Lecture 10 CHAPTER 7 Gene transfer in Bacterium(1) PDF

CHAPTER 7 GENETIC TRANSFER and MAPPING in BACTERIA Bacteria do possess allelic differences that affect their cellular traits However, these allelic differences (such as different sensitivity to antibiotics) are between different strains of bacteria because Bacteria are usually haploid Th...

CHAPTER 7 GENETIC TRANSFER and MAPPING in BACTERIA Bacteria do possess allelic differences that affect their cellular traits However, these allelic differences (such as different sensitivity to antibiotics) are between different strains of bacteria because Bacteria are usually haploid This fact makes it easier to identify loss-of-function mutations in bacteria than in eukaryotes These usually recessive mutations are not masked by dominant genes in haploid species Bacteria reproduce asexually Therefore crosses are not used in the genetic analysis of bacterial species Rather, researchers rely on a similar phenomenon called genetic transfer Genetic Transfer and Mapping in Bacteria © Eye of science/Science Source © McGraw-Hill Education. 7-3 Genetic Transfer in Bacteria Transfer of genetic material from one bacterium to another can occur in three ways: Conjugation Involves direct physical contact Transduction Involves viruses Transformation Involves uptake from the environment Mechanism: Conjugation Description: Requires direct contact between a donor and a recipient cell. The donor cell transfers a strand of DNA to the recipient. In the example shown here, DNA known as a plasmid is transferred to the recipient cell. Mechanism: Transduction Description: When a virus infects a donor cell, it incorporates a fragment of bacterial chromosomal DNA into a newly made virus particle. The virus then transfers this fragment of DNA to a recipient cell, which incorporates the DNA into its chromosome by recombination.  Mechanism: Transformation Description: When a bacterial cell dies, it releases a fragment of its DNA into the environment. This DNA fragment is taken up by a recipient cell, which incorporates the DNA into its chromosome by recombination. 7.2 Bacterial Conjugation (1 of 2) Genetic transfer in bacteria was discovered in 1946 by Joshua Lederberg and Edward Tatum They were studying strains of Escherichia coli that had different nutritional growth requirements Auxotrophs cannot synthesize a needed nutrient Prototrophs make all their nutrients from basic components © McGraw-Hill Education. 7-8 7.2 Bacterial Conjugation (2 of 2) One strain was designated met – bio– thr + leu + thi + It required one vitamin (biotin) and one amino acid (methionine) It could produce the amino acids phenylalanine, leucine and threonine Another strain was designated met + bio+ thr – leu – thi – It required one vitamin (thiamine) and two amino acids, leucine and threonine © McGraw-Hill Education. 7-9 Experiment of Lederberg and Tatum (Figure 7.1) © McGraw-Hill Education. 7-10 Bacteria can Transfer Genetic Material during Conjugation The genotype of the bacterial cells that grew on the plates has to be met + bio+ thr + leu+ thi + Lederberg and Tatum reasoned that some genetic material was transferred between the two strains Either the met – bio– thr + leu+ thi + strain got the ability to synthesize biotin and methionine (bio+ met +) Or the met + bio+ thr – leu – thi – strain got the ability to synthesize threonine and leucine and thiamine (thr + leu+ thi +) The results of this experiment cannot distinguish between these two possibilities © McGraw-Hill Education. 7-11 Conjugation Requires Direct Physical Contact Bernard Davis later showed that the bacterial strains must make physical contact for transfer of genetic material to occur Used an apparatus known as U-tube (see Figure 7.2) It contains a filter at the bottom which has pores that were Large enough to allow the passage of the genetic material But small enough to prevent the passage of bacterial cells © McGraw-Hill Education. 7-12 Conjugation Requires Direct Physical Contact Davis placed the two strains in question on opposite sides of the filter Application of pressure or suction promoted the movement of liquid through the filter The term conjugation now refers to the transfer of DNA from one bacterium to another following direct cell-to-cell contact Many, but not all, species of bacteria can conjugate Moreover, only certain strains of a bacterium can act as donor cells © McGraw-Hill Education. 7-13 Conjugation Requires Direct Physical Contact In E. coli those strains contain a small circular piece of DNA termed the F factor (for Fertility factor) Strains containing the F factor are designated F+ Those lacking it are F – Plasmid is the general term used to describe extra-chromosomal DNA © McGraw-Hill Education. 7-14 Conjugation Requires Direct Physical Contact Figure 7.2 © McGraw-Hill Education. 7-15 Plasmids Most plasmids are circular, although some are linear Present in many bacteria and a few eukaryotic cells Range in size from a few thousand to 500,000 bp Plasmids have their own replication of origin Replicate independent of bacterial chromosome Depending on ‘strength’ of origin, can exist as single copy or up to 100 copies Plasmids are usually not required for survival Can provide growth advantages to the bacteria © McGraw-Hill Education. 7-16 Five Different Categories of Plasmids (1 of 2) 1. Fertility plasmids Allow conjugation 2. Resistance Plasmids Known as R factors Contain genes conferring resistance to antibiotics 3. Degradative plasmids Carry genes allowing digestion of unusual substances © McGraw-Hill Education. 7-17 Five Different Categories of Plasmids (2 of 2) 4. Col-plasmids Contain genes that kill other bacteria 5. Virulence plasmids Carry genes that turn bacterium into pathogenic strains © McGraw-Hill Education. 7-18 F factors Carry Genes that are Required for Conjugation Plasmids, such as F factors, which are transmitted via conjugation are termed conjugative plasmids Genes that play a role in the transfer of DNA: Named tra or trb followed by a capital letter © McGraw-Hill Education. 7-19 F factors Carry Genes that are Required for Conjugation (2 of 2) Figure 7.3 © McGraw-Hill Education. 7-20 Contact Initiates Conjugation The first step in conjugation is the contact between donor and recipient cells This is mediated by sex pili (or F pili) which are made only by F+ strains These pili act as attachment sites for the F– bacteria Once contact is made, the pili shorten Donor and recipient cells are drawn closer together A conjugation bridge is formed between the two cells The successful contact stimulates the donor cell to begin the transfer process © McGraw-Hill Education. 7-21  Mechanism of Transfer (Figure 7.4) Exporter is a complex of 10-15 proteins encoded by the F factor that span both inner and outer membranes After cutting, most accessory proteins of the relaxosome are released One protein, relaxase, remains bound to the end of the T-DNA © McGraw-Hill Education. 7-22 Mechanism of Transfer (Figure 7.4) © McGraw-Hill Education. 7-23 Mechanism of Transfer (Figure 7.4) The result of conjugation is that the recipient cell has acquired an F factor Thus, it is converted from an F– to an F+ cell The F+ cell remains unchanged © McGraw-Hill Education. 7-24 Conjugation and Mapping via Hfr Strains In the 1950s, Luca Cavalli-Sforza discovered a strain of E. coli that was very efficient at transferring chromosomal genes He designated this strain as Hfr (for High frequency of recombination) Hfr strains are derived from F+ strains (Figure 7.5a) © McGraw-Hill Education. 7-25 Conjugation and Mapping via Hfr Strains (a) When an F factor integrates into the chromosome, it creates an Hfr cell. © McGraw-Hill Education. 7-26 F Factors can be Excised Imprecisely (1 of 2) In some cases, the integrated F factor is excised in an imprecise fashion (Figure 7.5b) may carry genes that were once found on the bacterial chromosome These types of F factors are called F’ factors © McGraw-Hill Education. 7-27 F Factors can be Excised Imprecisely (b) When an F factor excises imprecisely, an F’ factor is created © McGraw-Hill Education. 7-28 Conjugation between Hfr and F− Strains William Hayes demonstrated that conjugation between an Hfr and an F– strain involves the transfer of a portion of the Hfr bacterial chromosome The origin of transfer of the integrated F factor determines the starting point and direction of the transfer process When the DNA is cut, or nicked, at this site it becomes the starting point for the transfer of the Hfr chromosome to the F– cell Then, a strand of bacterial DNA begins to enter the recipient cell in a linear manner © McGraw-Hill Education. 7-29 Conjugation between Hfr and F− Strains It generally takes about 1.5-2 hours for the entire Hfr chromosome to be passed into the F– cell Most matings do not last that long Only a portion of the Hfr chromosome gets into the F– cell Since the nick is internal to the integrated F factor, only part of the plasmid is transferred and the F– cells does not become F+ © McGraw-Hill Education. 7-30  Conjugation between Hfr and F− Strains The F– cell does pick up chromosomal DNA from the Hfr cell This DNA can recombine with the homologous region on the chromosome of the recipient cell This may provide the recipient cell with a new combination of alleles © McGraw-Hill Education. 7-31 Transfer of Bacterial Genes from an Hfr Cell to an F− Cell Genotype of Hfr cell is: lac+  Ability to metabolize lactose pro+  Ability to synthesize proline Genotype of F- cell is: lac–  Inability to metabolize lactose pro–  Inability to synthesize proline © McGraw-Hill Education. 7-32 Transfer of Bacterial Genes from an Hfr Cell to an F− Cell © McGraw-Hill Education. 7-33 Transfer of Bacterial Genes from an Hfr Cell to an F− Cell © McGraw-Hill Education. 7-34 Experiment- Interrupted Mating Technique Developed by Elie Wollman and François Jacob in the 1950s The rationale behind this mapping strategy The time it takes genes to enter the recipient cell is directly related to their order along the bacterial chromosome The Hfr chromosome is transferred linearly to the F– recipient cell Therefore, interrupting matings at different times would lead to various lengths being transferred The order of genes along the chromosome can be deduced by determining the genes transferred during short matings vs. those transferred during long matings © McGraw-Hill Education. 7-35 Strains for Interrupted Mating The donor (Hfr) strain genetic composition thr + : Able to synthesize the essential amino acid threonine leu + : Able to synthesize the essential amino acid leucine azi s : Sensitive to killing by azide (a toxic chemical) ton s : Sensitive to infection by T1 (a bacterial virus) lac + : Able to metabolize lactose and use it for growth gal + : Able to metabolize galactose and use it for growth str s : Sensitive to killing by streptomycin (an antibiotic) © McGraw-Hill Education. 7-36 Strains for Interrupted Mating The recipient (F–) strain had the opposite genotype thr - : Unable to synthesize the essential amino acid threonine leu - : Unable to synthesize the essential amino acid leucine azi r : Resistant to killing by azide (a toxic chemical) ton r : Resistant to infection by T1 (a bacterial virus) lac - : Unable to metabolize lactose and use it for growth gal - : Unable to metabolize galactose and use it for growth str r : Resistant to killing by streptomycin (an antibiotic) © McGraw-Hill Education. 7-37 The Goal (Discovery-Based Science) Wollman and Jacob already knew that The thr + and leu+ genes were transferred first, in that order Both were transferred within 5-10 minutes of mating The chromosome of the donor strain in an Hfr mating is transferred in a linear manner to the recipient strain The order of genes along the chromosome can be deduced by determining the time various genes take to enter the recipient cell © McGraw-Hill Education. 7-38  The Goal (Discovery-Based Science) Therefore their main goal was to determine the times at which genes azi s, ton s, lac +, and gal + were transferred The transfer of the str s was not examined Streptomycin was used to kill the donor (Hfr) cell following conjugation The recipient (F– cell) is streptomycin resistant © McGraw-Hill Education. 7-39 Achieving the Goal (1 of 2) 1. Mix together a large number of Hfr donor and F- recipient cells. 2. After different periods of time, take a sample of cells and interrupt conjugation in a blender. 3. Plate the cells on solid growth medium lacking threonine and leucine but containing streptomycin. Note: The general methods for growing bacteria in a laboratory are described in the Appendix A. 4. Pick each surviving colony, which would have to be thr+ leu+ strr, and test to see if it is sensitive to killing by azide sensitive to infection by T1 bacteriophage, and able to metabolize lactose or galactose. © McGraw-Hill Education. 7-40 © McGraw-Hill Education. 7-41 The Data Number of Percentage Minutes Percentage of Percentage Percentage of That of Surviving Percentage of Surviving of Surviving Surviving Bacterial Bacterial Surviving Bacterial Bacterial Bacterial Cells Were Colonies Bacterial Colonies Colonies Colonies with Colonies with Allowed to with the with the Following with the the Following the Following Conjugate Following Genotypes: Following Genotypes: Genotypes: Before Genotypes: gal+ Genotypes: azis tons Blender lac+ thr+ leu+ Treatment 5* — — — — — 10 100 12 3 0 0 15 100 70 31 0 0 20 100 88 71 12 0 25 100 92 80 28 0.6 30 100 90 75 36 5 40 100 90 75 38 20 50 100 91 78 42 27 60 100 91 78 42 27 *There were no surviving colonies within the first 5 minutes of conjugation. Source: Data from Francois Jacob and Elie Wollman (1961), Sexuality and the Genetics of Bacteria, Academic Press, New York. © McGraw-Hill Education. 7-42  The Data (2 of 2) KEY RESULTS: There were no surviving colonies after 5 minutes of mating. After 10 minutes, the thr+ leu+ genotype was obtained. Next, the azis gene is transferred, Next, the tons gene, The lac+ gene enters between 15 and 20 minutes, The gal+ gene enters between 20 and 25 minutes. © McGraw-Hill Education. 7-43 A Genetic Map was Constructed From these data, Wollman and Jacob constructed the following genetic map: They also identified various Hfr strains in which the origin of transfer had been integrated at different places in the chromosome Comparison of the order of genes among these strains, demonstrated that the E. coli chromosome is circular © McGraw-Hill Education. 7-44  The E. coli Chromosome Conjugation experiments have been used to map more than 1,000 genes on the E. coli chromosome The E. coli genetic map is 100 minutes long Approximately the time it takes to transfer the complete chromosome in an Hfr mating Refer to Figure 7.8 © McGraw-Hill Education. 7-45 Genetic Map of the E. coli Chromosome (Figure 7.8) (1 of 2) © McGraw-Hill Education. 7-46 Genetic Map of the E. coli Chromosome (Figure 7.8) (2 of 2) The starting point, 0 minutes, is arbitrarily assigned. Units are minutes Refer to the relative time it takes for genes to first enter an F– recipient during a conjugation experiment © McGraw-Hill Education. 7-47 Interrupted Mating Experiment The distance between genes is determined by comparing their times of entry during an interrupted mating experiment The approximate time of entry is computed by extrapolating the time back to the x-axis In this example, these two genes are approximately 9 minutes apart along the E. coli chromosome © McGraw-Hill Education. 7-48 Interrupted Mating Experiment © McGraw-Hill Education. 7-49 Bacterial Transduction Transduction is the transfer of DNA from one bacterium to another via a bacteriophage A bacteriophage is a virus that specifically attacks bacterial cells It is composed of genetic material surrounded by a protein coat Depending on the type of virus, it may follow one of two different cycles, or both Lytic Lysogenic © McGraw-Hill Education. 7-50 Transduction P22, which infects Salmonella typhimurium P1, which infects Escherichia coli Both are temperate phages Cotransduction Mapping There is a maximum size to the DNA that can be packaged by bacteriophages during transduction P1 can pack up to 2-2.5% of the E. coli chromosome P22 can pack up to 1% of the S. typhimurium chromosome Cotransduction refers to the packaging and transfer of two closely-linked genes It is used to determine the order and distance between genes that lie fairly close together Cotransduction Mapping Researchers select for the transduction of one gene They then monitor whether a second gene is cotransduced Consider for example the following two E. coli strains The donor strain with genotype arg+ met + str s The recipient strain with genotype arg – met – str r The steps in a cotransduction experiment are presented in Figure 7.11 © McGraw-Hill Education. 7-54 Cotransduction Mapping Tai Te Wu derived a relationship between cotransduction frequency and map distances obtained from conjugation experiments Cotransduction frequency = (1 – d/L)3 Where d = distance between two genes in minutes L = the size of the transduced DNA (in minutes) For P1 transduction, this size is ~ 2% of the E. coli chromosome, which equals about 2 minutes © McGraw-Hill Education. 7-55  Steps in Cotransduction Donor cell: arg + met + strs © McGraw-Hill Education. 7-56 Steps in Cotransduction Mapping Recipient cells arg – met – strr. © McGraw-Hill Education. 7-57 Steps in Cotransduction Mapping (3 of 3) Results Number of Number of colonies that colonies that Nonselected Cotransduction Selected Gene grew on grew on medium gene frequency medium + - arginine arginine met+ arg+ 50 21 0.42 © McGraw-Hill Education. 7-58 Cotransduction Frequency Calculation 0.42 = (1 − d 2) 3 (1 − d 2) = 0.42 3 1 − d 2 = 0.75 d 2 = 0.25 d = 0.5 minute Therefore, the distance between the met+ and arg+ genes is approximately 0.5 minutes © McGraw-Hill Education. 7-59 Bacterial Transformation Transformation is the process by which a bacterium will take up extracellular DNA released by a dead bacterium It was discovered by Frederick Griffith in 1928 while working with strains of Streptococcus pneumoniae His experiments are outlined in Chapter 9 His experiments are outlined in Chapter 9 © McGraw-Hill Education. 7-60 Bacterial Transformation There are two types Natural transformation DNA uptake occurs without outside help Artificial transformation DNA uptake occurs with the help of special techniques © McGraw-Hill Education. 7-61 Transformation Natural transformation occurs in a wide variety of bacteria Bacterial cells able to take up DNA are termed competent cells They carry genes that encode proteins called competence factors These proteins facilitate the binding, uptake and subsequent incorporation of the DNA into the bacterial chromosome The steps of bacterial transformation are presented in Figure 7.12 © McGraw-Hill Education. 7-62 Transformation Sometimes, the DNA that enters the cell is not homologous to any genes on the chromosome It may be incorporated at a random site on the chromosome This process is termed nonhomologous or illegitimate recombination Some bacteria preferentially take up DNA bacteria from the same or a related species Streptococcus pneumoniae secretes a competence-stimulating peptide which leads to competence only when many cells of the same species are nearby © McGraw-Hill Education. 7-63 Transformation Other species use DNA uptake signal sequences 9 or 10 bp long repeated 1-2,000 times throughout genome DNA with this sequence is preferentially taken up © McGraw-Hill Education. 7-64 Steps of Transformation DNA in the extracellular environment is lys+ Cellular DNA is lys- Heteroduplex is a region of mismatch caused by sequence differences between the two alleles Repaired by DNA repair enzymes © McGraw-Hill Education. 7-65 Steps of Transformation © McGraw-Hill Education. 7-66 Cotransformation Can be used to map genes Method is similar to cotransduction Cotransformation frequency is high for two genes that are close together Will be very low or even zero for distant genes Used only to map genes that are relatively close together © McGraw-Hill Education. 7-67 Artificial Transformation Laboratory method commonly used to get plasmids into cells Common method is to treat with calcium chloride and a high temperature shock Makes the cells permeable to small DNA molecules Electroporation makes cells permeable with external electrical field © McGraw-Hill Education. 7-68 Medical Relevance of Bacterial Genetic Transfer Horizontal Gene Transfer Process in which an organism receives genes from another organism without being a direct offspring Conjugation, transformation and transduction are examples of horizontal gene transfer Can occur within and also between species The types of genes acquired through horizontal gene transfer are quite varied and include genes that confer antibiotic resistance © McGraw-Hill Education. 7-69 Acquired Antibiotic Resistance Horizontal gene transfer has dramatically contributed to the phenomenon of acquired antibiotic resistance Antibiotics are widely used to treat human infections and increase health and size of livestock Bacteria can acquire genes that breakdown the antibiotic, pump it out of the cell or block its inhibiting effects © McGraw-Hill Education. 7-70 Acquired Antibiotic Resistance (2 of 2) Bacterial resistance to antibiotics is a serious problem worldwide Some Staphylococcus aureus strains are resistant to methicillin and all penicillins Resistance probably acquired by horizontal transfer, possibly from Enterococcus faecalis These MRSA strains cause serious skin infections © McGraw-Hill Education. 7-71 Methicillin-Resistant Staphlococcus aureus (MRSA) © McGraw-Hill Education. 7-72