Microbial
Genetics
Transduction,Bacteriophages, and Gene Transfer
MI 505 –
1
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
2
Classification of
Bacteriophages

based on two major criteria


phage morphology
nucleic acid properties
3
Major
phage
families
and
genera
Figure 17.1
4
Reproduction of DoubleStranded 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
5
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
6
latent period – no
viruses released
from host
no virions –
either free or
within host
rise period –
viruses released
free viruses
Figure 17.2
7
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
8
Plaque assay
9
Focus on T4 replication


complex process
highly regulated



some genes expressed early
some genes expressed late
early genes and late genes clustered
separately
10
early
genes
late
genes
Figure 17.7
11
adsorption and
penetration
Figure 17.6a
12
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.
13
T4
Figure 17.3
empty capsid
remains outside
of host cell
tail tube may form pore
in host membrane through
which DNA is injected
penetration mechanism
differs from that of other
bacteriophages
14
Bacteriophage structure
15
Phage Tour

www.mansfield.ohiostate.edu/.../bgnws020.htm
16
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
17
some by
regular
host RNA
polymerase
others by
modified
host RNA
polymerase
Figure 17.6
some
products
needed
for DNA
replicati
18
on
Synthesis of T4 DNA

contains
hydroxymethylcytosine (HMC)
instead of
cytosine


synthesized by
two phage
encoded enzymes
then HMC
glucosylated
Figure 17.8
19
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
20
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
21
An example of terminal
redundancy
sticky ends
units linked together
Figure 17.9
22
during assembly –
concatemers are
cleaved, generating
circularly
permuted
genomes
Figure 17.10
23
synthesized
by host RNA
polymerase
under direction
of virus-encoded
sigma factor
Figure 17.6
encode
capsid
proteins
and
proteins
needed
for
assembly
24
The Assembly of Phage
Particles
scaffolding proteins –
aid in construction of
procapsid
Figure 17.11
25
Figure 17.6b2
26
Figure 17.6
27
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
28
Reproduction of SingleStranded DNA Phages

focus on two phages

filamentous phages
X174
29
X174
by usual
DNA replication
method
by rolling-circle
mechanism
Figure 17.12
new
virions
released
by lysis
of host
30
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)
31
Bacteriophage PhiX174.
32
33
Reproduction of RNA Phages


most are plus strand RNA viruses
only one (6) is double-stranded RNA
virus

also unusual because is envelope phage
34
ssRNA
phages
Figure 17.14
35
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
36
Temperate Bacteriophages
and Lysogeny

lysogeny


nonlytic relationship between a phage and its
host
usually involves integration of phage genome
into host DNA


lysogens (lysogenic bacteria)


prophage – integrated phage genome
infected bacterial host
temperate phages

phages able to establish lysogeny
37
Induction


process by which phage reproduction is
initiated
results in switch to lytic cycle
38
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
39
rate of production
of cro and cI gene
products determines if
lysogeny or lytic cycle
occurs
Figure 17.17
40
Focus on lambda phage


doublestranded
DNA phage
linear genome
with cohesive
ends

circularizes
upon entry
into host
Figure 17.16
41
Lambda repressor


product of cI
gene
blocks
transcription of
lytic cycle genes,
including cro gene
Figure 17.18
42
Cro protein


involved in
regulating
lytic cycle
genes
blocks
synthesis of
lambda
repressor
Figure 17.20
43
The
choice
the race
lambda
repressor
wins
cro wins
Figure 17.19
lysogeny
lysis
44
If lambda repressor wins…

lambda genome inserted into E. coli
genome

integrase

catalyzes integration
45
Figure 17.21
46
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
47
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
48
M 13 Filamentous Phage
49
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)
50
51


Gene VIII codes
for the major
structural protein
of the
bacteriophage
particles
Gene III codes for
the minor coat
protein
52
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
53
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)
54
Transcription

Transcription begins

Series of promoters


Two terminators



Provides a gradient of transcription such that gene
nearest the two transcription terminators are
transcribed the most
One at the end of gene VIII
One at the end of gene IV
Transcription of all 10 genes proceeds in same
direction
55
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
56
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)
57
Summary of Repliation
58
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
59
M13
60
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 acomplementation 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
61
M13 Cloning Vector
62
Polylinker Cloning Region
63
Medicine and Phages

www.intralytix.com/sciencemag.htm
64
Bacteriophage PhiX174.
65
66
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.
67
Adsorption and penetration by
other phages PRD1)
68
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.
69
Genome


The genome is not segmented and
contains a single molecule of linear
double-stranded DNA.
The complete genome is 147000157000 nucleotides long, is fully
sequenced and encodes gene 8 for DNA
terminal proteins and genes for protein
P15 (lytic enzyme).
70
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
71
M18 strain of GAS


Significant part of genome contains
phage genes
Difference in phage genes accounts for
differences in pathogenicity
72
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
73
Listeria phages



The Gram-positive bacterium
Listeria monocytogenes can be
found in raw food and causes
human disease, The immunecompromised 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.
74
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)
75
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
76
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
77
Plaque assay
78
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/unit1/control/genrec/u4fg2
1a.html
79
80
81
Specialized transduction

http://www.cat.cc.md.us/courses/bio14
1/lecguide/unit4/genetics/recombinati
on/transduction/spectran.html
82