Continued

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Chapter 10

• Continued

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Transfection

• Cells transformed by bacterial viruses that carry
  DNA
• Measure the amount by a plaque assay
• Use to study transformation and recombination
  because we will know the virus that was used
• standard transformation is random DNA sequence
  and is more complicated to study

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Transduction

• Transfer of DNA from cell to cell by virus
• 2 methods
• generalized transduction host DNA from any
  portion of host genome becomes part of DNA of
  virus in place of viral genome
• no integration DNA is lost as no longer part of
  virus or in bacteria
• specialized transduction only in some temperate
  viruses (may not cause lysis but lysogenic
  infection)
• recombination may occur but also get 1) DNA
  integrating into chromosome during lysogenization
  or 2) DNA may replicate in recipient during lytic
  infection
• DNA form specific regionof host chromosome is
  integrated into viral genome usually replacing
  viral genome
• virus is usually not infectious as they lost a
  chunk of their DNA

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Generalized Transduction

• Formation of transducing particles (TP)
  accidental packaging of bacterial DNA into virus
• TP are not infectious and but bacterial DNA can
  be injected into another host cell which may
  then undergo recombination, leading to genetic
  diversity

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Specialized Transduction

• More efficient transfer of small bacterial DNA
• Done in E coli with ? phage inserts near genes
  for galactose utilization under control of
  bacterial replication
• Induction reverses integration
• rarely - ? genes removed incorrectly and take
  galactose genes and leave behind viral genes - ?
  defective, galactose
• cant make infectious virus without a helper
  virus present to replace the missing viral genes

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Plasmids General Principles

• Plasmids are genetic elements that replicate
  independently of host chromosome
• No extra-cellular form, usually ds-circular DNA
• Carry non-essential but often helpful genes
• 1000s isolated
• Plasmid contains genes responsible for
  controlling own replication and distribution to
  daughter cells
• GNB plasmids use the ? intermediate and GPB use
  the rolling circle method of DNA replication

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F (Fertility) Plasmid

• 1 region controls regulating DNA replication,
  number of transposable elements
• Functions as an episome integrate into
  bacterial chromosome
• tra region allows for transfer from cell to cell

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Cell to Cell Transfer

• Must transfer by conjugation which is encoded in
  the plasmid
• called a conjugative plasmid
• tra encodes for proteins that encode for DNA
  transfer and replication and others that function
  in mating pair formation
• If plasmid integrates, then bacterial chromosome
  can move from cell to cell
• may have broad host range GNB to GPB, bacteria
  to plant, bacteria to fungi
• may not be able to replicate in new host but can
  recombine with host DNA evolutionary
  consequences

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Know the Phenotype classes and what each means
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Plasmid R100

• Contains several antibiotic resistance genes
• sulfonamides, streptomycin, spectinomycin,
  fusidic acid, chloramphenicol and tetracyclin
• also resistant to mercury
• Can pass to Escherichia, Klebsiella, Proetus,
  Shigella and Salmonella but not Pseudomonas
• Genes are also transposable elements

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Toxins and Other Virulence Factors

• 2 major characteristics involved in virulence of
  pathogens
• 1) ability of pathogen to attach to and colonize
  specific host tissue
• 2) formation of substances toxins, enzymes and
  others that damage host
• Genes for substances can be carried on plasmids

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E coli Diarrhea

• E coli colonizes small intestine produces a
  toxin that causes symptoms of diarrhea
• Cell surface protein encoded by plasmid called
  the colonization factor antigen helps it attach
  to cell wall of intestine
• At least 2 additional proteins are made that act
  as toxins
• hemolysin causes RBC to lyse
• enterotoxin induces H2O and salt movement into
  the bowel (diarrhea)
• Other virulence factors encoded by mobile genetic
  elements like transposons, bacteriophages and
  some are chromosomal

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Engineered Plasmids

• Can make countless new, artificial plasmids
• introduce any genetic material across any species
  barrier
• Requirements
• contain genes controlling own replication
• is conjugation contain tra region that
  facilitates this function
• they are stably maintained in host cell

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Conjugation

• Bacterial mating genetic transfer that involves
  cell-to-cell contact
• Plasmid controlled but other genetic material can
  be mobilized to a new host other plasmid or
  host chromosome itself
• Most plasmids in GNB employ mechanisms similar to
  F plasmid

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Pilus Formation

• Requires donor cell with conjugative plasmid and
  recipient cell which doesnt
• Genes contained in tra region are involved in
  mating pair formation make a sex pilus from
  donor cell
• region may differ slightly so pili may be
  different
• Pilus makes contact with receptor in recipient
  and then retracts and pulls cells together
• contacts become stable probably because of fusion
  of outer membranes and then DNA transfer

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Mechanism
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Mechanism

• DNA synthesis is necessary for transfer use
  rolling circle method
• 1 strand of plasmid DNA circle is nicked and
  transferred to recipient
• nicking enzyme is Tra I on tra operon also has
  helicase activity so unwinds strand
• complementary strand is made in recipient now 2
  F cells
• recipient can act as donor now
• If plasmid has a selective advantage, can spread
  and be of ecological significance

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Hfr Strains

• F plasmids can cause chromosome mobilization
  under certain conditions
• integrates into chromosome and can move genes
  when it moves
• creates a Hfr cell high frequency of
  recombination have higher rates of
  recombination (cells with F plasmid not
  integrated are F cells)
• Both Hfr and F cells can act as donors but
  cannot pick up a 2nd copy of F plasmid
• Conjugation between F- and Hfr cell will lead to
  donor chromosomal genes to be transferred to
  recipient cell which may express a new phenotype

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3 Distinct Alterations to F- Cell

• 1) can synthesize F pilus
• 2) mobilization of DNA for transfer to new cell
• 3) alteration of surface receptors

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Integration of F

• Integration occurs at specific sites called
  insertion sequence (IS) sites
• Following insertion, plasmid no longer controls
  own replication
• tra still functions pili and conjugation
• DNA transfer initiates at oriT (origin of
  transfer)

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Transfer

• Since oriT is part of chromosome get transfer
  of chromosomal genes
• requires DNA replication in the rolling circle
  method
• Donor remains Hfr because still integrated into
  the chromosome

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Distinct Hfr Cells

• Distinct because of insertion of F plasmid in
  different parts of genome
• Get different genes transferred
• DNA strand usually breaks in the transfer process
  so entire chromosome rarely completely
  transferred
• cant replicate in cell because didnt get a full
  copy of F plasmid
• rarely convert to F or Hfr
• F- cell gets new genes and maybe a new phenotype

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Uses of Hfr

• Can select recombinants from conjugation if
  recipient is different from parent
• Usually use a recipient resistant to an
  antibiotic and auxotrophic to something and the
  donor is sensitive to antibiotic but prototrophic
  for same substance
• Grow on minimal medium

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Transfer of Chromosomal Genes to F Plasmid

• Integrated plasmid may excise from chromosome and
  can take some genomic material with it
• remember number of identical IS sites in genome
• F plasmid with bacterial genes is an F plasmid
  with identifiable chromosomal genes
• transfer with high frequency to recipients
• F plasmid transfer can make bacteria diploid for
  some genes
• merodiploids

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Complementation

• If new genetic material is not integrated, it
  will be lost as it is unable to replicate on its
  own
• 2 instances where diploidy can be stably
  maintained
• specialized transducing phage genes as part of
  a phage genome
• use F plasmid where donor genes have become part
  of F plasmid genome on episome

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Complementation Test

• 2 mutant strains are crossed with homologous
  recombination making a wt possible it mutations
  are not in the same base pair
• 2 different Trp- E coli auxotrophs are crossed
  and get Trp recombinant but cant tell if same
  gene
• use the complementation test
• Get complementation mutation is in 2 separate
  genes, no complementation mutation in the same
  gene

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Cis Mutations

• Mutations that are on different DNA molecules are
  said to be in trans
• If the mutation is on the same DNA molecule it is
  called cis used as a positive control in
  complementation test
• cis-trans test will determine if 2 mutations are
  in the same genetic unit cistron (essentially a
  gene)
• 2 mutants occur in genes for different enzymes or
  even in different subunits complementation is
  possible and mutation is not in same cistron
• usually use sequencing rather than
  complementation test

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Transposons

• Order of genes on chromosome not necessarily
  permanent some genes can move
• movement from 1 place to another is transposition
  important in evolution and genetic analysis
• rare event frequency 10-5 to 10-7 per
  generation
• genes are relatively stable and not all have the
  ability to move linked to transposable elements
  which are special genetic elements
• originally found in corn

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Transposable Elements

• 3 types of transposable elements in Bacteria
• 1) insertion sequences (IS)
• 2) transposons
• 3) some special virus
• 1 and 2 have important features in common
• each carry genes encoding transposases (enzyme
  necessary for transposition)
• both have short inverted terminal repeats at ends
  of DNA each IS has a specific number of base
  pairs in the terminal repeat

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Insertion Sequences

• IS 2 is a common insertion element and transposon
  Tn5
• IS are the simplest transposable elements and no
  genes except for those necessary for moving in
  DNA
• 1000 bp long and can integrate into specific
  sites of genome chromosomal and plasmid
• several hundred SI indentified
• vary in number and frequency
• homologous recombinant of identical IS on F
  plasmid and chromosome, not transposition that
  allows plasmid to integrate in chromosome and
  then mobilize

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Transposons Extrachromosomal

• Larger than IS and carry other genes
• Confer important properties to organism such as
  drug resistance and other selectable genes
• Conjugative transposons transposons that can
  move between species by conjugation
• genes to move and tra gene
• may be composite genetic element gene or group
  gene between 2 identical insertion sequences

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Transposition

• Inverted repeats and transposases are essential
  for transposition
• recognize/cut/ligates DNA
• see short sequence duplication during insertion

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Evolutionary Advantages

• Transposase breaks ssDNA and attaches transposon
  to ss ends and repair ss portions in duplication
• If duplicated DNA is an entire gene then get
  multiple copies of gene
• mutation in one copy doesnt affect the other
• allows organism experimentation with the 2nd
  copy to see if can create a better gene

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Mechanism

• 2 mechanisms
• conservative excise from one location and
  insert in a 2nd location
• copy number is 1
• replicative a new copy is produced during
  transposition and inserted at another location
• copy number is 2 one at original site and one
  at new site

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Conservative and Replicative

• Conservative donor site is cut and replication
  repair fills in the gaps at target site
• forms direct repeats in target site at ends
• Replicative replication repair takes place
  while element is still attached to original
  target making co-integrate
• followed by resolution of co-integrate release
  original transposon and presence of new copy at
  target site

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