Microbial genetics
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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