Microbial Genetics Lectures

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Microbial 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

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Microbial Genetics Lectures

• Lecture 2
• Bacterial viruses (372-386)
• Classification
• Reproduction
• Transduction
• Recombinant Technology (312-333)
• Recombinant DNA
• Vectors and Cloning
• Applications

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• Bacterial viruses (372-386)
• Classification
• Reproduction
• Transduction

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Bacteriophages (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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Morphology
Head / Capsid
DNA
Sheath (Tail)
Tail fibres
Base plate
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Classification 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

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Classification 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)
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dsDNA Phage Life Cycle

• Vast majority of phages
• Two life styles
• Lytic (T4)
• Lysogenic (Lambda)

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Lytic 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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Lytic 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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Lytic 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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Lytic 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

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T4 phage 22 min 300 particles
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Single-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

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RNA 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

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Measuring 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)

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Measuring 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)

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Applications of Phage Biology
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Applications 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

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dsDNA 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.

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Temperate 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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Establishment 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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Notes

• 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

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Lysogenic 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

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Transduction

• 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.

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• 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 phageís 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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• Recombinant Technology (312-333)\
• Recombinant DNA
• Vectors and Cloning
• Applications

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• 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

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The 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)

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Restriction 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
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G 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
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CONSTRUCTION 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

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EcoRI
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
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BamH1
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
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A 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
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A 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
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BamH1
EcoRI
EcoRI
GFP
EcoRI
HindIII
Ampicillin resistance gene
Cloning Vector
Origin of replication
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BamH1
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
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Selection 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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Applications 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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• Any questions?

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Cloning 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

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G 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