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

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

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: • Transfer of DNA into the recipient cell, forming a • merozygote (various gene transfer mechanisms) • 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. Conjugation in E. coli is based on the F (fertility) plasmid Replication-coupled transfer of F Fig. 5-7

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

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

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. Hfr azir tonr lac+ gal+ strsX F-azis tons lac- gal- strr Fig. 5-11

13. Hfr azir tonr lac+ gal+ strsX F-azis tons lac- gal- strr Fig. 5-11

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

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

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. Bacteriophage lytic cycle Fig. 5-23

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. 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 Fig. 5-30

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

31. Fig. 5-

32. Fig. 5-

33. Fig. 5-

34. Fig. 5-