Chapter8 Microbial genetics

1. Chapter8 Microbial genetics 8.4 Plasmids 8.5 Genetic Conjugation, Transformation, transduction 8.6 Transposons and Insertion Sequences

2. 8.4 Plasmids Circular genetic elements that reproduce autonomously and have an extra-chromosomal existence: • 1-1000 KB in size • Typical plasmid 1/20 of chromosome • Most are circular double-stranded DNA, some linear ds DNA • Transmitted from cell to cell via conjugation process • Some can integrated into chromosome • Can carry a variety of genes for production of toxin, resistance to antibiotics and heavy metals et al.

3. Plasmid • Conjugative: plasmids which govern their own transfer by cell-to-cell contact are called conjugative • Tra region: a set of genes within the plasmid that control the transmissability by conjugation • Hfr (high frequency of recombination): strains of bacteria that transfer large amounts of chromosomal DNA during conjugation • Supercoil: plasmids isolated from the cells are in supercoiled configuration • Plasmid separation: by ultracentrifugation or electrophoresis • Curing of plasmids: elimination of plasmids from host cells by various treatments.

4. Plasmids • Replication: Most plasmids of gram-positive bacteria replicate by a rolling circle mechanism. • Copy number: The number of plasmids in a cell, can range from only 1-3 copies to 100 copies. • Incompatibility: Two different types of plasmids can not coexist in a cell. • Episomes: Plasmids having the ability to integrate into host chromosome

5. Col plasmids • Bacteria also harbor plasmids with genes that may give them a competitive advantage in the microbial word. • Bacteriocins are bacterial protein that destroy other bacteria. Usually act only against closely related strains

6. F-Plasmid-Fertility Plasmids • 100 KB • Can be cured with acridine orange • Incompatibility (inc) • Origin of replication (oriS) • Transposable elements (Tn) • tra region • phi: phage inhibition • IS (insertion sequence) • rep: replication functions

7. Cell to Cell Transfer of Plasmids • Conjugative: Plasmids that govern their own transfer by cell-to-cell contact are called conjugative (not all plasmids are conjugative) • Some conjugative plasmids can transfer genetic information between distintly related organisms (between gram-positive and gram-negative bacteria, between bacteria and plant cells, and between bacteria and fungi), it is important for evolution. Conjugation (接合作用）

8. Plasmid Biology Episomes

9. Plasmid Biology

10. Types of Plasmids and Their Biological Significance • The presence of plasmids in a cell can have a profound influence on the cell’s phenotype: • the ability of conjugation • the ability of Rhizobium to interact with plants • the resistance to antibiotics and heavy metals • the degradation of octane, camphor et al • the production of enterotoxin • the applications in genetic engineering

11. Resistance Plasmids (R-Plasmids): the most well studied plasmids • The emergence of bacteria resistant to several antibiotics is medically significant • Resistance can be transferred via cell-to-cell contact • This could be one of the reasons for the rapid rise of multiply resistant strains • Plasmid recombination is one mean by which multiply resistant organisms might have first arisen • Infectious nature of the R plasmids permits rapid spread of the characteristic through populations • Typical example: plasmid R100

12. The presence of multiple antibiotic resistance is due to the fact that a single R plasmid contains a variety of genes coding for different antibiotic inactiviation enzymes Biochemical mechanism of resistance mediated by R plasmids

13. 8.5 Three main processes of genetic recombination in prokaryotes fragments of homologous DNA from a donor chromosome are transferred to a recipient cell (1) Transformation, which involves donor DNA free in the environment (2) Transduction, in which the donor DNA transfer is mediated by a virus (3) Conjugation, in which the transfer involves cell-to-cell contact and a conjugative plasmid in the donor cell

14. DNA Transfer in Bacteria transformation transduction conjugation

15. 8.5.1 Conjugation • Conjugative plasmids possess genetic information to code for sex pili and for some proteins needed for DNA transfer. • Rolling circle replication occurs for DNA transfer during conjugation. F plasmid of E. coli has the special property of being able to mobilize the chromosome so that it can be transferred during cell-to-cell contact.

16. Conjugation and Chromosome Mobilization:F+ and F- strains • F+ strains: cells possessing an unintegrated F plasmid. • F- strains: cells which can act as recipients for F+ or Hfr, F- strains lack F plasmid. • F plasmid provides its host cell with: • ability to synthesize the F pilus • mobilization of DNA for transfer to another cell • alteration of surface receptors so that the cell is no longer able to behave as a recipient in conjugation

17. Integration of an F plasmid into the chromosome with the formation of an Hfr. IS elements are the sites of insertion. homology

18. Hfr strain • Hfr strains arise as a result of the integration of the F plasmid into the chromosome

19. Important Concept: F’ plasmids • Integrated F plasmids may be occasionally excised from the chromosome and bring some chromosomal genes with itself into the liberated F plasmid. • F’-mediated transfer resembles specialized transduction in that only a restricted group of chromosomal genes can be transferred.

22. Result of selected conjugation

24. Transfer of plasmid DNA by conjugation • The F plasmid of an F+ cell is being transferred to a F- recipient cell

25. Details of the replication and transfer process

26. Detection of Genetic Conjugation

28. Manner of formation of different Hfr strains • The direction in which the F factor is inserted determines which of the chromosomal genes will be inserted first into the recipient

29. Interrupted Mating • Mixing Hfr and F- cells. • Shake the mixture violently at various time. • Plate the samples on selective medium for recombinant to grow. Mapping the order of genes

31. Conjugation involves a donor cell, which contains a particular type of conjugative plasmid, and a recipient cell, which does not. The genes that control conjugation are contained in the tra region of the plasmid (see Section 9.8 in your text ). Many genes in the tra region have to do with the synthesis of a surface structure, the sex pilus . Only donor cells have these pili, The pili make specific contact with a receptor on the recipient and then retract, pulling the two cells together. The contacts between the donor and recipient cells then become stabilized, probably from fusion of the outer membranes, and the DNA is then transferred from one cell to another.

32. Mechanism of DNA Transfer DuringConjugation A mechanism of DNA synthesis in certain bacteriophages, called rolling circle replication, was presented here to explains DNA transfer during conjugation . if the DNA of the donor is labeled, some labeled DNA is transferred to the recipient but only a single labeled strand is transferred. Therefore, at the end of the process, both donor and recipient possess completely formed plasmids.

33. Genetic Recombination • Homologous or General Recombination • RecA protein participation • Homologous DNA sequences have the same or nearly the same sequence • New genotypes only arise when two homologous sequences are genetically distinct

34. Detection of Recombination • Requirement: reverse mutation for the selected characteristic must be low. This problem can often be overcome by using double mutants.

35. Complementation Test:cis-tran test • trans configuration: two mutations are each on separate DNA molecules • cis configuration: Two mutations were on the same DNA molecule • Complementation does not involve recombination

36. DNA transformation • 1928, Fred Griffith • Competent: cells able to take up a molecule of DNA.Competency is a complex phenomeono and is dependent on several conditions. 1. Bacteria need to be in a certain stage of growth. 2. Secrete a small protein called the competence factor that stimulates the production of 8 to 10 new proteins reauired for transformation. • Natural transformation has been discovered so far only in certain genera: Streptococcus, Bacillus, Thermoactinomytes, Haemophilus, Neisseria, Moraxella, Acinetobacter, Azotobacter, Pseudoomonas

37. The mechanism of Transformationin S. pneumoniae 1. A competent cell binds a ds DNA fragment 2. The DNA is cleaved by endonucleases to 5-15kb. 3. One stand is hydrolyzed by an envelop-associated exonuclease, the other strand associate with small proteins and moves through the plasma membrane. 4. Integration of transforming DNA

38. The transformation of Haemophilus influenzae • Difference: 1. Haemophilus does not produce a competence factor to stimulate the development of competence. • 2. It takes up DNA from only closed related species. • 3. Ds DNA, complexed with proteins, is taken in by membrane vesicles. • 4. DNA must have a special sequence (5’AAGTGCGGTCA3’) to be bound by a competent cell.

39. Transformation A number of prokaryotes have been found to be naturally transformable, including certain species of both gram-negative and gram-positive Bacteria and some species of Archaea. However, even within transformable genera, only certain strains or species are transformable

40. Competence A cell that is able to take up a molecule of DNA and be transformed is said to be competent. Competence in most naturally transformable bacteria is regulated, and special proteins play a role in the uptake and processing of DNA. These competence-specific proteins may include a membrane-associated DNA binding protein, a cell wall autolysin, and various nucleases. Competent cells bind much more DNA than do noncompetent cells as much as 1000 times more

41. Artificially Induced Competence High efficiency natural transformation is found only in a few bacteria; Azotobacter, Bacillus, Streptococcus,, for example, are easily transformed. Many prokaryotes are transformed only poorly or not at all under natural conditions. Determination of how to induce competence in such bacteria may involve considerable empirical study, with variation in culture medium, temperature, and other factors when E. coli is treated with high concentrations of calcium ions and then stored in the cold, the transformation by plasmid DNA is relatively efficient.

42. The introduction of DNA into cells by mixing the DNA and the cell • Binding of free DNA by a membrane-bound DNA binding protein. • (b) Passage of one of the two strands into the cell while nuclease activity degrades the other strand. • (c) The single strand in the cell is bound by specific proteins, and recombination with homologous regions of the bacterial chromosome mediated by RecA protein occurs. Transformed cell

43. DNA Transfer by Electroporation for artificial induction of competence are being supplanted by a new method termed electroporation. Small pores are produced in the membranes of cells exposed to pulsed electric fields. When DNA molecules are present outside the cells during the electric pulse, they can then enter the cells through these pores. This process is called electroporation.

44. The mechanism of bacterial transformation

45. Other methods for introducing DNA into bacterial cells • Transfection: transformed DNA is extracted from a bacterial virus • Artificially induced competence: e.g treat E. coli with high concentration of Ca ions, and then stored the cells at low T, the E. coli will become competent at low efficiency • Electroporation: pulsed electrical fields generate pores in the cell membranes, allowing DNA molecules to enter the cells. • DNA from any source can be introduced into bacteria by splicing it into a plasmid before transformation

46. Transformation (transfection) of eukaryotic cells • Transfection: introducing DNA into mammalian cells • phagocytosis in animal cells • Yeast: spheroplasts added with Ca ions plus polyethylene glycol • Electroporation • Particle gun, or gens gun

47. Agrobacterium and Plant Interactions: Crown gall and Hairy Root • Crown gall: caused by Agrobacterium tumefaciens whichcarries a Ti (Tumor induction) plasmid that promotes the crown gall formation • Hairy Root: caused by Agrobacterium rhizogenes which carries a Ri plasmid that leads to hairy roots formation

48. Overview of events of crown gall disease following infection of A. tumefaciens

49. Ti plasmid of Agrobacterium tumefaciens

50. Mechanism of transfer of T-DNA to the plant cell