Microbial Genetics Genomic structure Replication of chromosomal DNA Regulation of gene expression Mutation, repair and recombination Gene exchange in bacteria Genetic engineering 微免所何漣漪老師

Genomic structure 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) Eukaryotic microbes: fungi, yeasts Eukaryotic genome Chromosomal DNA Mitochondrial DNA Plasmids in yeasts

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

Control of gene expression A bacterial cell may regulate the expression of a certain set of its own genes in response to an environmental change by a variety of mechanisms. Alternative  factors (e.g. sporulation) Regulators (activators or repressors) activated or inactivated by an inducer or corepressor (e.g., cAMP in utilization of lactose; auto-inducers in quorum-sensing) Attenuation

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

Positive control Activator Inducer Lactose operon

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

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 which lead to replication errors

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. Mismatch repair 4. SOS response 5. Error-prone repair Thymine-thymine dimer formed by UV radiation

Excision repair Nucleotide excision repair Base excision repair

Nucleotide excision repair

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

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

Lysogenic phaseLytic phase Life cycle Phage as an example

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.

Natural transformation Transformation Artificial transformation (conventional method and electroporation)

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

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

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

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)

E Conjugational transposon

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

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 (transgenic pants or animals) Production of useful proteins (e.g. insulin, interferon, etc.) in bacteria, yeasts, insect and mammalian cells Recombinant protein (e.g. HBsAg) vaccines and DNA vaccines

Double-strand break repair (postreplication repair)

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