Chapter 15 - Genetics of Bacteria and Bacteriophages:

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• Chapter 15 - Genetics of Bacteria and
  Bacteriophages
• Mapping bacteria, 3 different methods
• Conjugation
• Transformation
• Transduction
• Bacteriophage mapping
• Bacteriophage gene mapping
• Cis-trans complementation test

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• Bacteria transfer (or receive) genetic material 3
  different ways
• Conjugation
• Transformation
• Transduction
• Transfer of DNA always is unidirectional, and no
  complete diploid stage forms.

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• Conjugation
• Discovered by Joshua Lederberg and Edward Tatum
  in 1946.
• Unidirectional transfer of genetic material
  between donor and recipient bacteria cells by
  direct contact.
• Segment (rarely all) of the donors chromosome
  recombines with the homologous recipient
  chromosome.
• Recipients containing donor DNA are called
  transconjugants.

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Fig. 15.2, Lederberg Tatum (1946) Experiment
demonstrating recombination in E. coli.
Recombination of 2 complimentary auxotrophs gives
rise to a strain that can synthesize all
nutrients.
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Fig. 15.3, Bernard Davis experiment demonstrated
that physical contact is required for bacterial
recombination.
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• Conjugation-transfer of the sex factor F
• William Hayes (1953) demonstrated that genetic
  exchange in E. coli occurs in only one direction.
• Genetic transfer is mediated by sex factor F.
• Donor is F and recipient is F-.
• F is a self-replicating, circular DNA plasmid
  (1/40 the size of the main chromosome).
• F plasmid contains an origin sequence (O), which
  initiates DNA transfer. It also contains genes
  for hair-like cell surface (F-pili or sex-pili),
  which aid in contact between cells.
• No conjugation can occur between cells of the
  same mating type.
• Conjugation begins when the F plasmid is nicked
  at the origin, and a single strand is transferred
  using the rolling circle mechanism.
• When transfer is complete, both cells are F
  double-stranded.

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Figs. 15.4 15.5a Transfer of the F factor
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• Conjugation of high-frequency recombinant
  strains
• No chromosomal DNA is transferred by standard sex
  factor F.
• Transfer of chromosome DNA is facilitated by
  special strains of F integrated into the
  bacteria chromosome by crossing over.
• Hfr strains high frequency recombination
  strains.
• Discovered by William Hayes and Luca
  Cavalli-Sforza.
• Hfr strains replicate F factor as part of their
  main chromosome.
• Conjugation in Hfr strains begins when F is
  nicked at the origin, and F and bacteria
  chromosomal DNA are transferred using the rolling
  circle mechanism.
• Complete F sequence (or complete chromosomal
  DNA) is rarely transferred (1/10,000) because
  bacteria separate randomly before DNA synthesis
  completes.
• Recombinants are produced by crossover of the
  recipient chromosome and donor DNA containing F.

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Fig. 15.5b Transfer of the Hfr F factor
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• Fig. 15.6
• Excision of the F factor also occurs
  spontaneously at low frequency.
• Begin with Hfr cell containing F.
• Small section of host chromosome also may be
  excised, creating an F plasmid.
• F plasmid is named for the gene it carries,
  e.g., F (lac)

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• Using conjugation to map bacterial genes
• Begin with two different Hfr strains selected
  from F x F- crosses and perform an interrupted
  mating experiment.
• HfrH thr leu aziR tonR lac gal strR
• F- thr leu aziS tons lac gal strS
• Mix 2 cell types in medium at 37C.
• Remove at experimental time points and agitate to
  separate conjugating pairs.
• Analyze recombinants with selective media.
• Order in which genes are transferred reflects
  linear sequence on chromosomes and time in media.
• Frequency of recombinants declines as donor gene
  enters recipient later.

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Fig. 15.7 Interrupted mating experiment
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Fig. 15.7b
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Fig. 15.7c, Genetic map-results of interrupted E.
coli mating experiment.
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• Generating a map for all of E. coli
• Location and orientation of the Hfr F in the
  circular chromosome varies from strain to strain.
• Overlap in transfer maps from different strains
  allow generation of a complete chromosomal map.

Fig. 15.8
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Circular genetic map of E. coli Total map units
100 minutes Time required for E. coli
chromosome to replicate at 37C.
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• Transformation
• Unidirectional transfer of extracellular DNA into
  cells, resulting in a phenotypic change in the
  recipient.
• First discovered by Frederick Griffith (1928).
• DNA from a donor bacteria is extracted and
  purified, broken into fragments, and added to a
  recipient strain.
• Donor and recipient have different phenotypes and
  genotypes.
• If recombination occurs, new recombinant
  phenotypes appear.

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• More about transformation
• Bacteria vary in their ability to take up DNA.
• Bacteria such as Bacillus subtilis take up DNA
  naturally.
• Other strains are engineered (i.e., competent
  cells).
• Competent cells are electroporated or treated
  chemically to induce E. coli to take up
  extracellular DNA.

http//medicalphysicsweb.org/cws/article/research/
27152
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Fig. 15.9, Transformation of Bacillus subtilus
Heteroduplex DNA
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• Mapping using transformation
• Recombination frequencies are used to infer gene
  order.
• p q o x p q o
• If p and q frequently cotransform, order is
  p-q-o.
• If p and o frequently cotransform, order is
  p-o-q.

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• Transduction
• Bacteriophages (bacterial viruses) transfer genes
  to bacteria (e.g., T2, T4, T5, T6, T7, and ?).
• Generalized transduction transfers any gene.
• Specialized transduction transfers specific
  genes.
• Phages typically carry small amounts of DNA, 1
  of the host chromosome.
• Viral DNA undergoes recombination with homologous
  host chromosome DNA.

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Fig. 15.12 Life cycle of phage ?
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Fig. 15.13 Generalized transduction of E. coli
by phage P1
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• Transduction mapping is similar to transformation
  mapping
• Gene order is determined by frequency of
  recombinants.
• If recombination rate is high, genes are far
  apart.
• If recombination rate is low, genes are close
  together.

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• Mapping genes of bacteriophages (see Fig. 15.15)
• Infect bacteria with phages of different
  genotypes using two-, three-, or four-gene
  crosses ? crossover.
• Count recombinant phage phenotypes by determining
  differences in cleared areas (no bacteria growth)
  on a bacterial lawn.
• Different phage genes induce different types of
  clearing (small/large clearings with
  fuzzy/distinct borders).

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Fig. 15.16 15.17
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• Fine structure gene-mapping of bacteriophages
• Same principles of intergenic mapping also can be
  used to map mutation sites within the same gene,
  intragenic mapping.
• First evidence that the gene is sub-divisible
  came from C. P. Oliver s (1940) work on
  Drosophila.
• Seymour Benzers (1950-60s) study of the rII
  region of bacteriophage T4.

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• Seymour Benzers (1950-60s) study of the rII
  region of T4
• Studied 60 independently isolated rII mutants
  crossed in all possible combinations.
• Began with two types of traits plaque
  morphology and host range property.
• Growth in permissive host E. coli B all four
  phage types grow.
• Growth in non-permissive host E. coli K12(?)
  rare r recombinants grow (rare because the
  mutations are close to each other and crossover
  is infrequent).
• Benzer studied 3000 rII mutants showing
  nucleotide deletions at different levels of
  subdivision (nested analyses).
• Was able to map to T4 to level equivalent to 3 bp
  (the codon).
• Ultimately determined that the rII region is
  sub-divisible into gt300 mutable sites by series
  of nested analyses and comparisons.

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Benzer identified recombinants of two rII mutants
of T4 using different strains of E. coli.
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Fig. 15.18, Benzers map of the rII region
generated from crosses of 60 different mutant T4
strains.
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Fig. 15.19 Benzers deletion analysis of the rII
region of T4 No recombinants can be produced if
mutant strain lacks the region containing the
mutation.
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Fig. 18.20 (2nd edition), Benzers deletion map
divided the rII region into 47 segments.
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Fig. 15.20, Benzers composite map of the rII
region indicating gt300 mutable sites on two
different genes. Small squares indicate point
mutations mapping to a given site.
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• Seymour Benzers cis-trans complementation test
• Used to determine the number of functional units
  (genes) defined by a given set of mutations, and
  whether two mutations occur on the same unit or
  different units.
• If two mutants carrying a mutation of different
  genes combine to create a wild type function, two
  mutations compliment.
• If two mutants carrying a mutation of the same
  gene create a mutant phenotype, mutations do not
  compliment.

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Fig. 15.21, Seymour Benzers cis-trans
complementation test.
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Example of complementation in Drosophila