1 Bacterial Genetics Part II
Dec 31, 2015
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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 cAMPlow or no glucose- high cAMP)
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CRP
CRP
CRPCRP
CRP
Polycistronic mRNA
(No cAMP)
(No cAMP)
Repressor doesn’t bind because of allolactose
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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 bindsTranscription of 5 genes in operon
[Trp] Repressor protein binds to operatorPrevents binding of RNA polymerase
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Trp Operon textbookofbacteriology.netR = repressor O = operatorP = promoter
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
Trp L regulatory geneCodes for the repressor protein
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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
textbookofbacteriology.net
Repressor protein is “inactive” until tryptophan binds.
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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
Transcription and translation occur in same place
Ribosomes can associate with transcript while it is still being made
Results in coupled transcription/translation
chromosome
RNA polymerase
transcript
chromosome
RNA polymerase
transcript
ribosome protein
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Multiple Ribosomes can Associate with the Growing TranscriptHighly 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 functionWild-type:
normal or non-mutant form of a species or gene
Mutant:aberrant form of a species or geneThere is a change in DNA sequences
MutationA randomly or intentionally-produced, heritable change in the DNA sequence
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One Gene – One Enzyme 1941
• Neurospora, a red orange bread mold• Minimal medium
– Sucrose– Minerals– Vitamins
• Induced mutations using X-rays
• Screened for auxotrophs
Beadle & Tatum
Wild-type strain can synthesizeall its own amino acids
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AuxotrophA mutant that requires a nutrientfor growth
X
Each mutant lacked a different enzyme along a pathwayGenes code for enzymesOne gene for one enzyme
X X
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Wild-type Missense mutation
Nonsense mutation
Frameshift mutation
Silent mutationAlters a base but does not change the amino acid
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Loss of function mutationsReduce or eliminate the activity of a gene
Gain of function mutationsMight 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
mutagenpresent
Solid media containing penicillin
Wish to generate a mutant that is resistant to penicillin
Grow normal bacteria in presence of mutagenChemical / physical agent/ irradiation that induces changes in DNA sequence
Plate on solid media that contains penicillin or penicillin analog
Only bacteria that have acquired a mutation that confers resistance to penicillin will survive
Termed positive selection for desired mutation
VERY powerful experiment
penicillin resistant colonies
Gain of function mutationNew function = resistance to penicillin
Mutagens discussed on page 277, Table 9.4
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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 StrategyGrow bacteria in presence of mutagen
Plate on rich media
Transfer colonies using velvet
Plate on media containing and lacking histidineMinimal mediumStrategy employed by Beadle and Tatum
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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 NOTproduce a capsule.
Loss of function
Grow each “rough” mutantInoculate 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.
Dead mice are not consistent with the hypothesis.Dead mice indicate that there may be other factors (gene products) involved in virulence.
Studies of this type generally result in living and a smaller percentage of dead mice.
This experiment needs a control.Inoculating mice with a wild-type culture of smooth, virulent S. pneumonia is an
appropriate control. What would be the expected outcome?
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CONTROL GROUPWildtype, smoothS. pneumoniaCapsule present
EXPERIMENTAL GROUPMutant “rough” strainsNo visible capsule
Inoculate mice Inoculate miceAnalyze Results
ConclusionRough mutants have greatly reduced virulence as compared to the wildtype strain
Capsule production by Streptococcus pneumonia is a virulence factor
Perform Experiment
48 of 50 mice (96.0%) die
10 of 50(20%) mice die
Significant difference
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Interruption of capsule production in Streptococcus pneumonia serotype 3 by insertion of transposon Tn916.D. A. Watson and D. M. Musher
Infect Immun. 1990 September; 58(9): 3135–3138.
LD50 of the wildtype was 1 CFU
LD50 of 3 selected rough mutants was 1 x 1.3 105, 1.4 x 106 CFU and 1.3 x 105 CFU
“…capsule was the principle virulence factor…”
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
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.
Red area represents the gene(s) controlling capsule production.
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Identify the transformants that produce a capsule.
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.
Poor drawing of capsule production by transformants.