Business

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Business

• Chapter 7 Problems 1-20,
• Master Solutions Problems 1 and 2,
• Master Concepts in action all,
• Questions about anything?

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Bacteria Predominate
Bacteria Do Almost Everything

• Metabolism
• Phototrophs,
• Chemotrophs,
• Biochemistry
• fix or synthesize a huge range of molecules,
• break down almost anything,
• adapt to just about anything.
• Molecular Biology
• Clone,
• Gene therapy,
• Eugenics,
• Biotechnology,
• Etc.

• 10,000 Species,
• Mycoplasma genetalium
• 200 nm
• Thiomargarista namibiensis
• 750 mm
• soil, water, air, symbionts,
• have adapted to aquatic and terrestrial extremes,
• 100 grams/person,
• 1014 bacteria.

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Bacterial Chromosome

• ...a circular molecule of double helical DNA,
• 4 - 5 Mb long in most species studied,
• 1.6 mm long if broken and stretched out.
• Inside the cell, the circular chromosome is
  condensed by super coiling and looping into a
  densely packed body termed the nucleoid.

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Extra Chromosomal DNA

• Plasmids circular double stranded DNA molecule
  that replicates independently,
• containing one or more (non-essential) genes,
  smaller than the bacterial chromosome,
• may carries genes for pathogenicity,
• may carry genes for adaptation to the
  environment, including drug resistance genes,
• 1000s of base pairs long.

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Bacterial Model Organism Escherichia coli
E. coli

• Enteric bacteria inhabits intestinal tracts,
• generally non-pathogenic,
• grows in liquid,
• grows in air,
• E. coli has all the enzymes it needs for
  amino-acid and nucleotide biosynthesis,
• can grow on minimal media (carbon source and
  inorganic salts),
• Divides about every hour on minimal media,
• up to 24 generations a day,

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Growth Equals Cell Division
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DNA Replication
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Bacterial mitosis. Why dont bacteria do meiosis?
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The (Awesome) Power of Bacterial Genetics

• ... is the potential for studying rare events.

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Counting Bacteria
10-3
10-5
10-4
(Serial) Dilution is the Solution
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Model Model Organism

• Ease of cultivation,
• Rapid Reproduction,
• Small size,
• Fecund (large brood size),
• Mutants are available, stable and easy to
  identify?
• Literature?
• PubMed Listings Eubacteria 612,471,
  Archaebacteria 9,420

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Bacteria Phenotypes

• colony morphology,
• large, small, shiny, dull, round or irregular,
• resistance to bactericidal agents,
• cells unable to synthesize certain raw materials
  from a minimal media,
• cells unable to break down complex molecules,
• essential genes, usually studied as conditional
  mutants.

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Prototroph

• a cell that is capable of growing on a defined,
  minimal media (all essentials salts, carbon
  source),
• can synthesize all essential organic compounds,
• usually considered the wild-type strain.

Auxotrophs
a cell that requires a substance for growth that
can be produced by a wild-type cell, his-
...cant synthesize histidine (his wt) leu-
...cant synthesize leucine (leu wt) arg-
...cant synthesize arginine (his wt) bio-
...cant synthesize biotin (bio wt)
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Bacterial Nomenclature I

• genes not specified are considered wild-type,
• Strain A met- bio-
• x
• Strain B thr- leu- thi-

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Complete Media Contains all essential
nutrients, plus a carbon source, and supplemented
with specific complex molecules, - bacteria
uptake molecules that it can not
synthesize.
Minimal Media Contains all essential nutrients
(usually as salts), plus a carbon source, -
bacteria synthesizes the rest.
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1 in 107awesome

• Strain A met- bio- tri leu thi
• x
• Strain B met bio thr- leu- thi-

Conjugation
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Conjugation

• ...temporary fusion of two single-celled
  organisms for the transfer of genetic material,
• the transfer of genetic material is
  unidirectional.

F Cells(F for Fertility)
F- Cells(F for Fertility)
F cells donate genetic material.
F- cells receive genetic material, there is
no reciprocal transfer.
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Contact is Required For Mating
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F Pilus
a filament-like projection from the surface of a
bacterium.
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F Factor
a plasmid whose presence confers F, or donor
ability.
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F Pilus Attaches to F- Cell
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Cells are Drawn Together
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DNA is Nicked, One Strand is Transferred, Both
Strands Replicate
Single Strand Moves Across Pilus
The single stranded plasmids synthesize
complementary strands in their respective cells .
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Cell Separation
Both cells are now F
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F Factor Replicates During Binary Fission
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Properties of the F Factor

• Can replicate its own DNA,
• Carries genes required for the synthesis of pili,
• F and F- cells can conjugate,
• the F factor is copied to the F- cell, resulting
  in two F cells,
• F cells do not conjugate with F cells,
• F Factor sometimes integrates into the bacterial
  chromosome.

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Hfr Cells
F factor
Bacterial Chromosome
Inserted F plasmid
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...the bacterial chromosome contains many
integration sites, ...thus, the F factor
inserts in different regions of the bacterial
chromosome.
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High Frequency of Recombination(Hfr)

• ...bacteria exhibiting a high frequency of
  recombination,
• an alteration of DNA sequence such that the
  genotype of subsequent individuals differs from
  the parent,
• HFR cells strains with a chromosome integrated
  F factor that is able to mobilize and transfer
  part of the chromosome to the F- cell.

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F Pilus Attaches to F- Cell
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Hfr DNA is Cut
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F factor and Chromosomal DNA are Transferred
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Recombination Requires Crossing over
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DNA not Incorporated into Chromosome are Digested
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Incomplete Transfer of DNA

• Interrupted Mating a break in the pilus during
  conjugation stops the transfer of DNA,
• Transfer occurs at a constant rate,
• provides a means to map bacterial genes.

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Hfr to F- Cells

• Hfr transfer begins in the middle of the F
  factor,
• Only 1/10,000 cells transfer the complete
  chromosome, including the remainder of the F
  factor,
• Thus, most recipient cells remain F-.

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FCells

• an F factor from an Hfr cell excises out of the
  bacterial genome and returns to plasmid form,
• often carries one or more bacterial genes along,
• Fcells behave like an F cells,
• merizygote partially diploid for genes copied on
  the Fplasmid,
• Fplasmids can be easily constructed using
  molecular biology techniques (i.e.vectors).

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Mutation in Bacteria

• the ultimate source of variation in bacteria is
  spontaneous mutation,
• generally errors in DNA replication,
• mutations occur in specific genes at a rate of 1
  in 106 to 1 in 107 cells,
• adaptive mutations are quickly replicated and
  adaptive colonies predominate.

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Spontaneous Mutations

• DNA replication in E. coli occurs with an error
  every 109 bases.
• The E. coli genome is 4.6 x 106 bases.
• An error occurs once per 2000 replications.
• If a single colony has 107 bacteria,
• 5,000 cells carry a mutation,
• or, one mutation every 1,000 bases (across a
  colony),
• or, a mutation in about every gene.

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Selective Media

• wild-type bacteria grow on minimal media,
• media supplemented with selected compounds
  supports growth of mutant strains,
• minimal media leucine supports leu- cells,
• minimal media leucine arginine supports leu-
  arg-
• etc.
• Selective Media a media in which only the
  desired strain will grow.

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Selection

• ...the process that establishes conditions in
  which only the desired mutant will grow.

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Problem
You want to create a culture of cells that are
only methionine auxitrophs?
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The Wrong Answer
Strain 1 x Strain 3 (complete media)
You have a recombinant that is met- only.
How do you get rid of Strain 1?
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Correct Answer
Strain 2 x Strain 3 (complete media)
Grow on Minimal Media Plus Methionine
Strain 2 dies because there is no arginine.
Strain 3 dies because there is no threonine or
thimine.
The new exconjugate lives.
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F factor Review