Business

2. Business • Chapter 7: Problems 1-20, • Master Solutions: Problems 1 and 2, • Master Concepts in action: all, • Questions about anything?

5. Bacteria Do Almost Everything Bacteria Predominate • Metabolism; • Phototrophs, • Chemotrophs, • Biochemistry; • ‘fix’ or synthesize a huge range of molecules, • break down almost anything, • adapt to just about anything. • Molecular Biology; • Clone, • Gene therapy, • Eugenics, • Biotechnology, • Etc. • 10,000+ “Species”, • Mycoplasma genetalium • 200 nm • Thiomargarista namibiensis • 750 mm • soil, water, air, symbionts, • have adapted to aquatic and terrestrial extremes, • 100 grams/person, • 1014 bacteria.

8. Bacterial Chromosome ...a circular molecule of double helical DNA, • 4 - 5 Mb long in most species studied, • 1.6 mm long if broken and stretched out. • Inside the cell, the circular chromosome is condensed by super coiling and looping into a densely packed body termed the nucleoid.

9. Extra Chromosomal DNA • Plasmids: circular double stranded DNA molecule that replicates independently, • containing one or more (non-essential) genes, smaller than the bacterial chromosome, • may carries genes for pathogenicity, • may carry genes for adaptation to the environment, including drug resistance genes, • 1000’s of base pairs long.

10. Bacterial Model OrganismEscherichia coli = E. coli • Enteric bacteria: inhabits intestinal tracts, • generally non-pathogenic, • grows in liquid, • grows in air, • E. coli has all the enzymes it needs for amino-acid and nucleotide biosynthesis, • can grow on minimal media (carbon source and inorganic salts), • Divides about every hour on minimal media, • up to 24 generations a day,

11. Growth Equals Cell Division

12. DNA Replication

13. Bacterial mitosis. Why don’t bacteria do meiosis?

14. Liquid Cultures, • 109cells/microliter, • Colonies on Agar, • 107+ cells/colony The (Awesome) Power of Bacterial Genetics ... is the potential for studying rare events.

15. Counting Bacteria 10-3 10-4 10-5 (Serial) Dilution is the Solution

16. Model Model Organism • Ease of cultivation, • Rapid Reproduction, • Small size, • Fecund (large brood size), • Mutants are available, stable and easy to identify? • Literature? • PubMed Listings: Eubacteria: 612,471, Archaebacteria: 9,420

17. Bacteria Phenotypes • colony morphology, • large, small, shiny, dull, round or irregular, • resistance to bactericidal agents, • cells unable to synthesize certain raw materials from a minimal media, • cells unable to break down complex molecules, • essential genes, usually studied as conditional mutants.

18. Prototroph …a cell that is capable of growing on a defined, minimal media (all essentials salts, carbon source), • can synthesize all essential organic compounds, • usually considered the ‘wild-type’ strain. Auxotrophs …a cell that requires a substance for growth that can be produced by a wild-type cell, his- ...can’t synthesize histidine (his+ = wt) leu- ...can’t synthesize leucine (leu+ = wt) arg- ...can’t synthesize arginine (his+ = wt) bio- ...can’t synthesize biotin (bio+ = wt)

19. thr+ leu+ thi+ met+ bio+ Bacterial Nomenclature I • genes not specified are considered wild-type, Strain A: met- bio- x Strain B: thr- leu- thi-

20. Complete Media… • Contains all essential nutrients, plus a carbon source, and supplemented with specific complex molecules, • - bacteria uptake • molecules that it can • not synthesize. • Minimal Media… • Contains all essential nutrients (usually as salts), plus a carbon source, • - bacteria synthesizes the • rest.

21. exconjugate Strain C: met+ bio+ thr+ leu+ thi+ 1 in 107awesome Strain A: met- bio- tri+ leu+ thi+ x Strain B: met+ bio+ thr- leu- thi- Conjugation

22. Conjugation ...temporary fusion of two single-celled organisms for the transfer of genetic material, …the transfer of genetic material is unidirectional. F+ Cells(F for Fertility) F- Cells(F for Fertility) … F+ cells donate genetic material. … F- cells receive genetic material, …there is no reciprocal transfer.

23. Contact is Required For Mating

24. F+ F- F Pilus …a filament-like projection from the surface of a bacterium.

25. F Factor …a plasmid whose presence confers F+, or donor ability.

26. F Pilus Attaches to F- Cell

27. Passageway Forms Cells are Drawn Together

28. DNA is Nicked, One Strand is Transferred, Both Strands Replicate Single Strand Moves Across Pilus The single stranded plasmids synthesize complementary strands in their respective cells .

29. Cell Separation Both cells are now F+

30. F Factor Replicates During Binary Fission

31. Properties of the F Factor • Can replicate its own DNA, • Carries genes required for the synthesis of pili, • F+ and F- cells can conjugate, • the F factor is copied to the F- cell, resulting in two F+ cells, • F+ cells do not conjugate with F+ cells, • F Factor sometimes integrates into the bacterial chromosome.

32. ...F factor integration site, ...host (bacteria chromosome) integration site. Hfr Cells F factor Bacterial Chromosome Inserted F plasmid

34. ...the bacterial chromosome contains many integration sites, ...thus, the F factor inserts in different regions of the bacterial chromosome.

35. High Frequency of Recombination(Hfr) ...bacteria exhibiting a high frequency of recombination, • an alteration of DNA sequence such that the genotype of subsequent individuals differs from the parent, …HFR cells; strains with a chromosome integrated F factor that is able to mobilize and transfer part of the chromosome to the F- cell.

36. F Pilus Attaches to F- Cell

37. Hfr DNA is Cut

38. F factor and Chromosomal DNA are Transferred

39. Double Crossover Recombination Requires Crossing over

40. DNA not Incorporated into Chromosome are Digested

41. Incomplete Transfer of DNA • Interrupted Mating: a break in the pilus during conjugation stops the transfer of DNA, • Transfer occurs at a constant rate, • provides a means to map bacterial genes.

42. Hfr to F- Cells • Hfr transfer begins in the middle of the F factor, • Only 1/10,000 cells transfer the complete chromosome, including the remainder of the F factor, • Thus, most recipient cells remain F-.

43. F’Cells • an F factor from an Hfr cell excises out of the bacterial genome and returns to plasmid form, • often carries one or more bacterial genes along, • F’cells behave like an F+ cells, • merizygote: partially diploid for genes copied on the F’plasmid, • F’plasmids can be easily constructed using molecular biology techniques (i.e.vectors).

44. Mutation in Bacteria …the ultimate source of variation in bacteria is spontaneous mutation, • generally errors in DNA replication, …mutations occur in specific genes at a rate of 1 in 106 to 1 in 107 cells, …adaptive mutations are quickly replicated and adaptive colonies predominate.

45. Spontaneous Mutations • DNA replication in E. coli occurs with an error every ~ 109 bases. • The E. coli genome is 4.6 x 106 bases. • An error occurs once per ~ 2000 replications. • If a single colony has 107 bacteria, • 5,000 cells carry a mutation, • or, one mutation every ~ 1,000 bases (across a colony), • or, a mutation in about every gene.

46. Selective Media • wild-type bacteria grow on minimal media, • media supplemented with selected compounds supports growth of mutant strains, • minimal media + leucine supports leu- cells, • minimal media + leucine + arginine supports leu- arg- • etc. • Selective Media: a media in which only the desired strain will grow.

47. Selection ...the process that establishes conditions in which only the desired mutant will grow.

48. Problem You want to create a culture of cells that are only methionine auxitrophs?

49. The Wrong Answer Strain 1 x Strain 3 (complete media) You have a recombinant that is met- only. How do you get rid of Strain 1?

50. Correct Answer Strain 2 x Strain 3 (complete media) Grow on Minimal Media Plus Methionine Strain 2 dies because there is no arginine. Strain 3 dies because there is no threonine or thimine. The new exconjugate lives.