1. Bacterial Genetics Pin Lin (凌 斌), Ph.D.
Departg ment of Microbiology & Immunology, NCKU
ext 5632
References:
1. Chapters 5 in Medical Microbiology (Murray, P. R. et al; 5th edition)
2. Chapter 25 in Biochemistry (Nelson, D. et al; 4th edition)

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

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

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

6. Replication of Bacterial DNA-I
Features:
1.Semi-conservative
2. Multiple growing
forks
3. Bidirectional
4. Proofreading
(DNA polymerase)
Bacterial DNA is the storehouse of information.
=> Essential to replicate DNA correctly => Daughter cells

7. Discovery of DNA synthesis

8. Replication of Bacterial DNA-II
Replication of bacterial genome requires several enzymes:
- Helicase, unwind DNA at the replication origin (OriC)
- Primase, synthesize primers to start the process
- DNA polymerase, synthesize a copy of DNA, first found by Arthur Kornberg
- DNA ligase, link two DNA fragements
- Topoisomerase, relieve the torsional strain during the
process, found by James Wang

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

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

12. Lactose Operon
E Coli can use either Glucose or other sugars (ex: lactose) as the source of carbon & energy.
In Glu-medium, the activity of the enzymes need to metabolize Lactose is very low.
Switching to the Lac-medium, the Lac-metabolizing enzymes become increased for this change .
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

13. Lactose Operon-Negative Control
Lac Operon:
- Lac metabolism
- Under pos & neg control
Negative ctrl
- Repressor
- Inducer (Allolactose)
- Operator
In presence of Lactose

14. Lactose Operon-Positive Control
Activator:
CAP-cAMP
(catabolite gene-activator protein)
CAP RNA pol
In absence of Lactose

16. Tryptophan Operon Negative control - Repressor
- Corepressor (Tryptophan)
- Operator

17. Transcription termination signal
Attenuation
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
Transcription termination signal

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

19. Types of mutations 1. Base substitutions
Silent mutation – No change of amino acid
Missense mutation – Switch to another amino acid
Nonsense mutation – Change to a stop codon
2. Deletion & Insertion - Change more base pairs in DNA
=> frameshift => truncated gene product
3. Rearrangements - Duplication, Inversion, Transposition

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

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

22. Excision repair
Base excision repair
Nucleotide excision repair

23. Double-strand break repair
(postreplication repair)

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

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

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

30. Gene exchange by Plasmids
Extrachromosomal
Autonomously replicating
Circular or linear (rarely)
May encode drug resistance or toxins
Various copy numbers
Some are self-transmissible

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

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

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

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

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

36. Mechanisms of evolution of Vancomycin-resistant Staphylococcus Aureus

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

39. Library construction
Genomic library
cDNA library

40. Applications of genetic engineering
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)

41. Take-Home Question: Mutations are good or bad for bacteria
The End & Thank You

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

43. E Conjugational transposon

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

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

46. Library construction
Genomic library
cDNA library

47. 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)

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

49. Life cycle
Phage l as an example
Lytic phase
Lysogenic phase

50. 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.