1. Gene Transfer Mechanisms – Conjugation (cont.) Transformation and Transduction

2. Insertion sequences and transposons provide regions of homology between plasmids and chromosomes Brock Biology of Microorganisms, vol. 9, Chapter 9

3. Plasmids can integrate into the host genome at sites of homology insertion sequence

4. Fig. 5.6 Snyder and Champness An integrated conjugal plasmid can drive the transfer of chromosomal genes – initiates directionally at oriT Called a High-frequency of recombination (Hfr) donor – results in many recombinant products Conjugates the a portion of the integrated plasmid and a section of the chromosome The recipient does not receive an intact plasmid

5. Fig. 5.7 Snyder and Champness This transfer process is directional from oriT – genes closer to oriT are transferred relatively efficiently Transfer of genes becomes less efficient as their location is less close to oriT

7. Brock Biology of Microorganisms, vol. 9, Chapter 9 The map of the circular E. coli chromosome was initially established using multiple Hfr strains

8. Fig. 5.8 Snyder and Champness Integrated plasmids can excise from the chromosome at high frequency Aberrant excision events can often lead to a plasmid that now stably carries chromosomal genes This is called a “prime” (eg. F’)

9. Some plasmids, while not self- conjugal, can piggy-back along with conjugal plasmids – these are called mobilizable. Requires that they have a functional oriT that is recognized by the conjugal plasmids Can be engineered to create a mobilizable cloning vector

10. Conjugative Transposons These genetic elements share attributes with conjugal plasmids and transposons A transposition event between two cells Requires all the functions found in conjugation plus those of transposition Scott, 1992, J. Bacteriol. 174:6005

11. Transformation Tranformation is process of taking up naked DNA in a stably inherited form. Two major types of transformation 1.Natural transformation (only a subset of microbes do this) - usually linear DNA 2. Artificially-induced (most, but not all microbes can be induced to take up DNA - usually plasmid DNA A cell that is proficient to take up DNA is described as competent

12. One of the most basic and important techniques in molecular biology is the ability to introduce foreign DNA into a bacterial host. Often achieved by preparing artificially-competent cells E. coli will take up and replicate foreign circular DNA Two types of artificial transformation Chemical competence Electroporation Artificial competence

13. Chemical competence In some bacteria, including E. coli, treatment of cells with divalent cations at low temperature, facilitates the uptake of plasmid DNA into the cell (linear DNA can be taken up, but is shredded by cytoplasmic DNases before it can do anything) Remains unclear how this works Hanahan and Bloom, 1996, Chapter 132, Escherichia coli and Salmonella, ASM Press Uptake channels made of polyP, PHB, and Ca

14. High field strengths result in very transient holes in the cellular envelope Under the appropriate conditions, DNA leaks in and DNA leaks out. A high concentration of plasmid outside results in a rapid influx of plasmids into the cell. Electroporation cuvette Cells go here High voltage shock Electroporation

15. How well has your transformation worked? Transformation efficiency Saturating cells (# of transformants/ug of DNA) 10 6 -10 9 /ug of pBR322 app. 10 11 plasmids/ug pBR322 can also be analyzed as % of cells that receive plasmid Saturating DNA % of DNA molecules that successfully transform cells ProtocolSat. cellsSat. DNA Chemical1%12% Electro10%90%

16. Natural transformation in Gram positives Examples: Streptococcus pneumoniae Bacillus subtilis -no base specificity -limited # of uptake sites (30-75) -nicked internally -complement is degraded during transport -recombines in recipient Dubnau. 1999. Ann. Rev. Microbiol. 53:217

17. Natural transformation in Gram negatives Examples: Haemophilus influenzae Neisseriae gonorrhoeae -sequence specific – uptake sequences -4-8 sites/cell -no cell bound intermediate -import of ds DNA to periplasm -complement is degraded during transport into cytoplasm -recombines in recipient Dubnau. 1999. Ann. Rev. Microbiol. 53:217

18. Gram positive uptake machinery -dedicated machinery for the transport of DNA into the cell Dubnau. 1999. Ann. Rev. Microbiol. 53:217 the reverse of a conjugal transfer system - some components similar to Tra functions

19. Gram-negative uptake machinary -dedicated machinery for the transport of DNA into the cell - must cross periplasm and outer membrane Dubnau. 1999. Ann. Rev. Microbiol. 53:217

20. Energy for driving the process? 1.Intracellular ATP hydrolysis 2.pH gradient – PMF? 3.Complement degradation Function for natural transformation 1.Nutrition 2.DNA repair 3.Genetic diversification

21. Transduction Genetic exchange mediated by bacterial viruses (bacteriophage) Two basic types of bacterial viruses Lytic viruses – infect cells, multiply rapidly, lyse cells Lysogenic viruses – infect cells, can integrate into genome and go dormant (a prophage) - at some point, can excise, multiply and lyse cells

22. Bacteriophage have a range of morphologies from simple filaments to large complex structures May contain either RNA or DNA associated with a protein coat Almost all bacteria have phage associated with them Brock Biology of Microorganisms, vol. 9, Chapter 8

23. Smithsonian (Oct 2000) Attach to specific receptors on the surface of their host bacteria Transfer their nucleic acid into the host cell T4 bacteriophage on the surface of an E. coli cell

24. Lysogeny of bacteriophage Integrate into host genome - Enter a semi-dormant state (eg. Lambda phage) Brock Biology of Microorganisms, vol. 9, Chapter 8

25. Dormant prophage – integrated bacteriophage – carries genes that alter the phenotype of the microbe - best examples are pathogens and toxin production toxin prophage insertion site Corynebacterium diptheriaea Phage produces diptheria toxin This is what makes people sick C.diptheriaea without phage strain produces no toxin Does not cause diptheria Phage conversion