Genetic Analysis and Mapping in Bacteria and Bacteriophages Active Lecture PowerPoint® Presentation for Essentials of Genetics Seventh Edition Klug, Cummings, Spencer, Palladino Chapter 8b Genetic Analysis and Mapping in Bacteria and Bacteriophages Copyright © 2010 Pearson Education, Inc.

Outline Transformation Bacteriophages Lytic cycle Lysogenic cycle Transduction Genetic mapping in bacteriophages

Genetic Recombination in Bacteria Conjugation Transformation Transduction

Transformation In transformation, small pieces of naked extracellular DNA are taken up by a living bacterial cell Bacterial cell has to be competent The DNA taken in is integrated stably into the chromosome

Transformation FIGURE 8-13 Proposed steps for transforming a bacterial cell by exogenous DNA. Only one of the two entering DNA strands is involved in the transformation event, which is completed following cell division.

FIGURE 8-13 part 1 Proposed steps for transforming a bacterial cell by exogenous DNA. Only one of the two entering DNA strands is involved in the transformation event, which is completed following cell division.

FIGURE 8-13 part 2 Proposed steps for transforming a bacterial cell by exogenous DNA. Only one of the two entering DNA strands is involved in the transformation event, which is completed following cell division.

FIGURE 8-13 part 3 Proposed steps for transforming a bacterial cell by exogenous DNA. Only one of the two entering DNA strands is involved in the transformation event, which is completed following cell division.

FIGURE 8-13 part 4 Proposed steps for transforming a bacterial cell by exogenous DNA. Only one of the two entering DNA strands is involved in the transformation event, which is completed following cell division.

FIGURE 8-13 part 5 Proposed steps for transforming a bacterial cell by exogenous DNA. Only one of the two entering DNA strands is involved in the transformation event, which is completed following cell division.

Transformation Once integrated into the chromosome, recombinant region contains one host strand (present originally) and one mutant strand Because these strands are from different sources, this region is referred to as a heteroduplex Two strands of DNA are not perfectly complementary in heteroduplex region

Transformation & Gene Mapping Genes that are close together and can undergo cotransformation are linked Linkage in prokaryotes refer to the close proximity of genes Cotransformation of linked genes is used to map bacterial chromosomes Bacteria have only one chromosome. Therefore, linkage has a different meaning in bacteria.

Bacteriophages Bacteriophages: viruses that can infect a host bacterium and inject their DNA into its chromosome Infected bacterium then produces more phage particles, which are released when host cell is lysed

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.

Multiplication of Bacteriophages Lytic cycle: Phage causes lysis and death of host cell. Lysogenic cycle: Phage DNA incorporated in host DNA. Two modes of multiplication of phage:

Life cycle of Bacteriophage T4 Lytic cycle FIGURE 8-15

FIGURE 8-15 part 1 Life cycle of bacteriophage T4.

FIGURE 8-15 part 2 Life cycle of bacteriophage T4.

FIGURE 8-15 part 3 Life cycle of bacteriophage T4.

FIGURE 8-15 part 4 Life cycle of bacteriophage T4.

FIGURE 8-15 part 5 Life cycle of bacteriophage T4.

Growing a Bacteriophage Bacteriophages form plaques on a lawn of bacteria.

Plaque Assay # of phages produced following infection of bacteria can be determined by plaque assay Technique entails performing serial dilutions of virally infected bacteria, which are then poured onto agar plates By counting # of plaques (areas clear of bacteria) on plates, # of phages in original culture can be determined

Figure 8-16 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 8-16 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 8-16 Copyright © 2006 Pearson Prentice Hall, Inc.

Lysogeny Lysogeny occurs when phage DNA integrates into bacterial chromosome, is replicated along with chromosome, and is passed to daughter cells Bacteria containing a prophage and can grow and divide stably until viral reproduction is induced Bacterium harboring a prohphage is lysogenic Prophage genes are expressed in the same way bacterial genes are expressed. Prophage can confer new properties to the host such as toxin production. Many deadly toxins are produced by prophage genes. Eg: Botulinum toxin produced by Clostridium botulinum, diptheria toxin produced by Corynebacterium diptheriae.

The Lysogenic Cycle This figure is not from your text.

Transduction Gene transfer between bacteria via a bacteriophage Transferred genes recombine with host DNA in the recepient Two forms of transduction Generalized transduction Specialized transduction

Transduction In generalized transduction, bacterial DNA instead of phage DNA is packaged in a phage particle and is transferred to a recipient host In specialized transduction, a small piece of bacterial DNA is packaged along with the phage DNA

Generalized Transduction FIGURE 8-18 Generalized transduction.

FIGURE 8-18 part 1 Generalized transduction.

FIGURE 8-18 part 2 Generalized transduction.

FIGURE 8-18 part 3 Generalized transduction.

FIGURE 8-18 part 4 Generalized transduction.

FIGURE 8-18 part 5 Generalized transduction.

FIGURE 8-18 part 6 Generalized transduction.

Transduction Generalized transduction results in transfer of a large number of bacterial genes Specialized transduction results in transfer of only a few bacterial genes Like transformation, generalized transduction can be used in linkage and chromosomal mapping

Bacteriophages Undergo Mutations Phage mutations often affect plaque morphology Such mutations have been important in understanding genetic phenomena in phages

Bacteriophages Undergo Intergenic Recombination When two distinct mutant strains infect the same bacterial culture, recombination takes place between the two strains Such mixed infection experiments allow genetic mapping in phage. As with eukaryotes, recombination frequency is used to estimate distance between genes. Bird flu situation: We are afraid that recombination can take place between bird flu virus and a human virus inside a host cell like pig, which can be infected with both types.

Simultaneous infection of E. coli by two phage T2 strains allows phage to recombine Parents: h+r and hr+ h+r, hr+ hr, h+r+ hr h+r+ h- locus that determines host range; r- rapid lysis strain that gives larger plaques. FIGURE 8-19 Plaque morphology phenotypes observed following simultaneous infection of E. coli by two strains of phage T2, h+r and hr+. In addition to the parental genotypes, recombinant plaques h r and h+r+ are shown. h+r hr+

TABLE 8.2 Results of a Cross Involving the h and r Genes in Phage T2 (hr+ x h+r)

Mapping in Bacteriophages Probability of recombination varies with the distance between two genes along the phage chromosome As with eukaryotes, recombination frequency is used to estimate distance between genes. Two and three point mapping are possible Data are analyzed in much the same way as in eukaryotic mapping.