1 transformation and transduction discovery of transformation (griffiths, 1928) streptococcus...

16
1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod discover DNA is the transforming agent (1944) cell capsule Living smooth (S) Living rough (R) X Heat killed S Heat killed S + living R

Upload: marilyn-barnett

Post on 02-Jan-2016

218 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

1

Transformation

and

Transduction

Discovery of transformation (Griffiths, 1928)

Streptococcuspneumoniae

Livingsmooth(S)

+

Avery, McCarty and MacLeod discoverDNA is the transforming agent (1944)

cell

capsule

Livingsmooth (S)

Livingrough (R)

X

Heatkilled S

Heat killed S+ living R

Page 2: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

3. Recombination

Bacterial Transformation

1. Lysis

2. DNAUptake

and exchange

Cell B: Recipient

Bacterial Transformation1. DNA originating from donor bacteria

Cell death lysis release of DNA

2. Uptake of DNA by recipient bacteria

Prior to uptake the cell must be competent

Competence - ability of cell to take up DNA

changes in cell wallformation/activation of DNA receptor proteins

natural competence eg. Bacillus, Neisseria spp.artificially induced competence -

CaCl2/cold treatment - low efficiency,Heat shock / Electrophorationused routinely in DNA cloning eg. E. coli

2

Page 3: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

3

3. Integration of transforming DNA

homologous recombination,ie. exchange of related sequences

Bacterial Transformation (cont.)

Related Mechanisms

Electroporation

production of small pores in themembrane of cells exposed topulsed electric fields

natural occurrence?- lightning

Particle gunhigh velocity mini-projectile gunused for a variety of plants, yeast, algae

Page 4: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

4

Transfectionuptake of DNA by eukaryotic cells

Natural uptake systemsphagocytosis in animals

artificially induced competence in yeast

Related Mechanisms (cont.)

Natural Transformation:General step

[1] Binding of double-stranded DNA to the outer cell surface

[2] Movement of the DNA across the membrane and cell wall

[3] Degradation of one of the DNA strand

[4] Translocation of the remaining single strand of DNA into thethe cytoplasm across the inner membrane

[5] Stable integration by homologous recombination

Page 5: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

5

Structure DNA uptake competence system in bacteria

Transformation

Page 6: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

6

Competence in GramPositive Bacteria

~Bacillus subtilisStreptococcus pneumoniae

~Competence: involve com genescomAcomK

~comEcomFcomG

~Operon comEA : encodes protein that directly bind DNAcomF

comG

~com E, F, G,

: encodes protein that translocate DNA into cell: encodes protein that provide pore or channel

-like structure that allow DNA to move throughthe peptydolgycan cell wall and acrosscytoplasmic membrane

: under the transcriptional control of comK.

Well-studied

Involved in regulation of competence

Encode structural protein forDNA uptake

comK is regulated by comA

Competence in GramNegative Bacteria

Acinetobacter calcoaceticusHelicobacter pyloriNeisseria sppHaemophilus spp

Take up DNA only ofthe same spesies

Utilize two different type of DNA uptake system~involves protein with structural similarities to the proteinused in gram positive

~uses protein related to type IV secretion-conjugation protein~PSTC:Pilus formationSecretionTwiching motilityCompetence

Pili

Overal proteins involved in DNA or proteinacross cell wall and membrane

Page 7: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

Sensor protein : ComP in the membrane (protein kinase)

Regulation of Competence inBacillus subtilis

[1] Regulation is achieved through two componentregulatory system

High cell density sensor kinase==Autophosphorylation

Regulator protein: ComA is phosphorylated from ComP (P)Receive PO4 from ComP phosphorylated.ComA-P become transcriptional activator of genes includingoperon srfA (required for competance).

[2] Competence pheromone~High density of pheromone (small peptide)Pheromone peptide is cut out (product of comX gene).ComQ upstream of comX, is also required for synthesis of thecompetence pheromone==cut pheromoneComX pheromone phosphorylate ComP

~B. subtilis also produces CSF (Competence stimulating factor),product of phrC gene.CSF peptide is transported into the cell by oligopeptidepermease SpoOK.CSF ==activate ComA-P.

Sensor

Regulator

7

Page 8: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

Role of NaturalTransformation

[1] Nutrition : C, N sources[2] DNA Repair: UV iradiationcell died

DNA is released==taken up by other cellto repair the DNA damage

[3] Recombination: DNA take up==Recombination

8

Page 9: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

9

Page 10: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

10

Transduction by Bacteriophage

Bacteriophage and Transduction

Bacteriophageviruses that infect prokaryotic cellsobligate intracellular parasitestwo types:

I) Lytic Phage- replicate and subsequently lysehost cell

eg. phage T4 and T7

(involved in generalised transduction)

Transduction –

bacteriophage-mediated gene transfer

Page 11: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

11

Bacteriophage and Transduction (cont.)

II) Temperate phagephage DNA is integrated into the hostchromosome (prophage), where it can bestably maintained.

phage DNA can be excised, and the phagecan enter the lytic cycle

eg. phage lambda (λ) and P2

(involved in specialised transduction)

Lytic and temperate bacteriophages of E. coli

Bacteriophage T4 (lytic) Bacteriophage lambda (λ)temperate: both lytic andlysogenic cycles

Page 12: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

12

Virulent phage are strictly lytic: followinginfection, they destroy the host chromosomeand use host enzymes to replicate viral DNAand make viral proteins.

Infected cell then lyses, releasing progeny phage.

Rarely, a piece of fragmented host DNAis packaged inside phage head instead ofphage DNA.

This phage can transduce another cell byintroducing an exogenote -- recombinationmay follow -- generalized transduction.

Page 13: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

13

The Lytic Cycle and Generalised Transduction2. Insertionof phage

1. Attachment DNA into cellof phage 3. Replication of

phage DNAand phagepackaging

Lyticcycle

Lytic phage

Transducing phage

4. Cell lysis andrelease of lytic andtransducing phages

Generalised transduction (cont.)

5. Attachment oftransducingphage to new cell

7. Homologous exchangeof incoming DNA withchromosomal of recipient

6. Insertion of chromosomalDNA fragment into cell

8. Transduced cell

Page 14: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

14

Temperate phage cycle between lytic andlysogenic cycles.

In lysogeny, phage genome is incorporatedinto host chromosome as a prophage, and isreplicated along with the host DNA.

Integration occurs at specific sites.

In its lytic cycle, a prophage separates fromthe host chromosome and initiates viralreplication and host lysis.

Once in a while, the “outlooping” will beabnormal, taking part of the host genomealong with it.

Limited to loci close to integration site:specialized transduction.

Page 15: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

15

Lysogeny and Specialised Transduction1. Attachment

of phage

3. Integration of phageDNA into chromosome

prophage prophage

2. Insertion of phageDNA into cell

Prophage induction leading toexcision of phage from chromosome

chromosome

aberrantexcisionexcision

Cell containing

5. Attachmentof phageparticles

to new cells

Normalphage

integrated prophage

4, Synthesis of phage coats,phage excision packaging ofDNA into phage coats, cell lysis

Defectivetransducingphage

SpecialisedTransduction (cont.)

Page 16: 1 Transformation and Transduction Discovery of transformation (Griffiths, 1928) Streptococcus pneumoniae Living smooth (S) + Avery, McCarty and MacLeod

16