Download Bacterial Genetics Part II

Bacterial Genetics Part II
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Review of the Lac Operon
• Repressors turn off gene
– Lac repressor
• Inducers bind to and inactivate repressors
– Allolactose
• Activators turn on genes
– CRP (cAMP receptor protein) binds to cAMP for it
to be activator
(remember high glucose-low cAMP
low or no glucose- high cAMP)
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CRP
CRP
(No cAMP)
CRP
CRP
(No cAMP)
CRP
Repressor doesn’t bind because of allolactose
Polycistronic mRNA
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Tryptophan Operon
•Escherichia coli operon
•Five genes involved in the synthesis of
tryptophan
–Trp A, B,C,D & E
•Regulatory elements
– Promoter
– Operator
– Repressor
– Attenuator
– Corepressor
textbookofbacteriology.net
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[Trp] 
RNA pol binds
Transcription of 5 genes in operon
[Trp] 
Repressor protein binds to operator
Prevents binding of RNA polymerase
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Trp Operon
R = repressor
O = operator
P = promoter
textbookofbacteriology.net
Trp L regulatory gene
Codes for the repressor protein
Attenuator
DNA sequence between the operator and the structural genes
RNA polymerase must cross the attenuator to transcribe the structural genes
 [Trp] RNApolymerase molecules dissociate from the DNA
 [Trp] RNApolymerase navigates the attenuator sequence and transcribe the
trp genes
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 [Trp] RNAp navigates the attenuator sequence and transcribe the trp genes
textbookofbacteriology.net
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 [Trp] RNApolymerase dissociates from the DNA
Repressor protein is “inactive”
until tryptophan binds.
textbookofbacteriology.net
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Two Types of Regulation (Promoters)
• Constitutive
– Allows continuous transcription of its genes
• lacI
• trpL
• “House keeping” genes involved in basic metabolism
–
–
–
–
Glycolysis
RNA polymerases
DNA repair enzymes
Ribosomal proteins
• Inducible
– Transcription is linked to a special circumstance
• Presence of a sugar
• Concentration of a metabolite
• Stress
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Transcription and Translation are
Coupled in Prokaryotes
No compartmentalization in
prokaryotic cells
RNA polymerase
transcript
Transcription and translation occur in
same place
chromosome
Ribosomes can associate with
transcript while it is still being made
Results in coupled
transcription/translation
ribosome
protein
RNA polymerase
transcript
chromosome
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Multiple Ribosomes can Associate
with the Growing Transcript
Highly expressed genes
require high levels of
translation
Multiple ribosomes associate
with growing transcripts to
accomplish this
Resulting structure is called a
poly-some
Allows prokaryotes to make a lot of protein very quickly.
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Experimental Bacterial Genetics
Generate mutations to determine gene
function Wild-type:
normal or non-mutant form of a species or gene
Mutant:
aberrant form of a species or gene
There is a change in DNA sequences
Mutation
A randomly or intentionally-produced, heritable change in
the DNA sequence
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One Gene – One Enzyme
1941
Beadle & Tatum
• Neurospora, a red orange bread mold
• Minimal medium
– Sucrose
– Minerals
– Vitamins
Wild-type strain can synthesize
all its own amino acids
• Induced mutations using X-rays
• Screened for
auxotrophs
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Auxotroph
X
A mutant that requires a nutrient
for growth
X
X
Each mutant lacked a different enzyme
along a pathway
Genes code for enzymes
One gene for one enzyme
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Wild-type
Missense mutation
Nonsense mutation
Frameshift mutation
Silent mutation
Alters a base but does not change the amino acid
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Loss of function mutations
Reduce or eliminate the activity of a gene
Gain of function mutations
Might increase the activity of a gene
“overexpressed gene”
but might be active at inappropriate circumstances
Extremely rare, but sometimes confers a new function to
gene… produces a protein that oes something new that
might be advantageous
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Positive Selection for Mutants
Wish to generate a mutant that is resistant to
penicillin
Mutagens discussed on page 277, Table 9.4
Grow normal bacteria in presence of mutagen
Chemical / physical agent/ irradiation that induces
changes in DNA sequence
mutagen
present
Plate on solid media that contains penicillin or
penicillin analog
Only bacteria that have acquired a mutation that
confers resistance to penicillin will survive
penicillin
resistant
colonies
Termed positive selection for desired mutation
VERY powerful experiment
Gain of function mutation
New function = resistance to penicillin Solid media containing
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penicillin
Positive selection is not always possible
Positive selection cannot identify
loss of function mutations
Wish to identify a mutant that cannot synthesize histidine
No positive selection
Alternate Strategy
Grow bacteria in presence of mutagen
Plate on rich media
Transfer colonies using velvet
Plate on media containing and lacking histidine
Minimal medium
Strategy employed by Beadle and Tatum
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minimal medium
Auxotrophic
mutant
His
no His
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Loss of Function Mutant Hunt
• Hypothesis
– Capsule production by Streptococcus
pneumonia is a virulence factor
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Mutagenize culture of a smooth,
virulent strain of S. pneumonia
Likelihood of
mutation should be
equal for all genes.
Plate out mutants on blood agar plates
Identify bacterial colonies that DO NOT
produce a capsule.
Loss of function
Grow each “rough” mutant
Inoculate mice
Screen for virulence
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Are the “rough” mutants avirulent?
Living mice is consistent
with the hypothesis that
capsule is a necessary
virulence factor.
Studies of this type
generally result in living and
a smaller percentage of dead
mice.
Dead mice are not consistent with
the hypothesis.
Dead mice indicate that there may
be other factors (gene products)
involved in virulence.
This experiment needs a control.
Inoculating mice with a wild-type culture of
smooth, virulent S. pneumonia is an
What would be
the expected outcome?
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appropriate control.
Capsule production by Streptococcus pneumonia
is a virulence factor
CONTROL GROUP
Wildtype, smooth
S. pneumonia
Capsule present
EXPERIMENTAL GROUP
Mutant “rough” strains
No visible capsule
Inoculate mice
Perform Experiment
Inoculate mice
Analyze Results
48 of 50 mice
(96.0%) die
10 of 50
(20%) mice die
Significant difference
Conclusion
Rough mutants have greatly reduced
virulence as compared to the wildtype
strain
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LD50 of 3 selected rough
mutants was 1 x 1.3 105, 1.4 x
106 CFU and 1.3 x 105 CFU
LD50 of the wildtype
was 1 CFU
Interruption of capsule production in Streptococcus pneumonia serotype 3 by
insertion of transposon Tn916.
“…capsule was the principle virulence factor…”
D. A. Watson and D. M. Musher
Infect Immun. 1990 September; 58(9): 3135–3138.
Transposons page 285
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Perform additional experiments to
identify the gene controlling capsule
production
Locate the gene from the wild-type
genome that will restore or “complement”
the mutation.
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Isolate DNA from the wild-type strain
Red area represents the gene(s) controlling capsule production.
Cut up genomic DNA with a restriction enzyme.
Page 290
Ligate fragments of DNA into specialized plasmids.
Page 290
Transform a “rough” mutant strain
with the different plasmids.
Screen transformants for capsule production.
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Identify the transformants
that produce a capsule.
Poor drawing of capsule
production by transformants.
Gene(s) on plasmid
“complements” the mutation on
the chromosome.
Inoculate mice with the complemented mutants.
This experiment needs a control.
Use rough mutants, a wild-type
strain and water.
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Percentage of Mice that Die after Inoculation
Transformants
(capsule)
45/50 = 90.0%
Rough
Wild-type
mutants
(capsule)
(no capsule)
10/50 =
20.0%
49/50 =
98.0%
Water
0/50 =
0%
Conclusion
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