Transcript Document

Business
• Chapter 7: Problems 1-20,
– Master Solutions: Problems 1 and 2,
– Master Concepts in action: all,
• Questions about anything?
Bacteria
Predominate
Bacteria Do Almost
Everything
• Metabolism;
• 10,000+ “Species”,
– Phototrophs,
– Chemotrophs,
– Mycoplasma genetalium
• 200 nm
– Thiomargarita namibiensis
• 750 mm
– soil, water, air, symbionts,
– have adapted to aquatic and
terrestrial extremes,
• 100 grams/person,
– 1014 bacteria.
• 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.
Metagenomics
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.
Extra Chromosomal DNA
• Plasmids: circular double
stranded DNA molecule that
replicates independently,
– containing one or more (nonessential) genes, smaller than
the bacterial chromosome,
– may carries genes for
pathogenicity,
– may carry genes for adaptation
to the environment, including
drug resistance genes,
– 1,000’s of base pairs long.
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,
Growth Equals Cell Division
DNA Replication
Binary Fission
Bacterial mitosis. Why don’t bacteria do meiosis?
The (Awesome) Power of Bacterial Genetics
... is the potential for studying rare events.
Liquid Cultures,
• 109cells/microliter,
Colonies on Agar,
• 107+ cells/colony
Counting Bacteria
10-3
10-4
10-5
(Serial) Dilution is the Solution
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
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.
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,
hisleuargbio-
...can’t synthesize histidine (his+ = wt)
...can’t synthesize leucine (leu+ = wt)
...can’t synthesize arginine (his+ = wt)
...can’t synthesize biotin (bio+ = wt)
Bacterial Nomenclature I
• genes not specified are considered wildtype,
-
-
Strain A: met bio thr+ leu+ thi+
x
+
+
Strain B: thr leu thi met bio
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.
1 in 10
7
awesome
Strain A: met- bio- tri+ leu+ thi+
x
+
+
-
-
Strain B: met bio thr leu thi
-
Conjugation
exconjugate
+
+
+
+
Strain C: met bio thr leu thi
+
Conjugation
...temporary fusion of two single-celled organisms
for the transfer of genetic material,
…the transfer of genetic material is unidirectional.
+
-
F Cells
F Cells
(F for Fertility)
(F for Fertility)
… F+ cells donate
genetic material.
… F- cells receive genetic material,
…there is no reciprocal transfer.
Contact is Required For Mating
F+
F Pilus
-
F
…a filament-like projection from
the surface of a bacterium.
F Factor
…a plasmid whose presence confers F+, or donor ability.
-
F Pilus Attaches to F Cell
Cells are Drawn Together
Passageway Forms
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 .
Cell Separation
+
Both cells are now F
F Factor Replicates During
Binary Fission
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.
Hfr Cells
F factor
...F factor
integration site,
...host (bacteria
chromosome)
integration site.
Bacterial Chromosome
Inserted F plasmid
...the bacterial chromosome
contains many integration
sites,
...thus, the F factor inserts in
different regions of the
bacterial chromosome.
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.
-
F Pilus Attaches to F Cell
Hfr DNA is Cut
F factor and Chromosomal DNA
are Transferred
Recombination Requires
Crossing over
Double Crossover
DNA not Incorporated into
Chromosome are Digested
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.
-
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-.
’
F Cells
• 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,
• F’cells behave like an F+ cells,
– merizygote: partially diploid for genes copied
on the F’plasmid,
• F’plasmids can be easily constructed using
molecular biology techniques (i.e.vectors).
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.
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.
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.
Selection
...the process that establishes conditions in
which only the desired mutant will grow.
Problem
Strain
Strain 1
Strain 2
Strain 3
Mating
Type
F'
F'
F-
Chromosome Geno type
F factor Genotype
wild-typ e
argthr- ; thi- ; met-
thr+ ; thi+
thr+ ; thi+
-
You want to create a culture of cells that are only methionine
auxitrophs?
The Wrong Answer
Strain
Strain 1
Strain 2
Strain 3
Mating
Type
F'
F'
F-
Chromosome Geno type
F factor Genotype
wild-typ e
argthr- ; thi- ; met-
thr+ ; thi+
thr+ ; thi+
-
Strain 1 x Strain 3 (complete media)
You have a recombinant that is met- only.
How do you get rid of Strain 1?
Correct Answer
Strain
Strain 1
Strain 2
Strain 3
Mating
Type
F'
F'
F-
Chromosome Geno type
F factor Genotype
wild-typ e
argthr- ; thi- ; met-
thr+ ; thi+
thr+ ; thi+
-
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.
F factor Review
+
Now F
Now F+