Bacterial Genetics & Bacteriophage

• 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. 醫用微生物學 ( 王聖予 等編譯 , 4th edition)

Outline

• Introduction

• Replication of DNA

• Bacterial Transcription

• Other Genetic Regulation

(Mutation, Repair, &

Recombination)

Introduction

• DNA:

the genetic material• Gene:

a segment of DNA (or chromosome),

the fundamental unit of information in a cell• Genome:

the collection of genes• Chromosome:

the large DNA molecule associated with proteins or other components

Why 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.

Human vs Bacterial Chromosome

E Coli:

1. Single circular chromosome,

double-stranded; one copy (haploid)

2. Extrachromosomal genetic

elements:

Plasmids (autonomously self- replicating)

Bacteriophages (bacterial viruses)

3. Structurally maintained by, ex polyamines, spermine & spermidine

Human:

1. 23 chromosomes, two copies

(diploid)

2. Extrachromosomal genetic ele

ments:

- Mitochondrial DNA

- Virus genome

3. Maintained by histones

Replication of Bacterial DNA

1. Bacterial DNA is the storehouse of information.

=> It is essential to replicate DNA correctly and pass into the daughter cells.

2. Replication of bacterial genome requires several enzymes:

- Replication origin (oriC), a specific sequence in the

chromosome

- Helicase, unwind DNA at the origin

- Primase, synthesize primers to start the process

- DNA polymerase, synthesize a copy of DNA

- DNA ligase, link two DNA fragements

- Topoisomerase, relieve the torsional strain during the

process

Replication of Bacterial DNA

Features:

1.Semiconservative

2. Multiple growing forks

3. Bidirectional

4. Proofreading

(DNA polymerase)

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 transcriptional regulation

Negative control

Repressor

Inducer

Operator

Lactose operon:

Lactose metabolism

Under positive or negative control

Positive control

Activator: CAP (catabolite gene-activator protein)

CAP RNA pol

Inducer

Lactose Operon- Positive Control

Tryptophan operon

Transcriptional Regulation (Example II)

-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

MutationTypes of mutations1. Base substitutions

Silent vs. neutral; missense vs. nonsense2. Deletions3. Insertions4. Rearrangements: duplication, inversion, transposition

May cause frameshift or null mutation

Induced mutationsPhysical 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. Postreplication repair

4. SOS response: induce many genes

5. Error-prone repair: fill 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)

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 bacteriaMediated by plasmids and phages

PlasmidExtrachromosomal

Autonomously replicating

Circular or linear (rarely)

May encode drug resistance or toxins

Various copy numbers

Some are self-transmissible

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.

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)

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