1. 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

2. 3. Recombination Bacterial Transformation 1. Lysis 2. DNA Uptake and exchange Cell B: Recipient Bacterial Transformation 1. DNA originating from donor bacteria Cell deathlysisrelease 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 wall  formation/activation of DNA receptor proteins  natural competence eg. Bacillus, Neisseria spp.  artificially induced competence - CaCl 2 /cold treatment - low efficiency, Heat shock / Electrophoration used routinely in DNA cloning eg. E. coli 2

3. 3 3. Integration of transforming DNA homologous recombination, ie. exchange of related sequences Bacterial Transformation (cont.) Related Mechanisms Electroporation  production of small pores in the membrane of cells exposed to pulsed electric fields natural occurrence? - lightning Particle gun  high velocity mini-projectile gun  used for a variety of plants, yeast, algae

4. 4 Transfection uptake of DNA by eukaryotic cells Natural uptake systems phagocytosis 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 the the cytoplasm across the inner membrane [5] Stable integration by homologous recombination

5. 5 Structure DNA uptake competence system in bacteria Transformation

6. 6 Competence in Gram Positive Bacteria ~Bacillus subtilis Streptococcus pneumoniae ~Competence: involve com genes comA comK ~comE comF comG ~Operon comEA : encodes protein that directly bind DNA comF 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 through the peptydolgycan cell wall and across cytoplasmic membrane : under the transcriptional control of comK. Well-studied Involved in regulation of competence Encode structural protein for DNA uptake comK is regulated by comA Competence in Gram Negative Bacteria Acinetobacter calcoaceticus Helicobacter pylori Neisseria spp Haemophilus spp Take up DNA only of the same spesies Utilize two different type of DNA uptake system ~involves protein with structural similarities to the protein used in gram positive ~uses protein related to type IV secretion-conjugation protein ~PSTC: Pilus formation Secretion Twiching motility Competence Pili Overalproteins involved in DNA or protein across cell wall and membrane

7. Sensor protein: ComP in the membrane (protein kinase) Regulation of Competence in Bacillus subtilis [1] Regulation is achieved through two component regulatory system High cell density sensor kinase==  Autophosphorylation Regulator protein: ComA is phosphorylated from ComP (P) Receive PO 4 from ComP phosphorylated. ComA-P become transcriptional activator of genes including operon 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 the competence pheromone==  cut pheromone ComX pheromone phosphorylate ComP ~B. subtilis also produces CSF (Competence stimulating factor), product of phrC gene. CSF peptide is transported into the cell by oligopeptide permease SpoOK. CSF ==  activate ComA-P. Sensor Regulator 7

8. Role of Natural Transformation [1] Nutrition: C, N sources [2] DNA Repair: UV iradiationcell died DNA is released==taken up by other cell to repair the DNA damage [3] Recombination: DNA take up ==  Recombination 8

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10. 10 Transduction by Bacteriophage Bacteriophage and Transduction Bacteriophage  viruses that infect prokaryotic cells  obligate intracellular parasites  two types: I) Lytic Phage - replicate and subsequently lyse host cell eg. phage T4 and T7 (involved in generalised transduction) Transduction – bacteriophage-mediated gene transfer

11. 11 Bacteriophage and Transduction (cont.) II) Temperate phage phage DNA is integrated into the host chromosome (prophage), where it can be stably maintained. phage DNA can be excised, and the phage can 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 and lysogenic cycles

12. 12 Virulent phage are strictly lytic: following infection, they destroy the host chromosome and use host enzymes to replicate viral DNA and make viral proteins. Infected cell then lyses, releasing progeny phage. Rarely, a piece of fragmented host DNA is packaged inside phage head instead of phage DNA. This phage can transduce another cell by introducing an exogenote -- recombination may follow -- generalized transduction.

13. 13 The Lytic Cycle and Generalised Transduction 2. Insertion of phage 1. Attachment DNA into cell of phage 3. Replication of phage DNA and phage packaging Lytic cycle Lytic phage Transducing phage 4. Cell lysis and release of lytic and transducing phages Generalised transduction (cont.) 5. Attachment of transducing phage to new cell 7. Homologous exchange of incoming DNA with chromosomal of recipient 6. Insertion of chromosomal DNA fragment into cell 8. Transduced cell

14. 14 Temperate phage cycle between lytic and lysogenic cycles. In lysogeny, phage genome is incorporated into host chromosome as a prophage, and is replicated along with the host DNA. Integration occurs at specific sites. In its lytic cycle, a prophage separates from the host chromosome and initiates viral replication and host lysis. Once in a while, the “outlooping” will be abnormal, taking part of the host genome along with it. Limited to loci close to integration site: specialized transduction.

15. 15 Lysogeny and Specialised Transduction 1. Attachment of phage 3. Integration of phage DNA into chromosome prophage 2. Insertion of phage DNA into cell Prophage induction leading to excision of phage from chromosome chromosome aberrant excision Cell containing 5. Attachment of phage particles to new cells Normal phage integrated prophage 4, Synthesis of phage coats, phage excision packaging of DNA into phage coats, cell lysis Defective transducing phage SpecialisedTransduction (cont.)

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