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
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
9 min
his+
39 min
34 min
20 min
63 min
thr+
51 min
44 min
74 min
59 min
71 min
78 min
68 min
80 min
95 min
88 min

12. Genetic Analyses Using Conjugation
A. Determining linkage from interrupted mating experiments
Problem 1, page 2-4
gal
lac
his
arg
xyl
ilv
thr 9 27 24 11 4 17 8 2 1 3

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+
Select for leu+ Test 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-
leu-
arg- F-

24. Genetic Analyses Using Conjugation
Problem 3, page 2-4
Leu  met = .1 = 10 map units
500
Met  arg = .16 = 16 map units
leu met arg
10 map units
16 map units

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-
his+
met+
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-
his+
met+
his+
met+

31. Genetic Analysis Using Transformation
Determining genetic distance with transformation mapping
Problem 4, page 2-5
Rf = = 62 = .24 = 24 map units

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 A+B+C+ 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 A+ Test 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
A+B+C
A+B+C
125/426 = .29
Cotransduction of A and C
A+B+C
A+B-C
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
lawn of bacterial cells
Plaque for
one genotype
Plaque for
alternate
genotype

44. Genetic Analysis for Infection With Bacteriophage
ra- h+ h-
ra+ X
Parental Types
ra h+
ra h-
Recombinant Types
ra h-
ra h+

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 r+xh-, where x=a, 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+
r+x h-
r+x h+
r-x h-
r-a h+ x r+a h-
340
420
120
r-b h+ x r+b h-
320
560 60
r-c h+ x r+c 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 = = 240 = .24 = 24 map units

48. Gene Transfer Processes for Bacteria and Their Viruses
Infection with bacteriophage
One possible map: h rc rb ra 2 10 12