Title: Microbial Genetics Lectures
1Microbial Genetics Lectures
- John Buchanan
- Research Scientist
- School of Medicine
- Department of Pediatric Infectious Diseases
- Research Projects
- The genetics of bacterial virulence
- Alternatives to antibiotics to treat bacterial
infections
2Microbial Genetics Lectures
- Lecture 2
- Bacterial viruses (372-386)
- Classification
- Reproduction
- Transduction
- Recombinant Technology (312-333)
- Recombinant DNA
- Vectors and Cloning
- Applications
3- Bacterial viruses (372-386)
- Classification
- Reproduction
- Transduction
4Bacteriophages (Phages)
- Viruses that infect bacteria
- Bacteriophages cannot reproduce and survive on
their own, must take over host cell - Fundamentaly important microbes
- Ecologically- 1031 total phages
- 108 - 109 / ml in water
- Controlling bacteria populations and energy
cycling - Gene shuffling in the environment
- Tools for molecular biology and recombinant
technology
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6Morphology
Head / Capsid
DNA
Sheath (Tail)
Tail fibres
Base plate
7Classification of Bacteriophages
- The most important criteria used for
classification are phage morphology and nucleic
acid properties - dsDNA
- Contractile tails
- Noncontractile tails
- Tailless
- Filamentous
- Head shape
- ssDNA
- ssRNA
- dsRNA
8Classification of Bacteriophages
- The most important criteria used for
classification are nucleic acid properties and
phage morphology - dsDNA
- Contractile tails
- Noncontractile tails
- Tailless
- Filamentous
- Head shape
- ssDNA
- ssRNA
- dsRNA
Flexible tail (lambda)
Contractile tail (T4)
Filamentous (fd)
Tailless (SSV-1)
9dsDNA Phage Life Cycle
- Vast majority of phages
- Two life styles
- Lytic (T4)
- Lysogenic (Lambda)
10Lytic Life Cycle - 1
- Adsorption to the host cell and penetration
- Specificity of phage infection
- 10 phages for every type of bacteria
- Viruses attach to specific receptor sites
- Proteins
- Lipopolysaccharides
- Teichoic acids and cell wall components
- Carbohydrates
- Sex pilus
- Phages then inject DNA into the cell
- Tail contraction (T4)
- Injection (PRD1)
- Unknown mechanisms
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12Lytic Life Cycle - 2
- Synthesis of phage nucleic acids and proteins
- mRNA molecules transcribed early in the infection
are synthesized using host RNA polymerase (1 min) - Make viral enzymes required to take over the host
cell - Degradation of host DNA (3 min)
- Transcription of viral genes (5-9 min)
- Phage DNA is replicated (5 min)
- Phage DNA sometimes modified protect the phage
DNA from host enzymes that would degrade the
viral DNA - The assembly of phage particles
- Phage mRNA directs the synthesis of capsid
proteins and other proteins involved in assembly
and release of the virus (12 min) - DNA packaged into the head (13 min)
- Phage pieces assembled (15 min)
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14Lytic Life Cycle - 3
- Release of phage particles (22 min 300 new
phage particles) - Many phages lyse their host by damaging the cell
membrane and cell wall - Holin enzyme which destabilizes the host cell
membrane (pokes holes) - Lysin phage enzyme which breaks host cell wall
(lyses host bacteria)
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16Lytic Life Cycle - Summary
- Adsorption to the host cell and penetration
- Viruses attach to specific receptor sites
(proteins, lipopolysaccharides, teichoic acids,
etc.) on the host cell - Many viruses inject DNA into the host cell,
leaving an empty capsid outside - Synthesis of phage nucleic acids and proteins
- mRNA molecules transcribed early in the infection
(early mRNA) are synthesized using host RNA
polymerase - make viral enzymes required to take over the host
cell - Transcription of viral genes follows
- Phage DNA is replicated
- Phage DNA sometimes modified protect the phage
DNA from host enzymes that would degrade the
viral DNA - The assembly of phage particles
- Phage mRNA directs the synthesis of capsid
proteins and other proteins involved in assembly
and release of the virus - Phage pieces assembled
- DNA packaged into the head
- Release of phage particles
- Many phages lyse their host by damaging the cell
wall or the cytoplasmic membrane - A few phages (e.g., filamentous fd phages) are
released without lysing the host cell secreted
instead
17T4 phage 22 min 300 particles
18Single-Stranded DNA phages
- ssDNA is converted to double-stranded form by
host DNA polymerase - Double-stranded form directs phage protein
synthesis - Two different strategies for lysis
- Similar to T4
- Secreted from the host cell (filamentous phages)
- Parasitic relationship
19RNA Phages
- Single-stranded RNA phages
- Codes for RNA replicase (enzyme for replicating
the RNA genome) - The RNA genome can usually act as mRNA to direct
the synthesis of the replicase - RNA is then converted to dsRNA
- dsRNA is then used as a template for production
of multiple copies of the genomic RNA - Capsid proteins are made, and ssRNA is packaged
into new virions - Very small genomes
- Lyses host through inhibition of cell wall
formation - Only one dsRNA phage has so far been discovered
(f6) it infects Pseudomonas phaseolicola and
possesses a membranous envelope
20Measuring Phage Number Plaque Assays
- Plaque assay method for enumerating the number
of phage particles in a sample results are
giving in plaque forming units (PFU)
21Measuring Phage Number Plaque Assays
- Plaque assay method for enumerating the number
of phage particles in a sample results are
giving in plaque forming units (PFU)
22Applications of Phage Biology
23Applications of Phage Biology
- Need for alternative therapies for treating
bacterial infections - Resistance exists to every antibiotic we have
- Phages are potent antibacterials
- Self-replicating (smart drugs?)
- Narrow specificity so dont damage the normal
flora - Resistance not as significant
- Resurgent interest in the application of phages
to agriculture and human health - Used for years in Eastern Europe and Russia
24dsDNA Phage Life Cycle
- Vast majority of phages
- Two life styles
- Lytic (T4) lyses host cell
- Lysogenic (Lambda) - Instead of destroying host
to produce virus progeny, the viral genome
remains within the host cell and replicates with
the bacterial chromosome.
25Temperate Bacteriophages and Lysogeny
- Temperate phages are capable of lysogeny, a
nonlytic relationship with their hosts (virulent
phages lyse their hosts - lytic) - Temperate lysogenic
- Virulent lytic
- In lysogeny, the viral genome (called a prophage)
remains in the host (usually integrated into the
host chromosome) but does not kill (lyse) the
host cell It may switch to the lytic cycle at
some later time - The switching to a lytic cycle is called
induction
Lambda phages
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27Establishment of lysogeny
- DNA is double stranded with cohesive ends (cos
sites) which are ss stretches of DNA that are
complementary to each other - Circularizes immediately after injection into the
host - Once a closed circle is formed transcription by
host RNA polymerase is initiated - The BIG Decision Lytic or Lysogenic life cycle?
- Battle between two repressors, cI or cro which
compete for the same binding sites (operators) on
phage DNA - If cI binds, represses synthesis of all genes
Lysogenic - If cro binds, represses synthesis of cI Lytic
- If cI repressor wins the circular DNA is inserted
into the chromosome via a process called
integration and is maintained there - At this stage it is called a prophage
- If cI levels drop, cro takes over and the phage
becomes lytic - Environmental factors, such as UV light or
chemical mutagens, that damage host DNA causes a
host protein, recA, to act as a protease and
cleave the cI repressor - Decrease in cI stops repression of phage genes
and balance shifts to cro and the lytic cycle
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29Notes
- For lambda and most temperate phages the viral
genome integrates into the host chromosome
however, some temperate phages can establish
lysogeny without integration - Most bacteriophages are temperate indicating that
this life strategy is advantageous - 1 T4 phage 300 new phages (may exterminate
hosts) - 1 lambda phage infects one host
- Host produces 1000 daughter cells (can live with
hosts) - Lambda emerges with 100 phages per cell 100,000
new phages
30Lysogenic conversion
- Lysogenic conversion is a change that is induced
in the host phenotype by the presence of a
prophage - Not directly related to the completion of the
viral life cycle - Expression of additional genes from prophage
- Production of diphtheria toxin only by
lysogenized strains of Corynebacterium
diphtheriae - Toxins that make Vibrio cholerae pathogenic are
carried on a phage
31Transduction
- Transduction is the transfer of bacterial genes
by phages. - Bacterial genes are incorporated into a phage
capsid due to errors made during the virus life
cycle. - The virus containing these genes then injects
them into another bacteria - Mistakes in bacteriophage replication can
generate diversity at the genomic level and
shuffle the genes of bacteria into novel
combinations - Most common mechanism for gene exchange and
recombination in bacteria.
32- Transducing particle- the phage which injects
bacterial DNA into a new recipient. - Generalized transduction Transfer of random
portions of host genomic DNA by bacteriophages
during the lytic cycle of virulent or temperate
phages - Any part of the bacterial genome can be
transferred - The phage degrades host chromosome into randomly
sized fragments - During assembly, fragments of host DNA can be
mistakenly packaged into a phage head - When the next host is infected, the bacterial
genes are injected - Preservation of the transferred genes requires
their integration into the host chromosome - Specialized transduction - transfer of only
specific portions of the bacterial genome by
temperate phages that have integrated their DNA
into the host chromosome - The prophage is sometimes excised incorrectly and
contains portions of the bacterial DNA that was
adjacent to the phageÃs integration site on the
chromosome - The excised phage genome is defective because
some of its own genes have been replaced by
bacterial genes therefore, the bacteriophage
cannot reproduce - When the next host is infected, the donor
bacterial genes are still injected and can become
incorporated
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34- Recombinant Technology (312-333)\
- Recombinant DNA
- Vectors and Cloning
- Applications
35- Genetic engineering - the deliberate modification
of an organism's genetic information by directly
changing its nucleic acid - Recombinant DNA technology - the collection of
methods used to accomplish genetic engineering - Recombinant DNA - DNA with a new sequence formed
by joining fragments from different sources
36The Polymerase Chain Reaction (PCR)
- PCR is used to synthesize large quantities of a
specific DNA fragment in vitro (in a test tube) - Synthetic DNA molecules with sequences identical
the target sequence are created during the
reaction - Made possible by bacteria - Replication is
carried out in successive heating-cooling cycles
using a heat-stable DNA polymerase from a
thermophilic bacteria - PCR has proven valuable in molecular biology,
medicine (e.g., PCR-based diagnostic tests) and
in biotechnology (e.g., use of DNA fingerprinting
in forensic science)
37Restriction enzymes
- Restriction enzymes (endonucleases) - bacterial
enzymes that recognize and cleave specific
sequences of DNA (4-8 bp long) - Bacteria use them to destroy foreign DNA
- Valuable molecular biology tools
- Enzyme EcoR1 (Restriction enzyme R1 from E. coli)
- Cuts at GAATTC (palindrome)
- Leaves a cleaved DNA molecule with specific ends
G A A T T C C T T A A G
G C T T A A
A A T T C G
Eco RI overhang
Eco RI overhang
38G C T T A A
A A T T C G
Eco RI overhang
Eco RI overhang
DNA Ligase
G A A T T C C T T A A G
39CONSTRUCTION OF A RECOMBINANT DNA MOLECULE
- Isolate gene of interest
- For example, create many copies of a gene by PCR
- Digest the ends of the gene with restriction
enzymes - Use DNA ligase to link the gene to a cloning
vector - Progate cloning vector and proceed with
applications with cloned gene - Cloning vector genetic element used to
propogate and express genes of interest in
bacteria - Plasmids, phages, cosmids, artificial chromosomes
40EcoRI
EcoRI
Gene of Interest Green Fluorescent Protein
G A A T T C C T T A A G
G A A T T C C T T A A G
Digest with EcoR1
A A T T C G
Green Fluorescent Protein
G C T T A A
41BamH1
EcoRI
HindIII
Ampicillin resistance gene
Cloning Vector
Origin of replication
Digest with EcoR1
A A T T C G
G C T T A A
42A A T T C G
Green Fluorescent Protein
G C T T A A
Add DNA Ligase
A A T T C G
G C T T A A
43A A T T C G
Green Fluorescent Protein
G C T T A A
Add DNA Ligase
A A T T C G
G C T T A A
BamH1
GFP
HindIII
Ampicillin resistance gene
Origin of replication
44BamH1
EcoRI
EcoRI
GFP
EcoRI
HindIII
Ampicillin resistance gene
Cloning Vector
Origin of replication
45BamH1
EcoRI
EcoRI
GFP
EcoRI
HindIII
Ampicillin resistance gene
Cloning Vector
BamH1
Origin of replication
EcoRI
GFP
HindIII
EcoRI
Ampicillin resistance gene
GFP Expression Plasmid
Origin of replication
46Selection for Bacteria with Gene of Interest
TRANSFORMATION
SELECTION FOR BACTERIA WITH PLASMID
Only bacteria containing the resistance gene grow
Medium contains Ampicillin
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48Applications of Recombinant Technology
- Use similar techniques for bacterial expression
of medically important proteins - Insulin
- Interleukins
- Growth hormone
- Industrial and agricultural application
- Use recombinant technology to understand the
genetics of organisms - Recombinant technology is the alteration of DNA
- Genetically modified organisms
- Increased efficiency and economic value
- Risks and social concerns?
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51Cloning Vectors
- Cloning vector - small, well-characterized DNA
molecule that contains at least one replication
origin, can be replicated within the appropriate
bacterial host, and code for a phenotype that is
easily detected - Antibiotic resistance, color change
- Plasmids vectors
- Easy to isolate and purify
- Can be introduced into bacteria by transformation
- Often bear antibiotic resistance genes that can
be used to select recombinants - Phage vectors
- Are more conveniently stored for long periods
- Contain insertion sites that do not interfere
with replication when foreign DNA is inserted - Recombinant phage DNA can be packaged into viral
capsids and used to infect a host cell - Cosmids - plasmids with lambda phage cos sites
- Cosmids can be packaged into lambda capsids and
then manipulated as a phage - Can also exist in the cell like a plasmid
- Can be used to clone very large pieces of DNA
- Artificial chromosomes - can be yeast or
bacterial have all of the elements necessary to
propagate as a chromosome they can be used to
clone DNA fragments from 100kb to 2000kb in
length
52G C T T A A
A A T T C G
G C T T A A
A A T T C G
EcoRI
EcoRI
EcoRI
EcoRI
Ligate with DNA ligase
Ligate with DNA ligase
G A A T T C C T T A A G
G A A T T C C T T A A G
Eco RI
Eco RI