Gene Transfer in Bacteria and Bacteriophage

1
Gene Transfer in Bacteria and Bacteriophage

• Using Gene Transfer Between Bacteria As a Means
  for Studying Bacterial Genes

2
Types of Traits Studied

• For bacteria
• -need for nutrients
• prototropic can grow on minimal medium
• auxotropic must have specific nutrients
  added to medium
• -morphology of colonies
• -resistance/sensitivity to antibiotics
• For bacteriophage
• -host range (ability to infect specific
  bacteria)
• -appearance of plaques (shows growth)

3
Testing for Nutritional Requirements
Replica plating transfers the pattern of
bacterial colonies to test plates.
4
DNA of Prokaryotic Cells

• Bacterial cells have a single, circular
  chromosome and therefore have one copy of each
  gene.
• Partial diploids (merozygotes) can be formed by
  the introduction of genetic material from another
  cell.

5
Gene Transfer Processes for Bacteria and Their
Viruses

• Conjugation
• Transformation
• Transduction
• Infection with bacteriophage

6
Gene Transfer Processes for Bacteria and Their
Viruses

• 1. Conjugation
• Transfer of DNA from one bacterial cell to
  another
• Donor cell (F or Hfr) transfers DNA to
  recipient cell (F-)

7
Conjugation
8
Genetic Analyses Using Conjugation

• Determining linkage from interrupted mating
  experiments
• Determining gene order from gradient of transfer
• Higher-resolution mapping by recombination
  frequency

9
Genetic Analyses Using Conjugation

• Determining linkage from interrupted mating
  experiments
• Combine Hfr strain (Strs) and F- strain.
• Remove samples at specific time intervals.
• Use blender to disrupt mating.
• Plate on streptomycin to kill donor cells.
• Test recipient cells for genes from Hfr strain.

10
Genetic Analyses Using Conjugation

• Determining linkage from interrupted mating
  experiments
• Problem 1, page 2-4

11
Genetic Analyses Using Conjugation

• Determining linkage from interrupted mating
  experiments
• Problem 1, page 2-4

12
Genetic Analyses Using Conjugation

• 2. Determining gene order from gradient of
  transfer
• Combine Hfr and F- strains.
• Allow for natural disruption of conjugated
  pairs.
• Select for earliest transferred marker.
• Test for markers transferred later in
  conjugation.
• Problem 2, page 2-4

13
Genetic Analyses Using Conjugation

• 2. Determining gene order from gradient of
  transfer
• Problem 2, page 2-4

Select for galTest for lac, pro, xyl
Gene order Gal---Lac---Pro---Xyl
14
Recombination to Integrate Transferred Genes
15
Genetic Analyses Using Conjugation

• 3. Higher-resolution mapping by recombination
  frequency
• Combine Hfr and F- strains.
• Allow for natural disruption of conjugated
  pairs.
• Select for marker that enters LAST.
• Test for unselected markers.
• Problem 3, page 2-4

16
Genetic Analyses Using Conjugation

• 3. Higher-resolution mapping by recombination
  frequency
• Problem 3, page 2-4

Select for leuTest for met, arg
17
Genetic Analyses Using Conjugation

• 3. Higher-resolution mapping by recombination
  frequency
• Problem 3, page 2-4

leu met arg
Hfr
met-
arg-
leu-
F-
18
Genetic Analyses Using Conjugation

• Problem 3, page 2-4
• Smallest number of offspring represents 4
  crossovers, identifies middle gene.
• Genotype will be leu met- arg.

leu met arg
Hfr
met-
arg-
leu-
F-
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Genetic Analyses Using Conjugation

• Problem 3, page 2-4
• Recombination between leu and met gives
• leu met- arg- offspring.

leu met arg
Hfr
met-
arg-
leu-
F-
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Genetic Analyses Using Conjugation

• Problem 3, page 2-4
• Recombination between met and arg gives
  leu met arg- offspring.

leu met arg
Hfr
met-
arg-
leu-
F-
21
Genetic Analyses Using Conjugation

• Problem 3, page 2-4
• Leu ? met 50 .1 10 map units
• 500
• Met ? arg 80 .16 16 map units
• 500

22
Gene Transfer Processes for Bacteria and Their
Viruses

• 2. Transformation
• DNA taken up from external environment

23
Genetic Analysis Using Transformation

• 1. Determining genetic distance with
  transformation mapping
• Transform bacteria with DNA containing two
  markers (eg. his-, met-) in addition to
  penicillin sensitivity.
• Select transformants on minimal medium
  penicillin to kill non-transformants.
• Plate survivors on complete medium to test
  for his-, met-.

24
Genetic Analysis Using Transformation

• 1. Determining genetic distance with
  transformation mapping
• Problem 4, page 2-5
• Rf number of single transformants
• total number of transformants

25
Genetic Analysis Using Transformation

• 1. Determining genetic distance with
  transformation mapping

26
Genetic Analysis Using Transformation

• Determining genetic distance with transformation
  mapping
• Single transformants, his- met and his
  met-,
• represent crossovers between the genes.

his- met-
his- met-
met
his
met
his
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Genetic Analysis Using Transformation

• Determining genetic distance with transformation
  mapping
• Problem 4, page 2-5
• Rf 35 27 62 .24 24 map units
• 256 256

28
Gene Transfer Processes for Bacteria and Their
Viruses

• 3. Transduction Transfer of
  bacterial genes with a
  bacteriophage

29
Transduction
30
Genetic Analysis Using Transduction

• Determining cotransduction frequency with
  three-factor transduction.
• Cotransduction frequency tendency for
  genes to be transferred together on same piece of
  transducing DNA

31
Genetic Analysis Using Transduction

• Three-factor transduction
• Transducing bacteriophage are used to
  transfer DNA with three markers to bacterial
  cells.
• Bacteria are selected for one of the
  markers and tested for the presence of the other
  two markers.
• Gene order and cotransduction frequency can
  be determined.
• Problem 6, page 2-5

32
Gene Transfer Processes for Bacteria and Their
Viruses

• Three-factor transduction
• Problem 6, Page 2-5

Select for ATest for B and C
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Genetic Analysis Using Transduction
Problem 6, page 2-5
Smallest number of offspring represents 4
crossovers, identifies middle gene. Genotype will
be A B- C.
A B C
B-
C-
A-
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Genetic Analysis Using Transduction
Problem 6, page 2-5
Cotransduction of A and B ABC 45 ABC-
80 125/426 .29
Cotransduction of A and C ABC 45 AB-C
1 46/426 .11
35
Genetic Analysis Using Transduction
Problem 6, page 2-5
Cotransduction of A and B .29 Cotransduction of
A and C .11 The higher the cotransduction
frequency, the closer the genes are to each
other. Therefore A and B are closer than A and C.
36
Gene Transfer Processes for Bacteria and Their
Viruses

• 4. Infection with bacteriophage
• In a mixed infection,recombination can be
  detected between bacteriophage carrying
  different genes.

37
Gene Transfer Processes for Bacteria and Their
Viruses

• 4. Infection with bacteriophage
• Infect bacteria with bacteriophage of two
  different genotypes.
• Recombination can occur between
  bacteriophage genes.
• Determine genotypes of resulting
  bacteriophage.
• Rf number of recombinant plaques
• total number of plaques

38
Gene Transfer Processes for Bacteria and Their
Viruses

• 4. Infection with bacteriophage

lawn of bacterial cells
Plaque for one genotype
Plaque for alternate genotype
39
Genetic Analysis for Infection With Bacteriophage
X
40
Gene Transfer Processes for Bacteria and Their
Viruses

• 4. Infection with bacteriophage
• Problem 5, Page 2-5

41
Gene Transfer Processes for Bacteria and Their
Viruses

• 4. Infection with bacteriophage
• Rf number of recombinant plaques
• total number of plaques
• Rf 120 120 240 .24 24 map units
• 1000 1000

42
Gene Transfer Processes for Bacteria and Their
Viruses

• 4. Infection with bacteriophage
• One possible map

rc
rb
h
ra
10
12
2