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Chapter 9: Genetics of Bacteria Student Learning Objectives Upon completion of this chapter you should be able to: 1. 2. 3. 4. Know the three mechanisms by which gene transfer can occur in bacteria. Understand the key experiment that initiated gene mapping in bacteria. Understand how gene transfer in bacteria can be used to map bacterial genes. Recognize the significance of horizontal gene transfer. 9.1 Overview of Genetic Transfer in Bacteria Overview The previous chapters in this course have primarily examined the patterns of inheritance between parents and offspring, or what is called vertical gene transfer. This chapter explores various types of gene transfer in bacteria, all of which are examples of horizontal gene transfer. Bacteria may transfer genetic information primarily by one of three methods: conjugation, transduction, and transformation. Study Table 9.1 carefully as it provides a very good introduction to the material in the rest of the chapter. Outline of Key Terms Genetic transfer Conjugation Transduction Transformation Focal Points Three mechanisms of genetic transfer found in bacteria (Table 9.1) Exercises and Problems For questions 1 to 3, match the method of genetic transfer to its correct description. a. transformation b. transduction c. conjugation _____ 1. Donor cell physically contacts recipient cell and transfers its DNA _____ 2. Donor cell dies releasing its DNA in the environment; recipient cell then picks it up _____ 3. Virus infects donor cells, picks up DNA, then transfers it to recipient cell 79 9.2 Bacterial Conjugation Overview Conjugation is the transfer of genetic information between two bacteria that are in contact with one another. The process was discovered by Joshua Lederberg and Edward Tatum, who analyzed auxotrophic strains of E. coli (Refer to Figure 9.1). The requirement for cell-to-cell contact was established by Bernard Davis, in a very clever experiment employing a U-tube apparatus (Figure 9.2). After this historical introduction, the section examines the steps of conjugation at the molecular and cellular levels. This transfer of genetic information usually involves the movement of small circular pieces of DNA, called plasmids (e.g. the F factor). While bacteria do not have sexes, they are classified as donor (F+) and recipient (F-) strains, which differ slightly in their genetic composition. The two cells make contact with each other via the sex pilus of the donor cell. A conjugation bridge then forms, which provides a passageway for the transfer of DNA (Refer to Figure 9.4). Outline of Key Terms F+ cell F– cell Sex pilus Conjugation bridge Origin of transfer Relaxosome Minimal medium Auxotroph Prototroph Conjugation Plasmids F factor Episome Focal Points Discovery of conjugation by Lederberg and Tatum (Figure 9.1) Genes on the F factor that play a role in conjugation (Figure 9.3) The transfer of an F factor during bacterial conjugation (Figure 9.4) Exercises and Problems Complete the following sentences with the most appropriate term(s): 1. A(n) ____ is a bacterial strain that cannot make a particular nutrient which must be given to it. 2. The sex pilus of E. coli is made up of the protein ________. 3. The movement of DNA by conjugation begins at a site on the plasmid called the ________. 4. The F factor encodes a protein complex called the _________, which cuts the DNA molecule, and catalyzes the separation of the strands, so that only the cut DNA strand is transferred. 5. Plasmids such as the F factor are ________, and so can integrate into bacterial chromosomes. 6. The result of conjugation is that the recipient cell has acquired an F factor, converting it from a(n) ______ cell to a(n) ______. 80 9.3 Conjugation and Mapping via Hfr Strains Overview In addition to F+ cells, there exists two other types of donor cells in E. coli: 1) Hfr strains (High frequency of recombination), which are produced when the F factor integrates into the bacterial chromosome; 2) F′ factors, which are created via an imprecise excision of the F factor from the chromosome, thereby producing a plasmid that carries a small part of the bacterial chromosome (Figure 9.5). Hfr strains can actually transfer portions of the bacterial chromosome during conjugation (Figure 9.6). Indeed, this process is the basis of the interrupted mating technique, which was developed by Elie Wollman and François Jacob as a way to map genes along the E. coli chromosome (Figure 9.7). You should familiarize yourself with this classic experiment in the history of genetics. Outline of Key Terms F′ factors Interrupted mating Hfr strains Minutes Focal Points Formation of Hfr cell and subsequent excision of F′ factor (Figure 9.5) Transfer of bacterial genes from an Hfr strain to an F– cell (Figure 9.6) Conjugation experiment used to map bacterial genes (pages 198-201) Exercises and Problems Complete the following sentences with the most appropriate term(s): The F factor can integrate into the bacterial chromosome, thus creating an (1) ________ strain. If the F factor is excised properly, the cell goes back to being (2) _______. However, an imprecise excision creates an (3) _______ plasmid, which carries some bacterial genes on it. (4) _________ is a technique that can be used to map genes along the bacterial chromosome. This technique allows researchers to determine the relative (5) ________ of genes based on the (6) ______ it takes for each gene to be transferred to the recipient cell. In this type of conjugation experiment, the recipient cell remains (7) ________ after conjugation because not all of the (8) ________ is transferred. Because the bacterial chromosome is (9) _________, a starting point on the map must be arbitrarily assigned. Researchers scale genetic maps from such bacterial conjugation studies in units of (10) _________. - 80 - 9.4 Bacterial Transduction Overview The second mechanism of gene transfer to be discussed is transduction. This involves the transfer of genetic material from one bacterium to another using a bacterial virus (or bacteriophage) as an intermediate. In order to understand how this is possible, it is important to look a little ahead to Chapter 10, Figure 10.3, which describes the reproductive cycles of bacteriophages. There are two types of transduction: 1) Generalized transduction involves the transfer of any piece of the bacterial chromosome from the donor to the recipient cell (Refer to Figure 9.10); and 2) Specialized transduction, in which only certain bacterial genes can be transferred to recipient cells (Refer to Solved Problem S5 at the end of the chapter). A rare form of transduction, called cotransduction, can be used to map bacterial genes that are closely linked on the chromosome (Figure 9.11). Outline of Key Terms Generalized transduction Co-transduction Focal Points Transduction in bacteria (Figure 9.10) The steps in a cotransduction experiment (Figure 9.11) Exercises and Problems Complete the following sentences with the most appropriate term(s): Transduction is the transfer of DNA from one bacterium to another, via a bacterial virus or (1) ________. These viruses can undergo either the (2) ________ or the (3) ________ cycles. In the (4) _______ cycle, when the virus infects the bacterial cell, the host DNA is (5) ________. When new viral particles are made, any piece of the bacterial chromosome can be incorporated into the virus and transferred to a recipient cell. This process is called (6) ________ transduction. This is in contrast to (7) ________ transduction, in which only certain bacterial genes can be transferred to a recipient cell. In all cases, if two genes are close together along the chromosome, a bacterial virus may package them both into a single piece and transfer them to a recipient cell. This phenomenon, called (8) ________, can be used to map genes that are within (9) ________ of each other on the bacterial chromosome. - 81 - 9.5 Bacterial Transformation Overview The third major form of gene transfer in bacteria is transformation. During transformation, bacteria acquire DNA from their environment (Figure 9.12). Not all bacteria are capable of transformation. Those that possess the ability are called competent cells. Transformation is a natural event, but has been exploited by geneticists to introduce genes of interest into bacteria. This process is called artificial transformation. Transformation may be used to map two genes that are closely linked by a process called cotransformation. Outline of Key Terms Homologous recombination Heteroduplex Nonhomologous recombination Illegitimate recombination Cotransformation Natural transformation Artificial transformation Competent cells Competence factors Competence-stimulating peptide (CSP) DNA-uptake signal sequence Focal Points The steps of bacterial transformation (Figure 9.12) Exercises and Problems Complete the following sentences with the most appropriate term(s): 1. A technique known as ________ uses an electric current to allow bacteria to uptake the DNA. 2. Bacterial cells that are able to take up DNA from the environment are called ________ cells. 3. During homologous recombination, alignment of foreign and host DNA results in a region called a ________ that contains one or more base sequence mismatches. 4. In Streptococcus pneumoniae, transformation is triggered by the secretion of a short peptide called the ________. 5. Some bacterial species, such as Haemophilus influenzae, promote the uptake of DNA among members of their own species via ________, which are 9-10 bp long. 6. If the DNA that enters the cell is not homologous to any host gene, the DNA strand may be incorporated at a random site in the chromosome. This process is known as nonhomologous, or ________, recombination. 7. If two genes are close together on the same chromosome, their ________ frequency is expected to be high. - 82 - 9.6 Horizontal Gene Transfer Overview Organisms can undergo two types of gene transfer. In vertical gene transfer, the genetic information is transferred from mother cell to daughter cell, or from parent to offspring. In contrast, horizontal gene transfer is a process in which an organism incorporates genetic material from another organism without being the offspring of that organism. Examples include all three mechanisms of genetic transfer that have been already discussed in this chapter. Horizontal gene transfer has great medical importance because it can lead to acquired antibiotic resistance. Outline of Key Terms Vertical gene transfer Horizontal gene transfer Acquired antibiotic resistance Focal Point Acquisition of antibiotic resistance via horizontal gene transfer (page 206) Exercises and Problems The following statements apply to the three types of bacterial horizontal gene transfer. Indicate if the statements are true (T) or false (F). If false, change the statement to make it true. _____ 1. Horizontal gene transfer is the movement of DNA from parent to offspring. _____ 2. Genetic distance obtained from conjugation studies is measured in total nucleotides. _____ 3. Bacteria that can uptake DNA from the environment are called competent cells. _____ 4. Artificial conjugation may be used to introduce DNA into a bacterium from its environment using laboratory procedures. _____ 5. The movement of genetic material by conjugation begins at a site on the plasmid called the start site. _____ 6. Interrupted mating is a form of transformation study used to map bacterial genes. _____ 7. Cotransformation and cotransduction experiments can be used to map closely linked genes in bacteria. - _____ 8. Both Hfr and F+ cells can transfer bacterial genes to F cells during conjugation. _____ 9. Plasmids that can integrate into the bacterial chromosome are called episomes. _____ 10. The two types of bacteriophage life cycles are termed lytic and lysogenic. - 84 - Chapter Quiz 1. Which of the following mechanisms of gene transfer in bacteria involves the use of a viral intermediate? a. transduction b. transformation c. conjugation d. co-transformation e. none of the above 2. Which of the following processes are sensitive to extracellular DNases? a. transduction b. transformation c. conjugation d. both transduction and conjugation e. both conjugation and transformation 3. In which of the following do bacteria have to be in physical contact for gene transfer to occur? a. transduction b. transformation c. conjugation d. both transduction and conjugation e. both conjugation and transformation 4. Which of the following bacteria are naturally competent? a. Streptococcus penumoniae b. Haemophilus influenzae c. Both A and B d. Neither A nor B 5. Which of the following procedures are used for genetic mapping in bacteria? a. interrupted mating b. cotransduction c. cotransformation d. two of the above e. all of the above 6. The transfer of genetic material between two different species is called _______. a. vertical gene transfer b. horizontal gene transfer c. conjugation d. sexual reproduction 7. A bacterial strain that is a methionine _________ cannot grow on a minimal medium unless methionine the medium contains methionine. a. autotroph b. auxotroph c. prototroph d. phototroph e. paratroph - 85 - - 8. After mating with an Hfr strain, what happens to an F cell? a. It becomes F+ and picks up some chromosomal genes b. It becomes Hfr and picks up some chromosomal genes c. It becomes F+ but does not pick up any chromosomal genes d. It stays F but picks up some chromosomal genes e. It stays F and does not pick up any chromosomal genes 9. In which type of transduction does the transducing particle carry only specific portions of the bacterial genome? a. general transduction b. abortive transduction c. specialized transduction d. natural transduction e. lytic transduction 10. The _________ is a protein complex that cuts the DNA during conjugation to initiate its transfer. a. episome b. transformasome c. nucleosome d. relaxosome e. phagosome Answer Key for Study Guide Questions This answer key provides the answers to the exercises and chapter quiz for this chapter. Answers in parentheses ( ) represent possible alternate answers to a problem, while answers marked with an asterisk (*) indicate that the response to the question may vary. 9.1 1. c 2. a 3. b 9.2 1. auxotroph 2. pilin 3. origin of transfer 4. relaxosome 5. episomes + 6. F to F 9.3 1. Hfr + 2. F ′ 3. F 4. Interrupted mating 5. order 6. time 7. F 8. F factor (F plasmid) 9. circular 10. minutes - 86 - 9.4 1. bacteriophages or phage 2. lytic (or lysogenic) 3. lysogenic (or lytic) 4. lytic 5. fragmented (digested, hydrolyzed, broken up, etc.) 6. generalized 7. specialized 8. cotransduction 9. 2 minutes 9.5 1. electroporation 2. competent 3. heteroduplex 4. competence-stimulating peptide 5. DNA-uptake signal sequences 6. illegitimate 7. cotransformation 9.6 1. F, vertical gene transfer 2. F, measured in minutes 3. T 4. F, artificial transformation 5. F, called the origin of transfer 6. F, conjugation study 7. T 8. F, only Hfr cells 9. T 10. T Quiz 1. a 2. b 3. c 4. c 5. e 6. b 7. b 8. d 9. c 10. d - 84 -