Chapter8 Microbial genetics
8.4 Plasmids
8.5 Genetic Conjugation,
Transformation, transduction
8.6 Transposons and Insertion Sequences
8.4 Plasmids
Circular genetic elements that
reproduce autonomously and have
an extra-chromosomal existence:
• 1-1000 KB in size
• Typical plasmid 1/20 of chromosome
• Most are circular double-stranded
DNA, some linear ds DNA
• Transmitted from cell to cell via
conjugation process
• Some can integrated into
chromosome
• Can carry a variety of genes for
production of toxin, resistance to
antibiotics and heavy metals et al.
Plasmid
• Conjugative: plasmids which govern their own transfer by
cell-to-cell contact are called conjugative
• Tra region: a set of genes within the plasmid that control the
transmissability by conjugation
• Hfr (high frequency of recombination): strains of bacteria
that transfer large amounts of chromosomal DNA during
conjugation
• Supercoil: plasmids isolated from the cells are in supercoiled
configuration
• Plasmid separation: by ultracentrifugation or electrophoresis
• Curing of plasmids: elimination of plasmids from host cells
by various treatments.
Plasmids
• Replication: Most plasmids of gram-positive
bacteria replicate by a rolling circle
mechanism.
• Copy number: The number of plasmids in a
cell, can range from only 1-3 copies to 100
copies.
• Incompatibility: Two different types of
plasmids can not coexist in a cell.
• Episomes: Plasmids having the ability to
integrate into host chromosome
Col plasmids
• Bacteria also harbor plasmids with
genes that may give them a
competitive advantage in the microbial
word.
• Bacteriocins are bacterial protein that
destroy other bacteria. Usually act only
against closely related strains
F-Plasmid-Fertility Plasmids
• 100 KB
• Can be cured with
acridine orange
• Incompatibility (inc)
• Origin of replication
(oriS)
• Transposable elements
(Tn)
• tra region
• phi: phage inhibition
• IS (insertion sequence)
• rep: replication
functions
Cell to Cell Transfer of Plasmids
• Conjugative: Plasmids that govern their own transfer
by cell-to-cell contact are called conjugative (not all
plasmids are conjugative)
• Some conjugative plasmids can transfer genetic
information between distintly related organisms
(between gram-positive and gram-negative bacteria,
between bacteria and plant cells, and between bacteria
and fungi), it is important for evolution.
Conjugation (接合作用）
Plasmid Biology
Episomes
Plasmid Biology
Types of Plasmids and Their
Biological Significance
• The presence of plasmids in a cell can have a
profound influence on the cell’s phenotype:
–
–
–
–
–
–
the ability of conjugation
the ability of Rhizobium to interact with plants
the resistance to antibiotics and heavy metals
the degradation of octane, camphor et al
the production of enterotoxin
the applications in genetic engineering
Resistance Plasmids (R-Plasmids):
the most well studied plasmids
• The emergence of bacteria
resistant to several antibiotics is
medically significant
• Resistance can be transferred via
cell-to-cell contact
• This could be one of the reasons
for the rapid rise of multiply
resistant strains
• Plasmid recombination is one
mean by which multiply
resistant organisms might have
first arisen
• Infectious nature of the R
plasmids permits rapid spread of
the characteristic through
populations
• Typical example: plasmid R100
The presence of multiple antibiotic resistance is due to the fact that a
single R plasmid contains a variety of genes coding for different
antibiotic inactiviation enzymes
Biochemical
mechanism
of resistance
mediated by
R plasmids
8.5 Three main processes of genetic recombination in
prokaryotes fragments of homologous DNA from a donor
chromosome are transferred to a recipient cell
(1) Transformation, which involves donor DNA free
in the environment
(2) Transduction, in which the donor DNA transfer
is mediated by a virus
(3) Conjugation, in which the transfer involves cellto-cell contact and a conjugative plasmid in the donor
cell
DNA Transfer in Bacteria
transformation
transduction
conjugation
8.5.1 Conjugation
• Conjugative plasmids possess genetic
information to code for sex pili and for some
proteins needed for DNA transfer.
• Rolling circle replication occurs for DNA
transfer during conjugation.
F plasmid of E. coli has the
special property of being able
to mobilize the chromosome
so that it can be transferred
during cell-to-cell contact.
Conjugation and Chromosome Mobilization:
F+ and F- strains
• F+ strains: cells possessing an unintegrated F plasmid.
• F- strains: cells which can act as recipients for F+ or
Hfr, F- strains lack F plasmid.
• F plasmid provides its host cell with:
– ability to synthesize the F pilus
– mobilization of DNA for transfer to another cell
– alteration of surface receptors so that the cell is no
longer able to behave as a recipient in conjugation
Integration of an F plasmid
into the chromosome with the
formation of an Hfr. IS elements
are the sites of insertion.
homology
Hfr strain
• Hfr strains
arise as a
result of the
integration
of the F
plasmid into
the
chromosome
Important Concept: F’ plasmids
• Integrated F plasmids may be occasionally
excised from the chromosome and bring
some chromosomal genes with itself into
the liberated F plasmid.
• F’-mediated transfer resembles specialized
transduction in that only a restricted group
of chromosomal genes can be transferred.
Result of selected conjugation
Donor Recipient Molecules transferred
Product
F+
F-
F plasmid
F+ Cell
Hfr
F-
F- with variable
quantity of
chromosomal DNA
F+
F-
Initiating segment of F
plasmid and variable
quantity of
chromosomal DNA
F+ plasmid and some
chromosomal genes it
carries with it
F+ Cell with some
duplicate gene pairs:
one on chromosom,
one on plasmid
Transfer of
plasmid DNA
by conjugation
• The F plasmid of
an F+ cell is
being transferred
to a F- recipient
cell
Details of the replication
and transfer process
Detection of
Genetic
Conjugation
Manner of formation
of different Hfr strains
• The direction in
which the F factor is
inserted determines
which of the
chromosomal genes
will be inserted first
into the recipient
Interrupted
Mating
Mapping the
order of genes
• Mixing Hfr
and F- cells.
• Shake the
mixture
violently at
various time.
• Plate the
samples on
selective
medium for
recombinant
to grow.
Conjugation involves a donor cell, which contains a particular
type of conjugative plasmid, and a recipient cell, which does not.
The genes that control conjugation are contained in the tra region
of the plasmid (see Section 9.8 in your text ). Many genes in the
tra region have to do with the synthesis of a surface structure, the
sex pilus . Only donor cells have these pili,
The pili make specific contact with a receptor on the recipient
and then retract, pulling the two cells together. The contacts
between the donor and recipient cells then become stabilized,
probably from fusion of the outer membranes, and the DNA is then
transferred from one cell to another.
Mechanism of DNA Transfer During Conjugation
A mechanism of DNA synthesis in
certain bacteriophages, called rolling
circle replication, was presented here
to explains DNA transfer during
conjugation .
if the DNA of the donor is labeled, some
labeled DNA is transferred to the
recipient but only a single labeled strand
is transferred. Therefore, at the end of
the process, both donor and recipient
possess completely formed plasmids.
Genetic Recombination
• Homologous or General Recombination
– RecA protein participation
– Homologous DNA sequences have the same or nearly
the same sequence
– New genotypes only arise when two homologous
sequences are genetically distinct
Detection of
Recombination
• Requirement:
reverse mutation
for the selected
characteristic
must be low.
This problem can
often be
overcome by
using double
mutants.
Complementation Test:
cis-tran test
• trans configuration: two
mutations are each on
separate DNA molecules
• cis configuration: Two
mutations were on the
same DNA molecule
• Complementation does not
involve recombination
DNA transformation
• 1928, Fred Griffith
• Competent: cells able to take up a molecule of DNA.
Competency is a complex phenomeono and is
dependent on several conditions.
1. Bacteria need to be in a certain stage of growth.
2. Secrete a small protein called the competence
factor that stimulates the production of 8 to 10 new
proteins reauired for transformation.
• Natural transformation has been discovered so far
only in certain genera: Streptococcus, Bacillus,
Thermoactinomytes, Haemophilus, Neisseria,
Moraxella, Acinetobacter, Azotobacter,
Pseudoomonas
The mechanism of Transformation
in S. pneumoniae
1. A competent cell binds a ds
DNA fragment
2. The DNA is cleaved by
endonucleases to 5-15kb.
3. One stand is hydrolyzed by an
envelop-associated exonuclease,
the other strand associate with
small proteins and moves
through the plasma membrane.
4. Integration of transforming
DNA
The transformation of Haemophilus
influenzae
• Difference: 1. Haemophilus does not produce a
competence factor to stimulate the development
of competence.
• 2. It takes up DNA from only closed related
species.
• 3. Ds DNA, complexed with proteins, is taken
in by membrane vesicles.
• 4. DNA must have a special sequence
(5’AAGTGCGGTCA3’) to be bound by a
competent cell.
Transformation
A number of prokaryotes have been found to be
naturally transformable, including certain species of
both gram-negative and gram-positive Bacteria and
some species of Archaea. However, even within
transformable genera, only certain strains or species
are transformable
Competence
A cell that is able to take up a molecule of DNA and be
transformed is said to be competent. Competence in
most naturally transformable bacteria is regulated,
and special proteins play a role in the uptake and
processing of DNA. These competence-specific
proteins may include a membrane-associated DNA
binding protein, a cell wall autolysin, and various
nucleases.
Competent cells bind much more DNA than do
noncompetent cells as much as 1000 times more
Artificially Induced Competence
High efficiency natural transformation is found only in a few
bacteria; Azotobacter, Bacillus, Streptococcus,, for example,
are easily transformed. Many prokaryotes are transformed
only poorly or not at all under natural conditions.
Determination of how to induce competence in such bacteria
may involve considerable empirical study, with variation in
culture medium, temperature, and other factors
when E. coli is treated with high concentrations of calcium ions
and then stored in the cold, the transformation by plasmid DNA
is relatively efficient.
The introduction of DNA into cells
by mixing the DNA and the cell
(a) Binding of free DNA by a membranebound DNA binding protein.
(b) Passage of one of the two strands into
the cell while nuclease activity
degrades the other strand.
(c) The single strand in the cell is bound
by specific proteins, and
recombination with homologous
regions of the bacterial chromosome
mediated by RecA protein occurs.
Transformed cell
DNA Transfer by Electroporation
for artificial induction of competence are being
supplanted by a new method termed electroporation.
Small pores are produced in the membranes of cells
exposed to pulsed electric fields. When DNA
molecules are
present outside the cells during the electric pulse,
they can then enter the cells through these pores.
This process is called electroporation.
The mechanism of bacterial transformation
Other methods for introducing
DNA into bacterial cells
• Transfection: transformed DNA is extracted from
a bacterial virus
• Artificially induced competence: e.g treat E. coli
with high concentration of Ca ions, and then
stored the cells at low T, the E. coli will become
competent at low efficiency
• Electroporation: pulsed electrical fields generate
pores in the cell membranes, allowing DNA
molecules to enter the cells.
• DNA from any source can be introduced into
bacteria by splicing it into a plasmid before
transformation
Transformation (transfection) of
eukaryotic cells
• Transfection: introducing DNA into mammalian
cells
– phagocytosis in animal cells
– Yeast: spheroplasts added with Ca ions plus
polyethylene glycol
– Electroporation
– Particle gun, or gens gun
Agrobacterium and Plant Interactions:
Crown gall and Hairy Root
• Crown gall: caused by
Agrobacterium tumefaciens
which carries a Ti (Tumor
induction) plasmid that
promotes the crown gall
formation
• Hairy Root: caused by
Agrobacterium rhizogenes
which carries a Ri plasmid
that leads to hairy roots
formation
Overview of events of
crown gall disease
following infection of
A. tumefaciens
Ti plasmid of Agrobacterium
tumefaciens
Mechanism of transfer
of T-DNA to the plant
cell
Wonder of the
Genetic Engineering
• Expression of
luciferase gene in a
plant.
Background of transduction
• Lytic cucle: end in lysis of the host
• Lysogeny: after adsorption and penetration, viral
genome remains within the host cell and is
reporduced along with the bacterial
chromosome.thia relationship between the phage
and its host is called lysogeny.
• Lysogens or lysogenic: bacteria that can produce
phage particles under some conditions.
• Temperate phages: phages able to eatablish this
relationship.
• Prophage: the latent form of the virus genome that
remains within the host without destroy it.
Virus Life Cycle
1. Attachment (adsorption)
2. Penetration (injection)
3. Early steps in replication
4. Replication
5. Synthesis of protein subunits
6. Assembly and packaging
7. Release
Temperate Bacterial Viruses:
Lysogeny and Lambda
• Virulent Viruses
• Temperate Viruses
– Lysogeny (溶原性）：Viruses can enter a state
called lysogeny, where most phage genes are
not expressed, and the phage genome is
replicated in synchrony with the host
chromosome
– Prophage or provirus
– Can be induced (lysogenic induction) by UV
radiation, nitrogen mustards or X-ray.
Temperate Bacterial
Viruses:
Lysogeny and
Lambda
Transduction
• Generalized transduction: host genes
derived from virtually any portion of the
host genome become part of the DNA of
the mature virus genome.
• Specialized transduction: occurs only in
some temperate viruses: a specific group of
host genes is integrated directly into virus
genome-usually replacing some of the virus
genes-and is transferred to the recipient
during lysogenization
Generalized transduction
Abortive transduction
About 70 to 90%of transferred DNA is not integrated but
often is able to survive and express itself. Abortive
transductants are bacteria that contain this nonintrgrated,
transduced DNA and are partial diploids.
Specialized
Transduction
• Under rare
conditions, the
phage genome is
excised incorrectly.
• Lambda dgal
(defective galactose)
under the assistance
of helper, the
defective phage can
be replicated and
can transduce the
galactose genes.
Specialized Transduction
• Low-frequency transduction (LFT) lysates: lysates
contain only a few transducing particle, the phage
genome is excised incorrectly.
• Helper phage: defective lambda phages carring the
gal gene can integrate if there is a normal lambda
phage in the same cell. This normal phage is termed
the help phage.
• High-frequent transduction (HFT) lysate: a lysate
containing a fairly equal mixture of defective
lambda dgal phage and normal helper phage.
Phage conversion
• A prophage is immune to further infection by the
same type of phage.
– Change in structure of a polysaccharide on the
cell surface of Salmonella anatum upon
lysogenization with e15,
– Conversion of nontoxin producing strains of
Corynebacterium diphtheriae to toxin producing
(pathogenic) strains.
• Information for production of these new
materials is apparently an integral part of the
phage genome and is automatically and
exclusively transferred upon infection by the
phage and lysogenization.
Transformation
Transduction
Conjugation
Transduction
Concept
Transduction involves transfer of host genes from one
bacterium to another by viruses. In generalized
transduction, defective virus particles randomly
incorporate fragments of the cell's chromosomal DNA;
virtually any gene of the donor can be transferred, but
the efficiency is low. In specialized transduction, the
DNA of a temperate virus excises incorrectly and
brings adjacent host genes along with it; only genes
close to the integration point of the virus are
transferred, but the efficiency may be high.
In transduction, DNA is transferred from cell
to cell through the agency of viruses. Genetic
transfer of host genes by viruses can occur in
two ways.
Generalized transduction
And
Specialized transduction
Generalized transduction: host DNA derived from
virtually any portion of the host genome becomes a
part of the DNA of the mature virus particle in
place of the virus genome.
Specialized transduction: occurs only in some
temperate viruses; DNA from a specific region of the
host chromosome is integrated directly into the virus
genome - usually replacing some of the virus genes.
Transduction has been found to occur in a variety of
prokaryotes, including certain species of the
Bacteria: Desulfovibrio, Escherichia, Pseudomonas,
Rhodococcus, Rhodobacter, Salmonella,
Staphylococcus, and Xanthobacter, as well as the
archaean Methanobacterium thermoautotrophicum.
Not all phages can be transducer and not all bacteria are transducible
Generalized transduction
Generalized transduction
In generalized transduction, virtually any genetic marker
can be transferred from donor to recipient
During a lytic infection, the
enzymes responsible for
packaging viral DNA into the
bacteriophage sometimes
accidentally package host DNA.
This DNA cannot replicate, it
can undergo genetic
recombination with the DNA of
the new host.
Specialized Transduction
the DNA of lambda is inserted into the host DNA
at the site adjacent to the galactose genes
On induction, Under rare conditions, the phage
genome is excised incorrectly
A portion of host DNA is exchanged for phage
DNA, called lambda dgal ( dgal means
"defective galactose“ )
Phage synthesis is completed
Cell lyses and releases defective phage
capable of transducing galactose genes
Transfection
Bacteria can be transformed with DNA extracted
from a bacterial virus rather than from another
bacterium, a process known as transfection.
Transformation
Transduction
Conjugation
8.6 Transposons and Insertion
Sequences
• Transposition: the process by which gene moves
from one place to another in the genome.
• Transposable elements: transposition of genes is
linked to the presence of special genetic elements
called transposable elements.
• Three types of transposable elements in bacteria:
– Insertion sequences (IS)
– Transposons (Tn)
– Some special viruses (such as Mu)
Three types of transposable elements in bacteria
• Insertion sequences (IS): about 1000 nucleotides,
carry only information to move them to new
location (IS1, IS2 and IS3).
• Transposons (Tn): larger than IS, carry genes,
such as drug resistance markers and other
selectable genes.
• Some special viruses (such as Mu)
Insertion of a transposable
element generates a duplication
Both IS and Tn have short
inverted terminal repeats (IR)
at the ends of their DNA, IR are
involved in the transposition
process
How is the targeted
sequence duplicated?
Transposon mutagenesis
• Insertion of transposon within a gene leads to
mutation.
• Transposon with antibiotic-resistant marker can be
used for selection purposes.
• Two tranposons widely used: Tn 5 (neomycin and
kanamycin resistance), Tn10 (tetracycline resistance).
Invertible DNA and the phenomenon of phase variation
Salmonella
flagella
synthesis
When a DNA segment is oriented in one direction, a particular
gene is expressed. Whereas when it is oriented in the opposite
direction, a different gene is expressed.
Questions
for
Microbial
Genetics
Describe as much as you know about plasmids.
•
• What is the difference between a plasmid and an episome?
• What are Hfr strain? F+ or F-, or F’ strain?
• Draw the F plasmid and describe functions of various DNA
regions.
• Why is it said that conjugative plasmid contributes to
evolution?
• How many types of plasmids and their functions you have
learned?
• Schematically describe R100 plasmid and its functions.
• How do R plasmids inactivate antibiotics?
• What is an engineered plasmid?
• What points do F plasmid provide to its host?
• How to detect genetic recombination. Please cite one
example.
• How is bacterial genome mapped? What are the three types
of transposable elements?
• Explain transposon mutagenesis and its possible application.
• Give an example to explain conversible DNA and phase
variation.