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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
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
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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
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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
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Structure DNA uptake competence system in bacteria
Transformation
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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
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
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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
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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
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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
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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.
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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
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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.
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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.)
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