The Bacterial

Genetics
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, c) Space-filling
model of DNA
showing helical sugar-phosphate
backbones of the two strands held
together by hydrogen bonding
between the basis
The Bacterial Genome
 DNA Forms

a) The DNA double helix of most b) The circular DNA strands, already
procaryotes has the shape of a coiled in a double helix, are twisted a
closed circle second time to produce supercoils

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

Vegetative

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

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
F-Plasmid them.
F - fertility factor
R - antibiotic resistance
Col - colicin production
Virulence plasmids:
Ent - enterotoxin production
Hly - hemolysin production
CFA-I; CFA-II - adhesins production
The Bacterial Genome
 Bacteriophages

Litic
cycle

Lysogenic
cycle

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 Nonhomologous
(legitimate) (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:

4. Transformation
5. Conjugation
6. 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

Conjugation
IS3
γδ

IS3
tra
F
F-plasmid IS2

100 kb

oriT
oriV

The Bacterial Genome

 Gene Transfer

Conjugation
Donor Types:
• F+ cells with free F-plasmid
F+ x F- F+

• Hfr cells with integrated

plasmid
Hfr x F- F- (F+100)

• F’ free F-plasmid with a

portion of chromosomal
genes
F’ x F- F’

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
Transduction

The Bacterial Genome

 Genetic Engineering

Genetic Engineering – a combination of methods which allows

to conduct artificial recombination of DNA and produce chimerical
molecules, non-typical for nature

The Bacterial Genome

 Genetic Engineering

Main aims of Genetic Engineering:

3. The production of medically useful proteins (somatostotine,
insulin, human growth harmone, interferons, interleukin-2,
etc.)
4. Recombinant vaccines (hepatitis B vaccine)
5. Gene therapy (involves the insertion of a normal gene into
cell to correct a defective gene

The Bacterial Genome

Genetic Engineering

The basic tools of Genetic

Engineering:
3. Cloning vectors which can be
used to deliver the DNA
sequences into receptive
bacteria
4. Restriction enzymes which
are used to cleave DNA at
defined sequences
5. DNA – ligase – the enzyme
that links the fragment to the
cloning vector

The Bacterial Genome

 Genetic Engineering

Types of vector:
3. Plasmid (pUC, pBR322, pGEM) are used for DNA
fragments up to 20Kb
4. Bacteriophages (λ, T7) are used for larger fragments up to
25Kb
5. Cosmid (combination of plasmid and phage genes,
pJC720) for fragments up to 45Kb
6. Viruses (poxvaccine)

The Bacterial Genome

 Genetic Engineering

The specific properties of plasmid cloning vectors:

3. Small size to be easy inserted into bacteria
4. High number of copies to be easily purified in sufficient
quantities
5. Ability to replicate within a host cell
6. Selectable traits (resistance to an antibiotic)
7. One or few sites for restriction endonucleases which cut
DNA and allow the insertion of foreign DNA

The Bacterial Genome

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

The Bacterial Genome

Thank you for your attention!
Questions?