Transduction

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Transcript Transduction

Microbial genetics
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• Microbes have been important in genetic
research
– Short reproductive cycles
– Millions of progeny in a short time
– Studied in pure culture, variants can be examined
– Single piece of DNA usually; no masking of traits
– Easy to create, isolate, identify mutants
– Bacteria are the source of restriction
endonucleases
• Bacteriophages: viruses that infect bacteria
– Typically destroy the bacterium, release new virions
Terminology in microbial genetics
• Prototroph: “original” and “feed”, a wild type
strain, one able to synthesize all needed
compounds from a simple carbon source such
as glucose.
• Auxotroph: a mutant that has lost the ability to
make some necessary organic compound; it
must be added to the culture medium.
• Bacteria show horizontal gene transmission
– Acquire new genetic information naturally
– Acquire genetic info from genetic engineering
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Plasmids
• Plasmids are small, circular DNA molecules
– Plasmids are found in the cytoplasm of many
bacteria
– Plasmids are not essential for survival of the cell
– They may exist singly or in many copies
– Plasmids have a variety of functions
• Examples: metabolic, resistance, cryptic
• Fertility plasmids, such as F factor, allow
conjugation (direct cell-cell gene exchange)
• F plasmids are found in E. coli
– F+ strains are considered male, F- are female
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Conjugation
• Mechanism by which one bacterium transfers
genes to another
– Can occur be related and unrelated bacteria
– Usually involves transfer of a plasmid
– Involves attachment between bacteria w/ a pilus
A pilus is a protein
appendage that
connects the cells.
Direct contact is
required.
http://www.cbs.dtu.dk/staff/
dave/roanoke/fig10_10.jpg
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Conjugation: F plasmids
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• The “feminist’s nightmare”: male cells transfer the F
plasmid to F- cells, changing them to F+ (male)
– F plasmid codes for genes that produce a pilus and
other genes that promote transfer of genetic
material
– DNA synthesis occurs in donor cell, makes ds DNA.
– F+ cells attach to F- cells w/ pilus; ssDNA is
transferred.
– Genetic recombination: replacement of old genes
w/ new ones
• Fertility plasmids “mobilize” other genes
Hfr bacteria
• Hfr = high frequency of recombination
– Instead of gene exchange at rate of 1 in 107, rate
improves to 1 in 104.
– F plasmid is inserted into E. coli chromosome
– F plasmid not transferred, rather, E.coli
chromosomal genes at high frequency.
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Hfr strains-2
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•In any particular Hfr strain, same genes transferred
•Genes transferred determined by where in chromosome
the F plasmid was inserted.
• If plasmid is inserted near a, b genes, those are
transferred during conjugation.
• If plasmid is inserted near g, h genes, those are
transferred during conjugation.
Genetic mapping in E. coli
• Conjugation between prototroph and/or
antibiotic resistant Hfr strain and auxotroph
strain.
– Hfr strain should transfer genes that will “cure”
auxotroph.
• Interrupted mating technique
– Hfr (donor strain) mixed with recipient strain.
– Samples removal at various times, placed in
blender to shear off pili and break up mating.
– Cells were plated onto medium and tested for
prototrophy, that is, are they “cured”
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Mapping-2
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• Data was collected based on how many
minutes of conjugation (standard conditions) it
took for a gene to be transfer and thus “cure”
the recipient.
– This allowed the genes to be placed in order: the
longer it took for transfer, the farther away the gene.
http://www.mun.ca/biochem/courses/4103/figures/Snyder-Champness/F14-3.jpg
Mapping-3
• These data were collected for several different Hfr
strains and pooled.
– The order came up the same, but one end
overlapped the other. Conclusion: E. coli has a
circular chromosome.
– Circular DNA is the rule for bacteria.
– Map units are in minutes, reflecting the
methodology used.
http://www.cbs.dtu.dk/staff/dave/roanoke/fig10_33.jpg
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More about plasmids and conjugation
• R plasmids
– Code for resistance to
antibiotics, heavy metals, etc.
– Usually contain RTF (resistance
transfer factor)
• Responsible for transfer of
plasmid to other bacteria,
transferring antibiotic
resistance.
– Major factor in the spread of
resistance among bacteria
http://www.med.sc.edu:85/mayer/trans-14.jpg
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Mechanisms of horizontal gene transmission
• Conjugation
– Bacteria make direct contact with pilus
– Transfer genes directly
– Both related and unrelated partners
• Transformation
– “naked” DNA in solution
• Transduction.
– Requires bacteriophage,
virus that infects bacteria
www.nature.com/.../
031013/full/031013-2.html
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Transformation
• “Naked” DNA taken up from solution
– Bacteria must be “competent”
• E. coli treated with high [Ca2] for example
– DNA binds to receptor sites on surface
– DNA brought into cell by active transport process
• One DNA strand is used
– One strand is digested leaving ssDNA
– ss DNA forms heteroduplex with recipient DNA
• Recombination event, one old strand degraded
• Transformation between close relatives only.
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Transformation-2
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• When bacterium divides, each strand of
heteroduplex is copied
– One bacterium has old phenotype, one shows new
phenotype from the newly acquired DNA
• Transformation can be used for some mapping
– Genes are said to be “linked” if they are close
enough together to be on same piece of DNA
• 10,000- 20,000 bp, enough for several genes
• If several mutant phenotypes are cured
simultaneously, genes are close together.
Transformation w/ recombination
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Viral life cycles
• Transduction is gene transfer by
bacteriophages
– Bacteriophages (“phage”) are viruses that infect
bacteria
• Understanding the action of viruses:
• The Lytic Cycle
– Phage attaches to bacteria surface, injects DNA
– Viral DNA takes over cell, uses cell machinery to
• Produce new copies of viral DNA
• Synthesize viral proteins
• Destroy host DNA by cutting it into pieces
– Viruses self-assemble
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Viral life cycles (continued)
• Lytic cycle (continued)
– After self-assembly, viruses lyse cell, escape,
spread to neighboring bacteria and infect them.
• Such viruses are called virulent or lytic phage.
• Alternative pathway to reproduction: lysogeny
– Carried out by “temperate” phages
– Once in cell, viral DNA incorporates into host
DNA
– When the bacterium reproduces, viral DNA is
copied.
– Harmful stimuli (e.g. UV light) causes viral DNA to
excise, begin lytic cycle.
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Transduction
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• Generalized transduction
– Occurs when host DNA piece is incorporated into
phage “head” instead of viral DNA
– Binding of virus particle to recipient, injection of
DNA: bacterial DNA is injected instead.
• Specialized transduction
– Prophage: the viral DNA while it exists only as a
piece of DNA with the bacterial DNA.
– First, prophage excises, begins lytic cycle usually
because of damage to host DNA, pulls part of host
DNA from “next door” with it when it excises
– DNA containing phage and host DNA is packaged.
Transduction visual
Red: phage DNA;
Blue: bacterial DNA
http://fig.cox.miami.edu/Faculty/Dana/transduction.jpg
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Summary: Gene transfer in bacteria
• Conjugation: direct contact via pilus
– Mediated by plasmids
– Doesn’t necessarily require close relationships
• R plasmids: no recombination, so no DNA
homology needed.
• Transformation: naked DNA from solution
– Competent cells only
– Recombination takes place; DNA homology
needed.
• Transduction: DNA carried by a virus
– For greatest effect, DNA homology needed.
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