the bacterial genetics - university of baghdad bacterial genome plasmids plasmids is a small genetic...

The Bacterial

Genetics

Lectures of Dr.Mohammad Alfaham

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)


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


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


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


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)


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


DNA Replication

An autoradiograph of a replicating E.coli chromosome; about one-third of the

chromosome has been replicated


Replication of circular DNA

Vegetative


Replication of circular DNA

Conjugative

Mechanism used by

some bacterial

viruses, plasmids

and in chromosomal

transfer


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



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


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


Bacteriophages

Litic

cycle

Lysogenic

cycle


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


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


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


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)


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


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


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


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)


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


IS2

oriT

oriV

tra

F

100 kb

IS3

IS3γδ

F-plasmid

Gene Transfer

Conjugation


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


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


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


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


Transduction


Genetic Engineering – a combination of methods which allows

to conduct artificial recombination of DNA and produce chimerical

molecules, non-typical for nature

Genetic Engineering


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 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


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 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


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

Thank you for your attention!

Questions?

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