1. Fig. 5-2 Plating bacteria and growing colonies

2. Commonly used genetic markers Prototrophic markers: wild-type bacteria are prototrophs (grow on minimal medium) Auxotrophic markers: mutants that require additional nutrient (fail to grow on minimal medium) Antibiotic-sensitivity: wild-type bacteria are susceptible (fail to grow on antibiotic-containing medium) Antibiotic-resistance: mutants that grow in presence of antibiotic (grow on antibiotic-containing medium)

4. Fig. 5-1 Chapter 5: Genetics of bacteria and their viruses

5. Gene transfer mechanisms in bacteria (especially E. coli) Conjugation: orderly, deliberate transfer of DNA from one cell to another; programmed by specialized genes and organelles. Transformation: uptake of environmental DNA into a cell Transduction: transfer of DNA from one cell to another mediated by a virus

6. Properties of gene transfer in bacteria All are unidirectional (donor – recipient) Recombination requires two steps: 1.Transfer of DNA into the recipient cell, forming a merozygote (various gene transfer mechanisms) 2.Crossing over that replaces a portion of the recipient genome (endogenote) with the homologous portion of the donor genome (exogenote) Transfer is always partial

7. Conjugating E. coli pili Fig. 5-6

8. Fig. 5-7 Conjugation in E. coli is based on the F (fertility) plasmid Replication-coupled transfer of F

9. Fig. 5-8 F can integrate into the bacterial chromosome Hfr: high frequency recombination derivative

10. Fig. 5-10 Transfer of integrated F includes donor chromosome Crossing over of exo/endogenote results in recombinant genome (replacement of a segment of recipient genome with the homologous segment of the donor genome) Unidirectional transfer…… Recombination….. Partial transfer…..

11. DNA transfer during conjugation is time-dependent Transfer of an entire E. coli donor genome requires about 1 hour (F sequence is last to transfer) Therefore, can map the chromosome as a time function: Mix donor Hfr and recipient F- cells Interrupt transfer of DNA at various times (violent mixing in a Waring blendor works!) Plate out cells to determine which genes were transferred within each timeframe

12. Fig. 5-11 Hfr azi r ton r lac + gal + str s X F - azi s ton s lac - gal - str r

13. Fig. 5-11 Hfr azi r ton r lac + gal + str s X F - azi s ton s lac - gal - str r

14. Genetic map generated by interrupted mating experiment

15. Conjugation map depends upon: site of F factor insertion within Hfr chromosome (original F insertion can occur at any one of many sites within chromosome) direction/orientation of the F factor within that Hfr strain (clockwise or counter-clockwise) Mapping using different Hfr strains can provide a map of the entire bacterial chromosome

16. Fig. 5-13

17. Fig. 5-16 Mapping of small regions by recombination

18. Fig. 5-17 F integration by recombination of IS element Excision using another IS element results in F bearing chromosome fragment (F’) Transfer create partial diploid

20. …at least 10 species ancestors. Fig. 5-18

21. Transformation: DNA in the environment of a cell is taken into the recipient cell forming a merozygote; then recombination occurs occurs naturally in some bacteria (e.g., Pneumococcus) occurs rarely in others, but can be promoted by treating cells to destabilize their membranes (e.g., in recombinant DNA work) can map genes by co-transformation (frequency with which two genes are simultaneously transferred

22. Fig. 5-19

23. Transduction: Transfer of DNA from one cell to another mediated by a virus; followed by recombination to integrate the DNA into the recipient cell can map genes by the frequency of co-transduction (frequency of simultaneous transfer of two genes)

24. Fig. 5-22

25. Fig. 5-23 Bacteriophage lytic cycle

26. Plaques (infection bursts) of bacteriophage  on a lawn of E. coli Fig. 5-24

27. Generalized transduction Random DNA fragments are transferred Fig. 5-27

28. Linkage mapping of a segment of the E. coli chromosome by co-transduction experiments with phage P1 Fig. 5-28

29. Fig. 5-30 Lysogenic infection: integration of a viral genome into one of many sites within the host cell chromosome where it quiescently resides Upon specific cues, the process may be reversed, resulting in lytic infection

30. Fig. 5-31 Specialized transduction (genes nearest the insertion site are most efficiently transferred)

31. Fig. 5-