Bacteria Predominate

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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 supercoiling 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,
• auxitrophs,
• unable to synthesize raw materials from minimal
  media,
• cells unable to break down complex molecules,
• essential genes, usually studied as conditional
  mutants.

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Bacteria Phenotypes

• colony morphology,
• large, small, shiny, dull, round or irregular,
• resistance to bactericidal agents,
• vital dyes,
• auxitrophs,
• unable to synthesize or use raw materials from
  the growth media.

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Prototroph

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

Auxotrophs
a cell that requires a substance for growth that
can be synthesized 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

• genes not specifically referred to are considered
  wild-type,
• Strain A met bio (require methionine and
  biotin)
• Strain B thr leu thi
• bacteriacide resistance is a gain of function,
• Strain C strA (can grow in the presence of
  strptomycin).

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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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F Pilus
a filamentlike 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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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 creating Hfr cells.

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Hfr Cells
F factor
Bacterial Chromosome
Inserted F plasmid
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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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• Strain F genotype Chromosome Genotype
• CSH23 Flac proA proB D(lacpro)supE
  spc thi
• x
• CSH 50 ara D(lacpro)strA thi

Conjugation
Recombinant Strain Flac proA proB
ara D(lacpro)strA thi
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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,
• Selective Marker a genetic mutation that allows
  growth in selective medium.

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Selection

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

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Genetic Screen

• A system that allows the identification of rare
  mutations in large scale searches,
• unlike a selection, undesired genotypes are
  present, the screen provides a way of screening
  them out.

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Procedure I

• Day 0 Overnight cultures of the CSH23 and CSH50
  will be set up in L broth (a rich medium).
• Day 1 These cultures will be diluted and grown
  at 37o until the donor culture is 2-3 X 108
  cell/ml. What is the quickest way to quickly
  determine cells per ml? (This will be done for
  you.)
• ?Prepare a mating mixture by mixing 1.0 ml of
  each culture together in a small flask. Rotate at
  30 rpms in a 37o shaking incubator for 60
  minutes.
• At the end of the incubation
• Do serial dilutions
• Fill 6 tubes with 4.5 ml of sterile saline.
  Transfer 0.5 ml of the undiluted mating culeture
  to one of the tubes. This is a 10-1 dilution.
• Next make serial dilutions of 10-2, 10-3, 10-4,
  10-5 10-6. Always change pipets and mix well
  between dilutions.

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Procedure II

• Plate 0.1 ml of a 10-2, 10-3 and 10-4 dilution
  onto minimal glucose streptomycin thiamine.
  
• Plate 0.1 ml of a 10-5 and 10-6 dilution onto a
  MacConkey streptomycin plates. A MacConkey
  plate is considered a rich media. It has lactose
  as well as other carbon sources. The phenol red
  dye is present to differentiate lac colonies
  (red) from lac- colonies (white).
• Controls
• Plate 0.1 ml of a 10-1 dilution of donor (CSH23)
  cells on minimal glucose strep thiamine
  plates. Repeat for the recipient (CSH50) cells.
  
• Plate 0.1 ml of a 10-5 dilution of the
  recipient on a MacConkey strep plate.
• Plate 0.1 ml of a 10-1 dilution of donor on a
  MacConkey strep plate.
• Place all plates at 37o overnight.
• Day 2 Remove the plates from the incubator the
  next day and count the number of white-clear
  colonies on the MacConkey plates (optional but
  easier). Store plates at 4oC. NOTE MacConkey
  color reactions fade after several days or
  rapidly in the cold, so plates need to be scored
  soon after incubation.

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Extra Credit

• On another piece of paper, answer the dilution
  problems on the last page of your handout (2 pts).