Bacterial Genetics

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Chapter 11

• Bacterial Genetics

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Prokaryotic diversity

• Why are prokaryotes so diverse when they do not
  reproduce sexually?
• Mutation
• Inherited change in genotype that can lead to
  change in the phenotype
• Small gradual change
• Recombination
• Integration of DNA (from another organism or
  genetic element) into chromosomal DNA
• Sometimes very large changes

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Mutants

• A different gene sequence than parent
• Often silent
• Change in nucleic acid, but no change in amino
  acid coded for
• Sometimes phenotypic changes
• Requires change in amino acid
• Lethal, neutral, beneficial
• Sometimes a change in amino acid (so not silent)
  but no change in protein so no phenotypic change

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Wild type versus mutant

• Wild type typically refers to strain isolated
  from nature
• hisC gene codes for HisC protein
• Mutation in the hisC gene are called hisC1,
  hisC2 etc.
• Phenotype His or His
• His capable of making histidine
• His- not capable of making histidine

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Isolation of mutants

• Selectable mutants can select for a phenotype
  by subjecting population to a selection factor
• Give the mutant a growth advantage under certain
  environmental conditions
• Selectable antibiotic resistance
• Only certain bacteria will grow on a particular
  antibiotic
• Used for cloning

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Isolation of mutants

• Non-selectable usually does not have an advantage
  or disadvantage over parent
• Loss of color (may still have a selective
  advantage in a natural ecosystem, but cannot
  easily select for the trait in culture)
• Non-selectable mutants have to be screened
• Some will have a different color but all will
  grow

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Isolation of mutants

• Screening is always more tedious than selection
• Replica plating is one method to screen for
  nutritional mutants
• His C mutant cannot make histidine
  (auxotroph)
• Auxotroph a nutritional mutant (requires a
  growth factor that the WT parent did not require)
• Prototroph the WT parent from which the
  auxotroph was derived

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Replicate plating to isolate auxotrophic mutants
grow with His but not without His
Figure 11.5
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Isolation of mutants

• Penicillin selection method is another
• Select against non mutants first then grow mutant
  organisms left in culture
• Penicillin used to enrich His mutants
• Then replicate plate
• Penicillin kills only growing cells
• Penicillin added to a culture lacking the
  nutrient desired by the mutant parent cells
  killed, mutant cells unaffected
• Transfer to a plate containing the nutrient
  isolate desired mutants

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Penicillin Selection Method
Inoculate new media containing no histidine
Culture containing Histidine
His- mutant (auxotroph) which cannot grow
without Histidine
His wild type (prototroph) which can without
Histidine

• Penicillin only kills growing cells
• Auxotrophic mutants will not grow (will not die
  in presence of penicillin)
• Transfer culture to media with histidine and
  survivors (mutants) grow

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Molecular Basis of Mutation

• Induced mutations
• Those made deliberately
• Spontaneous mutations
• Those that occur without human intervention
• Can result from exposure to natural radiation or
  oxygen radicals
• Point mutations
• Mutations that change only one base pair
• Can lead to single amino acid change in a protein
  or no change at all

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Point mutations

• Involving one base pair
• Nucleic acid base substitution
• Missense changes the codon (1st or 2nd
  base)?wrong amino acid
• Changes protein
• Sometimes a phenotypic change and sometimes not
• Nonsense changes the codon and codes for a stop
  codon
• Translation terminated early? protein often
  non-functional
• Silent changes last base in codon?same amino
  acid usually
• Degeneracy of the code

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Point mutation Base substitution
Figure 11.6
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Point mutations

• Transitions
• One purine base (A or G) is substituted for
  another purine or one pyrimidine base (C or T) is
  substituted for another pyrimidine
• Transversions
• A purine is substituted for a pyrimidine or a
  pyrimidine is substituted for a purine

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Point mutations

• Frameshift mutation
• Insertion or deletion of a few nucleotides
  causing a reading frame shift and disruption of
  translation
• Insertion 1 frameshift and deletion -1
  frameshift
• Often result in complete loss of gene function

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Point Mutation Frameshift
Figure 11.7
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Point Mutation Revertants

• Point mutations are typically reversible
  (reversion)
• Revertants those strains in which WT phenotype
  is restored
• True revertants back to original genotype
• Second site revertants (supressor mutations)
  response to compensate for mutation
• Occur at different site in the DNA
• In same gene?restores enzyme function (frameshift
  back)
• In another gene?restores phenotype but not enzyme
  directly
• Replace original enzyme
• Alter original damaged enzyme so that it is
  functional
• Large scale deletions are nonrevertable

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Other Mutations More Bases

• Large deletions more likely lethal
• Can only be restored by recombination
• Large insertions often inactivate gene
• Can only be reverted by large deletion
• Translocations movement of a large segment from
  one area to another (ex. Transposons)
• Inversion Orientation of DNA reversed

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Types Of Mutagens

• Chemical
• Nucleotide base analogs faulty base pairing
• Alkalating agents causes changes in base which
  causes faulty base pairing
• Intercalating agent insertion between DNA
  strands which causes insertions and deletions
  (ethidium bromide)
• Cause frameshift mutations

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(No Transcript)
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Types Of Mutagens

• Radiation Two Types
• Nonionizing causes pyrimidine (thymine) dimers,
  which causes problems with replication and
  transcription
• Ex UV light
• Ionizing
• More energy
• Penetrates through glass
• Ionize water and produce free radicals which
  disrupt base pairing by putting breaks in and
  damages the DNA
• Ex X-rays and Gamma radiation
• Must use at low levels or cell will die

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Nucleotide incision Repair mechanism

• Nucleotide excision and repair of a thymine dimer
• Remove the thymine dimer
• DNA polymerase fills the gap
• Ligase seals the gap

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SOS regulatory system

• Activated as a result of some types of DNA
  damage, initiates a number of DNA repair
  processes, and error-prone
• The repair process introduces mutations
• Does not use a template

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SOS Regulatory System

• SOS regulatory system is repressed by LexA
• LexA is inactivated by RecA
• DNA damage causes the cell to produce RecA
• Distress signal
• One of RecAs functions is as a protease that
  destroyed LexA
• When RecA inactivates LexA the SOS system is
  expressed
• SOS system
• umuC and umuD encode DNA polymerase V, which is
  error prone (no use of template)

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SOS Regulatory System
Figure 11.10
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Homologous recombination

• Closely related DNA sequences from two distinct
  genetic elements are combined in a single element
• The greater the distance 2 genes are on a
  chromosome the greater the probability for
  recombination
• New combination of genes increase diversity and
  fitness of the microorganisms
• Also used in DNA recombinant technology

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• Recombination
• DNA nicked (nuclease)
• Strand separation (helicase)
• Single stranded binding protein binds
• RecA protein binds
• Strand invasion single strand invades recipient
  duplex and DNA pairing occurs
• Crossover event recipient nicked, crossover and
  ligation
• Nuclease and polymerase replace mismatched
  strands
• Resolution cutting and reannealing

Figure 11.13
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Detection rate of recombination

• To detect a change DNA must express a different
  phenotype
• Antibiotic or nutritional selection

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Genetic exchange in prokaryotes

• Vertical vs. Horizontal
• Donor DNA is transferred to recipient cell in 3
  possible ways
• Transformation free DNA released from one cell
  is taken up by another
• Transduction DNA transfer is mediated by a
  virus
• Conjugation plasmid transfer with cell to cell
  contact

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Genetic Exchange

• Recombination has to occur after transfer for
  chromosomal change to occur
• For transformation and transduction the
  transferred DNA cannot be expressed unless it is
  within the chromosome
• Plasmid DNA can be expressed outside of the
  chromosome
• Not always a change in genotype or phenotype

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Transformation

• Never a whole chromosome 1-15 kb pieces
• Competence ability of the bacteria to take up
  the DNA only certain strains are transformable
• Competence can be induced in the laboratory
• Ca treatment
• Electroporation makes holes in the membrane and
  force the cells to take up the DNA
• Particle gun

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Griffith, Avery, MacLeod, McCartyEvidence for
Transformation
S. pneumoniae phenotype S (capsule) versus R
(no capsule) blue red Kill some S cells and
mix with live R cells?Transformants with S
phenotype
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Transformation

• Binding of DNA
• Double stranded DNA binds best
• Uptake of DNA
• Single strand for most
• Nuclease degrades one strand
• Binding of competence-specific SSBP (protects
  from restriction enzymes)

Figure 11.16
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Transformation, continued

• Recombination with the chromosome is mediated by
  RecA
• Chromosome genetically altered as compared to the
  parent cell

Figure 11.16
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Transduction

• Transduction occurs when phage (virus) transfers
  DNA
• Transducing phage contains only bacterial DNA
  virus genes have been removed
• It cannot produce progeny phage particles when it
  infects a host.
• Two types of transduction
• Generalized
• DNA from any part of the host genome transferred
• Specialized
• Specific gene

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Review Phage Infection of Prokaryotic Cell

• Lytic pathway to produce phage progeny
• Lysogenic pathway to pass phage DNA on to new
  generations
• Prophage
• When induction occurs the phage DNA is spliced
  out and replicated so progeny can be produced

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Phage Conversion

• Bacteria exhibit immunity!!!!!
• During prophage a cell cannot be re-infected with
  same phage
• Change in phenotype
• Some other changes have been observed during
  prophage as well
• Non-toxin producing strains of Corynebacterium
  diphtheriae when infected with phage ß become
  toxin producing!
• Phage conversion is defined as the phenomenon of
  lysogenic phage causing phenotypic changes in
  host cell when it is lysogenized.

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Generalized Transduction

• Lytic pathway to produce phage progeny
• Most phage have phage DNA
• By accident, a small of phage have bacterial
  DNA
• Then, virus infects new cells
• Small release bacterial DNA
• Integrate DNA into host cell homologous
  recombination of donor DNA with the bacterial
  chromosome

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Specialized Transduction

• Phage genome becomes integrated into the host DNA
  at a specific site
• Lambda type of virus
• Lamda integrates into the E. coli chromosome
  adjacent to a cluster of genes involved in
  galactose utilization

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Specialized Transduction

• Lysogenized cell
• Phage DNA integrated into bacterial DNA
• Phage DNA separates from host DNA
• Sometimes, phage DNA is excised with some
  bacterial DNA
• Phage can transduce galactose genes

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Conjugation Genetic transfer involving cell to
cell contact

• Donor cell
• Contains a conjugative plasmid
• Produces a sex pilus
• F plasmid produces F pilus
• Pili make contact with recipient cell and pull it
  closer
• Only donor cells produce pili
• Replication and transfer of F plasmid by rolling
  circle replication (semiconservative)

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Mechanism of Conjugation