Transcript Microbial Genetics - University of Montana

Microbial Genetics
MICB404, Spring 2006
Lecture #17
Bacteriophage I
Lytic phage
• Announcements
• Today we finish conjugation
• Bacteriophage I: Lytic phage
Hfr gene mapping
E. coli chromosome genetic map
Selected
Marker
hisG
Selected Markers
argH
rif
trpA
7%
6%
1%
Plot frequency of unselected
markers
Selected = argH
Selected = hisG
89%
100
7
12%
6
1
100
90
argH
10
20
30
trpA
rif
Prime factor selection
Prime Factors
• Selection
– Early transfer of distal
markers
• Markers that were far from
oriT in chromosome of Hfr
will be close in F’, and
transferred early
• Recipient is merodiploid
and transconjugant, not recombinant
• Capable of conjugation
Prime Factors
• Selection
– Replicons
• Capable of replication
independent of chromosome
• If recipient is defective
in recombination, prime
factor transconjugants can
acquire and transmit selected
marker but no recombinants
from Hfr individuals form
Bacteriophage
• Viruses that infect bacteria
Bacteriophage
• Important in the history of genetic research
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replication
transcription
recombination
gene regulation
DNA is the genetic material
mRNA is an intermediate in translating genetic information
from DNA to protein
– http://www.asm.org/division/m/blurbs/Secrets.html
• Important tools in molecular biology
– Enzymes (T4 DNA Ligase, T7 RNA polymerase, λ
exonuclease, cre recombinase)
– λ DNA
– phage cloning vectors
– phage display
Confirmation that genes consist
of DNA
Bacteriophage
• Bacterial parasites
– Exploits host metabolism for replication and
production of new phage particles
• amino acids, cellular energy, translation
– Phage genes encode factors required to re-direct
cellular activity to phage manufacture
• Minimum functions for survival
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Protection of genome in environment
Delivery of genome into bacterium
Conversion of infected bacterium to phage factory
Release of progeny phage
Bacteriophage
• 10-100+ genes
• Structure
– Icosahedral tailless
– Icosahedral tailed
– Filamentous
Tail Fibers
Bacteriophage
• Head: Coat or Capsid
– Contains nucleic acid
• Tail & fibers
– Interaction with
bacterial surface
– Injection of DNA
into bacterium
Bacteriophage
• Nucleic acid
– single-stranded DNA
– double-stranded DNA
• linear or circular molecules
– RNA
• generally, linear & single-stranded
– Genome size
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M13: 7200 bases ssDNA
λ: 49,000 bp
T4: 160,000 bp
packaging
Lytic Phage Life Cycle
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Adsorption
Injection
Transcription
Take over
Production
Packaging
Assembly
Lysis
Phage Life Cycle
• Adsorption
– binding to receptors in bacterial
cell surface
• receptors play various roles for
cells, e.g. solute uptake
• reversible phase
• irreversible phase
Phage Life Cycle
• Injection
– DNA passes through bacterial
cell wall and membrane
• Medium exposure
– None for tailed phage
– Occurs with tailless phage
• Transcription
– use host-like promoters for
expression of early genes
Phage Life Cycle
• Take over
– phage shut down host replication,
transcription, and translation
– re-program cell to produce phage
coat proteins and genome
• alter activity of host enzymes
– phage-specific sigma factors
• express phage-specific enzymes
– DNA & RNA polymerases, etc.
• evade host defense mechanisms
• Production
Phage Life Cycle
– phage nucleic acids and proteins
synthesized in host cell
• structural proteins
• catalytic proteins (maturation)
• Packaging
– Nucleic acid inserted into heads
• Assembly
– coat proteins assembled into
capsids
– tail proteins assembled
• Lysis
Phage Life Cycle
– Lysozyme or endolysin one of the
late-expressed enzymes
– Disrupts cell wall
– Phage released to environment
– Note: filamentous phage are
extruded through cell wall without
lysis or cell death
• Yield: 10-1000 phage per cell
Lytic Development Cycle
• Early genes
• Middle genes
• Late genes
Lytic Development Cycle
• Early genes
• Middle genes
• Late genes
Studying phage
• Plaques: clear zones in lawns of plated
bacteria caused by cell lysis
– Plaque morphology
affected by phage
genotype
• small, large
• sharp, haloed
Studying phage
Bacteria
Phage
Plate 108 cells and 1 phage
Infection and lysis  100 phage
2nd round of infection & lysis
 10,000 phage
Lawn
Plaque
Result: all cells are killed and
lysed surrounding site of original
phage particle
1 phage results in 1 plaque (106
phage)
Studying phage
• Multiplicity of Infection MOI
– Average number of phage adsorbed per
bacterium
• mixing 107 E. coli with 3x107 T4 phage: MOI = 3
– High, >1
– Low, <1
Studying phage
• Calculate the proportion of cells infected by a
specific number of phage
• Poisson distribution
• P(n) = (mn * e-m)/n!
where: m = MOI
n = number of phage infecting a
cell
P(n) = probability cell will be
infected with n phage
(e.g. MOI =1: at most 63% cells infected)
Studying phage
• Burst size
– Number of phage produced by infected cell
= (number of phage produced after lysis)
(number of infected cells)
– Typical burst size is 100 for wildtype phage
• mutants with burst size <10 can produce plaques
Host defense
• Restriction-Modification
• Expression of surface receptors
• Activity
Phage genetics
• Bacteriophage T4
– Life cycle & replication
– Genetic analysis
Phage genetics
• Bacteriophage T4
• > 200 genes
T series of bacteriophage
Morphology
Name
Plaque size
Head (nm)
Tail (nm)
Latent period (min) Burst size
T1
medium
50
150 x 15
13
180
T2
small
65 x 80
120 x 20
21
120
T3
large
45
invisible
13
300
T4
small
65 x 80
120 x 20
23.5
300
T5
small
100
tiny
40
300
T6
small
65 x 80
120 x 20
25.5
T7
large
45
invisible
13
T-even and T-odd phage
The T-even phages, T2, T4 and T6, are all related
serologically and all have large genomes; T4 has a genome
168,895 bp in length
The T-odd phages fall into three serological
groups: T3 and T7 are related to each other but not to T1 or
to T5, which are unrelated. The T7 genome was sequenced
in 1983; it is 39,937 bp in length
200-300
300
T4
• Seymour Benzer, 1950’s
– Fine-structure mapping of rII (rapid lysis
mutants type II) locus
• rII will complete life cycle in E. coli B but not on
K12λ
– but can infect K12λ
• Complementation indicated rII has 2 genes or
“complementation groups”
T4
• r mutants distinguished based on plaque
morphology
Complementation
Co-infect K12 with 2 rII mutant phage:
A
a1
a
b
B
Defects in different genes:
infection has functional copy of
each gene: complementation.
Massive lysis with progeny
mostly of parental Ab or aB
genotypes
a2
B
B
Overlapping defects in same
gene: no complementation.
No lysis, no progeny phage
Recombination
Recombination frequency
• The closer two sequence regions are to
one another, the less room for, and less
likelihood of, crossover.
• Frequency of recombinants is therefore
a measure of how far apart mutations
are in the sequences.
• Recombination frequency.
Recombination
Co-infect E. coli B with 2 rII mutant phage:
a1
a2
B
B
a1
a2
B
A
B
Non-overlapping defects in same gene: recombination.
Low degree of K12 lysis, progeny are almost entirely AB, wild-type
Collect
phage
lysate
Recombination
Co-infect E. coli B with 2 rII mutant phage:
a1
a2
B
B
a1
a2
B
Mapping
Infect both B and K12
B: plaques represent total number of progeny phage
K12: wildtype are ½ of total recombinants
Map distance:
2 x f(wild-type progeny) x 100
A
B
• Monday’s lecture:
– Bacteriophage II
– Reading
• Continue with Snyder and Champness, Chapter 7