Microbial Genetics Eukaryotic microbes: fungi, yeasts Eukaryotic genome Chromosomal DNA Mitochondrial DNA Plasmids in yeast Prokaryotic microbes: bacteria Prokaryotic genome Chromosomal DNA: double-stranded; circular; haploid. Extrachromosomal genetic elements Plasmids (autonomously self- replicating) Phages (bacterial viruses) Transposons (DNA sequences that move within the same or between two DNA molecules)

Replication of chromosomal DNA Replication of bacterial genome requires: Replication origin (oriC) DNA polymerase Primase Helicase Topoisomerase Semiconservative Bidirectional

Regulation of gene expression at transcriptional level (Example I) Operon Negative control Repressor Inducer Operator Lactose operon

Positive control Activator Inducer Lactose operon

Regulation of gene expression at transcriptional level (Example II) Negative control Repressor Corepressor Operator Tryptophan operon

Attenuation Transcription termination signal

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 Cuased by tautomeric shift of the nucleotides; replication errors

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

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

Excision repair Base excision repair Nucleotide 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

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

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

Bacteriophage (bacterial viruse) 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.

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

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

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

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

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

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.

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

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)