GENETIC of BACTERIA

Professor M.Boychenko
Bacterial genome
DNA molecules that replicate as
discrete genetic units in bacteria are
called replicons. In some bacterial
strains, the chromosome is the only
replicon present in the cell. Other
bacterial strains have additional
replicons, such as plasmids and
bacteriophages.
Chromosomal DNA

Bacterial genomes vary in size from

about 0.4 x 109 to 8.6 x 109 daltons
(Da),
Most bacteria have a haploid
genome, a single chromosome
consisting of a circular, double
stranded DNA molecule.
Bacterial genome
However linear chromosomes have been
found in Gram-positive Borrelia and
Streptomyces spp.,
and one linear and one circular
chromosome is present in the Gram-
negative bacterium Agrobacterium
tumefaciens.
V.cholerae possesses 2 circular
chromosomes
plasmids

The term plasmid was first introduced by

the American molecular biologist Joshua
Lederberg in 1952
Plasmids are considered transferable
genetic elements, or "replicons", capable
of autonomous replication within a suitable
host. Plasmids can be found in all three
major kingdoms, Archea, Bacteria and
Eukarya
plasmids
A plasmid is an extra-
chromosomal DNA
molecule separate
from the chromosomal
DNA,which is capable
of replicating
independently of the
chromosomal DNA. In
many cases, it is
circular and double-
stranded. Plasmids
usually occur naturally
in bacteria, but are
sometimes found in
eukaryotic organism.
 PLASMIDS
Plasmid size
varies from 1 to
over 200 kilobase
pairs (kbp). The
number of
identical plasmids
within a single
cell can range
anywhere from
one to even
thousands
types of plasmids
There are two types
of plasmid
integration into a
host bacteria: Non-
integrating plasmids
replicate as with the
top instance;
whereas episome
integrate into the
host chromosome.
types of plasmids
One way of grouping plasmids
is by their ability to transfer to
other bacteria.
Types of plasmids

Conjugative
plasmids contain so-
called tra-genes,
which mediate
process of
conjugation, the
transfer of plasmids
to another bacterium
F + x F-
Types of plasmids

Non-conjugative plasmids are

incapable of initiating conjugation,
hence they can only be
transferred with the assistance of
conjugative plasmids.,
Types of plasmids

It is possible for plasmids of different types

to coexist in a single cell. Seven different
plasmids have been found in E.coli. But
related plasmids are often incompatible, in
the sense that only one of them survives
in the cell line, due to the regulation of vital
plasmid functions. Therefore, plasmids
can be assigned into compatibility groups.
Types of plasmids
.
Another way to classify plasmids is by
function. There are five main classes:
Fertility-F-plasmid, which contain tra-
genes. They are capable of conjugation
(transfer of genetic material between
bacteria which are touching).
Resistance-(R)plasmids, which contain
genes that can build a resistance
against antibiotics or poisons.
Historically known as R-factors, before
the nature of plasmids was understood.
Types of plasmids
Col-plasmids, which contain genes that
code for (determine the production of)
bactericins proteins, that can kill other
bacteria.
Degradative plasmids, which enable the
digestion of unusual substances, e.g.,
toluene or salicylic acid.
Virulence plasmids, which turn the
bacterium into a pathogen (one that
causes disease).
Using of plasmids
Another major use of plasmids is to
make large amounts of proteins. In this
case, researchers grow bacteria
containing a plasmid harboring the gene
of interest. Bacteria can be induced to
produce large amounts of proteins from
the inserted gene. This is a cheap and
easy way of mass-producing a gene or
the protein it then codes for, for
example, insulin or even antibiotics.
Plasmids are now being used to
manipulate DNA and may possibly
be a tool for curing many diseases.
Using of plasmids
Major use of plasmids
is to make large
amounts of proteins. In
this case, researchers
grow bacteria
containing a plasmid
harboring the gene of
interest. Bacteria can
be induced to produce
large amounts of
proteins from the
inserted gene.
Plasmids profile

Comparing plasmid
profiles is a useful
method for assessing
possible relatedness of
individual clinical
isolates of a particular
bacterial species for
epidemiological studies.
Mobile genetics elements

Transposons are segments of DNA

.

that can move from one site in a

DNA molecule to other target sites
in the same or a different DNA
molecule.
The process is called transposition
and occurs by a mechanism that is
independent of generalized
recombination.
Mobile genetics elements
Transposons are not self-
replicating genetic elements,
however, and they must integrate
into other replicons to be
maintained stably in bacterial
genomes
Mobile genetics elements
Insertion of a transposon often interrupts
the linear sequence of a gene and
inactivates it.
Transposons have a major role in causing
deletions, duplications, and inversions of
DNA segments as well as fusions between
replicons.
Mobile genetics elements
Most transposons share a number of
common features. Each transposon
encodes the functions necessary for
its transposition, including a
transposase enzyme that interacts
with specific sequences at the ends of
the transposon.
Mobile genetics elements
Complex transposons vary in length
from about 2,000 to more than 40,000
nucleotide pairs and contain insertion
sequences (or closely related
sequences) at each end, usually as
inverted repeats.
 IS-elements
Insertion sequences
are simplest in
structure and encode
only the functions
needed for
transposition. Inverted
repeats are at their
endsThe DNA
between the inverted
terminal repeats
contains transposase
genes
During transposition a short
sequence of target DNA is
duplicated,
The duplication is presumed to
involve asymmetric cleavage of DNA
at the target site.

If the transposon at a donor

site is replicated and a copy is
inserted into the target site,
however, the process is called
replicative transposition
Mobile genetics elements
Resolution of the cointegrate into its
component replicons is often accomplished
by a transposon-encoded resolvase that
catalyzes site-specific recombination
between the transposons.

Transposition differs from site-specific

recombination by duplicating a segment of
the target sequence and by using a variety of
different target sequences for a single donor
sequence.
Role of transposone in bacterial evolution

Some of the multiple antibiotic resistant

plasmids have individual transposons with
several resistance determinants. The
stepwise acquisition of resistance
determinants can lead, in some cases, to
the formation of composite transposons
that encode multiple resistance
determinants.
Role of transposone in bacterial evolution

After a plasmid carrying a transposon is

introduced into a new bacterial host, the
transposon and its determinants can jump
into the chromosome or indigenous
plasmids of the new host. Therefore,
stability of the mobilizing plasmid in a new
bacterial host is not essential for
persistence of genetic determinants
located on a transposon.
.The acquisition of resistance gene arrays involves
genetic mobile elements like :
Plasmids
Transposons
Integrons are a system of gene capture
and expression composed of an intI
gene encoding an integrase, a
recombination site attI, and a
promoter.
 P
attI
5'conserved
segment
cassette1
intI
attC1
attI attC1

cassette2

attC2

attI attC2 attC1

Integrons are a system of gene capture and expression

composed of an intI gene encoding an integrase, a
recombination site attI, and a promoter.
The integrase is able to integrate or
excise gene cassettes, by a site-
specific system of recombination.

Cassette mobility results in a very

efficient system of dissemination of
resistance genes.
Recombination
Recombination is the
rearrangement of donor and
recipient genomes to form new,
hybrid genomes
Recombination involves breakage
and joining of parental DNA
molecules to form hybrid,
recombinant molecules.

.
Recombination
Several distinct kinds of recombination
have been identified that depend on
different features of the participating
genomes and require the activities of
different gene products. Specific
enzymes that act on DNA (for example,
exonucleases, endonucleases,
polymerases, ligases) participate in
recombination
Generalized recombination
Generalized recombination involves
donor and recipient DNA molecules
that have homologous nucleotide
sequences.
The product of the recA gene is
essential for generalized
recombination, but other gene
products also participate.
Site-specific recombination
Site-specific recombination involves
reciprocal exchanges only between
specific sites in donor and recipient
DNA molecules.
The recA gene product is not required
for site-specific recombination.
Integration of the temperate bacteriophage l into
the chromosome of E coli is a well-studied
example of site-specific recombination
.
Site-specific recombination
In phage l the product of the int gene
(integrase) is required for the site-specific
integration event in lysogenization;
The products of the int and xis
(excisionase) genes are both needed for
the complementary site-specific excision
event that occurs during induction of lytic
phage development in lysogenic cells.
Site-specific recombination
The specific
attachment (att) sites
on the E coli
chromosome and l
phage DNA have a
common core
sequence of 15
nucleotides, within
which reciprocal
recombination
occurs
Exchange of Genetic Information
Genetic exchanges among bacteria occur by
several mechanisms.
In transformation, the recipient bacterium
takes up extracellular donor DNA.
In transduction, donor DNA packaged in a
bacteriophage infects the recipient
bacterium.
In conjugation, the donor bacterium transfers
DNA to the recipient by mating.
Transfer of genetics information
transformation
Historically, characterization of
"transforming principle" from S
pneumoniae provided the first
direct evidence DNA is genetic
material.
transformation
In transformation, pieces of DNA released
from donor bacteria are taken up directly
from the extracellular environment by
recipient bacteria. To be active in
transformation, DNA molecules must be at
least 500 nucleotides in length, and
transforming activity is destroyed rapidly
by treating DNA with deoxyribonuclease.
transformation
Molecules of transforming DNA
correspond to very small fragments of the
bacterial chromosome.
Transformation was discovered in
S.pneumoniae and occurs in other
bacterial genera including Haemophilus,
Neisseria, Bacillus, and Staphylococcus
transformation
The ability of bacteria to take up
extracellular DNA and to become
transformed, called competence,
varies with the physiologic state
of the bacteria.
transformation
Many bacteria that are not usually
competent can be made to take
up DNA by laboratory
manipulations, such as calcium
shock or exposure to a high-
voltage electrical pulse
(electroporation).
transformation
Competent bacteria may also take up
intact bacteriophage DNA (transfection) or
plasmid DNA, which can then replicate as
extrachromosomal genetic elements in the
recipient bacteria. Recombination
occurs between single molecules of
transforming DNA and the
chromosomes of recipient bacteria.
transformation
Conjugation
F+ x F-
In matings between F+ and F- bacteria,
only the F plasmid is transferred with high
efficiency to recipients.
In matings between F+ and F- strains, the
F plasmid spreads rapidly throughout the
bacterial population, and most
recombinants are F+.
conjugation
Conjugation
Donor strains with integrated F
factors can transfer
chromosomal genes to
recipients with high efficiency,
they are called Hfr (High
frequency recombination)
strains.
Hfr x F-
In matings between Hfr and F-
strains, the segment of the F
plasmid containing the tra region
is transferred last, after the entire
bacterial chromosome has been
transferred.
Conjugation
Formation of recombinant
progeny requires recombination
between the transferred donor
DNA and the genome of the
recipient bacterium.
Conjugation
Most recombinants from matings
between Hfr and F- are
phenotypically F-.
In matings between F+ and F- strains,
the F plasmid spreads rapidly
throughout the bacterial population,
and most recombinants are F+.
conjugation
General transduction
In transduction,
bacteriophages function as
vectors to introduce DNA from
donor bacteria into recipient
bacteria by infection.
General transduction
For some phages, called
generalized transducing phages,
a small fraction of the virions
produced during lytic growth
contain a random fragment of the
bacterial genome instead of
phage DNA.
General transduction
Each individual transducing phage carries
a different set of closely linked genes,
representing a small segment of the
bacterial genome.
When a generalized transducing phage
infects a recipient cell, expression of the
transferred donor genes occurs
General transduction
Specialized transduction
Specialized transduction differs from
generalized transduction in several
ways.
It is mediated only by specific
temperate phages,
and only a few specific donor genes
can be transferred to recipient
bacteria.
Specialized transduction
Specialized transducing
phages are formed only when
lysogenic donor bacteria enter
the lytic cycle and release
phage progeny.
Specialized transduction
The specialized transducing
phages lack part of the normal
phage genome and contain part
of the bacterial chromosome
located adjacent to the prophage
attachment site.
Specialized transduction
Specialized transduction
results from lysogenization of
the recipient bacterium by the
specialized transducing phage
and expression of the donor
genes.
Specialized transduction
Phage conversion and specialized
transduction have many
similarities, but the origin of the
converting genes in temperate
converting phages is unknown.
68

Practical genetic
Molecular methods in microbial
laboratory

1. PCR
2. Micro array technique Identification of
microbes without
isolation of pure culture
3.Ribotyping
4.Finger printing Intra species
5.Plasmids profile identification
8.PCR-RT
DNA microarrays are:
glass slides with
hundreds to thousands of
DNA probes bound

100-200 m
 Methodology
Community DNA

Specific PCR product

Oligonucleotide probes
(i.e., 16S rRNA gene)
Length: between 15 and 30 nt

Fluorescently labelled target Microarray with the above probes

(DNA or RNA)

Hybridisation

Data analysis
 Emitted light (fluorescence)

Attached fluorophore

Labelled target DNA

Many target molucules No target A few target molecules
High signal No signal Low signal
DNA probes

Glass slide

The principle of detection of specific DNA sequences

Real time pcr
Branch-pcr