga toxin can be released by a variety of bacteria)

76. 76 E. Coli  Shiga toxin is integrated into E. coli DNA – the gift of a phage  When it becomes active – E. coli’s food poisoning becomes more severe

77. 77 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

78. 78 Plaque assay

79. 79 Generalized transduction  http://www.cat.cc.md.us/courses/bio14 1/lecguide/unit4/genetics/recombinati on/transduction/gentran.html http://www.cat.cc.md.us/courses/bio14 1/lecguide/unit4/genetics/recombinati on/transduction/gentran.html  http://www.cat.cc.md.us/courses/bio14 1/lecguide/unit1/control/genrec/u4fg2 1a.html http://www.cat.cc.md.us/courses/bio14 1/lecguide/unit1/control/genrec/u4fg2 1a.html

80. 80

81. 81

82. 82 Specialized transduction  http://www.cat.cc.md.us/courses/bio14 1/lecguide/unit4/genetics/recombinati on/transduction/spectran.html http://www.cat.cc.md.us/courses/bio14 1/lecguide/unit4/genetics/recombinati on/transduction/spectran.html