Bacterial Genetics 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; 5th edition) 2. Chapter 25 in Biochemistry (Nelson, D. et al; 4th edition)

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

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

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.

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: 1. 23 chromosomes, two copies (diploid) 2. Extrachromosomal genetic elements: - Mitochondrial DNA - Virus genome 3. Maintained by histones

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

Discovery of DNA synthesis

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

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

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.

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

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

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

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

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

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

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

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

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

Excision repair Base excision repair Nucleotide excision repair

Double-strand break repair (postreplication repair)

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

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

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.

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

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

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

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

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

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

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

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

Mechanisms of evolution of Vancomycin-resistant Staphylococcus Aureus

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

Library construction Genomic library cDNA library

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)

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

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

E Conjugational transposon

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

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

Library construction Genomic library cDNA library

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)

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

Life cycle Phage l as an example Lytic phase Lysogenic phase

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.