William S. Klug Michael R. Cummings Charlotte A William S. Klug Michael R. Cummings Charlotte A. Spencer Concepts of Genetics Eighth Edition Chapter 6 Genetic Analysis and Mapping in Bacteria and Bacteriophages Copyright © 2006 Pearson Prentice Hall, Inc.

Bacteria Mutate Spontaneously and Grow at an Exponential Rate

Figure 6-1 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-1 Typical bacterial population growth curve illustrating the initial lag phase, the subsequent log phase where exponential growth occurs, and the stationary phase that occurs when nutrients are exhausted. Figure 6-1 Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-2 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-2 Results of the serial dilution technique and subsequent culture of bacteria. Each of the dilutions varies by a factor of 10. Each colony was derived from a single bacterial cell. Figure 6-2 Copyright © 2006 Pearson Prentice Hall, Inc.

Conjugation Is One Means of Genetic Recombination in Bacteria

Figure 6-3 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-3 Genetic recombination involving two auxotrophic strains producing prototrophs. Neither auxotrophic strain will grow on minimal medium, but prototrophs do, suggesting that genetic recombination has occurred. Figure 6-3 Copyright © 2006 Pearson Prentice Hall, Inc.

Conjugation Is One Means of Genetic Recombination in Bacteria F+ and F- Bacteria

Figure 6-4 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-4 When strain A and strain B auxotrophs are grown in a common medium, but separated by a filter, no genetic recombination occurs and no prototrophs are produced. The apparatus shown is a Davis U-tube. Figure 6-4 Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-5 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-5 An electron micrograph of conjugation between an E. coli cell and an cell. The sex pilus linking them is clearly visible. Figure 6-5 Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-6 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-6 An mating, demonstrating how the recipient cell is converted to During conjugation, the DNA of the F factor is replicated with one new copy entering the recipient cell, converting it to To indicate the clockwise rotation during replication, a bar is shown on the F factor. Figure 6-6 Copyright © 2006 Pearson Prentice Hall, Inc.

Conjugation Is One Means of Genetic Recombination in Bacteria Hfr Bacteria and Chromosome Mapping

Figure 6-7 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-7 The progressive transfer during conjugation of various genes from a specific Hfr strain of E. coli to an strain. In this strain certain genes (azi and ton) are transferred sooner than others and recombine more frequently. Others (lac and gal) take longer to transfer and recombine with a lower frequency. Still others (thr and leu) are always transferred and are used in the initial screen for recombinants, but not shown in the histograms above. Figure 6-7 Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-8 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-8 A time map of the genes studied in the experiment depicted in Figure 6–7. Figure 6-8 Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-9 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-9 (a) The order of gene transfer in four Hfr strains, suggesting that the E. coli chromosome is circular. (b) The point where transfer originates (O) is identified in each strain. Note that transfer can proceed in either direction, depending on the strain. The origin is determined by the point of integration of the F factor into the chromosome, and the direction of transfer is determined by the orientation of the F factor as it integrates. Figure 6-9 Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-9a Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-9a (a) The order of gene transfer in four Hfr strains, suggesting that the E. coli chromosome is circular. (b) The point where transfer originates (O) is identified in each strain. Note that transfer can proceed in either direction, depending on the strain. The origin is determined by the point of integration of the F factor into the chromosome, and the direction of transfer is determined by the orientation of the F factor as it integrates. Figure 6-9a Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-9b Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-9b (a) The order of gene transfer in four Hfr strains, suggesting that the E. coli chromosome is circular. (b) The point where transfer originates (O) is identified in each strain. Note that transfer can proceed in either direction, depending on the strain. The origin is determined by the point of integration of the F factor into the chromosome, and the direction of transfer is determined by the orientation of the F factor as it integrates. Figure 6-9b Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-10 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-10 Conversion of to an Hfr state occurs by integration of the F factor into the bacterial chromosome (Step 1). The point of integration determines the origin (O) of transfer. During a subsequent conjugation (Steps 2–4), an enzyme nicks the F factor, now integrated into the host chromosome, initiating the transfer of the chromosome at that point. Conjugation is usually interrupted prior to complete transfer. Only the A and B genes are transferred to the cell (Steps 3–5), which may recombine with the host chromosome. Figure 6-10 Copyright © 2006 Pearson Prentice Hall, Inc.

Conjugation Is One Means of Genetic Recombination in Bacteria Recombination in F+ X F- Matings: A Reexamination

The F' State and Merozygotes

Figure 6-11 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-11 Conversion of an Hfr bacterium to and its subsequent mating with an cell. The conversion occurs when the F factor loses its integrated status. During excision from the chromosome, the F factor may carry with it one or more chromosomal genes (A and E). Following conjugation with an cell, the recipient cell becomes partially diploid and is called a merozygote. It also behaves as an donor cell. Figure 6-11 Copyright © 2006 Pearson Prentice Hall, Inc.

Mutational Analysis Led to the Discovery of the Rec Proteins Essential to Bacterial Recombination

F Factors Are Plasmids

Figure 6-12 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-12 (a) Electron micrograph of a plasmid isolated from E. coli. (b) Diagrammatic representation of an R plasmid containing resistance transfer factors (RTFs) and multiple r-determinants (Tc, tetracycline; Kan, kanamycin; Sm, streptomycin; Su, sulfonamide; Amp, ampicillin; and Hg, mercury). Figure 6-12 Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-12a Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-12a (a) Electron micrograph of a plasmid isolated from E. coli. (b) Diagrammatic representation of an R plasmid containing resistance transfer factors (RTFs) and multiple r-determinants (Tc, tetracycline; Kan, kanamycin; Sm, streptomycin; Su, sulfonamide; Amp, ampicillin; and Hg, mercury). Figure 6-12a Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-12b Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-12b (a) Electron micrograph of a plasmid isolated from E. coli. (b) Diagrammatic representation of an R plasmid containing resistance transfer factors (RTFs) and multiple r-determinants (Tc, tetracycline; Kan, kanamycin; Sm, streptomycin; Su, sulfonamide; Amp, ampicillin; and Hg, mercury). Figure 6-12b Copyright © 2006 Pearson Prentice Hall, Inc.

Transformation Is Another Process Leading to Genetic Recombination in Bacteria The Transformation Process

Figure 6-13 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-13 Proposed steps for transformation of a bacterial cell by exogenous DNA. Only one of the two strands of the entering DNA is involved in the transformation event, which is completed following cell division. Figure 6-13 Copyright © 2006 Pearson Prentice Hall, Inc.

Transformation Is Another Process Leading to Genetic Recombination in Bacteria Transformation and Linked Genes

Bacteriophages Are Bacterial Viruses Phage T4: Structure and Life Cycle

Figure 6-14 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-14 The structure of bacteriophage T4, including an icosahedral head filled with DNA, a tail consisting of a collar, tube, sheath, base plate, and tail fibers. During assembly, the tail components are added to the head and then tail fibers are added. Figure 6-14 Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-15 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-15 Life cycle of bacteriophage T4. Figure 6-15 Copyright © 2006 Pearson Prentice Hall, Inc.

Bacteriophages Are Bacterial Viruses Lysogeny

Transduction Is Virus-Mediated Bacterial DNA Transfer The Lederberg–Zinder Experiment

Figure 6-17 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-17 The Lederberg–Zinder experiment using Salmonella. After placing two auxotrophic strains on opposite sides of a Davis U-tube, Lederberg and Zinder recovered prototrophs from the side containing the LA-22 strain, but not from the side containing the LA-2 strain. These initial observations led to the discovery of the phenomenon called transduction. Figure 6-17 Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-18 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-18 Generalized transduction. Figure 6-18 Copyright © 2006 Pearson Prentice Hall, Inc.

Transduction Is Virus-Mediated Bacterial DNA Transfer The Nature of Transduction Transduction and Mapping

Bacteriophages Undergo Intergenic Recombination

Figure 6-16 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-16 The plaque assay for bacteriophage analysis. Serial dilutions of a bacteriophage culture are first made. Then, three of the dilutions ( and ) are analyzed using the plaque assay technique. In each case, 0.1 ml of the diluted culture is used. Each plaque represents the initial infection of one bacterial cell by one bacteriophage. In the dilution, so many phages are present that all bacteria are lysed. In the dilution, 23 plaques are produced. In the dilution, the dilution factor is so great that no phages are present in the 0.1 ml sample, and thus no plaques form. Figure 6-16 Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-19 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-19 Plaque morphology phenotypes observed following simultaneous infection of E. coli by two strains of phage T2, and In addition to the parental genotypes, recombinant plaques hr and were also recovered. Figure 6-19 Copyright © 2006 Pearson Prentice Hall, Inc.

Table 6-1 Copyright © 2006 Pearson Prentice Hall, Inc. Table 6.1 Some Mutant Types of T-Even Phages Table 6-1 Copyright © 2006 Pearson Prentice Hall, Inc.

Bacteriophages Undergo Intergenic Recombination Mapping in Bacteriophages

Table 6-2 Copyright © 2006 Pearson Prentice Hall, Inc. Table 6.2 Results of a Cross Involving the h and r Genes in Phage T2 (hr+ * h+r) Table 6-2 Copyright © 2006 Pearson Prentice Hall, Inc.

Intragenic Recombination Occurs in Phage T4 The rII Locus of Phage T4

Figure 6-20 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-20 Illustration of intragenic recombination between two mutations in the rII locus of phage T4. The result is the production of a wild-type phage that will grow on both E. coli B and K12 and a phage that has incorporated both mutations into the rII locus. It will grow on E. coli B, but not on E. coli K12. Figure 6-20 Copyright © 2006 Pearson Prentice Hall, Inc.

Intragenic Recombination Occurs in Phage T4 Complementation by rII Mutations

Figure 6-21a Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-21a Comparison of two rII mutations that either (a) complement one another or (b) do not complement one another. Complementation occurs when each mutation is in a separate cistron. Failure to complement occurs when the two mutations are in the same cistron. Figure 6-21a Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-21b Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-21b Comparison of two rII mutations that either (a) complement one another or (b) do not complement one another. Complementation occurs when each mutation is in a separate cistron. Failure to complement occurs when the two mutations are in the same cistron. Figure 6-21b Copyright © 2006 Pearson Prentice Hall, Inc.

Intragenic Recombination Occurs in Phage T4 Recombinational Analysis

Figure 6-22 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-22 The experimental protocol for recombinational studies between pairs of mutations in the same cistron. In this figure all phage infecting E. coli B (in the flask) contain one of two mutations in the A cistron, as shown in the depiction of their chromosomes to the left of the flask. Figure 6-22 Copyright © 2006 Pearson Prentice Hall, Inc.

Intragenic Recombination Occurs in Phage T4 Deletion Testing of the rII Locus

Figure 6-23 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-23 Demonstration that recombination between a phage chromosome with a deletion in the A cistron and another phage with a point mutation overlapped by that deletion cannot yield a chromosome with wild-type A and B cistrons. Figure 6-23 Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 6-24 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-24 Three series of overlapping deletions in the A cistron of the rII locus used to localize the position of an unknown rII mutation. For example, if a mutant strain tested against each deletion (dashed areas) in Series I for the production of recombinant wild-type progeny shows the results at the right ( or ), the mutation must be in segment A5. In Series II, the mutation is further narrowed to segment A5c, and in Series III to segment A5c3. Figure 6-24 Copyright © 2006 Pearson Prentice Hall, Inc.

Intragenic Recombination Occurs in Phage T4 The rII Gene Map

Figure 6-25 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 6-25 A partial map of mutations in the A and B cistrons of the rII locus of phage T4. Each square represents an independently isolated mutation. Note the two areas in which the largest number of mutations are present, referred to as “hot spots” (A6cd and B5). Figure 6-25 Copyright © 2006 Pearson Prentice Hall, Inc.