the bacterial genetics - university of baghdad bacterial genome plasmids plasmids is a small genetic...
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The Bacterial Genome
The Bacterial Genome
The Bacterial Genome is
the total collection of
genes carried by a
bacterium both on its
chromosome and on its
extrachromosomal genetic
elements (plasmids)
The Bacterial Genome
A Gene
A gene (unit of heredity) is a nucleotide sequence of DNA which
determines the synthesis of polypeptide, or replication of suitable
molecule of RNA
Genes essential for bacterial growth are carried on a single
chromosome - nucleoid
All the bacterial cells are therefore haploid
The Bacterial Genome
DNA Structure
a) A schematic, nonhelical model
b) A schematic drawing of the
Watson-Crick structure of DNA,
showing helical sugar-phosphate
backbones of the two strands held
together by hydrogen bonding
between the basis
c) Space-filling model of DNA
The Bacterial Genome
DNA Forms
b) The circular DNA strands, already
coiled in a double helix, are twisted a
second time to produce supercoils
a) The DNA double helix of most
procaryotes has the shape of a
closed circle
The Bacterial Genome
DNA Forms
The total length of E.coli chromosome is about 1 mm
The Bacterium itself is only several micrometers in length
The DNA is about 1000 times longer than bacterium and
must be condensed (supercoiling)
The Bacterial Genome
DNA Replication
Semiconservative
Replication of DNA
The replication fork of DNA
showing the synthesis of two
progeny strands. Each copy
contain one new and one old
strand.
Bacterial chromosome is
called Replicon
Replicon – a part of the
genome that contains an
origin site and is replicated
as a whole unit
The Bacterial Genome
DNA Replication
An autoradiograph of a replicating E.coli chromosome; about one-third of the
chromosome has been replicated
The Bacterial Genome
Replication of circular DNA
Conjugative
Mechanism used by
some bacterial
viruses, plasmids
and in chromosomal
transfer
The Bacterial Genome
Transposable elements
IS – Insertion sequences
Tn - Transposones
IS are small DNA pieces, about 1-2 kilobases long with 2
distinctive traits:
- CORE area containing genes for transposition
- Specific INVERTED REPEATS at their ends
Tn is a genetic element that containing genes for transposition
and additional extragenes, which provide resistance against
antibiotics or synthesis enzymes of specific metabolic
pathways
These elements can transfer from chromosomal location to
another, and from a chromosome to a plasmid or back
The Bacterial Genome
Transposable elements
Main roles:
• Cause deletion and inversions of DNA sequences (internal or biological mutagenic agents)
• Insert into genes and inactivate those genes
• Spread of antibiotic resistance genes
Mobile genetic elements are responsible for the major part of genetic variability in natural bacterial populations
Tn-s may enter other genera of bacteria during transfer of plasmids or via transducing phage
The Bacterial Genome
Plasmids
Plasmids is a small genetic elements
capable of independent replication in
bacteria.
Most plasmids are circular double-
stranded DNA molecule, but some are
linear.
Plasmids made their presence by
conferring phenotypes of cell harboring
them.
F - fertility factor
R - antibiotic resistance
Col - colicin production
Virulence plasmids:
Ent - enterotoxin production
Hly - hemolysin production
CFA-I; CFA-II - adhesins production
F-Plasmid
The Bacterial Genome
Bacteriophages
Virulent bacteriophage – a bacteriophage which always
causes the lytic cycle, resulting in a death of a cell and
production of new phage particles
Temperate (or lysogenic) bacteriophage – a bacteriophage
whose DNA integrates into bacterial chromosome, but doesn’t
cause the lysis of the cell and production new phage particles.
The integrative phage’s DNA is called prophage
A bacterial cell with prophage is called lysogenic cell
The Bacterial Genome
Bacteriophages
Lysogenic conversion is a state when bacterial cell exhibit
new properties, that are coded by the prophage genes
Example:
- Toxigenic (tox+) and nontoxigenic strains of Corynebacterium
diphtheriae
- Production of erythrogenic toxin by Streptococcus pyogenes
- Production botulinal toxin of Clostridium botulinum
The Bacterial Genome
Genetic Variation
Mutations are stable hereditary
changes in the coding sequence
of DNA
Occur spontaneously or are
induced by different mutagens
Various kind of mutations
The Bacterial Genome
Genetic Variation
Genetic recombination at the molecular level is the process by which DNA from a donor cell and DNA from a recipient cell combine to yield a new genome containing information from both sources
Molecular Mechanisms of
Recombination
Homologous
(legitimate)Nonhomologous
(illegitimate)
The Bacterial Genome
Genetic Variation
Homologues recombination occurs between closely related DNA sequences and generally substitutes one sequence to another.
The process requires a set of enzymes produced by the group “rec” genes, and homology in 100-200 n.p.
Nonhomologues recombination occurs between dissimilar DNA sequences and generally produces insertions or deletions or both.
This process usually requires specialized (site-specific) recombination enzymes, such as those produced by many transposons and lysogenic bacteriophages
The Bacterial Genome
Gene Transfer
The transfer of genetic material between procaryotes is
called horizontal (or lateral) gene transfer.
It takes place in one of 3 ways:
1. Transformation
2. Conjugation
3. Transduction
The Bacterial Genome
Gene Transfer
Transformation
Was discovered by Griffits in 1928 during the
experiments with Streptococcus pneumoniae.
Later Avery, MacLeod and McCarty identified
the DNA as the transforming agent and as the
genetic material
Transformation is the process by which
bacteria take up fragment of naked DNA and
incorporate this molecule into recipient
chromosome in a heritable form
The Bacterial Genome
Gene Transfer
Transformation
The requirement for the DNA fragment from the donor bacterium
Molecular weight (M) 105 -106 D
Double-stranded fragment of DNA
The state of recipient cell is called competence
Competency is a complex phenomenon which is dependent on
several conditions:
- Certain stage of bacterial culture’s growth (exponential
phase)
- Secretion of a small protein, called competence factor,
that stimulates the production of 8 to 10 new proteins is
required for transformation (DNA-biding protein,
endonucleases, autolysins)
The Bacterial Genome
Gene Transfer
Conjugation
Was discovered in 1946 by Lederberg and Tatum.
In 1952 Hayes demonstrated that the gene transfer was polar.
Polarity is mediated by plasmid, known as F-factor.
Donor (F+, or fertile) and recipient (F-, or nonfertile) were defined.
F plasmid contains tra-operon.
Genes of tra-operon encode proteins for building the sex pili and
proteins needed to construct the type four secretion system that
will transfer DNA from donor to the recipient
The Bacterial Genome
Gene Transfer
Donor Types:
1. F+ cells with free F-plasmid
F+ x F- F+
2. Hfr cells with integrated
plasmid
Hfr x F- F- (F+100)
3. F’ free F-plasmid with a
portion of chromosomal
genes
F’ x F- F’
Conjugation
The Bacterial Genome
Gene Transfer
Cell-to-cell
transfer of a
conjugative
plasmid
Integration of
conjugative
plasmid into
the bacterial
chromosome
an Hfr
The order of genes on the
bacterial chromosome
can be determined by the
time of entry of the genes
into a recipient cell
The Bacterial Genome
Gene Transfer
Conjugation is very useful for
genetic mapping of bacteria
A circular genetic map of E.coli K12
with the location of selected genes.
The map is divided into 100 minutes
The Bacterial Genome
Gene Transfer
Transduction
Was discovered by Zinder and Lederberg in 1951-1953.
Transduction is a process of transfer of the bacterial DNA with
bacteriophages.
Transduction can be classified as:
- Generalized
any gene of the host bacterium can be transferred and doesn’t
require lysogeny (Salmonella enterica – P-22)
- Specialized
only specific genes near the attachment sites of a lysogenic
phage in the host chromosome can be transferred (E.coli- λ)
- Abortive
the transferred DNA is not integrated but often is able to
temporary survive and express. The fragment inherits linearly
and lost in progeny
The Bacterial Genome
Genetic Engineering – a combination of methods which allows
to conduct artificial recombination of DNA and produce chimerical
molecules, non-typical for nature
Genetic Engineering
The Bacterial Genome
Main aims of Genetic Engineering:
1. The production of medically useful proteins (somatostotine,
insulin, human growth harmone, interferons, interleukin-2,
etc.)
2. Recombinant vaccines (hepatitis B vaccine)
3. Gene therapy (involves the insertion of a normal gene into
cell to correct a defective gene
Genetic Engineering
The Bacterial Genome
The basic tools of Genetic
Engineering:
1. Cloning vectors which can be
used to deliver the DNA
sequences into receptive
bacteria
2. Restriction enzymes which
are used to cleave DNA at
defined sequences
3. DNA – ligase – the enzyme
that links the fragment to the
cloning vector
Genetic Engineering
The Bacterial Genome
Types of vector:
1. Plasmid (pUC, pBR322, pGEM) are used for DNA
fragments up to 20Kb
2. Bacteriophages (λ, T7) are used for larger fragments up to
25Kb
3. Cosmid (combination of plasmid and phage genes,
pJC720) for fragments up to 45Kb
4. Viruses (poxvaccine)
Genetic Engineering
The Bacterial Genome
The specific properties of plasmid cloning vectors:
1. Small size to be easy inserted into bacteria
2. High number of copies to be easily purified in sufficient
quantities
3. Ability to replicate within a host cell
4. Selectable traits (resistance to an antibiotic)
5. One or few sites for restriction endonucleases which cut
DNA and allow the insertion of foreign DNA
Genetic Engineering
The Bacterial Genome
Steps in Cloning a Gene:
• Isolate a DNA to be cloned
• Use restriction enzyme to
generate fragments of DNA
• Generate a recombinant
molecule by inserting DNA
fragments into a cloning vector
• Introduce recombinant
molecule into new host
• Select bacterial clones carrying
specific genes
Genetic Engineering