Bacterial Genetics Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU...

Bacterial Genetics

• Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKUext 5632lingpin@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)

Introductio

n• 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 copi

es (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

• Introduction

• Replication of DNA

• Bacterial Transcription

• Other Genetic Regulation (Mutation,

Repair, & Recombination)

Transcriptional Regulation in Bacteria

1. Bacteria regulate 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.

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 => 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

Negative ctrl

- Repressor

- Inducer (Allolactose)

- Operator

Lac Operon:

- Lac metabolism

- Under pos & neg control

In presence of Lactose

Positive control

Activator:

CAP-cAMP

(catabolite gene-activator protein)

CAP RNA pol

Lactose Operon-Positive Control

In absence of Lactose

Tryptophan OperonNegative control- Repressor- Corepressor (Tryptophan)- Operator

Attenuation

Transcription termination signal

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

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 acidMissense 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 mutationsPhysical mutagens:

e.g., UV irradiation (heat, ionizing radiation)

Chemical mutagens

Base analog

Frameshift

intercalating agents

Base modification

Transposable elements

DNA Repair1. 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

Nucleotide excision repair

Base excision repair

Double-strand break repair(postreplication repair)

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

SOS repair in bacteria

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.

Natural transformation

Transformation

Artificial transformation(conventional method and electroporation)

Demonstration of

transformation

Avery, MacLeod, and McCarty (1944)

Gene exchange by Plasmids

PlasmidExtrachromosomal

Autonomously replicating

Circular or linear (rarely)

May encode drug resistance or toxins

Various copy numbers

Some are self-transmissible

Conjugationmediated 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

Transductionphage-mediated genetic recombination

Generalized v.s. specialized transduction

TransposonsMobile genetic elements

May carry drug resistance genes

Sometimes insert into genes and inactivate them (insertional mutation)

Trans-Gram gene transfer

Spread of transposon throughout a bacterial population

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

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)

Applications of genetic engineering

The End & Thank You

Take-Home Question: Mutations are good or bad for bacteria

Mechanism of Recombination

Homologous recombination Site-specific recombination

Transposition Illegitimate recombination

Intermolecular

Intramolecular

Double crossover

Homologous recombination

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

Production of useful proteins (e.g. insulin, interf

eron, etc.) in bacteria, yeasts, insect and mam

malian cells

Recombinant vaccines (e.g. HBsAg)

Bacteriophage (bacterial virus)

Icosahedral tailess

Icosahedral tailed

Filamentous

Structure and genetic materials of phages

Coat (Capsid)

Nucleic acid

Lysogenic phaseLytic phase

Life cyclePhage 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.