Transcript Recombination, Bacteriophages, and Horizontal Gene Transfer

Horizontal Gene Transfer and Genetic
Engineering
Conjugation, Transformation, and
Transduction
Genetics
Gene Transfer
• Refers to the movement of genetic
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information between organisms
When genes are transferred between two
bateria or a bacteria and a virus it involves a
combination of the DNA from two different
sources.
This is referred to as recombination
This type of transfer is referred to as
horizontal( lateral) gene transfer
Horizontal Gene Transfer
• Horizontal gene transfer is a driving
force in the development of drug
resistance in bacteria
• This type of transfer is different from
the transmission of genetic
chracteristics from one generation to
generation vertically
Gene transfer can occur
between bacteria and plants
• Agrobacterium tumefaciens lives in the soil
• It is able to transfer a plasmid from its cells into
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roots or stems through a scratch or injury to the
plant tissue
The plasmid integrates in host DNA and affects the
host to cause the growth of tumors called CROWN
GALLS
Gene transfer from Bacteria
to plant
Gene transfer can occur
between viruses and animals
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SV 40
Simian virus
Is a DNA virus
Transforms or alters
DNA and causes
cancer
Used to study
cancer and HIV
BCTERIAL CONJUGATION
Bacterial Conjugation
• transfer of DNA
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by direct cell to
cell contact
Contact with the
pili
discovered 1946
by Lederberg and
Tatum
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F
x
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F
Mating
• F+ = donor( contains the plasmid with the gene
for conjugation)
– This is referred to as the F factor or Fertility
Factor
• F– = recipient
– does not contain F factor
• F factor replicated by rolling-circle
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mechanism and duplicate is transferred
ACROSS the pilus from the + to the recipients usually become F+ after it receives a
copy of the DNA
donor remains F+
Gene transfer and recombination
• Genes are transferred in a linear
manner
• The F factor integrates into
chromosomes at different points
Mating
• When two strains were mixed
• There were incubated.
• At intervals of 5 minutes, samples were taken
of the F- cells
• The cells were centrifuged so that they would
know which genes were transferred.
• The distance between genes was measured by
the time that it took for the genes to be
transferred.
• During the first five minutes, the strains
were mixed there was no recombination
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F
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F
• In its
mating
extrachromosomal
state the factor has
a molecular weight
of approximately 62
kb
Conjugative Proteins
• Key players are the proteins that
initiate the physical transfer of ssDNA,
the conjugative initiator proteins
• They nick the DNA and open it to begin
the transfer
• Working in conjunction with the
helicases they facilitate the transfer of
ss RNA to the F- cell
- Formation of Hfr
Hfr - high frequency of
recombination
DNA Transformation
• Uptake of naked DNA molecule from
the environment and incorporation into
recipient in a heritable form
• Competent cell
– capable of taking up DNA
• May be important route of genetic
exchange in nature
Transformation
• Uptake of DNA can
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only occur at a
certain cell density
Cells need to be in
the log phase of
growth
A competence factor
is required for the
uptake of DNA from
the environment
Streptococcus pneumoniae
DNA binding
protein
competence-specific
protein
nuclease – nicks and degrades one
strand
Bacteria and transformation
• Not all bacteria can
be transformed in
nature
• Streptococcus
pneumonia,
Haemophilus
influenza, and
Neisseria gonorrhea
Lab protocol
Genetic recombination and
transformation in the
laboratory
• Plasmids are designed to
contain genes of
interest
• Transformation done in
laboratory with species
that are not normally
competent (E. coli)
• Variety of techniques
used to make cells
temporarily competent
– calcium chloride
treatment
• makes cells more
permeable to DNA
Cloning vectors
pAmp
pGlo and transformation
Microbial Genetics
Bacteriophages
Horizontal gene transfer
• Clearly this plays a central role in the
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diversity of E. coli
The greatest contributors are the
bacteriophages
Among the 18 prophage remnants on O157 –
12 resemble lambda phage
They all contain a variety of deletions and or
insertions
Some of the phages are so similar that they
contain a 20 kb segment tat is identical.
Recombinant phages
• It is believed that the phages have undergone
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recombination and diversification
They have been a major force in developing
resistance and pathogenicity in bacteria such
as E. coli and Streptococcus pyogenes
Recombination could occur with in a single cell
It could occur as the result of recombination
Bacteriophages
Bacteriophages
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Bacterial viruses
Obligate intracellular parasites
Inject themselves into a host bacterial cell
Take over the host machinery and utilize it for
protein synthesis and replication
T- 4 Bacteriophage
• Ds DNA virus
• 168, 800 base pairs
• Phage life cycles studied by Luria and
Delbruck
Bacteriophage structure
Bacteriophage structure(con)
• Most bacteriophages have tails
• The size of the tail varies.
• It is a tube through which the nucleic acid is
injected as a result of attachment of the
bacteriophage to the host bacterium
• In the more complex phages the tail is
surrounded by a contractile sheath for
injection of the nucleic acids
Bacteriophage structure
• Many bacteriophages have a base plate
and tail fibers
• Some have icosahedral capsids
• M13 has a helical capsid
Capsid
• The base plate requires 12 protein products
• The head or capsid requires 10 genes
• The capside requires scaffolding proteins for
assembly
• DNA packaging a mysterious process
• Many phages lyse their host cells at the end
of the intracellular phase
T even phages
Luria and Delbruck
• Four distinct periods in the release of phages from
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host cells
Latent period- follows the addition of phage( no
release of virions)
Eclipse period – virions were detectable before
infection and are now hidden or eclipsed
Rise or burst period – Host cells rapidly burst and
release viruses
The total number of phages released can be
determined by the burst size – the number of viruses
produced per
infected cell
General Steps
Steps in the life cycle
• Adsorption of the virus to the host
• This is mediated by tail fibers or some analagous
structure
• When the tail fibers make contact, the base plate
settles to the surface
• This connection which is maintianed by electrostatic
attraction and the ions Mg++ and Ca++
Attachment
• There is host specificity in the attachment
and adsorption of the bacteriophage
• There are receptors for the attachment.
They vary from bacteria to bacteria
• The receptors are on the bacteria for other
purposes: the bacteriophages evolved to
utilize them for their invasion
T even phages - Injection
• The phage sheath shortens from 24
rings to 12 rings
• The sheath becomes shorter and wider
• This causes the central tube to push
through the bacterial cell wall
Gp5
• The baseplate
contains the protein
gp5 with lysozyme
activity which made
aid in the
penetration of the
host
Early Genes
• E. coli RNA polymerase starts transcribing
genes( phage genes) within minutes of
entering the bacterial cell
• The early m RNA direct the synthesis of
proteins and enzymes that are needed for
hostile tack over
• Some early virus specific enzymes degrade
host DNA to nucleotides so that virus DNA
synthesis can commence
Late mRNA
• Phage structural structural proteins
• Proteins that help with phage assembly
• Proteins involved in cell lysis and release
Release
• When the bacteriophages are released
from the bacteria they can lyse the
bacterial cell and break it open
• They can be released through the cell
membrane
Irreversible attachment
• The attachment of the tail fibers to
the bacterium is a weak attachment
• The attachment of the bacteriophage is
also accompanied by a stronger
interaction usually by the base plate
Sheath contraction
• The irreversible binding results in the
sheath contraction
Injection
• When the irreversible attachment has
been made and the sheath contracts,
the nucleic acid passes through the tail
and enters the cytoplasm
Phage Multiplication Cycle – Lytic
phages
• Lytic phages or virulent phages enter the
bacterial cell, complete protein synthesis,
nucleic acid replication, and then cause lysis
of the bacterial cell when the assembly of the
particles has been completed.
Eclipse Period
• The bacteriophages may be seen inside or outside of
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the bacterial cells
The phages take over the cell’s machinery and phage
specific mRNA’s are made
Early mRNA’s are generally needed for DNA
replication
Later mRNA’s are required for the synthesis of phage
proteins
Intracellular accumulation phase
• The bacteriophage sub units accumulate
in the cytoplasm of the bacterial cell
and are assembled
Lysis or Release Phase
• A lysis protein is released
• The bacterial cell breaks open
• The viruses escape to invade other
bacterial cells
Plaque assay
• Phage infection and lysis can easily be
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detected in bacterial cultures grown on agar
plates
Typically bacterial cells are cultured in high
concentrations on the surface of an agar
plate
This produces a “ bacterial lawn”
Phage infection and lysis can be seen as a
clear area on the plate. As phage are
released they invade neighboring cells and
produce a clear area
Plaque assay
Lambda and Plaques
• The plaque produced by Lambda had a
different appearance on the Petri Dish.
• It is considered to be turbid rather than
clear
• The turbidiy is the result of the growth of
phage immune lysogens in the plaque
• The agar surface contains a ratio of about a
phage /107 bacteria
MOI
• Average number of phages /bacterium
• After several lytic cycles the MOI gets
higher due to the release of phage
particles
Transduction
• Transfer of bacterial genes by viruses
• Virulent bacteriophages
– reproduce using lytic life cycle
• Temperate bacteriophages
– reproduce using lysogenic life cycle
Generalized transduction
• http://www.cat.cc.md.us/courses/bio141
/lecguide/unit4/genetics/recombination
/transduction/gentran.html
• http://www.cat.cc.md.us/courses/bio141
/lecguide/unit1/control/genrec/u4fg21a
.html
Generalized transduction
• E. coli phage P21 or P22.
• As a part of the lytic cycle, the phage cuts the
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bacterial DNA into fragments
This fragmentation prevents the expression of
bacterial genes
Nucleotides can be used to make phage DNA
Occasionally these DNA fragments are about the
same size as phage DNA
They become mistakenly packaged into phage capsids
in place of phage DNA
Types of Lysogenic Cycle
• The most common type is the classic model of the
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Lambda phage
The DNA molecule is injected into a bacterium
In a short period of time, after a brief period of
transcription, an integration factor and a repressor
are synthesized
A phage DNA molecule typically a replica of the
injected molecules is inserted into the DNA
As the bacterium continue to grow and multiply and
the phage genes replicate as part of the bacterial
chromosome
Temperate
• A bacteriophage that can exist as a
lytic or lysogenic phage is referred to
as a temperate phage
• A bacterium containing a full set of
phage genes is a lysogen
• The process of infecting a bacterial
culture with a temperate phage is called
lysogenization
Immunization
• A bacterial cell or lysogen cannot be
reinfected by a phage of the same type
• This is resistance to superinfection is
called immunity
• More than 90% of the bacteriophages
are temperate
• These are unable to produce bursts
such as T4 and T7
Lysogenic Phage
Lambda Phage
• Temperate phage
• Alternate life cycle
• Ds DNA – linear then circularizes when it
enters the host
• 48,502 base pairs
• Molecular biology workhorse – because of its
life cycle
Genes
Lambda genes
• 46 genes have been identified
• 14 are non esswential to the lytic cycle
• Only 7 are nonessential to both the lytic
and lysogenic cycles
Life cycle of λ Phage
Latency
• Lysogenic conversion can lead to
virulence
• Botulism, cholera,and diptheria toxins
are encoded by prophages that convert
their host into a pathogenic bacterium
Terminology
• LEGEND
• att: an E.coli seqence for the "attachment" or integration of
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lambda's circular chromosome.
oriC: E.coli's origin of Chromosome replication (given here for
orientation only)
gal: E.coli's gene for galactose utilization
pe:prophage ends (site of integration)
cos: joined sticky ends of vegetative DNA; sometimes called ve
("vegetative ends")
int: gene for the enzyme integrase
c: gene for lambda repressor to maintain lysogeny
• Q: another gene concerned with lysogeny
• h: the last of the many capsomer genes.
Bacteriophages
Specialized transduction
Attachment site
• The E. coli chromosome contains one site at which
lambda integrates. The site, located between the gal
and bio operons, is called the attachment site and is
designated attB since it is the attachment site on the
bacterial chromosome.
• The site is only 30 bp in size and contains a conserved
central 15 bp region where the recombination
reaction will take place.
• The structure of the recombination site was
determined originally by genetic analyses and is
usually represented as BOB', where B and B'
represent the bacterial DNA on either side of the
conserved central element
Recombination site
• The bacteriophage recombination site - attP •
is more complex. It contains the identical
central 15 bp region as attB.
The overall structure can be represented as
POP'. However, the flanking sequences on
either side of attP are very important since
they contain the binding sites for a number of
other proteins which are required for the
recombination reaction. The P arm is 150 bp in
length and the P' arm is 90 bp in length.
Integration
• Integration of bacteriophage lambda requires one
phage-encoded protein - Int, which is the integrase and one bacterial protein - IHF, which is Integration
Host Factor.
• Both of these proteins bind to sites on the P and P'
arms of attP to form a complex in which the central
conserved 15 bp elements of attP and attB are
properly aligned.
• The integrase enzyme carries out all of the steps of
the recombination reaction, which includes a short 7
bp branch migration.
Normal Excision
Generalized Transduction
• Any part of bacterial genome can be
transferred
• Occurs during lytic cycle
• During viral assembly, fragments of
host DNA mistakenly packaged into
phage head
– generalized transducing particle
Generalized transduction
Specialized Transduction
• Also called restricted transduction
• carried out only by temperate phages
that have established lysogeny
• only specific portion of bacterial
genome is transferred
• occurs when prophage is incorrectly
excised
Specialized
transduction
Figure 13.20
Figure 13.20
Recombination and Genome
Mapping in Viruses
• viral genomes can also undergo recombination
events
• viral genomes can be mapped by determining
recombination frequencies
• physical maps of viral genomes can also be
constructed using other techniques
Specialized transduction
mapping
• provides distance of genes from viral
genome integration sites
• viral genome integration sites must first
be mapped by conjugation mapping
techniques
Recombination mapping
• recombination
frequency
determined
when cells
infected
simultaneously
with two
different
viruses
Figure 13.24