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Microbial genetics. 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

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microbial genetics
Microbial genetics
  • 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
    • Viruses used in Hershey-Chase experiments
bacteriophages in genetics research
Bacteriophages in Genetics research
  • Bacteriophages: viruses that infect bacteria
    • Typically destroy the bacterium, release new virions
    • Clear areas = “plaques” where viruses have replicated and killed bacteria.

Mutant plaques could be small, large, turbid, etc. = recognizable phenotype.

users.rcn.com/.../BiologyPages/L/Luria.html

terminology in microbial genetics
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
plasmids
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
conjugation
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.
  • Conjugation requires direct contact.

http://www.cbs.dtu.dk/staff/dave/roanoke/fig10_10.jpg

conjugation f plasmids
Conjugation: F plasmids
  • 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 for transfer of genetic material
    • F+ cells attach to F- cells w/ pilus;
    • DNA unwinds, and a ss DNA is transferred from the donor to the recipient cell.
    • DNA synthesis occurs in both, making ds DNA.
    • Genetic recombination: replacement of old genes w/ new ones
  • Fertility plasmids “mobilize” other genes
structure of f plasmid
Structure of F plasmid

www.langara.bc.ca/.../mario/Assets/Fplasmid.jpg

visual of conjugation with f plasmid
Visual of conjugation with F plasmid

http://www.chemistrydaily.com/chemistry/upload/8/8e/Conjugative_plasmids.png

hfr bacteria
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.
hfr strains 2
Hfr strains-2
  • 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
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?”
mapping 2
Mapping-2
  • 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
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

more about plasmids and conjugation
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

mechanisms of horizontal gene transmission
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,

Transfers genes from 1 bacterium

to another.

www.nature.com/.../ 031013/full/031013-2.html

transformation
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.
transformation 2
Transformation-2
  • 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.
viral life cycles
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
viral life cycles continued
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.
transduction
Transduction
  • 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
Transduction visual

Red: phage DNA;

Blue: bacterial DNA

http://fig.cox.miami.edu/Faculty/Dana/transduction.jpg

summary gene transfer in bacteria
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.
genetic notation in bacteria
Genetic notation in bacteria
  • leu - leu + etc.
  • LacZ is a protein, lacZ is the gene!!

Try these bacterial genetics problems:

http://www.bio.unc.edu/courses/2006Fall/biol621/Cannon/Cannon%20Problem%20Set%201.doc

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