MCB100 Introductory Microbiology March 11, 2019 Chapters 7 and 8

1. MCB100 Introductory Microbiology March 11, 2019 Chapters 7 and 8

2. Exam 2 will be on Wednesday, March 27, 2019. Place: If your last name starts with A – Pe: please take exam in room 112 Gregory If your last name starts with Ph – Z: please take exam in room 213 Gregory Covers chapters: 5, 6, 5, 7, 5, 9 &10 and 5 Review Session: 7:00 – 8:20 pm, Monday, March 25 Place: Room 124 Burrill Hall

3. Three Ways Genetic Recombination Occurs in Bacteria: Transformation – the recipient cell takes up naked DNA Conjugation – one-way transfer of DNA from a donor cell to a touched recipient Transduction – transfer of DNA from a virus-infected cell via a defective phage particle

4. In bacteria, there are 3 ways DNA can move from a donor strain to a recipient strain. 1)Transformation – uptake of naked DNA Transformation – DNA is released from a donor strain by lysis of dead cells, the recipient strain takes up the DNA.

5. Genetic Recombination in Bacteria - Transformation Transformation is a genetic change produced in a bacterium that is caused by the uptake of naked foreign DNA which becomes a part of the recipient cell's genome. Bacterial transformation was discovered by Griffith in 1928 inStreptococcus pneumonia. Discovery of bacterial transformation is described in the text on pgs 228 – 230. Note especially figure 7.30 on page 228 which shows the role of homologous recombination in the insertion and integration of the foreign DNA into the chromosome of the recipient cell. This allows the genotype of the recipient cell to become permanently changed so the trait can be passed on to the offspring of the transformed cell. Note figure 7.31 on page 229 which shows Griffith's experiment. The rough strain (R) of Streptococcus can be genetically transformed into the smooth strain (S) by DNA that is released by heat killed S cells. This can happen both in mice and in a test tube, indicating that it can occur in a natural environment, but the mouse is not required.

6. Genetic Recombination in Bacteria - Transformation The capsule is an important virulence factor in Streptococcus pneumonia. The bacterial capsule is a layer of polysaccharides that coats the outer surface of the cell. In Streptococcus pneumonia the capsule can protect the bacterium from the defensive white blood cells of the host animal. Thus a capsule makes Streptococcusa much more virulent pathogen. (virulent: dangerous, able to cause disease) Encapsulated strains of Streptococcus produce smooth and shiny appearing colonies when grown on an agar medium. Unencapsulated strains produce colonies that appear to be dry or rough.

7. Genetic Recombination in Bacteria - Transformation In 1944 Avery, MacLeod and McCarty showed that the transforming substance from the dead virulent strain was DNA.They observed that an enzyme that breaks down DNA destroyed the transforming agent but enzymes that break down proteins did not.

8. Genetic Recombination in Bacteria - Transformation Bacteria that can take up naked DNA are said to be competent. Some bacteria are naturally competent, including species within the genera: Streptococcus, Staphylococcus, Bacillus, Haemophilus and Pseudomonas. Transformation is most efficient when the size of the DNA is relatively small, 1000 – 2000 base pairs. This is just big enough to contain 1 – 3 genes. Circular supercoiled plasmid DNA works better than linear DNA. Transformation by plasmids that are 4000 – 5000 base pairs long works reasonably well. If one gives the recipient cells a good shock of electricity they are more likely to take up DNA, this procedure is called electroporation. It is not clear how electroporation works, but it is useful with a wide variety of bacteria. Above: general scheme for bacterial transformation with plasmid DNA.To right: device for electroporation, typically shock cells with 2 kV for a few milliseconds.

9. Genetic Recombination in Bacteria - Conjugation 2) Conjugation - DNA is transferred from the donor strain to the recipient strain by direct cell to cell contact, the DNA is transferred through a narrow cytoplasmic bridge. The donor cell is not killed by conjugation and in fact does not usually loose any DNA. Conjugation generally involves replication of DNA that is about to be transferred and donation of one copy to the recipient cell.

10. Conjugation

11. Genetic Transformation in Bacteria - Conjugation In conjugation the DNA travels from cell to cell through a cytoplasmic bridge. A sex pilus, such as the F-pilus, is an appendage that helps bacterial cells stick together so conjugation can occur. The genes for most sex pili are located on plasmids. The genes that encode all of the proteins needed to make the F-pilus are found on the F-plasmid. Most of the time the DNA that is transferred by conjugation is a plasmid, but some plasmids can mobilize the movement of chromosomal genes into a recipient cell. The plasmid is copied as it is transferred so the donor cell does not loose it.

12. Conjugation with Hfr cell

13. F+ strain vs. HFR strain An F+ strain of E. coli has a copy of the F plasmid inits cytoplasm. An HFR strain has the F factor integrated into its chromosome. An F+ strain will transfer genes that are on the F-plasmid but an HFR strain will transfer chromosomal genes that are near the insertion site of the F-factor. The transfer of chromosomal genes by conjugation from an HFR strain occurs in a linear order. Genes that are close to the integrated F-factor are transferred sooner while genes that are farther from the F-factor are transferred later. You can disrupt the transfer of genes by agitating the mating mix in a blender and until the cells separate. By measuring the time it takes for a particular marker gene to be transferred, you can determine the distance between the integrated F-factor and that marker gene. See pages 230 – 234 of your textbook for information about transduction and conjugation, especially figures 7.32 and 7.33. Figures 7.34 and 7.35 describe transfer of chromosomal genes by conjugation from HFR strains.

14. Genetic Recombination in Bacteria - Transduction 3) Transduction - DNA from a donor cell gets incorporated into a defective virus particle called a transducing phage particle. The defective virus attaches to a recipient cell and injects the DNA from the donor strain. Transduction kills the donor strain. General life cycle of a lytic DNA phage and how transducing particles form

15. Transfer of DNA Between Bacteria by Transducing Phages Generalized life cycle of a lytic DNA phage and how transducing particles form.

16. Transduction

17. Genetic Recombination in Bacteria Transformation, Conjugation & Generalized Transduction Choose the response that is TRUE for Conjugation but NOT for Transformation or Transduction. A. The DNA that is transferred from the donor cell to the recipient cell could be a plasmid. B. The transferred piece of DNA must have a replication origin. C. The transferred DNA is encapsulated within a defective bacteriophage particle. D. The recipient cell takes up naked DNA from the environment. E. The donor cell survives and keeps a copy of the DNA that is transferred to the recipient cell.

18. Genetic Recombination in Bacteria Transformation, Conjugation & Generalized Transduction Choose the response that is TRUE for Conjugation but NOT for Transformation or Transduction. A. The DNA that is transferred from the donor cell to the recipient cell could be a plasmid. B. The transferred piece of DNA must have a replication origin. C. The transferred DNA is encapsulated within a defective bacteriophage particle. D. The recipient cell takes up naked DNA from the environment. E. The donor cell survives and keeps a copy of the DNA that is transferred to the recipient cell. (Also: in conjugation the DNA donor cell and the recipient cell have to come into physical contact with each other, that isn’t the case for transformation or transduction.)

19. Genetic Recombination in Bacteria Integration of Foreign DNA Into the Genome of a Recipient Cell Foreign DNA will be degraded unless it can be replicated and maintained in the recipient cell. There are 3 ways foreign DNA can survive in a recipient cell. 1) The foreign DNA can survive if it is an autonomous replicon. DNA replication starts at a specific site called the origin of replication. If the foreign DNA has a functional origin of replication it can be copied within the host cell and passed on to future generations. Examples of autonomous replicons include: the chromosome, plasmids and temperate bacteriophages (a latent virus).

20. Genetic Recombination in Bacteria 3 ways foreign DNA can survive in a recipient cell 2) The foreign DNA integrates into the host chromosome or another autonomous replicon such as a plasmid by homologous recombination.

21. Genetic Recombination in Bacteria 3 ways foreign DNA can survive in a recipient cell 3) The foreign DNA integrates into the host chromosome or another autonomous replicon such as a plasmid by transposition.

22. Genetic Terms What is a “plasmid”? A. A sequence of DNA where the DNA polymerase complex binds and begins synthesis of new DNA B. A defective virus that can transfer bacterial DNA from a donor bacterial cell to a recipient bacterium C. A small circular piece of DNA with an autonomous origin of replication that is similar to a miniature chromosome that contains just a few genes D. A sequence of double-stranded DNA, usually about 1 – 2 thousand base pairs in length, that can be moved from place to place within the genome of an organism by an enzyme called a transposase

23. Genetic Terms What is a “plasmid”? A. A sequence of DNA where the DNA polymerase complex binds and begins synthesis of new DNAorigin of replication B. A defective virus that can transfer bacterial DNA from a donor bacterial cell to a recipient bacterium transducing phage or transducing particle C. A small circular piece of DNA with an autonomous origin of replication that is similar to a miniature chromosome that contains just a few genes D. A sequence of double-stranded DNA, usually about 1 – 2 thousand base pairs in length, that can be moved from place to place within the genome of an organism by an enzyme called a transposasetransposon

24. Regulation of Gene Expression in BacteriaBacterial genes are organized into physically linked coordinately expressed groups of genes called operons.

25. Tryptophan Operon The trp operon is involved with the synthesis of the amino acid tryptophan.A high level of tryptophan in the cytoplasm causes repression of the trp operon. The trp repressor protein is inactive in the absence of tryptophan. Tryptophan acts as an allosteric activator of the repressor activity.

26. Bacterial Operons Complete this sentence correctly: In E. coli, transcription of the trp operon: A. occurs at about the same level all of the time. B. is activated by high levels of tryptophan in the cell. C. is repressed by high levels of tryptophan in the cell. D. is all about the degradation of tryptophan so it can be used as a source of carbon. E. is required for growth on a rich broth that contains all 20 common amino acids.

27. Bacterial Operons Complete this sentence correctly: In E. coli, transcription of the trp operon: A. occurs at about the same level all of the time. B. is activated by high levels of tryptophan in the cell. C. is repressed by high levels of tryptophan in the cell. D. is all about the degradation of tryptophan so it can be used as a source of carbon. No, the trp operon contains genes for making enzymes that are needed to make tryptophan. E. is required for growth on a rich broth that contains all 20 common amino acids. No, if the cell has plenty of tryptophan it doesn’t need to make more. On rich broth, making enzymes that are needed to make tryptophan is a waste of energy.

28. The lac operon in E.coli consists of 3 genes that are involved in the breakdown of the sugar lactose. When lactose is present there is always a small amount of allolactose too. When lactose is absent, the lac repressor protein binds to the operon and blocks transcription. When lactose is present, allolactose binds to the repressor and inactivates it. This allows RNA polymerase to transcribe the operon.

29. Chapter 8 Recombinant DNA Technology Restriction enzymes recognize specific sequences of DNA and make breaks in the backbone chain, often producing sticky ends. Example: EcoRI recognizes: …GAATTC… and cuts between the G and A.

33. Diagram of the PCR cycle 1. Denaturing the DNA at 95oC. 2. Anneal DNA primers at 68oC. 3. Elongation reaction at 72oC. (P = Taq DNA polymerase) 4. 1st cycle complete. Repeat cycle many times. The number of copies of target DNA doubles with each cycle.