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

1. Determining linkage from interrupted mating
   experiments
2. Determining gene order from gradient of transfer
3. 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

• A. Determining linkage from interrupted mating
  experiments
• Problem 1, page 2-4
• Three Hfr strains for E. coli are mated
  individually with an auxotrophic F- strain using
  an interrupted mating procedure. Approximate
  times of entry of each gene are listed below.
  Determine the map of the E. coli chromosome and
  show the orientation of the F plasmid in each Hfr
  strain.

11
Genetic Analyses Using Conjugation

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

Strain 1 Strain 2 Strain 3
lac 3 min argG 19 min ilv 5 min
gal 12 min xyl 30 min xyl 9 min
his 39 min ilv 34 min argG 20 min
argG 63 min thr 51 min his 44 min
xyl 74 min lac 59 min gal 71 min
ilv 78 min gal 68 min lac 80 min
thr 95 min his 95 min thr 88 min
12
Genetic Analyses Using Conjugation

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

13
Genetic Analyses Using Conjugation

• B. 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.

14
Genetic Analyses Using Conjugation

• B. Determining gene order from gradient of
  transfer
• Problem 2, page 2-4
• An Hfr strain donates the genes xyl pro
  lac and gal to an F- strain. Recombinants are
  selected for gal. Tests are done to determine
  the presence of the other three genes in the gal
  recombinants. What is the gene order?

15
Genetic Analyses Using Conjugation

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

gal 100 of strains
lac 70 of strains
pro 30 of strains
xyl 10 of strains
Select for gal Test for lac, pro, xyl
Gene order Gal---Lac---Pro---Xyl
16
Recombination to Integrate Transferred Genes
17
Genetic Analyses Using Conjugation

• C. 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.

18
Genetic Analyses Using Conjugation

• C. Higher-resolution mapping by recombination
  frequency
• Problem 3, page 2-4
• An Hfr strain that is met arg leu strs
  is conjugated with an F- strain that is met- arg-
  leu- strr. Interrupted mating studies show that
  leu enters last. Recombinants that are leu strr
  are selected and then tested for the presence of
  met and arg. The following numbers of bacteria
  are found for each of the genotypes listed below.
  Determine the gene order and the distances
  between the genes in map units.

19
Genetic Analyses Using Conjugation

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

leu met- arg- 50
leu met arg- 80
leu met arg 370
leu met- arg 0
Select for leuTest for met, arg
20
Genetic Analyses Using Conjugation

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

leu met arg
Hfr
met-
arg-
leu-
F-
21
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-
22
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-
23
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-
24
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

25
Gene Transfer Processes for Bacteria and Their
Viruses

• 2. Transformation
• DNA taken up from external environment

26
Genetic Analysis Using Transformation

• 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-.

27
Genetic Analysis Using Transformation

• Determining genetic distance with
  transformation mapping
• Problem 4, page 2-5
• DNA is isolated from E. coli strain A
  (his- met- pens) and used to transform strain B
  (his met pens). Transformants are selected on
  minimal medium penicillin to kill his met
  cells and survivors are plated on complete
  medium. The classes and numbers of cells obtained
  are listed below. Determine the recombination
  frequency between the his and met genes.

28
Genetic Analysis Using Transformation

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

29
Genetic Analysis Using Transformation

• Determining genetic distance with
  transformation mapping

his- met 35
his met- 27
his- met- 194
30
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
31
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

32
Gene Transfer Processes for Bacteria and Their
Viruses

• 3. Transduction Transfer of
  bacterial genes with a
  bacteriophage

33
Transduction
34
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

35
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.

36
Gene Transfer Processes for Bacteria and Their
Viruses

• Three-factor transduction
• Problem 6, Page 2-5
• Transducing phages that infected an ABC
  cell are used to infect an A-B-C- cell.
  Transductants receiving the A marker were tested
  for the presence of B and C. The classes and
  numbers of transductants observed is shown below.
  Determine the gene order and the cotransduction
  frequencies for A with B and A with C.

37
Gene Transfer Processes for Bacteria and Their
Viruses

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

A B C 45
A B C- 80
A B- C 1
A B- C- 300
Select for ATest for B and C
38
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-
39
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
40
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.
41
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.

42
Gene Transfer Processes for Bacteria and Their
Viruses

• 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

43
Gene Transfer Processes for Bacteria and Their
Viruses

• Infection with bacteriophage

44
Genetic Analysis for Infection With Bacteriophage
X
45
Gene Transfer Processes for Bacteria and Their
Viruses

• Infection with bacteriophage
• Problem 5, Page 2-5
• Three different bacteriophage T2 strains
  carrying mutations in the r gene (ra, rb and rc)
  were each involved in a cross r-xh X rxh-,
  where xa, b or c. The numbers of bacteriophage
  of each type are listed below. Give any one of
  four possible linkage maps for these genes.

46
Gene Transfer Processes for Bacteria and Their
Viruses

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

r-x h rx h- rx h r-x h-
r-a h x ra h- 340 420 120 120
r-b h x rb h- 320 560 60 60
r-c h x rc h- 390 590 8 12
47
Gene Transfer Processes for Bacteria and Their
Viruses

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

48
Gene Transfer Processes for Bacteria and Their
Viruses

• Infection with bacteriophage
• One possible map

rc
rb
h
ra
10
12
2