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Microbial Genetics Lectures

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Title: Microbial Genetics Lectures


1
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

2
Microbial 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

4
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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6
Morphology
Head / Capsid
DNA
Sheath (Tail)
Tail fibres
Base plate
7
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

8
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)
9
dsDNA Phage Life Cycle
  • Vast majority of phages
  • Two life styles
  • Lytic (T4)
  • Lysogenic (Lambda)

10
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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12
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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14
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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16
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

17
T4 phage 22 min 300 particles
18
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

19
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

20
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)

21
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)

22
Applications of Phage Biology
23
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

24
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.

25
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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27
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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29
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

30
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

31
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.

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 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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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

36
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)

37
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
38
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
39
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

40
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
41
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
42
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
43
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
44
BamH1
EcoRI
EcoRI
GFP
EcoRI
HindIII
Ampicillin resistance gene
Cloning Vector
Origin of replication
45
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
46
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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48
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?

49
  • Any questions?

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51
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

52
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
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