5. Genetics of Bacteria and Viruses

Hfr strains that differ in integrated F factor orientation and site of integration are used to construct consolidated bacterial chromosome maps. The data below show the order of gene transfer for five strains.

Hfr Strain       Order of Gene Transfer (First → Last)
Hfr A                oriT–thr–leu–azi–ton–pro–lac–ade
Hfr B                oriT–mtl–xyl–mal–str–his
Hfr C                oriT–ile–met–thi–thr–leu–azi–ton
Hfr D                oriT–his–trp–gal–ade–lac–pro–ton
Hfr E                oriT–thi–met–ile–mtl–xyl–mal–str

Draw a consolidated map of the bacterial chromosome. (Hint: Begin by placing the insertion site for Hfr A at the 2 o'clock position and arranging the genes thr–leu–azi- . . . in clockwise order.)

Hfr strains that differ in integrated F factor orientation and site of integration are used to construct consolidated bacterial chromosome maps. The data below show the order of gene transfer for five strains.

Hfr Strain       Order of Gene Transfer (First → Last)
Hfr A                oriT–thr–leu–azi–ton–pro–lac–ade
Hfr B                oriT–mtl–xyl–mal–str–his
Hfr C                oriT–ile–met–thi–thr–leu–azi–ton
Hfr D                oriT–his–trp–gal–ade–lac–pro–ton
Hfr E                oriT–thi–met–ile–mtl–xyl–mal–str

Identify the overlaps between Hfr strains. Identify the orientations of integrated F factors relative to one another.

Five Hfr strains from the same bacterial species are analyzed for their ability to transfer genes to F⁻ recipient bacteria. The data shown below list the origin of transfer (oriT) for each strain and give the order of genes, with the first gene on the left and the last gene on the right. Use the data to construct a circular map of the bacterium.

Hfr Strain          Genes Transferred
Hfr 1                  oriT met ala lac gal
Hfr 2                  oriT met leu thr azi
Hfr 3                  oriT gal pro trp azi
Hfr 4                  oriT leu met ala lac
Hfr 5                  oriT trp azi thr leu met

An interrupted mating study is carried out on Hfr strains 1, 2, and 3 identified in Problem 17. After conjugation is established, a small sample of the mixture is collected every minute for 20 minutes to determine the distance between genes on the chromosome. Results for each of the three Hfr strains are shown below. The total duration of conjugation (in minutes) is given for each transferred gene.

Hfr strain 1           oriT     met     ala     lac     gal
Duration (min)       0          2        8       13      17
Hfr strain 2           oriT     met     leu     thr      azi
Duration (min)       0          2        7       10      17
Hfr strain 3           oriT     gal      pro     trp      azi
Duration (min)       0          3        8       14      19

In minutes, what is the total length of the chromosome in the donor species?

An interrupted mating study is carried out on Hfr strains 1, 2, and 3 identified in Problem 17. After conjugation is established, a small sample of the mixture is collected every minute for 20 minutes to determine the distance between genes on the chromosome. Results for each of the three Hfr strains are shown below. The total duration of conjugation (in minutes) is given for each transferred gene.

Hfr strain 1           oriT     met     ala     lac     gal
Duration (min)       0          2        8       13      17
Hfr strain 2           oriT     met     leu     thr      azi
Duration (min)       0          2        7       10      17
Hfr strain 3           oriT     gal      pro     trp      azi
Duration (min)       0          3        8       14      19

Write out the interrupted mating results you would expect after 20 minutes of conjugation for Hfr strains 4 and 5. Use the format shown at the beginning of this problem.

An interrupted mating study is carried out on Hfr strains 1, 2, and 3 identified in Problem 17. After conjugation is established, a small sample of the mixture is collected every minute for 20 minutes to determine the distance between genes on the chromosome. Results for each of the three Hfr strains are shown below. The total duration of conjugation (in minutes) is given for each transferred gene.

Hfr strain 1           oriT     met     ala     lac     gal
Duration (min)       0          2        8       13      17
Hfr strain 2           oriT     met     leu     thr      azi
Duration (min)       0          2        7       10      17
Hfr strain 3           oriT     gal      pro     trp      azi
Duration (min)       0          3        8       14      19

Explain why azi is the last gene of strain 2 to transfer in the 20 minutes of conjugation time. How many minutes of conjugation time would be needed to allow the next gene on the map to transfer from Hfr strain 2?

An Hfr strain with the genotype cys⁺ lue⁺ met⁺ strˢ is mated with an  strain carrying the genotype cys⁻ lue⁻ met⁻ strᴿ. In an interrupted mating experiment, small samples of the conjugating bacteria are withdrawn every 3 minutes for 30 minutes. The withdrawn cells are shaken vigorously to stop conjugation and then placed on three different selection media, composed as follows:

Medium 1: Minimal medium plus leucine, methionine, and streptomycin
Medium 2: Minimal medium plus cysteine, methionine, and streptomycin
Medium 3: Minimal medium plus cysteine, leucine, and streptomycin

The following table shows the number of colonies growing on each selection medium. The sampling time indicates how many minutes have passed since conjugation began.

Sampling Time (minutes)              Number of Colonies
                                            Plate 1         Plate 2         Plate 3
           3                                   0                  0                   0
           6                                   0                  0                   0
           9                                   0                 62                  0
         12                                   0                 87                  0
         15                                  51               124                 0
         18                                  79               210                 62
         21                                 109               250                85
         24                                 144               250               111
         27                                 152               250               122
         30                                 152               250               122

Suppose a fourth selection medium containing leucine and streptomycin is prepared. At what sampling time do you expect the first-growing colonies to appear? Explain your reasoning.

An Hfr strain with the genotype cys⁺ lue⁺ met⁺ strˢ is mated with an  strain carrying the genotype cys⁻ lue⁻ met⁻ strᴿ. In an interrupted mating experiment, small samples of the conjugating bacteria are withdrawn every 3 minutes for 30 minutes. The withdrawn cells are shaken vigorously to stop conjugation and then placed on three different selection media, composed as follows:

Medium 1: Minimal medium plus leucine, methionine, and streptomycin
Medium 2: Minimal medium plus cysteine, methionine, and streptomycin
Medium 3: Minimal medium plus cysteine, leucine, and streptomycin

The following table shows the number of colonies growing on each selection medium. The sampling time indicates how many minutes have passed since conjugation began.

Sampling Time (minutes)              Number of Colonies
                                            Plate 1         Plate 2         Plate 3
           3                                   0                  0                   0
           6                                   0                  0                   0
           9                                   0                 62                  0
         12                                   0                 87                  0
         15                                  51               124                 0
         18                                  79               210                 62
         21                                 109               250                85
         24                                 144               250               111
         27                                 152               250               122
         30                                 152               250               122

Determine the order of donor genes cys, leu, and met from the interrupted mating data.

An Hfr strain with the genotype cys⁺ lue⁺ met⁺ strˢ is mated with an  strain carrying the genotype cys⁻ lue⁻ met⁻ strᴿ. In an interrupted mating experiment, small samples of the conjugating bacteria are withdrawn every 3 minutes for 30 minutes. The withdrawn cells are shaken vigorously to stop conjugation and then placed on three different selection media, composed as follows:

Medium 1: Minimal medium plus leucine, methionine, and streptomycin
Medium 2: Minimal medium plus cysteine, methionine, and streptomycin
Medium 3: Minimal medium plus cysteine, leucine, and streptomycin

What is the purpose of adding streptomycin to each selection medium?

An Hfr strain with the genotype cys⁺ lue⁺ met⁺ strˢ is mated with an  strain carrying the genotype cys⁻ lue⁻ met⁻ strᴿ. In an interrupted mating experiment, small samples of the conjugating bacteria are withdrawn every 3 minutes for 30 minutes. The withdrawn cells are shaken vigorously to stop conjugation and then placed on three different selection media, composed as follows:

Medium 1: Minimal medium plus leucine, methionine, and streptomycin
Medium 2: Minimal medium plus cysteine, methionine, and streptomycin
Medium 3: Minimal medium plus cysteine, leucine, and streptomycin

List all possible bacterial genotypes growing on each medium.

An Hfr strain with the genotype cys⁺ lue⁺ met⁺ strˢ is mated with an  strain carrying the genotype cys⁻ lue⁻ met⁻ strᴿ. In an interrupted mating experiment, small samples of the conjugating bacteria are withdrawn every 3 minutes for 30 minutes. The withdrawn cells are shaken vigorously to stop conjugation and then placed on three different selection media, composed as follows:

Medium 1: Minimal medium plus leucine, methionine, and streptomycin
Medium 2: Minimal medium plus cysteine, methionine, and streptomycin
Medium 3: Minimal medium plus cysteine, leucine, and streptomycin

What donor gene is the selected marker in each medium?

Synthesis of the amino acid histidine is a multistep anabolic pathway that uses the products of 13 genes (hisA to hisM) in E. coli. Two independently isolated  E. coli mutants, designated his1⁻ and his2⁻ are studied in a conjugation experiment. A his⁺ F' donor strain that carries a copy of the hisJ gene on the plasmid is mated with a his1⁻ recipient strain in Experiment 1 and with a his2⁻ recipient in Experiment 2. The exconjugants are grown on plates lacking histidine. Growth is observed among the exconjugants of Experiment 2 but not among those of Experiment 1.

What is the genotype of exconjugants in Experiment 2?