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:
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. (1) Transformation, which involves donor DNA free in the environment
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
Donor
Recipient
Molecules transferred
Product F+ F-
F plasmid
F+ Cell
Hfr
Initiating segment of F plasmid and variable quantity of chromosomal DNA
F- with variable quantity of chromosomal DNA
F+ plasmid and some chromosomal genes it carries with it
F+ Cell with some duplicate gene pairs: one on chromosom, one on plasmid

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 During Conjugation
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 Transformation in 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 which carries 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

51. Wonder of the Genetic Engineering
Expression of luciferase gene in a plant.

52. Background of transduction
Lytic cucle: end in lysis of the host
Lysogeny: after adsorption and penetration, viral genome remains within the host cell and is reporduced along with the bacterial chromosome.thia relationship between the phage and its host is called lysogeny.
Lysogens or lysogenic: bacteria that can produce phage particles under some conditions.
Temperate phages: phages able to eatablish this relationship.
Prophage: the latent form of the virus genome that remains within the host without destroy it.

53. Virus Life Cycle 1. Attachment (adsorption) 2. Penetration (injection)
3. Early steps in replication
4. Replication
5. Synthesis of protein subunits
6. Assembly and packaging
7. Release

54. Temperate Bacterial Viruses: Lysogeny and Lambda
Virulent Viruses
Temperate Viruses
Lysogeny (溶原性）：Viruses can enter a state called lysogeny, where most phage genes are not expressed, and the phage genome is replicated in synchrony with the host chromosome
Prophage or provirus
Can be induced (lysogenic induction) by UV radiation, nitrogen mustards or X-ray.

55. Temperate Bacterial
Viruses: Lysogeny and
Lambda

56. Transduction
Generalized transduction: host genes derived from virtually any portion of the host genome become part of the DNA of the mature virus genome.
Specialized transduction: occurs only in some temperate viruses: a specific group of host genes is integrated directly into virus genome-usually replacing some of the virus genes-and is transferred to the recipient during lysogenization

57. Generalized transduction

58. Abortive transduction
About 70 to 90%of transferred DNA is not integrated but often is able to survive and express itself. Abortive transductants are bacteria that contain this nonintrgrated, transduced DNA and are partial diploids.

59. Specialized Transduction
Under rare conditions, the phage genome is excised incorrectly.
Lambda dgal (defective galactose) under the assistance of helper, the defective phage can be replicated and can transduce the galactose genes.

60. Specialized Transduction
Low-frequency transduction (LFT) lysates: lysates contain only a few transducing particle, the phage genome is excised incorrectly.
Helper phage: defective lambda phages carring the gal gene can integrate if there is a normal lambda phage in the same cell. This normal phage is termed the help phage.
High-frequent transduction (HFT) lysate: a lysate containing a fairly equal mixture of defective lambda dgal phage and normal helper phage.

61. Phage conversion
A prophage is immune to further infection by the same type of phage.
Change in structure of a polysaccharide on the cell surface of Salmonella anatum upon lysogenization with e15,
Conversion of nontoxin producing strains of Corynebacterium diphtheriae to toxin producing (pathogenic) strains.
Information for production of these new materials is apparently an integral part of the phage genome and is automatically and exclusively transferred upon infection by the phage and lysogenization.

62. Transformation
Transduction
Conjugation

63. Transduction
Concept
Transduction involves transfer of host genes from one bacterium to another by viruses. In generalized transduction, defective virus particles randomly incorporate fragments of the cell's chromosomal DNA; virtually any gene of the donor can be transferred, but the efficiency is low. In specialized transduction, the DNA of a temperate virus excises incorrectly and brings adjacent host genes along with it; only genes close to the integration point of the virus are transferred, but the efficiency may be high.

64. Specialized transduction
In transduction, DNA is transferred from cell to cell through the agency of viruses. Genetic transfer of host genes by viruses can occur in two ways.
Generalized transduction
And
Specialized transduction

65. Generalized transduction: host DNA derived from virtually any portion of the host genome becomes a part of the DNA of the mature virus particle in place of the virus genome.
Specialized transduction: occurs only in some temperate viruses; DNA from a specific region of the host chromosome is integrated directly into the virus genome - usually replacing some of the virus genes.

66. Transduction has been found to occur in a variety of prokaryotes, including certain species of the Bacteria: Desulfovibrio, Escherichia, Pseudomonas, Rhodococcus, Rhodobacter, Salmonella, Staphylococcus, and Xanthobacter, as well as the archaean Methanobacterium thermoautotrophicum.
Not all phages can be transducer and not all bacteria are transducible

67. Generalized transduction

68. Generalized transduction
In generalized transduction, virtually any genetic marker can be transferred from donor to recipient
During a lytic infection, the enzymes responsible for packaging viral DNA into the bacteriophage sometimes accidentally package host DNA. This DNA cannot replicate, it can undergo genetic recombination with the DNA of the new host.

69. Specialized Transduction
the DNA of lambda is inserted into the host DNA at the site adjacent to the galactose genes
On induction, Under rare conditions, the phage genome is excised incorrectly
A portion of host DNA is exchanged for phage DNA, called lambda dgal ( dgal means "defective galactose“ )
Phage synthesis is completed
Cell lyses and releases defective phage capable of transducing galactose genes

70. Transfection
Bacteria can be transformed with DNA extracted from a bacterial virus rather than from another bacterium, a process known as transfection.

71. Transformation
Transduction
Conjugation

72. 8.6 Transposons and Insertion Sequences
Transposition: the process by which gene moves from one place to another in the genome.
Transposable elements: transposition of genes is linked to the presence of special genetic elements called transposable elements.
Three types of transposable elements in bacteria:
Insertion sequences (IS)
Transposons (Tn)
Some special viruses (such as Mu)

73. Three types of transposable elements in bacteria
Insertion sequences (IS): about 1000 nucleotides, carry only information to move them to new location (IS1, IS2 and IS3).
Transposons (Tn): larger than IS, carry genes, such as drug resistance markers and other selectable genes.
Some special viruses (such as Mu)

74. Insertion of a transposable element generates a duplication
Both IS and Tn have short
inverted terminal repeats (IR)
at the ends of their DNA, IR are
involved in the transposition
process

75. How is the targeted
sequence duplicated?

76. Transposon mutagenesis
Insertion of transposon within a gene leads to mutation.
Transposon with antibiotic-resistant marker can be used for selection purposes.
Two tranposons widely used: Tn 5 (neomycin and kanamycin resistance), Tn10 (tetracycline resistance).

77. Invertible DNA and the phenomenon of phase variation
Salmonella
flagella
synthesis
When a DNA segment is oriented in one direction, a particular gene is expressed. Whereas when it is oriented in the opposite direction, a different gene is expressed.

78. Questions for Microbial Genetics
Describe as much as you know about plasmids.
What is the difference between a plasmid and an episome?
What are Hfr strain? F+ or F-, or F’ strain?
Draw the F plasmid and describe functions of various DNA regions.
Why is it said that conjugative plasmid contributes to evolution?
How many types of plasmids and their functions you have learned?
Schematically describe R100 plasmid and its functions.
How do R plasmids inactivate antibiotics?
What is an engineered plasmid?
What points do F plasmid provide to its host?
How to detect genetic recombination. Please cite one example.
How is bacterial genome mapped? What are the three types of transposable elements?
Explain transposon mutagenesis and its possible application.
Give an example to explain conversible DNA and phase variation.