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Modern Genetic Analysis Chapter 9. The Genetics of Bacteria and Phages
Problems

1. | Describe the state of the F factor in an Hfr, F+, and F strain.

2. | How does a culture of F+ cells transfer markers from the host chromosome to a recipient?

3. | Draw an analogy between gene transfer and integration of the transferred gene into the recipient genome in a. Hfr crosses by conjugation and generalized transduction. b. F derivatives such as F lac and specialized transduction.

4. | Why can generalized transduction transfer any gene, but specialized transduction is restricted to only a small set?

5. | A microbial geneticist isolates a new mutation in E. coli and wishes to map its chromosomal location. She uses interrupted-mating experiments with Hfr strains and generalized transduction experiments with phage P1. Explain why each technique, by itself, is insufficient for accurate mapping.

6. | In E. coli, four Hfr strains donate the following markers, shown in the order donated: All these Hfr strains are derived from the same F+ strain. What is the order of these markers on the circular chromosome of the original F+? See answer

7.

Four E. coli strains of genotype a+ b are labeled 1, 2, 3, and 4. Four strains of genotype a b+ are labeled 5, 6, 7, and 8. The two genotypes are mixed in all possible combinations and (after incubation) are plated to determine the frequency of a+ b+ recombinants. The following results are obtained, where M = many recombinants, L = low numbers of recombinants, and 0 = no recombinants.

On the basis of these results, assign a sex type (either Hfr, F+, or F) to each strain.

See answer

Pattern Recognition Problem

8. | Deduce the genotypes of the following four E. coli strains.

9.

An Hfr strain of genotype a+ b+ c+ d strs is mated with a female strain of genotype a b c d+ strr. At various times, the culture is shaken vigorously to separate mating pairs. The cells are then plated on agar of the following three types, where nutrient A allows the growth of a cells; nutrient B, of b cells; nutrient C, of c cells; and nutrient D, of d cells (a plus indicates the presence of streptomycin and a nutrient, and a minus indicates its absence):

a. What donor genes are being selected on each type of agar?
b. The following table shows the number of colonies on each type of agar for samples taken at various times after the strains are mixed. Use this information to determine the order of the genes a, b, and c.
c. From each of the 25-minutes plates, 100 colonies are picked and transferred to a dish containing agar with all of the nutrients except D. The numbers of colonies that grow on this medium are 89 for the sample from agar type 1, 51 for the sample from agar type 2, and 8 for the sample from agar type 3. Using these data, fit gene d into the sequence of a, b, and c.
d. At what sampling time would you expect colonies to first appear on agar containing C and streptomycin but no A or B?

(Problem 9 is from D. Freifelder, Molecular Biology and Biochemistry. Copyright © 1978 by W. H. Freeman and Company, New York.)

10.

In an interrupted conjugation experiment in E. coli, it is established that the pro gene enters after the thi gene. A pro+ thi+ Hfr is crossed with a pro thi F strain, and exconjugants are plated on medium containing thiamine but no proline. A total of 360 colonies are observed, and they are isolated and cultured on fully supplemented medium. These cultures are then tested for their ability to grow on medium containing no proline or thiamine (minimal medium), and it is found that 320 of the cultures can grow but the remainder cannot.
a. Deduce the genotypes of the two types of cultures.
b. Draw the crossover events required to produce these genotypes.
c. Calculate the distance between the pro and thi genes in recombination units.

Unpacking the Problem

a. What type of organism is E. coli?
b. What does a culture of E. coli look like? (Sketch one.)
c. On what sort of substrates does E. coli generally grow in its natural habitat?
d. What are the minimal requirements needed for E. coli cells to divide?
e. Define the terms prototroph and auxotroph.
f. Which cultures in this experiment are prototrophic and which are auxotrophic?
g. Given some strains of unknown genotype regarding thiamine and proline, how would you test their genotypes? Give precise experimental details, including equipment.
h. What kinds of chemicals are proline and thiamine? Does this matter in this experiment?
i. Draw a schematic diagram showing the full set of manipulations performed in the experiment of the problem.
j. Why do you think the experiment was done?
k. How was it established that pro enters after thi? Give precise experimental steps.
l. In what way does the interrupted mating experiment differ from the experiment described in this problem?
m. What is an exconjugant? How do you think that exconjugants were obtained? (This might involve genes not described in this problem.)
n. When the pro gene is said to enter after thi, does it mean the pro allele, the pro+ allele, either, or both?
o. What is "fully supplemented medium" in the context of this question?
p. Some exconjugants did not grow on minimal medium. On what medium would they grow?
q. What types of crossovers are involved in Hfr × F recombination? How do these crossovers differ from crossovers in eukaryotes?
r. What is a recombination unit in the context of the present analysis? How does it differ from the map units used in eukaryote genetics?

Now try to solve the problem.

11.

A cross is made between two E. coli strains: Hfr arg+ bio+ leu+ × F arg bio leu. Interrupted-mating studies show that arg+ enters the recipient last, so arg+ recombinants are selected on a medium containing bio and leu only. These recombinants are tested for the presence of bio+ and leu+. The following numbers of individuals are found for each genotype:

a. What is the gene order?
b. What are the map distances in recombination units?
See answer

12. | Linkage maps in an Hfr bacterial strain are calculated in units of minutes (the number of minutes between genes indicates the length of time that it takes for the second gene to follow the first during conjugation). In making such maps, microbial geneticists assume that the bacterial chromosome is transferred from Hfr to F at a constant rate. Thus, two genes separated by 10 minutes near the origin end are assumed to be the same physical distance apart as two genes separated by 10 minutes near the F attachment end. Suggest a critical experiment to test the validity of this assumption. See answer

13. | A particular Hfr strain normally transmits the pro+ marker as the last one during conjugation. In a cross of this strain with an F strain, some pro+ recombinants are recovered early in the mating process. When these pro+ cells are mixed with F cells, the majority of the F cells are converted into pro+ cells that also carry the F factor. Explain these results. See answer

14. | F strains in E. coli are derived from Hfr strains. In some cases, these F strains show a high rate of integration back into the bacterial chromosome of a second strain. Furthermore, the site of integration is often the same site that the sex factor occupied in the original Hfr strain (before production of the F strains). Explain these results.

15. | You have two E. coli strains, F strr ala and Hfr strs ala+, in which the F factor is inserted close to ala+. Devise a screening test to detect strains carrying F ala+.

16.

Five Hfr strains A through E are derived from a single F+ strain of E. coli. The following chart shows the entry times of the first five markers into an F strain when each is used in an interrupted-conjugation experiment:

a. Draw a map of the F+ strain, indicating the positions of all genes and their distances apart in minutes.
b. Show the insertion point and orientation of the F plasmid in each Hfr strain.
c. In using each of these Hfr strains, state which gene you would select to obtain the highest proportion of Hfr exconjugants.

17.

Streptococcus pneumoniae cells of genotype strs mtl are transformed by donor DNA of genotype strr mtl+ and (in a separate experiment) by a mixture of two DNAs with genotypes strr mtl and strs mtl+. The following table shows the results.

a. What does the first line of the table tell you? Why?
b. What does the second line of the table tell you? Why?

18.

A transformation experiment is performed with a donor strain that is resistant to four drugs: A, B, C, and D. The recipient is sensitive to all four drugs. The treated recipient cell population is divided up and plated on media containing various combinations of the drugs. The following table shows the results.

a. One of the genes obviously is quite distant from the other three, which appear to be tightly (closely) linked. Which is the distant gene?
b. What is the probable order of the three tightly linked genes?

(Problem 18 is from Franklin Stahl, The Mechanics of Inheritance, 2d ed. Copyright © 1969, Prentice-Hall, Englewood Cliffs, New Jersey. Reprinted by permission.)

See answer

19. | Although most l -mediated gal+ transductants are inducible lysogens, a small percentage of these transductants in fact are not lysogens (that is, they contain no integrated l ). Control experiments show that these transductants are not produced by mutation. What is the likely origin of these types?

20.

An ade+ arg+ cys+ his+ leu+ pro+ bacterial strain is known to be lysogenic for a newly discovered phage, but the site of the prophage is not known. The bacterial map is

The lysogenic strain is used as a source of the phage, and the phages are added to a bacterial strain of genotype ade arg cys his leu pro. After a short incubation, samples of these bacteria are plated on six different media, with the supplementations indicated in the following table. The table also shows whether colonies were observed on the various media. (note: A plus sign indicates the presence of a nutrient supplement; a minus sign indicates supplements are not present; N indicates that no colonies are present; and C indicates that colonies are present.)

a. What genetic process is at work here?
b. What is the approximate locus of the prophage?
See answer

21.

You have two strains of l that can lysogenize E. coli; the following figure shows their linkage maps:

The segment shown at the bottom of the chromosome, designated 123, is the region responsible for pairing and crossing-over with the E. coli chromosome. (Keep the markers on all your drawings.)
a. Diagram the way in which l strain X is inserted into the E. coli chromosome (so that the E. coli is lysogenized).
b. It is possible to superinfect the bacteria that are lysogenic for strain X by using strain Y. A certain percentage of these superinfected bacteria become "doubly" lysogenic (that is, lysogenic for both strains). Diagram how this will occur. (Don't worry about how double lysogens are detected.)
c. Diagram how the two l prophages can pair.
d. It is possible to recover crossover products between the two prophages. Diagram a crossover event and the consequences.

22.

A generalized transducing phage is used to transduce an a b c d e recipient strain of E. coli with an a+ b+ c+ d+ e+ donor. The recipient culture is plated on various media with the results shown in the following table. (Note that a determines a requirement for A as a nutrient, and so forth.) What can you conclude about the linkage and order of the genes?

See answer

23.

In a generalized transduction system using P1 phage, the donor is pur+ nad+ pdx and the recipient is pur nad pdx+. The donor allele pur+ is initially selected after transduction, and 50 pur+ transductants are then scored for the other alleles present. The results follow:

a. What is the cotransduction frequency for pur and nad?
b. What is the cotransduction frequency for pur and pdx?
c. Which of the unselected loci is closest to pur?
d. Are nad and pdx on the same side or on opposite sides of pur? Explain. (Draw the exchanges needed to produce the various transformant classes under either order to see which requires the minimum number to produce the results obtained.)

24.

In a generalized transduction experiment, phages are collected from an E. coli donor strain of genotype cys+ leu+ thr+ and used to transduce a recipient of genotype cys leu thr. Initially, the treated recipient population is plated on a minimal medium supplemented with leucine and threonine. Many colonies are obtained.
a. What are the possible genotypes of these colonies?
b. Strains from these colonies are then inoculated onto three different media: (1) minimal plus threonine only, (2) minimal plus leucine only, and (3) minimal. What genotypes could, in theory, grow on these three media?
c. It is observed that 56 percent of the original colonies grow on (1), 5 percent grow on (2), and no colonies grow on (3). What are the actual genotypes of the colonies on (1), (2), and (3)?
d. Draw a map showing the order of the three genes and which of the two outer genes is closer to the middle gene.

See answer

25.

You have infected E. coli cells with two strains of T4 virus. One strain is minute (m), rapid-lysis (r), and turbid (tu); the other is wild-type for all three markers. The lytic products of this infection are plated and classified. Of 10,342 plaques, the following numbers are classified as each genotype:

a. Determine the linkage distances between m and r, between r and tu, and between m and tu.
b. What linkage order would you suggest for the three genes?
c. What is the coefficient of coincidence in this cross, and what does it signify?

(Problem 25 is reprinted with the permission of Macmillan Publishing Co., Inc., from Monroe W. Strickberger, Genetics. Copyright © 1968 by Monroe W. Strickberger.)

© 1999 by W. H. Freeman and Company. All rights reserved.