1

Microbial GeneticsTransduction,Bacteriophages, and Gene Transfer

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

4Figure 17.1

Major phage families and genera

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

free viruses

no virions –either free orwithin host

latent period – noviruses releasedfrom host

rise period –viruses released

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

11Figure 17.7

earlygenes

lategenes

12

adsorption andpenetration

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

Figure 17.3

penetration mechanismdiffers from that of otherbacteriophages

empty capsidremains outsideof host cell

tail tube may form porein host membrane throughwhich DNA is injected

T4

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 byregularhost RNApolymerase

others bymodifiedhost RNApolymerase

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

22Figure 17.9

sticky ends

units linked together

An example of terminal redundancy

23Figure 17.10

during assembly – concatemers arecleaved, generatingcircularlypermuted genomes

24

synthesizedby host RNApolymeraseunder directionof virus-encodedsigma factor

encodecapsidproteinsandproteinsneededforassembly

Figure 17.6

25

The Assembly of Phage Particles

Figure 17.11

scaffolding proteins –aid in construction ofprocapsid

26

Figure 17.6b2

27Figure 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

Reproduction of Single-Stranded DNA Phages

focus on two phagesX174

filamentous phages

30

X174

Figure 17.12

newvirionsreleasedby lysisof host

by usualDNA replicationmethod

by rolling-circlemechanism

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.

33

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

40Figure 17.17

rate of productionof cro and cI geneproducts determines iflysogeny or lytic cycleoccurs

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

44Figure 17.19

The choice

the race

lambda repressor wins lysogeny

cro wins lysis

45

If lambda repressor wins… lambda genome inserted into E. coli

genome integrase

catalyzes integration

46Figure 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)

51

52

Gene VIII codes for the major structural protein of the bacteriophage particles

Gene III codes for the minor coat protein

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

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

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

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

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

Summary of Repliation

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

M13

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

M13 Cloning Vector

63

Polylinker Cloning Region

64

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

65

Bacteriophage PhiX174.

66

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

Adsorption and penetration by other phages PRD1)

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

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

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

M18 strain of GAS Significant part of genome contains

phage genes Difference in phage genes accounts

for differences in pathogenicity

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

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 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

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

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

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

Plaque assay

79

Generalized transduction http://www.cat.cc.md.us/courses/bio

141/lecguide/unit4/genetics/recombination/transduction/gentran.html

http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/control/genrec/u4fg21a.html

80

81

82

Specialized transduction http://www.cat.cc.md.us/courses/bio

141/lecguide/unit4/genetics/recombination/transduction/spectran.html