Microbial genetics

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. 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. 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. 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 • A pilus is a protein appendage that connects the cells. • Conjugation requires direct contact. http://www.cbs.dtu.dk/staff/dave/roanoke/fig10_10.jpg

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. Hfr bacteria • Hfr = high frequency of recombination • Instead of gene exchange at rate of 1 in 107, rate improves to 1 in 104. • F plasmid is inserted into E. coli chromosome • F plasmid not transferred, rather, E.coli chromosomal genes at high frequency.

7. Hfr strains-2 • In any particular Hfr strain, same genes transferred • Genes transferred determined by where in chromosome the F plasmid was inserted. • 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.

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. 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. 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. 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. 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. Transformation • “Naked” DNA taken up from solution • Bacteria must be “competent” • E. coli treated with high [Ca2] 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. 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. Transformation w/ recombination

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

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. 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/Cannon/Cannon%20Problem%20Set%201.doc