Course Title

MICROBIAL GENETICS AND MOLECULAR BIOLOGY

(MCB 307)

Name of Presenter: DR. (MRS.) O.B. SHITTU

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Mobile Genetic Elements

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What are mobile genetic elements(MGEs)?
Mobile genetic elements (MGEs) are genetic elements that
mediate the movement of DNA sequences within genomes
(intracellular mobility) or between bacterial cells (intercellular
mobility).

MGES are: Plasmids, Transposable Genetic elements (Insertion

sequences (Is) and Transposons (Tns) , Miniature Inverted
Repeats Transposable Elements (MITES), Tn3-derived
inverted-repeat miniature elements (TIMEs), Bacteriophages,
Gene Cassette/Integrons, Genomic Islands (GIs).
Intercellular movement of DNA (also known as horizontal gene transfer)
takes three forms in prokaryotes: Transformation, Conjugation and
Transduction.

Horizontal gene transfer (HGT) is the transfer of genetic material between

bacteria from the same generation.

Intracellular movement could be from the chromosome to a plasmid or

between plasmids.

MGEs are important sources of genetic diversity and play a fundamental

role in bacterial ecology, adaptation and evolution.

MGEs play important roles in infectious diseases, antibiotic resistance,

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5
Different resistance genes associated with different MGE are
represented by small arrows of various colors. Thin black arrows
indicate intracellular processes, with those mediated by a transposase
protein labeled Tnp and those mediated by a site-specific recombinase
protein labeled Ssr. Thick green arrows represent intercellular
(horizontal) transfer. 6

Successive insertions of the same IS on both sides of a resistance gene

may allow it to be captured and moved to another DNA molecule (e.g.,
from the chromosome to a plasmid) as part of a composite Tn.

(A). A unit Tn carrying a resistance gene may transpose between

plasmids .
(B) or from a plasmid to the chromosome or vice versa. A gene cassette
may move between In (a class 1 In/Tn structure is represented here) via
a circular intermediate.
(C). An ICE can be integrated into the chromosome or excised as a
circular element that can then conjugate into a recipient cell and
integrate (reversibly) into the chromosome at a specific recombination
site.
(D). A plasmid may be able to mediate its own intercellular transfer by
conjugation or, if it lacks a conjugation region, be mobilized by another
plasmid (or, alternatively, move horizontally by phage transduction or
transformation). Tn and/or In and associated resistance genes on an
incoming plasmid may move into the chromosome or other plasmid(s) in
the recipient cell.
(E), as illustrated here for class 1 In/Tn, which are known to target unit
Tn.
 PLASMIDS

Plasmids are small, circular or

linear self-replicating
(autonomous), extrachromosomal
genetic elements (DNA
molecules) that permit
microorganisms to store genetic
information (genes) that code for
specialized functions in addition to
that which is contained in the
chromosome and also transfer
genes from one bacterium to
another.
Characteristics of Plasmids
They are much smaller than the
chromosome (<1/20th the size),
ranging in size from 200 kb to 2
kb.

Many contain partitioning

systems which help partition
plasmids into daughter cells
during cell division.
 Characteristics of Plasmids (contd)
Copy number varies (1 –200) copies per cell depending on the plasmid

Sizes vary; the smallest plasmid is only 846 bp long and contains only
one gene while the largest plasmid known carries 1,674 genes.

Some plasmids can integrate into the chromosome.

The acquisition and loss of a plasmid alters the genotype of a

bacteria strain.
.
Characteristics of Plasmids (contd)
Most plasmids are closed circular and supercoiled like chromosome
although certain plasmids eg Borrelia burgdorferi are known carry
linear plasmids.

Different plasmids may exist together in a cell with different copy

number.

Larger plasmids are present in smaller numbers (1-2) and small

plasmids may be found in high copy numbers (~40).

Plasmids are capable of transfer to other cells but not all plasmids are
transferable.
Characteristics of Plasmids (contd)
Only plasmids carrying genes for self-transfer are transferable
to other cells but not all while those that do not have cannot.

The two prominent properties of plasmids are: the ability to

replicate and to partition themselves between the daughter
cells after cell division.

Plasmids don’t freely float in the cell cytoplasm, instead are

membrane bound.
Types of Plasmids
1. Fertility factor /conjugative plasmid
F (fertility) plasmids are those that have complete gene set to
mediate self-transfer by conjugation.
Cells possessing it (donor) are termed F+ and those lacking it
(recipient) are termed F-.
The F-plasmid is a conjugative plasmid that carries genes for sex
pili and for the transfer of the plasmid to another cell.
Conjugative plasmids are those that are able to mobilize
self-transfer.
Types of Plasmids (Contd.)
These tend to be larger because they have to accommodate
genes coding for self-transfer.
Conjugative plasmids in Gram positive bacteria tend to be smaller
than those in Gram-negative bacteria.
Conjugative plasmid carries genes for transfer to another
bacterium via conjugation and can integrate into the
chromosome.
Types of Plasmids (Contd.)
These tend to be larger because they have to accommodate
genes coding for self-transfer.
Conjugative plasmid carries genes for transfer to another
bacterium via conjugation and can integrate into the
chromosome.
Non-conjugative plasmids may be transferred from one cell to another by
inserting themselves into a conjugative plasmid or a chromosome or by
transformation when released from a dead cell. Insertion is made possible
by insertion sequences.
Types of Plasmids (Contd.)
2) R plasmids

These are plasmids that carry genes that code for antibiotic
resistance (drug resistance genes); heavy metals, or cellular
toxins.
R (resistance) plasmids are large conjugative plasmids that
carry one or more antibiotic resistance genes.

R plasmids code for their own replication.

 Types of Plasmids (Contd.)
Many resistance plasmids contain two groups of genes:
resistance transfer factor (RTF) and r-determinant.RTF includes
genes for plasmid replication and conjugation.

R-determinant has the resistance genes and codes for the

production of enzymes that inactivate some certain drugs or toxic
substances. E.g. R-plasmid R-100 carrying resistance genes for
sulfonamides, streptomycin, chloramphenicol and tetracycline as
well as genes for resistance to mercury.
Types of Plasmids (Contd.)
3) Colicin (Col) plasmids
Colicin plasmids are small plasmids which encode the genes to
synthesize colicins (bacteriocines).
Colicins are proteins which are toxic to only closely related bacteria.

4) Virulence plasmids
Virulence plasmids carry one or several genes that confer virulence
properties on the bacterial cell.
Allow for dissemination and survival of the organisms in the body.
Types of plasmids (Contd)
5). Degradative plasmids
Carry genes for degradation of a variety of toxic substances such as
toluene, camphor, and other organic hydrocarbons, herbicides, and
pesticides.
Such specialized capabilities permit the survival of those microbes in
very diverse and challenging environment.
Gives environmental adaptability & persistence, and metabolic
functions that allow utilization of different nutrients.
 TRANSPOSABLE GENETIC ELEMENTS (TGEs)
They are discrete DNA segments
that are able to move (to change
their position) themselves (and
associated resistance genes) almost
randomly to new locations in the
same or different DNA molecules
within a single cell.
They move from site to site within a
bacteria chromosome and undergo
non-homologous recombination.
● TGEs can be autonomous or
non-autonomous.
● Autonomous TGEs possesses
genes encoding transposing
enzymes while non-autonomous
can be defective from the lack of
genes encoding the enzyme to
transpose.
● The non-autonomous TGEs ,can
occasionally transpose via genes
encoding transposase located
elsewhere.
General Characteristics of Transposable Genetic Elements (Contd.)
● Their mobile nature has also
resulted in these elements being
referred to as jumping genes.
● By inserting themselves into
protein-coding genes, TGEs can
affect genes by either providing
novel regulatory sequences or via
epigenetic silencing of TGEs or
through the mutation of the
interrupted gene.
● TGEs can also cause mutation in
a larger scale through
chromosomal rearrangements
such as deletions, duplications,
inversions and translocations
through homologous
recombination and alternative
transposition.
● TGEs can be divided into two
major classes based on their
mechanism of transposition,
● and each class can be subdivided
into subclasses based on the
mechanism of chromosomal
 TRANSPOSABLE GENETIC ELEMENTS
Major TGEs are: Insertion sequences (IS) and
transposons (Tn) termed construction and
deconstruction agents.

IS elements consist of a single gene coding for

a site-specific recombinase (called a
transposase) and short terminal inverted
repeat sequences that are recognized by the
transposase for transposition.

a) carry gene(s) required for transposition

(transposase).

b) some carry conjugation genes.

 TRANSPOSABLE GENETIC ELEMENTS
c) "ends" are composed of inverted
repeats (similar sequences that are
inverted) that are recognized by
transposase during transposition.
__________> <___________

GGGGTCTAGAtransposonTCTAGACCCC

CCCCAGCTCTtransposonAGATCTGGGG

d) insertion site on chromosome is

duplicated during insertion so that
the element is flanked by direct
repeats of approx. 9 basepairs ).
 Insertion Sequences
IS carry little more than one (sometimes two) transposase (tnp) gene
required for transposition. Contain about 1,000 nucleotides.
The inverted repeats facilitate its non-reciprocal recombination.
Requires no sequence homology between it and the point where it inserts.
May be involved in specifying the location at which site specific
recombination occurs.
IS can insert into structural genes. IS may distrupt functional genes and give
rise to mutation.
The transposase enzyme recognizes, cuts and relegates the IS anywhere in
the bacteria genome.
 Transposons
Trasposons carry other genes in addition to transposase (contain
IS and structural genes).

Carry genes such as antibiotic resistance, toxins, and virulence

genes.
Can be formed by 2 insertion sequences that flank additional
genes (composite transposons).
Conjugative transposons have the ability to promote their own
transfer into other bacterial cells by conjugation since they carry
their own conjugation genes.
 Conservative and ReplicativeTransposition
Transposition can be conservative or replicative.

Conservative cut-and-paste mechanism: The TE is simply excised from the

donor and inserted into the recipient. Conservative involves the relocation of
the transposase enzyme from one place in the genome to another.

Replicative transposition: Either by a copy-and-paste mechanism (the TE is

replicated to join the donor and recipient in a cointegrate, which is then
resolved to give the original donor plus the recipient with the IS.

OR a copy-out-paste-in mechanism (the TE is replicated into a

double-stranded circular intermediate that then integrates into the recipient).
In replicative, the element remains in its original position and a copy is made
and inserted elsewhere in the genome.
 4.
Mechanisms of Replicative Transposition within circular DNA molecules
a) generation of staggered breaks in
target DNA and in the transposon.
b) joining of transposons to ends of
target DNA.
c) replication of transposon and
staggered breaks.
d) ligation to generate a cointergrate
(both molecules are joined).
e) recombination to resolve the
molecules apart.
 MITES AND TIMES

Miniature Inverted-repeat Transposable Elements (MITEs) are

nonautonomous (i.e., incapable of self-transposition) derivatives of bacterial
IS or transposons that retain the inverted repeats but which have lost central
parts, including the transposase gene(s).

While similar to transposons, a distinctive feature of MITEs is that they do not

encode a transposase; however, they can commandeer transposases from
other mobile elements for mobilization.

Pairs of MITEs, including Tn3-derived inverted-repeat miniature elements

(TIMEs) appear to have been involved in mobilization of resistance genes.

MITEs and TIMEs may provide an explanation for movement of resistance

 MITES AND TIMES

.
 4.
GENE CASSETTES AND INTEGRONS
Gene cassettes are discrete genetic
mobile element that may exist as
free, circular, non-replicative DNA
molecules when moving from one
genetic site to another, but they are
normally found as linear sequences
that constitute part of a larger DNA
molecule, such as plasmid or
bacteria chromosome.
 4.
GENE CASSETTES AND INTEGRONS
Gene cassette are small, normally
500-1000bp (0.5 to 1 kb) consisting of a
single gene (occasionally two), and an
attC recombination site, typically lacking a
promoter, and are expressed from a
promoter on the integron.

Usually consisting of a single open

reading frame (ORF) bounded by a
cassette-associated recombination site,
originally called a 59-base element but
now refers to as att1.
.
 4.
GENE CASSETTES AND INTEGRONS (Contd)
They are usually found inserted into
an integron, characterized by an intI
gene, an attI recombination site, and a
promoter (Pc).

The intI catalyzes recombination

between the attI site of the integron
and the attC site of a cassette. This
inserts the cassette into the integron
in the orientation that allows
expression of the cassette-borne gene
from the Pc promoter.
 4.
GENE CASSETTES AND INTEGRONS
Circular gene cassettes are integrated by
site specific recombination between att1
and attC, a process mediated by the
integron integrase.

The process is reversible, and cassettes

can be excised as free circular DNA
elements.
Multiple cassettes may be inserted into
the same integron to create a cassette
array (often incorrectly referred to as a
“cassette”) that may confer
multiresistance.
 4.
GENE CASSETTES AND INTEGRONS

Integrons are genetic elements that

contain a site-specific recombination
system, able to integrate, express
and exchange specific DNA elements
called gene cassette.

An integron allows efficient

acquisition, expression and
dissemination of exogenous genes
giving room for genomic complexity,
phenotypic diversity and adaptive
responses.
 4.
GENE CASSETTES AND INTEGRONS

Integrons have 3 essential structure:

1) int1, a gene encoding an integron

integrase (int1), a member of the
tyrosine recombinase family.

2) att1, an integron-associated
recombination site.

3) An integron –associated promoter.

Integron acquire new genes as part of
gene cassettes.
 4.
GENE CASSETTES AND INTEGRONS (Contd)

Different classes of integron have

been defined based on the sequence
of IntI (called IntI1, IntI2, IntI3, etc.,
with cognate attI1, attI2, and attI3
sites), with class 1 being the first
reported and most common in
antibiotic-resistant clinical isolates.

Integrons (In), use site-specific

recombination to move resistance
genes between defined sites.
 Integrons—gene capture systems

● Capture of DNA by an integron. The

integron core unit (A) is composed of
an integrase (intI) gene, a promoter
(PINT) to drive expression of intI, a
promoter (PC) to drive expression of
captured genes and asite for
integration of genes (attI).
● (B) Expression of the integrase
occurs during the SOS response and
the integrase protein (pale blue oval)
catalyses site-specific recombination
of circularised gene cassettes with an
attC site that matches attI so that
● (C) cassettes (cass) are incorporated
into the integron. More cassettes can
be integrated into attI and cassettes
 APPLICATION OF CLASS 1 INTEGRONS IN ENVIRONMENTAL POLLUTION MONITORING

Pollution with pesticides, heavy metals, pharmaceuticals, personal

care products and the microorganisms associated with human
waste streams and agriculture is a major challenge for monitoring.

The relative abundance of the clinical class 1 integron-integrase

gene, intI1, is a good proxy for diversity of pollutants, whose
concentration varies spatially and temporally.

.
APPLICATION OF CLASS 1 INTEGRONS IN ENVIRONMENTAL POLLUTION MONITORING
(CONTD)
Class 1 integrons could serve as a proxy for anthropogenic pollution because:
(1) intI1 is linked to genes conferring resistance to antibiotics, disinfectants
and heavy metals;
(2) It is found in a wide variety of pathogenic and nonpathogenic bacteria;
(3) Its abundance can change rapidly because its host cells can have rapid
generation times and it can move between bacteria by horizontal gene
transfer; and
(4) A single DNA sequence variant of intI1 is now found on a wide diversity of
xenogenetic elements, these being complex mosaic DNA elements fixed
through the agency of human selection.
GENOMIC ISLANDS

Genomic islands are clusters of genes within a bacterial genome that have
evidence to have been acquired by horizontal gene transfer.
GI may integrate into the chromosome of the host; excised and transferred to
a new host by transformation, conjugation or transduction.
Genomic islands (GIs) are of particular medical, environmental and/or
industrial interest, as they disproportionately encode virulence factors and
some antimicrobial resistance genes and may harbor entire metabolic
pathways that confer a specific adaptation (solvent resistance, symbiosis
properties, etc).
GENOMIC ISLANDS (Contd.)

Genomic islands are often named after the adaptive phenotypic properties
that they confer, such as pathogenicity islands, metabolic islands, antibiotic
resistance islands and symbiosis islands.

Symbiosis encodes genes that enable mutually beneficial association with a

eukaryotic host eg. saprophytic soil bacterium to form a nitrogen-fixing
mutualistic association with leguminous plants of the genus.

There is evidence that PAIs are remnants of bacteriophages, plasmids and

integrative conjugative elements.
INTEGRATIVE CONJUGATIVE ELEMENTS

Integrative Conjugative Elements (ICE) constitute a diverse group of mobile

elements found in bacteria that encode for mobility proteins.
Similar to plasmids, Integrative conjugative elements (ICE) are a diverse
group of chromosomally integrated, self-transmissible mobile genetic
elements (MGE) that are active in shaping the functions of bacteria and
bacterial communities.
ICE are self-transmissible by conjugation i.e. ICE have the ability to promote
their own transfer into other bacterial cells by conjugation since they carry
their own conjugation genes.
They are characterized by their ability to mediate and encode all
determinants for their own integration, excision, and transfer from one host
genome to another by a mechanism of site-specific recombination,
self-circularisation, and conjugative transfer.
 INTEGRATIVE CONJUGATIVE ELEMENTS

They are important in the evolution of bacterial genomes allowing

bacteria to rapidly acquire new phenotypic traits and adaptive functions
such as antimicrobial and heavy metals resistance, virulence,
degradation of xenobiotic pollutants and the bacteriophage infection
resistance.
The core set of genes required for a functionally active ICE are divided
into three distinct modules known as the maintenance, conjugation, and
regulation module.
The maintenance module contains the genes responsible for the stable
integration of an ICE into a host genome and is defined by the presence
of two genes int and xis.
 INTEGRATIVE CONJUGATIVE ELEMENTS

The ICE integrase (int) is a recombinase which catalyzes the recombination

reaction between specific recognition sites in a host genome (attB) and the
circular ICE element usually called attP (located on the ICE).

In addition to the integrase most ICEs encode a recombination directionality

factor (RDF) termed Xis which stimulates excision of ICE elements for
transfer). ICEs transfer via the conjugation module and utilize conjugation
mechanisms that are highly similar to those of conjugative plasmids.

ICE elements generally contain a regulatory module to control various

aspects of ICE metabolism.
 INTEGRATIVE CONJUGATIVE ELEMENTS