Chapter8 Microbial genetics
8.4 Plasmids
8.5 Genetic Conjugation,
Transformation,transduction
8.6 Transposons and Insertion Sequences
8.4 Plasmids
? 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.
Circular genetic elements that
reproduce autonomously and have
an extra-chromosomal existence:
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
(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 cell-
to-cell contact and a conjugative plasmid in the donor
cell
8.5 Three main processes of genetic recombination in
prokaryotes fragments of homologous DNA from a donor
chromosome are transferred to a recipient 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 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.
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.
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
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
Detection of
Genetic
Conjugation
Interrupted
Mating
? Mixing Hfr
and F- cells.
? Shake the
mixture
violently at
various time.
? Plate the
samples on
selective
medium for
recombinant
to grow.Mapping the
order of genes
Result of selected conjugation
Donor Recipient Molecules transferred Product
F+ F- F plasmid F+ Cell
Hfr F- Initiating segment of F
plasmid and variable
quantity of
chromosomal DNA
F- with variable
quantity of
chromosomal DNA
F+ F- F+ plasmid and some
chromosomal genes it
carries with it
F+ Cell with some
duplicate gene pairs,
one on chromosom,
one on plasmid
? 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,
? 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
? Natural transformation has been discovered so far only in certain
genera,Streptococcus,Bacillus,Thermoactinomytes,Haemophilus,
Neisseria,Moraxella,Acinetobacter,Azotobacter,Pseudoomonas
DNA transformation
(a) Binding of free DNA by a membrane-
bound 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.
The introduction of DNA into cells
by mixing the DNA and the cell
Transformed cell
The mechanism of bacterial transformation
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.
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 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.
Concept
Transduction
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
Generalizedtransduction
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.
Conjugation
Transformation
Transduction
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
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)
Both IS and Tn have short
inverted terminal repeats (IR)
at the ends of their DNA,IR are
involved in the transposition
process
Insertion of a transposable
element generates a duplication
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
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.
Salmonella
flagella
synthesis
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.
8.4 Plasmids
8.5 Genetic Conjugation,
Transformation,transduction
8.6 Transposons and Insertion Sequences
8.4 Plasmids
? 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.
Circular genetic elements that
reproduce autonomously and have
an extra-chromosomal existence:
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
(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 cell-
to-cell contact and a conjugative plasmid in the donor
cell
8.5 Three main processes of genetic recombination in
prokaryotes fragments of homologous DNA from a donor
chromosome are transferred to a recipient 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 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.
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.
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
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
Detection of
Genetic
Conjugation
Interrupted
Mating
? Mixing Hfr
and F- cells.
? Shake the
mixture
violently at
various time.
? Plate the
samples on
selective
medium for
recombinant
to grow.Mapping the
order of genes
Result of selected conjugation
Donor Recipient Molecules transferred Product
F+ F- F plasmid F+ Cell
Hfr F- Initiating segment of F
plasmid and variable
quantity of
chromosomal DNA
F- with variable
quantity of
chromosomal DNA
F+ F- F+ plasmid and some
chromosomal genes it
carries with it
F+ Cell with some
duplicate gene pairs,
one on chromosom,
one on plasmid
? 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,
? 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
? Natural transformation has been discovered so far only in certain
genera,Streptococcus,Bacillus,Thermoactinomytes,Haemophilus,
Neisseria,Moraxella,Acinetobacter,Azotobacter,Pseudoomonas
DNA transformation
(a) Binding of free DNA by a membrane-
bound 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.
The introduction of DNA into cells
by mixing the DNA and the cell
Transformed cell
The mechanism of bacterial transformation
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.
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 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.
Concept
Transduction
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
Generalizedtransduction
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.
Conjugation
Transformation
Transduction
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
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)
Both IS and Tn have short
inverted terminal repeats (IR)
at the ends of their DNA,IR are
involved in the transposition
process
Insertion of a transposable
element generates a duplication
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
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.
Salmonella
flagella
synthesis
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.
