1. 1 Microbial genetics Microbes have been important in genetic research –Short reproductive cycles –Millions of progeny in a short time –Studied in pure culture, variants can be examined –Single piece of DNA usually; no masking of traits –Easy to create, isolate, identify mutants –Bacteria are the source of restriction endonucleases Bacteriophages: viruses that infect bacteria –Typically destroy the bacterium, release new virions

2. 2 Terminology in microbial genetics Prototroph: “original” and “feed”, a wild type strain, one able to synthesize all needed compounds from a simple carbon source such as glucose. Auxotroph: a mutant that has lost the ability to make some necessary organic compound; it must be added to the culture medium. Bacteria show horizontal gene transmission –Acquire new genetic information naturally –Acquire genetic info from genetic engineering

3. 3 Plasmids Plasmids are small, circular DNA molecules –Plasmids are found in the cytoplasm of many bacteria –Plasmids are not essential for survival of the cell –They may exist singly or in many copies –Plasmids have a variety of functions Examples: metabolic, resistance, cryptic Fertility plasmids, such as F factor, allow conjugation (direct cell-cell gene exchange) F plasmids are found in E. coli –F+ strains are considered male, F- are female

4. 4 Conjugation Mechanism by which one bacterium transfers genes to another –Can occur be related and unrelated bacteria –Usually involves transfer of a plasmid –Involves attachment between bacteria w/ a pilus http://www.cbs.dtu.dk/staff/ dave/roanoke/fig10_10.jpg A pilus is a protein appendage that connects the cells. Conjugation requires direct contact.

5. 5 Conjugation: F plasmids The “feminist’s nightmare”: male cells transfer the F plasmid to F- cells, changing them to F+ (male) –F plasmid codes for genes that produce a pilus and other genes for transfer of genetic material –F+ cells attach to F- cells w/ pilus; –DNA unwinds, and a ss DNA is transferred from the donor to the recipient cell. –DNA synthesis occurs in both, making ds DNA. –Genetic recombination: replacement of old genes w/ new ones Fertility plasmids “mobilize” other genes

6. 6 Hfr bacteria Hfr = high frequency of recombination –Instead of gene exchange at rate of 1 in 10 7, rate improves to 1 in 10 4. –F plasmid is inserted into E. coli chromosome –F plasmid not transferred, rather, E.coli chromosomal genes at high frequency.

7. 7 Hfr strains-2 If plasmid is inserted near a, b genes, those are transferred during conjugation. If plasmid is inserted near g, h genes, those are transferred during conjugation. In any particular Hfr strain, same genes transferred Genes transferred determined by where in chromosome the F plasmid was inserted.

8. 8 Genetic mapping in E. coli Conjugation between prototroph and/or antibiotic resistant Hfr strain and auxotroph strain. –Hfr strain should transfer genes that will “cure” auxotroph. Interrupted mating technique –Hfr (donor strain) mixed with recipient strain. –Samples removal at various times, placed in blender to shear off pili and break up mating. –Cells were plated onto medium and tested for prototrophy, that is, are they “cured?”

9. 9 Mapping-2 Data was collected based on how many minutes of conjugation (standard conditions) it took for a gene to be transfer and thus “cure” the recipient. –This allowed the genes to be placed in order: the longer it took for transfer, the farther away the gene. http://www.mun.ca/biochem/courses/4103/figures/Snyder-Champness/F14-3.jpg

10. 10 Mapping-3 These data were collected for several different Hfr strains and pooled. –The order came up the same, but one end overlapped the other. Conclusion: E. coli has a circular chromosome. –Circular DNA is the rule for bacteria. –Map units are in minutes, reflecting the methodology used. http://www.cbs.dtu.dk/staff/dave/roanoke/fig10_33.jpg

11. 11 More about plasmids and conjugation R plasmids –Code for resistance to antibiotics, heavy metals, etc. –Usually contain RTF (resistance transfer factor) Responsible for transfer of plasmid to other bacteria, transferring antibiotic resistance. –Major factor in the spread of resistance among bacteria http://www.med.sc.edu:85/mayer/trans-14.jpg

12. 12 Mechanisms of horizontal gene transmission Conjugation –Bacteria make direct contact with pilus –Transfer genes directly –Both related and unrelated partners Transformation –“naked” DNA in solution Transduction. –Requires bacteriophage, virus that infects bacteria www.nature.com/.../ 031013/full/031013-2.html

13. 13 Transformation “Naked” DNA taken up from solution –Bacteria must be “competent” E. coli treated with high [Ca 2 ] for example –DNA binds to receptor sites on surface –DNA brought into cell by active transport process One DNA strand is used –One strand is digested leaving ssDNA –ss DNA forms heteroduplex with recipient DNA Recombination event, one old strand degraded Transformation between close relatives only.

14. 14 Transformation-2 When bacterium divides, each strand of heteroduplex is copied –One bacterium has old phenotype, one shows new phenotype from the newly acquired DNA Transformation can be used for some mapping –Genes are said to be “linked” if they are close enough together to be on same piece of DNA 10,000- 20,000 bp, enough for several genes If several mutant phenotypes are cured simultaneously, genes are close together.

15. 15 Transformation w/ recombination

16. 16 Viral life cycles Transduction is gene transfer by bacteriophages –Bacteriophages (“phage”) are viruses that infect bacteria Understanding the action of viruses: The Lytic Cycle –Phage attaches to bacteria surface, injects DNA –Viral DNA takes over cell, uses cell machinery to Produce new copies of viral DNA Synthesize viral proteins Destroy host DNA by cutting it into pieces –Viruses self-assemble

17. 17 Viral life cycles (continued) Lytic cycle (continued) –After self-assembly, viruses lyse cell, escape, spread to neighboring bacteria and infect them. Such viruses are called virulent or lytic phage. Alternative pathway to reproduction: lysogeny –Carried out by “temperate” phages –Once in cell, viral DNA incorporates into host DNA –When the bacterium reproduces, viral DNA is copied. –Harmful stimuli (e.g. UV light) causes viral DNA to excise, begin lytic cycle.

18. 18 Transduction Generalized transduction –Occurs when host DNA piece is incorporated into phage “head” instead of viral DNA –Binding of virus particle to recipient, injection of DNA: bacterial DNA is injected instead. Specialized transduction –Prophage: the viral DNA while it exists only as a piece of DNA with the bacterial DNA. –First, prophage excises, begins lytic cycle usually because of damage to host DNA, pulls part of host DNA from “next door” with it when it excises –DNA containing phage and host DNA is packaged.

19. 19 Transduction visual Red: phage DNA; Blue: bacterial DNA http://fig.cox.miami.edu/Faculty/Dana/transduction.jpg

20. 20 Summary: Gene transfer in bacteria Conjugation: direct contact via pilus –Mediated by plasmids –Doesn’t necessarily require close relationships R plasmids: no recombination, so no DNA homology needed. Transformation: naked DNA from solution –Competent cells only –Recombination takes place; DNA homology needed. Transduction: DNA carried by a virus –For greatest effect, DNA homology needed.

21. 21 Genetic notation in bacteria –leu - leu + etc. –LacZ is a protein, lacZ is the gene!! Try these bacterial genetics problems: http://www.bio.unc.edu/courses/2006Fall/biol621/Can non/Cannon%20Problem%20Set%201.doc