Bacterial Genetics

1. Bacterial Genetics

2. Overview • Two general mechanisms of genetic change in bacteria: • Mutation - alteration in existing DNA sequence • DNA transfer - acquisition of DNA from another source • Spontaneous • Induced (caused by mutagens)

3. Overview • Two general mechanisms of genetic change in bacteria: • Mutation - alteration in existing DNA sequence • DNA transfer - acquisition of DNA from another source • Spontaneous • Induced (caused by mutagens) • Why study bacterial genetics? • Model system • Spontaneous mutations occur in all cells at a very low frequency (≈one per billion nucleotides) • Bacteria quickly grow to high concentrations (109/ml) in culture, making it possible to study rare occurrences • Test chemicals for potential carcinogens • Understand bacterial adaptation • Resistance to antimicrobial drugs

4. Agency Urges Change in Antibiotics for Gonorrhea By LAWRENCE K. ALTMAN Published: NY Times April 13, 2007 The rates of drug-resistant gonorrhea in the United States have increased so greatly in the last five years that doctors should now treat the infection with a different class of antibiotics, the last line of defense for the sexually transmitted disease, officials said yesterday….. No new antibiotics for gonorrhea are in the pipeline, officials of the centers told reporters by telephone. “Now we are down to one class of drugs,” said Dr. Gail Bolan, an expert in sexually transmitted diseases at the California Department of Health Services. “That’s a very perilous situation to be in.”

5. Overview • Two general mechanisms of genetic change in bacteria: • Mutation - alteration in existing DNA sequence • DNA transfer - acquisition of DNA from another source • Spontaneous • Induced (caused by mutagens) • Why study bacterial genetics? • Model system • Spontaneous mutations occur in all cells at a very low frequency (≈one per billion nucleotides) • Bacteria quickly grow to high concentrations (109/ml) in culture, making it possible to study rare occurrences • Some mutagens are carcinogens • Understand bacterial adaptation • Resistance to antimicrobial drugs • Acquisition of disease-causing traits

6. Terms Phenotype Genotype

7. disrupt gene required for histidine synthesis Terms Phenotype - the observable characteristics of an organism Genotype - the sequence of nucleotides in the DNA of an organism Wild type - characteristics similar to the organism as it occurs in nature. Prototroph - requires the same nutrients as the wild type. Auxotroph - a strain that has lost the ability to synthesize a specific compound; as a consequence, that compound must be supplied as a nutrient in the growth medium. Prototroph His- auxotroph When studying mutations, you only see what you look for

8. Part I Mutation • How mutations occur, and their consequences • How cells can repair errors/damage • How we can select (and therefore, study) mutants

9. Spontaneous Mutation • Mistakes during replication • Base substitution TGT cysteine Silent mutation Missense mutation Nonsense mutation TGC cysteine TGG tryptophan TGA Stop codon No consequence Consequence varies Truncated protein; generally non-functional

10. Spontaneous Mutation • Mistakes during replication • Base substitution • Removal or addition of nucleotides TGTTTGACCTAGGT

11. TGT TTG ACC TAG GT TGT TGA CCT AGG T Spontaneous Mutation • Mistakes during replication • Base substitution • Removal or addition of nucleotides TGTTTGACCTAGGT TGTTGACCTAGGT • Frameshift mutation • Generates an entirely different set of triplets • Often, a stop codon is generated

12. Spontaneous Mutation • Mistakes during replication • Base substitution • Removal or addition of nucleotides

13. Spontaneous Mutation Transposons “jumping genes” • Insertional inactivation of the gene in which the transposon lands • A transposon can insert elsewhere in the same DNA molecule, or into an entirely different DNA molecule • Some transposons simply “hop”; others replicate then hop

14. Summary • Mutations • spontaneous • mistakes during replication • base substitution • addition/removal of nucleotides • transposable elements • induced

15. Induced Mutation • Chemical mutagens (potential carcinogens) • Chemicals that modify purines and pyrimidines • Alter the base-pairing properties

16. Induced Mutation • Chemical mutagens • Chemicals that modify purines and pyrimidines • Alter the base-pairing properties • Example: nitrous acid strips the amino group from nucleotides :A :G

17. Induced Mutation • Chemical mutagens • Chemicals that modify purines and pyrimidines • Alter the base-pairing properties • Example: nitrous acid strips the amino group from nucleotides • Base analogs • Resemble nucleotide bases; erroneously incorporated into DNA • Analog base-pairs with a different nucleotide T C

18. Induced Mutation • Chemical mutagens • Chemicals that modify purines and pyrimidines • Alter the base-pairing properties • Example: nitrous acid strips the amino group from nucleotides • Base analogs • Resemble nucleotide bases; erroneously incorporated into DNA • Analog base-pairs with a different nucleotide • Intercalating agents • Insert between base-pairs, pushing nucleotides apart; extra nucleotide may then be erroneously added during replication

19. Induced Mutation Transposons • Intentional use of an agent that naturally creates spontaneous mutations

20. Induced Mutation Radiation • Ultraviolet irradiation • Causes formation of covalent bonds (thymine dimers) between adjacent thymine bases • Distorted DNA can be repaired, but the process (SOS repair) may introduce errors • High doses are used to sterilize surfaces, lower doses to introduce mutations • X rays • Causes double- and single-stranded breaks in DNA

21. Summary • Mutations • spontaneous • mistakes during replication • base substitution • removal or addition of nucleotides • transposable elements • induced • chemical mutagens • radiation • transposons

22. DNA Repair • Repair of errors in base incorporation • DNA polymerase • proofreading • Mismatch repair • excision/replacement Repair of thymine dimmers

23. DNA Repair • Repair of errors in base incorporation • DNA polymerase • proofreading • Mismatch repair • excision/replacement Repair of thymine dimmers • Light reactivation (photorepair)

24. DNA Repair • Repair of errors in base incorporation • DNA polymerase • proofreading • Mismatch repair • excision/replacement Repair of thymine dimmers • Light reactivation (photorepair) • Excision repair (dark repair; light-independent repair)

25. DNA Repair • Repair of errors in base incorporation • DNA polymerase • proofreading • Mismatch repair • excision/replacement Repair of thymine dimers • Light reactivation (photorepair) • Excision repair (dark repair; light-independent repair) Repair of Modified Bases • Glycosylase removes oxidized guanine • SOS repair • Induction of SOS system • New polymerase (tolerates “slop”)

26. Prototroph (revertant) Auxotrophs Minimal medium (glucose-salts) Enriched complex medium Mutant Selection • Direct selection • Obtain resistant mutants (ex. antibiotic resistant) • Obtain prototrophs that have reverted from auxotrophs • Application of direct selection • Ames Test - screens for mutagens • (used to narrow down list of possible carcinogens)

27. The Ames Test Also do expt. with liver extract added

28. Mutant Selection • Indirect selection (replica plating) • Obtain auxotrophs 106 prototrophs 1 auxotroph

29. Indirect selection (replica plating) • Obtain auxotrophs

30. Indirect selection (replica plating) • Obtain auxotrophs Joshua and Esther Lederberg

31. Summary • Mutations • spontaneous • mistakes during replication • transposons • induced • chemical mutagens • radiation • transposons • Repair • repair of errors in base incorporation • repair of thymine dimmers • SOS repair • Selecting mutants • direct - obtain antibiotic resistant mutants, Ames test • indirect - obtain prototrophs

32. Part II DNA Transfer Donor Recipient Horizontal (lateral) transfer

33. DNA Transfer 1920s; Frederick Griffith-strains of Streptococcus pneumoniae that produce capsules kill mice “transforming principle” (DNA)

34. DNA Transfer 1/109 =10-9 10-9 x 10-9 = 10-18

35. DNA Transfer Donor Recipient To be stably maintained, transferred DNA must either be a plasmid (has an origin of replication), or integrate into the host cell’s genome

36. DNA Transfer Donor Recipient To be stably maintained, transferred DNA must either be a plasmid (has an origin of replication), or integrate into the host cell’s genome

37. DNA Transfer Donor Recipient Integrate into host genome by Homologous recombination (site-specific recombination)

38. DNA Transfer Donor Recipient Integrate into host chromosome by Homologous recombination (site-specific recombination)

39. DNA Transfer Donor Recipient Integrate into host chromosome by Homologous recombination (site-specific recombination)

40. replication DNA Transfer Donor Recipient Integrate into host chromosome by Homologous recombination (site-specific recombination) heteroduplex

41. DNA Transfer Donor Recipient Integrate into host chromosome by Homologous recombination (site-specific recombination)

42. Donor Recipient DNA Transfer Horizontal (lateral) gene transfer A+, B- A-, B+ B- A- A+, B+ A-, B- A+, B-

43. DNA Transfer Donor Recipient • DNA-mediated transformation • Transduction • Conjugation

44. DNA-Mediated Transformation Uptake of naked DNA • Process is sensitive to the addition of DNAse

45. Natural competence • Artificial competence DNA-Mediated Transformation Uptake of naked DNA • Process is sensitive to the addition of DNAse Recipient cell must be competent • Observed in only certain species • Example - Streptococcus pneumoniae (GPC) • Example - Haemophilus influenzae (GNR) • Becomes competent in late log phase • Competent cell binds ds DNA • Enzymes cut DNA into smaller fragments (5 - 15 kb) • Single strand is taken up by cell • Cell binds DNA only from related species • Takes up ds DNA • In the laboratory, treat cells with specific chemicals (plasmids taken up)

46. Conjugation Requires cell-to-cell contact • Involves a conjugative plasmid • F plasmid (fertility plasmid) serves as a model • Three types of donors: • F+ • Hfr • F’

47. Conjugation: F+ donor “male” “female”

48. Conjugation: F+ donor

49. Conjugation: F+ donor

50. Conjugation: F+ donor In donor cell, replication replaces strand that’s being transferred In recipient cell, complement to transferred strand is synthesized