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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 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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His no HisAuxotrophic mutant

minimal medium

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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.

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Percentage of Mice that Die after Inoculation

Transformants(capsule)

Rough mutants

(no capsule)

Wild-type(capsule)

Water

45/50 = 90.0%

10/50 = 20.0%

49/50 = 98.0%

0/50 = 0%

Conclusion