2. Bacterial Genetics & Bacteriophage Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References: 1. Chapters 5 in Medical Microbiology (Murray, P. R. et al; 5 th edition) 2. 醫用微生物學 ( 王聖予 等編譯, 4th edition)

3. Outline Introduction Replication of DNA Bacterial Transcription Other Genetic Regulation (Mutation, Repair, & Recombination)

4. Introduction DNA: the genetic material Gene: a segment of DNA (or chromosome), the fundamental unit of information in a cell Genome: the collection of genes Chromosome: the large DNA molecule associated with proteins or other components

5. Why we study Bacterial Genetics? Bacterial genetics is the foundation of the modern Genetic Engineering & Molecular Biology. The best way to conquer bacterial disease is to understand bacteria first.

6. Human vs Bacterial Chromosome E Coli: 1. Single circular chromosome, double-stranded; one copy (haploid) 2. Extrachromosomal genetic elements: Plasmids (autonomously self- replicating) Bacteriophages (bacterial viruses) 3. Structurally maintained by, ex polyamines, spermine & spermidine Human: 1. 23 chromosomes, two copies (diploid) 2. Extrachromosomal genetic elements: - Mitochondrial DNA - Virus genome 3. Maintained by histones

7. Replication of Bacterial DNA 1.Bacterial DNA is the storehouse of information. => It is essential to replicate DNA correctly and pass into the daughter cells. 2. Replication of bacterial genome requires several enzymes: - Replication origin (oriC), a specific sequence in the chromosome - Helicase, unwind DNA at the origin - Primase, synthesize primers to start the process - DNA polymerase, synthesize a copy of DNA - DNA ligase, link two DNA fragements - Topoisomerase, relieve the torsional strain during the process

8. Replication of Bacterial DNA Features: 1.Semiconservative 2. Multiple growing forks 3. Bidirectional 4. Proofreading (DNA polymerase)

9. Transcriptional Regulation in Bacteria 1.Bacteria regulate expression of a set of genes coordinately & quickly in response to environmental changes. 2.Operon: the organization of a set of genes in a biochemical pathway. 3.Transcription of the gene is regulated directly by RNA polymerase and “repressors” or “inducers”. 4.The Ribosome bind to the mRNA while it is being transcribed from the DNA.

10. Lactose Operon 1.E Coli can use either Glucose or other sugars (ex: lactose) as the source of carbon & energy. 2.In Glu-medium, the activity of the enzymes need to metabolize Lactose is very low. 3.Switching to the Lac-medium, the Lac-metabolizing enzymes become increased for this change. 4.These enzymes encoded by Lac operon: Z gene => b-galactosidase => split disaccharide Lac into monosaccharide Glu & Gal Y gene => lactose permease => pumping Lac into the cell A gene => Acetylase

11. Lactose Operon- Negative transcriptional regulation Negative control Repressor Inducer Operator Lactose operon: Lactose metabolism Under positive or negative control

12. Positive control Activator: CAP (catabolite gene-activator protein) CAP  RNA pol Inducer Lactose Operon- Positive Control

13. Tryptophan operon Transcriptional Regulation (Example II) -Tryptophan operon Negative control - Repressor - Corepressor (Tryptophan) - Operator

14. Attenuation Transcription termination signal Couple Translation w/ Transcription Sequence 3:4 pair -G-C rich stem loop - Called attenuator -Like transcriptional terminator Sequence2: 3 pair - weak loop won’t block translation

15. Mutation Types of mutations 1. Base substitutions Silent vs. neutral; missense vs. nonsense 2. Deletions 3. Insertions 4. Rearrangements: duplication, inversion, transposition May cause frameshift or null mutation

16. Induced mutations Physical mutagens: e.g., UV irradiation (heat, ionizing radiation) Chemical mutagens Base analog Frameshift intercalating agents Base modification Transposable elements Mutator strains

17. DNA Repair 1. Direct DNA repair (e.g., photoreactivation) 2. Excision repair Base excision repair Nucleotide excision repair 3. Postreplication repair 4. SOS response: induce many genes 5. Error-prone repair: fill gaps with random sequences Thymine-thymine dimer formed by UV radiation

18. Excision repair Nucleotide excision repair Base excision repair

19. Double-strand break repair (postreplication repair)

20. SOS repair in bacteria 1.Inducible system used only when error-free mechanisms of repair cannot cope with damage 2.Insert random nucleotides in place of the damaged ones 3.Error-prone

21. Gene exchange in bacteria Mediated by plasmids and phages Plasmid Extrachromosomal Autonomously replicating Circular or linear (rarely) May encode drug resistance or toxins Various copy numbers Some are self-transmissible

22. Bacteriophage (bacterial virus) Icosahedral tailess Icosahedral tailed Filamentous Structure and genetic materials of phages Coat (Capsid) Nucleic acid

23. Lysogenic phaseLytic phase Life cycle Phage as an example

24. Virulent phages: undergo only lytic cycle Temperate phages: undergo both lytic and lysogenic cycles Plaques: a hollow formed on a bacterial lawn resulting from infection of the bacterial cells by phages.

25. Mechanisms of gene transfer Transformation: uptake of naked exogenous DNA by living cells. Conjugation: mediated by self-transmissible plasmids. Transduction: phage-mediated genetic recombination. Transposons: DNA sequences that move within the same or between two DNA molecules

26. Importance of gene transfer to bacteria Gene transfer => a source of genetic variation => alters the genotype of bacteria. The new genetic information acquired allows the bacteria to adapt to changing environmental conditions through natural selection. Drug resistance (R plasmids) Pathogenicity (bacterial virulence) Transposons greatly expand the opportunity for gene movement.

28. Natural transformation Transformation Artificial transformation (conventional method and electroporation)

29. Demonstration of transformation Avery, MacLeod, and McCarty (1944)

30. Conjugation mediated by self-transmissible plasmids (e.g., F plasmid; R plasmids)

31. F’ plasmid Hfr strain F plasmid F plasmid can integrate into bacterial chromosome to generate Hfr (high frequency of recombination) donors Excision of F plasmid can produce a recombinant F plasmid (F’) which contains a fragment of bacterial chromosomal DNA F plasmid --an episome

32. Transduction phage-mediated genetic recombination Generalized v.s. specialized transduction

33. Transposons Mobile genetic elements May carry drug resistance genes Sometimes insert into genes and inactivate them (insertional mutation)

34. Trans-Gram gene transfer Spread of transposon throughout a bacterial population

35. Mechanisms of evolution of Vancomycin- resistant Staphylococcus Aureus

36. Cloning Cloning vectors plasmids phages Restriction enzymes Ligase In vitro phage packaging

37. Library construction Genomic library cDNA library

38. 1. Construction of industrially important bacteria 2. Genetic engineering of plants and animals 3. Production of useful proteins (e.g. insulin, interferon, etc.) in bacteria, yeasts, insect and mammalian cells 4. Recombinant vaccines (e.g. HBsAg) Applications of genetic engineering