1. Bacterial Genetics Pin Ling ( 凌 斌 ), Ph.D. Department of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw Reference: Murray, P. et al., Medical Microbiology (5 th edition)

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

3. 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

4. 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.

5. Human vs Bacterial Chromosome E Coli: 1. Single circular chromosome, double-stranded; one copy (haploid) 2. Extrachromosomal genetic elements: Plasmids (autonomously self- replicating) Phages (bacterial viruses) Transposons (DNA sequences that move within the same or between two DNA molecules) 3. Structurally maintained by polyamines, ex spermine & spermidine Human: 1. 23 chromosomes, two copies (diploid) 2. Extrachromosomal genetic elements: - Mitochondrial DNA - Virus genome 3. Maintained by histones

6. 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 - Topoisomerase, relieve the torsional strain during the process

7. Replication of Bacterial DNA Features: 1. Semiconservative 2. Bidirectional 3. Proofreading (DNA polymerase)

8. Transcriptional Regulation in Bacteria 1.Bacteria regulates 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.

9. 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  -galactosidase => split disaccharide Lac into monosaccgaride Glu & Gal Y gene => lactose permease => pumping Lac into the cell A gene => Acetylase

10. Transcriptional regulation of gene expression (Example I) Negative control Repressor Inducer Operator Lactose operon: Lactose metabolism Under positive or negative ctrl

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

12. Tryptophan operon Transcriptional Regulation of gene expression (Example II) Negative control Repressor Corepressor Operator

13. Attenuation Transcription termination signal

14. 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 Spontaneous mutations Caused by tautomeric shift of the nucleotides; replication errors

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

16. DNA Repair 1. Direct DNA repair (e.g., photoreactivation) 2. Excision repair Base excision repair Nucleotide excision repair 3. Mismatch repair 4. SOS response 5. Error-prone repair Thymine-thymine dimer formed by UV radiation

17. Excision repair Nucleotide excision repair Base excision repair

19. Nucleotide excision repair

20. Double-strand break repair (postreplication repair)

21. 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

22. End-joining (error-prone) Translocation Short deletion at the joining point

23. 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

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

25. Lysogenic phaseLytic phase Life cycle Phage as an example

26. 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.

27. Mechanisms of gene transfer Transformation: uptake of naked exogenous DNA by living cells. Conjugation: mediated by self-transmissible plasmids. Transduction: phage-mediated genetic recombination.

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. Mechanism of Recombination Homologous recombination Site-specific recombination Transposition Illegitimate recombination Intermolecular Intramolecular Double crossover Homologous recombination

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

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

36. E Conjugational transposon

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

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

39. Library construction Genomic library cDNA library

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

41. History of signaling transduction Adopted from Nobelprize.org