2. Extrachromosomal genetic elements

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Dec 14, 2012 (4 years and 7 months ago)

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Bacterial Genetics


Pin Ling (



),
Ph.D.


Department of Microbiology & Immunology, NCKU


ext 5632


lingpin@mail.ncku.edu.tw



Reference: Murray, P. et al., Medical Microbiology
(5
th

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)



Phages

(bacterial viruses)


Transposons

(DNA sequences that
move within the same or between
two DNA molecules)

3. Structurally 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

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



-

Topoisomerase, relieve the torsional strain during the


process

Replication of Bacterial DNA

Features:

1. Semiconservative

2. Bidirectional

3. Proofreading (DNA
polymerase)


Transcriptional Regulation in Bacteria

1.
Bacteria regulates 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




monosaccgaride Glu & Gal




Y gene => lactose permease => pumping Lac into the cell




A gene => Acetylase




Transcriptional regulation of gene
expression (Example I)

Negative control


Repressor


Inducer


Operator

Lactose operon:

Lactose
metabolism

Under positive or
negative ctrl

Positive control


Activator: CAP
(catabolite
gene
-
activator
protein)


CAP


RNA
pol


Inducer

Lactose Operon: Positive Control

Tryptophan operon

Transcriptional Regulation of gene
expression (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

Caused 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

Nucleotide
excision
repair

Base excision
repair

Base excision
repair

Nucleotide
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

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)

Icosahedral
tailess

Icosahedral
tailed

Filamentous

Structure and genetic materials of phages


Coat (Capsid)


Nucleic acid

Lysogenic phase

Lytic phase

Life cycle

Phage
l

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


Production of useful proteins (e.g. insulin,
interferon, etc.) in bacteria, yeasts, insect and
mammalian cells


Recombinant vaccines (e.g. HBsAg)

History of signaling transduction

Adopted from Nobelprize.org